Molecular Nutrition and Mitochondria: Metabolic Deficits, Whole-Diet Interventions, and Targeted Nutraceuticals 0323902561, 9780323902564

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Molecular Nutrition and Mitochondria Metabolic Deficits, Whole-Diet Interventions, and Targeted Nutraceuticals

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Molecular Nutrition and Mitochondria Metabolic Deficits, Whole-Diet Interventions, and Targeted Nutraceuticals

Edited by

Sergej M. Ostojic Department of Nutrition and Public Health, University of Agder, Kristiansand, Norway

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2023 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-323-90256-4 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

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Contents List of contributors .................................................................................................xix Preface ...................................................................................................................xxv Acknowledgments .............................................................................................. xxvii

Section 1 Mitochondria as a target in experimental and clinical nutrition CHAPTER 1 Targeting mitochondrial dysfunction with nutrients: challenges and opportunities....................................... 3 Marie-Ce´line Frantz 1.1 Introduction ....................................................................................3 1.2 Diseases involving mitochondrial dysfunction..............................3 1.3 Targeting mitochondrial dysfunction with nutrients .....................5 1.3.1 Vitamins and cofactors ....................................................... 5 1.3.2 Endogenous antioxidants .................................................. 31 1.3.3 Endogenous metabolites and transporters ........................ 33 1.3.4 Dietary fatty acids............................................................. 34 1.3.5 Carotenoids........................................................................ 35 1.3.6 Ginsenosides...................................................................... 36 1.3.7 Polyphenols ....................................................................... 37 1.3.8 Isothiocyanates .................................................................. 42 1.4 Challenges and limitations of using nutrients to target mitochondrial dysfunction............................................................43 1.5 Topical use of nutrients for dermo-cosmetic applications ..........45 1.6 Conclusion and perspectives ........................................................53 References.................................................................................... 54

CHAPTER 2 Mitochondrion at the crossroads between nutrients and the epigenome...................................... 71 Laura Bordoni and Domenico Sergi 2.1 Introduction ..................................................................................71 2.2 Epigenetic modifications..............................................................72 2.2.1 DNA methylation.............................................................. 72 2.2.2 Histone modifications and chromatin remodeling ........... 73 2.2.3 Noncoding RNA ............................................................... 73 2.3 Mitochondrial epigenetics and mito-epigenetics .........................74

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2.3.1 Mitochondrial epigenetics: how mitochondria affect epigenetic pathways .......................................................... 74 2.3.2 Mito-epigenetics: epigenetic regulations in the mitochondrial genome ...................................................... 76 2.4 Impact of diet on the epigenome: the mediation of mitochondria ............................................................................78 2.4.1 How diet modulates the epigenome ................................. 78 2.4.2 Focus on diet-related metabolic connections between mitochondria and cytoplasm able to affect the epigenome ......................................................................... 80 2.4.3 Effects of nutrients and diet on mitochondrial epigenetics and mito-epigenetics...................................... 82 2.5 Conclusions ..................................................................................84 References.................................................................................... 84

CHAPTER 3 Nutritional assessment and malnutrition in adult patients with mitochondrial disease.......................... 93 Heidi Zweers 3.1 Introduction ..................................................................................93 3.1.1 Gastro intestinal problems and BMI ................................ 95 3.1.2 Food intake........................................................................ 95 3.1.3 Prevalence of malnutrition in mitochondrial diseases ..... 95 3.1.4 The optimal method for nutritional assessment in adult mitochondrial diseases patients ............................... 96 3.1.5 Sex differences.................................................................. 99 3.2 Nutritional assessment and dietary interventions ........................99 3.3 Conclusion ..................................................................................100 References.................................................................................. 101

CHAPTER 4 Therapeutic potential and metabolic impact of alternative respiratory chain enzymes .................... 105 Sina Saari Introduction ................................................................................105 Alternative oxidase.....................................................................107 Alternative NADH dehydrogenase ............................................108 Transgenic models of alternative respiratory chain enzymes ...109 4.4.1 Mammalian cell models.................................................. 109 4.4.2 Drosophila melanogaster ................................................ 112 4.4.3 Rodent models................................................................. 113 4.5 Metabolic impact of alternative enzymes..................................115 4.1 4.2 4.3 4.4

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4.5.1 Nutrition .......................................................................... 115 4.5.2 Reactive oxygen species ................................................. 118 4.6 Therapeutic potential of alternative enzymes in mitochondria-related diseases ....................................................120 References.................................................................................. 121

Section 2 Essential nutrients in mitochondrial nutrition CHAPTER 5 Aging, mitochondrial dysfunctions, and vitamin E ...... 131 Gaetana Napolitano, Gianluca Fasciolo and Paola Venditti 5.1 Introduction ................................................................................131 5.1.1 Mitochondria, reactive oxygen species and the free radical theory of aging.................................................... 133 5.1.2 Mitocondrial DNA and aging ......................................... 135 5.1.3 Mitochondrial dynamics, mitophagy and aging ............. 136 5.1.4 Retrograde signaling: from mitochondria to nucleus..... 140 5.1.5 Mitochondria and the “inflammaging”........................... 142 5.2 Vitamin E ...................................................................................144 5.2.1 Vitamin E and antioxidant capacity ............................... 145 5.2.2 Uptake and cellular distribution of vitamin E................ 145 5.2.3 Vitamin E functions in mitochondria ............................. 146 5.2.4 Vitamin E, mitochondria, and aging .............................. 147 5.3 The necessity for an alternative theory......................................149 5.3.1 ROS signaling, aging, and lifespan ................................ 150 5.3.2 “The gradual ROS response hypothesis” ....................... 151 5.4 Concluding remarks ...................................................................152 References.................................................................................. 152

CHAPTER 6 The role of B vitamins in protecting mitochondrial function.............................................. 167 Sandip Mukherjee, Oly Banerjee and Siddhartha Singh 6.1 Introduction ................................................................................167 6.2 B vitamins and mitochondrial metabolism................................168 6.2.1 Vitamin B1 (thiamine) .................................................... 168 6.2.2 Vitamin B2 (riboflavin) .................................................. 168 6.2.3 Vitamin B3 (niacin) ........................................................ 170 6.2.4 Vitamin B5 (pantothenic acid) ....................................... 170 6.2.5 Vitamin B6 (pyridoxal phosphate) ................................. 171 6.2.6 Vitamin B8/B7 (biotin)................................................... 171

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6.2.7 Vitamin B11/B9 (folate) ................................................. 172 6.2.8 Vitamin B12 (cobalamin) ............................................... 173 6.3 Oxidative stress and mitochondrial toxicity: role of B vitamins...173 6.4 Role of B vitamins as mitochondrial nutrients..........................178 6.5 Mitochondrial signaling metabolites: impact of B vitamins.....181 6.5.1 B vitamins and HIF1 signaling....................................... 181 6.5.2 Impacts of B vitamin on methylation of histone and DNA ................................................................................ 182 6.5.3 B vitamin: as regulator of histone acetylation ............... 184 References.................................................................................. 185

CHAPTER 7 Analysis of the mitochondrial status of murine neuronal N2a cells treated with resveratrol and synthetic isomeric resveratrol analogs: aza-stilbenes............................................................. 195

7.1 7.2

7.3 7.4

Mohamed Ksila, Imen Ghzaiel, Aline Yammine, Thomas Nury, Anne Vejux, Dominique Vervandier-Fasseur, Norbert Latruffe, Emmanuelle Prost-Camus, Smail Meziane, Olfa Masmoudi-Kouki, Amira Zarrouk, Taoufik Ghrairi and Ge´rard Lizard Introduction ................................................................................195 Material and methods.................................................................198 7.2.1 Synthesis of aza-stilbenes I to VII ................................. 198 7.2.2 Cell culture and treatments............................................. 198 7.2.3 Measurement of cell viability with the fluorescein diacetate assay................................................................. 200 7.2.4 Evaluation of adherent cells with crystal violet staining assay .................................................................. 200 7.2.5 Flow cytometric quantification of cells with depolarized mitochondria with DiOC6(3) ...................... 201 7.2.6 Flow cytometric measurement of mitochondrial reactive oxygen species production with MitoSOX-Red ......................................................... 201 7.2.7 Statistical analysis ........................................................... 202 Results ........................................................................................202 Discussion and conclusion .........................................................207 Acknowledgments ..................................................................... 208 Conflict of interest..................................................................... 208 References.................................................................................. 208

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CHAPTER 8 Dietary eicosapentaenoic acid and docosahexaenoic acid for mitochondrial biogenesis and dynamics.......... 213 Sebastian Jannas-Vela and Mauricio Castro-Sepulveda 8.1 Introduction ................................................................................213 8.2 Mitochondrial biogenesis and dynamics....................................214 8.2.1 Mitochondrial biogenesis................................................ 214 8.2.2 Mitochondrial dynamics ................................................. 215 8.3 Effect of n-3 polyunsaturated fatty acids on mitochondrial biogenesis and dynamics............................................................218 8.4 Conclusion ..................................................................................220 References.................................................................................. 221

CHAPTER 9 Vitamin C and mitochondrial function in health and exercise.............................................................. 225 9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9

Michael J. Gonzalez, Jorge R. Miranda-Massari and Jose Olalde Vitamin C (ascorbic acid, ascorbate).........................................225 Mitochondria ..............................................................................226 Mitochondria structure and roles ...............................................226 Vitamin C and the mitochondria ...............................................227 Mitochondriopathies...................................................................228 Role of vitamin C in mitochondrial disease..............................229 Safety of vitamin C ....................................................................231 Vitamin C and exercise (physiology/inflammation/recuperation)....................................231 Vitamin C as an ergogenic factor (performance)......................234 References.................................................................................. 236

CHAPTER 10 Roles of dietary fiber and gut microbial metabolites short-chain fatty acids in regulating mitochondrial function in central nervous system ......................................................... 243 10.1 10.2 10.3 10.4

Huajun Pan and Zhigang Liu Introduction ................................................................................243 Gut microbiota and short-chain fatty acids ...............................243 Short-chain fatty acids regulate peripheral organizational activities......................................................................................244 Effects of short-chain fatty acids on modulating the central nervous system function.............................................................246 10.4.1 Short-chain fatty acids influence cognitive and psychological function on mitochondria in the brain....... 247

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10.4.2 Short-chain fatty acids influence appetitive function on mitochondria in the brain ........................................ 248 References.................................................................................. 249

Section 3 Dietary bioactive compounds and mitochondrial function CHAPTER 11 Mitochondria-targeted antioxidants: coenzyme Q10, mito-Q and beyond .......................... 255 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8

11.9

11.10 11.11

Guillermo Lo´pez-Lluch Introduction ................................................................................255 Importance of coenzyme Q in mitochondria.............................256 CoQ10 prevents oxidative damage .............................................257 Structure of coenzyme Q and mitochondrial-targeted coenzyme Q-related compounds................................................258 Idebenone reduces reactive oxygen species levels and bypasses complex I-deficiency ..................................................260 MitoQ a strong antioxidant that protects against apoptosis and induces mitophagy...............................................................261 Pharmacokinetics of mitochondrial-targeted antioxidant..........262 Therapeutic use of idebenone ....................................................264 11.8.1 Therapeutic use of idebenone in Friedreich ataxia ...... 264 11.8.2 Idebenone treatment of leber hereditary optic neuropathy and other neuropathic diseases .................. 265 11.8.3 Therapeutic use of idebenone in other oxidative-damage related diseases................................ 266 Therapeutic activity of MitoQ ...................................................267 11.9.1 MitoQ use in inflammation and immune response ...... 267 11.9.2 MitoQ as a treatment in neurodegenerative diseases ........................................... 269 11.9.3 Rare diseases ................................................................. 272 11.9.4 Ischemia/reperfusion and organ transplantation........... 273 11.9.5 Liver fibrosis ................................................................. 273 11.9.6 Metabolic syndrome and related diseases .................... 273 11.9.7 Therapeutic potential of MitoQ in the treatment of cardiovascular diseases ................................................. 275 11.9.8 Other uses of MitoQ ..................................................... 276 Other mitochondria-targeted compounds ..................................277 Conclusions ................................................................................279 References.................................................................................. 279

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CHAPTER 12 Flavonoids, mitochondrial enzymes and heart protection ........................................................ 303 12.1 12.2 12.3 12.4

12.5 12.6 12.7 12.8

12.9

Sneha Sivadas, Nandakumar Selvasudha, Pooja Prasad and Hannah R. Vasanthi Introduction ................................................................................303 Mitochondria and mitochondrial enzymes in cellular functions .....................................................................................304 Mitochondria as an essential organelle for cardiovascular health..................................................................305 Role of mitochondrial enzymes in cardiomyocytes ..................306 12.4.1 Mitochondrial enzymes for scavenging reactive oxygen species .............................................................. 306 12.4.2 Mitochondrial enzymes for apoptosis in cardiomyocytes ............................................................. 308 12.4.3 Mitochondrial enzymes in autophagy .......................... 309 Structure and function of dietary flavonoids.............................310 Pharmacokinetic profile (ADME) of flavonoids .......................311 Structure activity relationship of flavonoids for cardioprotective activity.............................................................314 Biological action of flavonoids in cardioprotection..................316 12.8.1 Antiplatelet activity....................................................... 318 12.8.2 Antioxidant activity ...................................................... 318 12.8.3 Anti-inflammatory activity ........................................... 319 12.8.4 Antihypertensive activity .............................................. 320 12.8.5 Antiatherogenic activity................................................ 321 12.8.6 Hypoxia, necrotic and apoptotic activity...................... 321 12.8.7 Mitophagy ..................................................................... 323 Concluding remarks ...................................................................323 References.................................................................................. 324

CHAPTER 13 Tea polyphenols stimulate mt bioenergetics in cardiometabolic diseases ........................................ 333 13.1 13.2 13.3 13.4 13.5 13.6 13.7 13.8

Ravichandran Srividhya An introduction to cardiometabolic diseases.............................333 Structure and bioenergetics of mitochondria.............................334 Mitochondria and its role in metabolism...................................337 Mitochondria and metabolic stress ............................................339 Mitochondrial fission and fusion ...............................................340 Polyphenols as functional food..................................................341 Tea and its health benefits .........................................................344 Cytoprotective actions of green tea polyphenols ......................346

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13.9 Effects of nutraceuticals on cardiometabolic disorders.............348 13.10 Molecular mechanisms of flavonoids in cardiometabolic diseases .......................................................................................349 13.11 Molecular mechanisms of action of tea polyphenols................350 References.................................................................................. 353

CHAPTER 14 A review of quercetin delivery through nanovectors: cellular and mitochondrial effects on noncommunicable diseases ..................................... 363 14.1 14.2 14.3

14.4 14.5

Omar Lozano, Diego Solis-Castan˜ol, Sara Cantu´-Casas, Paolo I. Mendoza Muraira and Gerardo Garcı´a-Rivas Introduction ................................................................................363 Quercetin metabolism, biodistribution and pharmacokinetics........................................................................364 Mechanism of protection of quercetin in noncommunicable diseases ........................................................365 14.3.1 Quercetin as an antioxidant compound ........................ 365 14.3.2 Quercetin as an anticancer agent .................................. 369 Nanomaterials for quercetin encapsulation ...............................372 Conclusions ................................................................................376 Acknowledgments ..................................................................... 376 References.................................................................................. 376

CHAPTER 15 Creatine monohydrate for mitochondrial nutrition....... 383 Maher A. Kamel, Yousra Y. Moussa and Mennatallah A. Gowayed 15.1 Creatine monohydrate ................................................................383 15.1.1 Structure ........................................................................ 383 15.1.2 De novo synthesis of creatine....................................... 384 15.1.3 Supplementation form................................................... 385 15.1.4 Tissue distribution of creatine ...................................... 385 15.1.5 Catabolism..................................................................... 386 15.2 Creatine in cellular and mitochondrial bioenergetics................387 15.2.1 Creatine kinase isoenzymes .......................................... 388 15.2.2 The phosphocreatine “shuttle” system in cell energy homeostasis ....................................................... 389 15.3 Creatine/mitochondrial creatine kinase system in health and disease..................................................................................389 15.3.1 In cardiac and skeletal muscles of athletes .................. 389 15.3.2 In muscle disorders ....................................................... 393 15.3.3 In pregnancy and gestation ........................................... 394

Contents

15.3.4 Creatine and central nervous system mitochondria ..... 395 15.3.5 Creatine and adipocyte-specific functions of the mitochondria ........................................................... 401 15.4 A promising future .....................................................................404 References.................................................................................. 404

CHAPTER 16 Arginine and neuroprotection: a focus on stroke.... 417 Yasutoshi Koga Introduction ................................................................................417 Mitochondrial angiopathy in MELAS .......................................418 Endothelial dysfunction in MELAS ..........................................419 Neuroimaging of stroke-like episodes in MELAS ....................420 Clinical study of L-arginine in MELAS....................................421 Superacute intervention by L-arginine ......................................422 Therapeutic regimen of L-arginine for MELAS .......................422 Contraindication in the treatment of MELAS ...........................425 Concluding remarks ...................................................................426 Applications to other neurological conditions...........................427 Key facts of arginine and neuroprotection: a focus on stroke ........................................................................427 16.11.1 Key fact of neuroprotection in MELAS..................... 427 16.12 Summary points..........................................................................427 References.................................................................................. 428 16.1 16.2 16.3 16.4 16.5 16.6 16.7 16.8 16.9 16.10 16.11

CHAPTER 17 Nutraceuticals for targeting NAD1 to restore mitochondrial function.............................................. 433 Antje Garten and Gareth G. Lavery 17.1 Nicotinamide adenine dinucleotide as redox cofactor and signaling molecule in mitochondria...........................................433 17.2 Cellular and mitochondrial nicotinamide adenine dinucleotide metabolism ............................................................434 17.3 Nicotinamide adenine dinucleotide and mitochondrial function.......................................................................................436 17.4 Nicotinamide adenine dinucleotide supplementation in human diseases ...........................................................................440 17.5 Conclusion ..................................................................................448 References.................................................................................. 449

CHAPTER 18 Curcumin for protecting mitochondria and downregulating inflammation ................................... 461 Ahmad Salimi, Zhaleh Jamali and Leila Rezaie Shirmard 18.1 Introduction ................................................................................461

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18.2 18.3 18.4 18.5 18.6 18.7 18.8 18.9 18.10 18.11 18.12 18.13 18.14

Inflammation and oxidative stress .............................................464 Mitochondria and inflammation ................................................465 Mitochondria and oxidative stress .............................................467 Mitochondrial inflammation and oxidative stress in inflammatory-related diseases....................................................470 Curcumin as antioxidant and antiinflammatory agent ..............475 Mitochondrial targeting for the reduction of oxidative stress and inflammation .............................................................479 Curcumin as a direct mitochondrial reactive oxygen species scavenger .......................................................................480 Curcumin enhances mitochondrial antioxidants........................482 Curcumin activates the Nrf2 signaling pathway and protects mitochondrial damage and oxidant generation............482 Targeting of mitochondrial uncoupling proteins by curcumin................................................................................484 Targeting of mitochondrial sirtuins by curcumin......................485 Targeting of mitochondrial p66shc by curcumin ......................487 Conclusion ..................................................................................487 Conflict of interest..................................................................... 488 References.................................................................................. 488

CHAPTER 19 Dihydrogen as an innovative nutraceutical for mitochondrial viability.............................................. 501 19.1 19.2 19.3 19.4 19.5 19.6 19.7

Sergej M. Ostojic Introduction ................................................................................501 Dietary sources of molecular hydrogen.....................................501 Hydrogen-rich water and mitochondrial function .....................502 Other dietary and complementary interventions with hydrogen .....................................................................................505 Dihydrogen and mitochondria: molecular mechanisms ............506 Open questions and future research...........................................507 Conclusion ..................................................................................508 References.................................................................................. 508

CHAPTER 20 Fucoxantin and mitochondrial uncoupling protein 1 in obesity .................................................. 513 Chunhong Yan 20.1 Three types of adipocytes ..........................................................514 20.2 The importance of uncoupling protein 1 in regulating energy homeostasis ....................................................................515

Contents

20.3 Fucoxanthin and uncoupling protein 1 ......................................516 References.................................................................................. 518

CHAPTER 21 Rice bran extract for the prevention of mitochondrial dysfunction ........................................ 521 21.1 21.2 21.3 21.4

21.5

Nancy Saji, Boris Budiono, Nidhish Francis, Christopher Blanchard and Abishek Santhakumar Introduction ................................................................................521 Role of mitochondrial function in disease.................................522 Rice bran extracts and the mitochondria ...................................522 Health properties of rice bran constituents associated with mitochondrial function.......................................................525 21.4.1 Proteins, nonproteogenic amino acids and derivatives .............................................................. 525 21.4.2 Fats and oils .................................................................. 525 21.4.3 Carbohydrates................................................................ 526 21.4.4 Fiber .............................................................................. 527 21.4.5 Small molecule antioxidants......................................... 527 21.4.6 Plant-based pigments and organic compounds ............ 528 21.4.7 Mitochondria-specific enzyme mimetics from food, administered either as monocomponent formulas or mitochondria-specific cocktails with synergetic potential......................................................................... 528 Conclusion ..................................................................................530 References.................................................................................. 530

CHAPTER 22 Silymarin as a vitagene modulator: effects on mitochondria integrity in stress conditions............. 535 Peter F. Surai 22.1 Introduction ................................................................................535 22.2 An integrated antioxidant defense system .................................535 22.3 Mitochondria as an important source of reactive oxygen species............................................................................537 22.4 Antioxidant properties of silymarin...........................................539 22.5 Protective effects of silymarin on mitochondria .......................539 22.5.1 In vitro evidence ........................................................... 539 22.5.2 In vivo evidence............................................................ 543 22.6 Effect of SM on vitagene expression.........................................546 22.7 Application of silymarin in poultry ...........................................547 22.8 Conclusions ................................................................................549 References.................................................................................. 551

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CHAPTER 23 Buckwheat trypsin inhibitors: novel nutraceuticals for mitochondrial homeostasis ........ 561 Si-Quan Li 23.1 Introduction ................................................................................561 23.2 Roles of mitochondrial proteases in maintaining mitochondrial homeostasis and deliberate regulation by protease inhibitors .................................................................562 23.2.1 Mitochondrial metabolisms and homeostasis............... 562 23.2.2 Proteases and their inhibitors are critical for health and mitochondrial homeostasis.......................... 564 23.3 Buckwheat, health benefits and presence of trypsin inhibitors ........................................................................567 23.3.1 Buckwheat as a food staple in some regions and its global presence as a functional food ................ 567 23.3.2 Potential health benefits from consuming buckwheat foods ........................................................... 567 23.3.3 Presence of buckwheat trypsin inhibitors, characteristics and physiological roles ......................... 568 23.4 Roles of mitochondrial homeostasis in healthy aging and improvement by presence of recombinant buckwheat trypsin inhibitor .......................................................570 23.4.1 Roles of mitochondrial homeostasis in healthy aging ................................................................. 570 23.4.2 Buckwheat trypsin inhibitor and recombinant buckwheat trypsin inhibitors: properties, functionality and their potential roles in maintaining stability of mitochondrial homeostasis .... 574 23.4.3 Potential future trends in research and studies............. 580 References.................................................................................. 580

SECTION 4 Whole-diet interventions and mitochondrial function CHAPTER 24 Diet restriction-induced mitochondrial signaling and healthy aging ..................................... 587 Meredith Pinkerton and Antoni Barrientos 24.1 Mitochondrial pathways induced by caloric restriction ............587 24.1.1 Caloric restriction, inhibition of insulin/insulin-like growth factor-1 signaling insulin-like growth factor 1 pathway, and mitochondria ............................. 589

Contents

24.1.2 Caloric restriction, inhibition of target of rapamycin signaling, and mitochondria ....................... 590 24.1.3 Caloric restriction, sirtuin activation, and mitochondria ................................................................. 592 24.1.4 Caloric restriction, AMP-activated protein kinase activation, and mitochondria ........................................ 594 24.1.5 Caloric restriction, PGC-1α activation, and mitochondria ................................................................. 595 24.1.6 Caloric restriction and mitochondrial signaling to the cell ........................................................................... 595 24.1.7 Mitochondria-mediated tissue-specific effects of caloric restriction .......................................................... 597 24.1.8 Effects of calorie restriction in mitochondrial biogenesis and energy metabolism in nonhuman primates and healthy humans ....................................... 600 24.2 Mitochondrial mechanisms underlying health span extension by popular restrictive diet regimes in mammals.......601 24.2.1 Ketogenic diet ............................................................... 602 24.2.2 Macronutrient restriction .............................................. 603 24.2.3 Intermittent fasting........................................................ 604 24.3 Mitochondrial pathways activated by caloric restriction mimetics .....................................................................................604 24.3.1 Multifunctional compounds: polyphenols and polyamines .................................................................... 605 24.3.2 NAD1 precursors .......................................................... 609 24.3.3 AMP-activated protein kinase agonists ........................ 610 24.3.4 Mammalian target of rapamycin inhibitors.................. 611 24.3.5 Mitochondrial uncouplers ............................................. 612 24.4 Concluding remarks ...................................................................613 Funding ...................................................................................... 613 References.................................................................................. 613

CHAPTER 25 Rejuvenation of mitochondrial function by time-controlled fasting ............................................. 633 Michael N. Sack 25.1 Introduction ................................................................................633 25.2 Strategies employed to study the effects of time-controlled fasting ...............................................................634 25.3 Time-controlled fasting and health ............................................634 25.4 Effects of time-controlled fasting on mitochondrial function ...............................................................635

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25.5 Temporal caloric restriction effects on mitochondrial biogenesis ...................................................................................635 25.6 Fasting effects on mitochondrial dynamics and turnover................................................................................637 25.7 Effects on mitochondrial energy metabolism............................638 25.8 Effects on reactive oxygen species handling.............................640 25.9 Effects on mitochondrial synthetic function..............................641 25.10 Fasting-mediated modulation of mitochondrial signaling.........641 25.11 Adverse effects on mitochondrial function in response to fasting .....................................................................................643 25.12 Time-controlled fasting strategies to boost mitochondrial fidelity and disease amelioration ...............................................643 25.13 Fasting and other organelles ......................................................644 25.14 Conclusion ..................................................................................644 References.................................................................................. 645

CHAPTER 26 Dietary modulation and mitochondrial DNA damage .............................................................. 651 Thiago de Souza Freire and Nadja C. de Souza-Pinto 26.1 Introduction ................................................................................651 26.2 Mitochondrial DNA damage accumulation and maintenance of the mitochondrial DNA....................................652 26.3 Caloric restriction and dietary restriction ..................................653 26.4 Dietary components with the potential to activate the nutrient sensing pathways ....................................................655 26.5 Impact of high-fat diets on mitochondrial DNA .......................657 26.6 Fructose and ethanol as potential metabolic toxins ..................658 26.7 Conclusion ..................................................................................659 References.................................................................................. 659 Index ......................................................................................................................667

List of contributors Oly Banerjee Department of Physiology, Serampore College, Hooghly, West Bengal, India Antoni Barrientos Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, United States; Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL, United States Christopher Blanchard School of Dentistry and Medical Sciences, Faculty of Science and Health, Charles Sturt University, Wagga Wagga, NSW, Australia; Gulbali Research Institute, Charles Sturt University, Wagga Wagga, NSW, Australia Laura Bordoni Unit of Molecular Biology and Nutrigenomics, School of Pharmacy, University of Camerino, Camerino, MC, Italy Boris Budiono School of Dentistry and Medical Sciences, Faculty of Science and Health, Charles Sturt University, Wagga Wagga, NSW, Australia; Gulbali Research Institute, Charles Sturt University, Wagga Wagga, NSW, Australia Sara Cantu´-Casas Tecnologico de Monterrey, School of Medicine and Health Sciences, Ca´tedra de Cardiologı´a y Medicina Vascular, Monterrey, Mexico Mauricio Castro-Sepulveda Laboratorio Ciencias del Ejercicio, Escuela de Kinesiologı´a, Universidad Finis Terrae, Santiago, Chile Thiago de Souza Freire Departmento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil Nadja C. de Souza-Pinto Departmento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil Gianluca Fasciolo Department of Biology, University of Naples “Napoli Federico II,” Complesso Universitario di Monte Sant’Angelo, Naples, Italy Nidhish Francis Gulbali Research Institute, Charles Sturt University, Wagga Wagga, NSW, Australia; School of Agricultural, Environmental and Veterinary Sciences, Faculty of Science and Health, Charles Sturt University, Wagga Wagga, NSW, Australia Marie-Ce´line Frantz L’Ore´al Research & Innovation, Advanced Research, Aulnay-sous-Bois, France

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Gerardo Garcı´a-Rivas Tecnologico de Monterrey, School of Medicine and Health Sciences, Ca´tedra de Cardiologı´a y Medicina Vascular, Monterrey, Mexico; Tecnologico de Monterrey, Hospital Zambrano Hellion, Biomedical Research Center, San Pedro Garza Garcı´a, Mexico; Tecnologico de Monterrey, The Institute for Obesity Research, Monterrey, Mexico; Tecnologico de Monterrey, Functional Medicine Center, Hospital Zambrano Hellion, TecSalud, San Pedro Garza Garcı´a, Mexico Antje Garten Hospital for Children and Adolescents, Center for Pediatric Research Leipzig, Leipzig, Germany Taoufik Ghrairi Laboratory of Neurophysiology, Department of Biologie, Faculty of Sciences, Cellular Physiopathology and Valorisation of BioMolecules, University Tunis-El Manar, Tunis, Tunisia Imen Ghzaiel Team “Biochemistry of the Peroxisome, Inflammation and Lipid Metabolism”, University Bourgogne Franche-Comte´, Dijon, France; Lab-NAFS “Nutrition— Functional Food & Vascular Health,” Faculty of Medicine, University of Monastir, Monastir, Tunisia; Faculty of Sciences of Tunis, University Tunis-El Manar, Tunis, Tunisia Michael J. Gonzalez School of Public Health, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico; Universidad Central del Caribe, School of Chiropractic Medicine, Bayamon, Puerto Rico Mennatallah A. Gowayed Faculty of Pharmacy, Department of Pharmacology and Therapeutics, Pharos University in Alexandria, Alexandria, Egypt Zhaleh Jamali Department of Addiction Studies, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran; Student Research Committee, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran Sebastian Jannas-Vela Instituto de Ciencias de la Salud, Universidad de O’Higgins, Rancagua, Chile Maher A. Kamel Department of Biochemistry, Medical Research Institute, Alexandria University, Alexandria, Egypt Yasutoshi Koga Department of Pediatrics and Child Health, Graduate School of Medicine, Kurume University, Kurume, Fukuoka, Japan; Cognitive and Molecular Research Institute of Brain Diseases, School of Medicine, Kurume University, Kurume, Fukuoka, Japan Mohamed Ksila Team “Biochemistry of the Peroxisome, Inflammation and Lipid Metabolism”, University Bourgogne Franche-Comte´, Dijon, France; Laboratory of

List of contributors

Neurophysiology, Department of Biologie, Faculty of Sciences, Cellular Physiopathology and Valorisation of BioMolecules, University Tunis-El Manar, Tunis, Tunisia Norbert Latruffe Team “Biochemistry of the Peroxisome, Inflammation and Lipid Metabolism”, University Bourgogne Franche-Comte´, Dijon, France Gareth G. Lavery Department of Biosciences, Nottingham Trent University, Nottingham, United Kingdom Si-Quan Li Michael Foods, Inc./Post Holdings, Hopkins, MN, United States Zhigang Liu College of Food Science and Engineering, Northwest A&F University, Yangling, China Ge´rard Lizard Team “Biochemistry of the Peroxisome, Inflammation and Lipid Metabolism”, University Bourgogne Franche-Comte´, Dijon, France Guillermo Lo´pez-Lluch Andalusian Centre for Developmental Biology (CABD-CSIC), CIBERER, Pablo de Olavide University, Seville, Spain Omar Lozano Tecnologico de Monterrey, School of Medicine and Health Sciences, Ca´tedra de Cardiologı´a y Medicina Vascular, Monterrey, Mexico; Tecnologico de Monterrey, Hospital Zambrano Hellion, Biomedical Research Center, San Pedro Garza Garcı´a, Mexico; Tecnologico de Monterrey, The Institute for Obesity Research, Monterrey, Mexico Olfa Masmoudi-Kouki Laboratory of Neurophysiology, Department of Biologie, Faculty of Sciences, Cellular Physiopathology and Valorisation of BioMolecules, University Tunis-El Manar, Tunis, Tunisia Paolo I. Mendoza Muraira Tecnologico de Monterrey, School of Medicine and Health Sciences, Ca´tedra de Cardiologı´a y Medicina Vascular, Monterrey, Mexico Smail Meziane Institut Europe´en des Antioxydants, Neuves-Maisons, France Jorge R. Miranda-Massari School of Pharmacy, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico Yousra Y. Moussa Faculty of Science, Department of Biochemistry, Alexandria University, Alexandria, Egypt Sandip Mukherjee Department of Physiology, Serampore College, Hooghly, West Bengal, India

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List of contributors

Gaetana Napolitano Department of Science and Technology, University of Naples “Parthenope”, Naples, Italy Thomas Nury Team “Biochemistry of the Peroxisome, Inflammation and Lipid Metabolism”, University Bourgogne Franche-Comte´, Dijon, France Jose Olalde Centro Me´dico Regenerativo (CMR), Bayamon, and Caguas, Puerto Rico Sergej M. Ostojic Department of Nutrition and Public Health, University of Agder, Kristiansand, Norway Huajun Pan College of Food Science and Engineering, Northwest A&F University, Yangling, China Meredith Pinkerton Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, United States; Neuroscience Graduate Program, University of Miami Miller School of Medicine, Miami, FL, United States Pooja Prasad Natural Products Research Laboratory, Department of Biotechnology, Pondicherry University, Pudhucherry, India Emmanuelle Prost-Camus LARA-Spiral Laboratories, Couternon, France Sina Saari Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland; Obesity Research Unit, Research Program for Clinical and Molecular Metabolism, Faculty of Medicine, University of Helsinki, Helsinki, Finland Michael N. Sack Laboratory of Mitochondrial Biology and Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, United States Nancy Saji School of Dentistry and Medical Sciences, Faculty of Science and Health, Charles Sturt University, Wagga Wagga, NSW, Australia Ahmad Salimi Department of Pharmacology and Toxicology, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran; Traditional Medicine and Hydrotherapy Research Center, Ardabil University of Medical Sciences, Ardabil, Iran Abishek Santhakumar School of Dentistry and Medical Sciences, Faculty of Science and Health, Charles Sturt University, Wagga Wagga, NSW, Australia; Gulbali Research Institute, Charles Sturt University, Wagga Wagga, NSW, Australia

List of contributors

Nandakumar Selvasudha Natural Products Research Laboratory, Department of Biotechnology, Pondicherry University, Pudhucherry, India Domenico Sergi Department of Translational Medicine, University of Ferrara, Ferrara, FE, Italy Leila Rezaie Shirmard Department of Pharmaceutics, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran Siddhartha Singh Department of Physiology, Serampore College, Hooghly, West Bengal, India Sneha Sivadas Natural Products Research Laboratory, Department of Biotechnology, Pondicherry University, Pudhucherry, India Diego Solis-Castan˜ol Tecnologico de Monterrey, School of Medicine and Health Sciences, Ca´tedra de Cardiologı´a y Medicina Vascular, Monterrey, Mexico Ravichandran Srividhya Department of Biotechnology, IIT Madras, Chennai, Tamil Nadu, India Peter F. Surai Department of Biochemistry, Vitagene and Health Research Centre, Bristol, United Kingdom; Department of Biochemistry and Physiology, SaintPetersburg State University of Veterinary Medicine, St. Petersburg, Russia; Department of Microbiology and Biochemistry, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria; Department of Animal Nutrition, Faculty of Agricultural and Environmental Sciences, Szent Istvan University, Go¨do¨llo, Hungary; Veterinary Medicine Department, Sumy National Agrarian University, Sumy, Ukraine; Department of Grain and Compound Technology, Odessa National Academy of Food Technologies, Odessa, Ukraine; Agricultural Department, Russian Academy of Sciences, Moscow, Russia Hannah R. Vasanthi Natural Products Research Laboratory, Department of Biotechnology, Pondicherry University, Pudhucherry, India Anne Vejux Team “Biochemistry of the Peroxisome, Inflammation and Lipid Metabolism”, University Bourgogne Franche-Comte´, Dijon, France Paola Venditti Department of Biology, University of Naples “Napoli Federico II,” Complesso Universitario di Monte Sant’Angelo, Naples, Italy Dominique Vervandier-Fasseur Team OCS, Institute of Molecular Chemistry of University of Burgundy (ICMUB UMR CNRS 6302), University of Bourgogne Franche-Comte´, Dijon, France

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List of contributors

Aline Yammine Team “Biochemistry of the Peroxisome, Inflammation and Lipid Metabolism”, University Bourgogne Franche-Comte´, Dijon, France Chunhong Yan School of Food Science and Technology, Dalian Polytechnic University, Dalian, Liaoning, China Amira Zarrouk Lab-NAFS “Nutrition—Functional Food & Vascular Health,” Faculty of Medicine, University of Monastir, Monastir, Tunisia; University of Sousse, Faculty of Medicine, Sousse, Tunisia Heidi Zweers Department of Gastroenterology and Hepatology-Dietetics, Radboud University Medical Centre, Nijmegen, The Netherlands

Preface Since mitochondria have been recognized as the cells’ critical organelles involved in many fundamental homeostatic pathways, targeting the organelle with dietary compounds has been identified as a viable opportunity to prevent and treat various inherited and acquired disorders accompanied by mitochondrial dysfunction. Besides whole-diet interventions, individual essential nutrients and specifically designed nonessential bioactive compounds appear to affect this hard-to-target subcellular structure and reduce mitochondrial imbalance in animal models and human trials. Over 100 novel mitochondria-specific nutraceuticals and nutritional interventions (e.g., ketogenic diet, small molecule antioxidants, polyphenols, thiols, amino acid derivatives, and nucleotides) have been discovered, developed, and scrutinized during the past decade or so, with many still waiting in the pipeline for efficacy-safety analyses in experimental and clinical nutrition. A broad PubMed search revealed over 5000 recent papers in this field, suggesting emerging scientific interest in mitochondrial nutrition. The main aim of this book is to critically review nutritional interventions targeted at mitochondria by emphasizing cellular and subcellular interactions between food components and mitochondria-specific molecular pathways and regulatory proteins/mtDNA. This book contains 26 chapters, divided into four major sections. The first section, Mitochondria as a target in experimental and clinical nutrition, describes the fundamentals of mitochondria (patho)physiology and outlines a possible nutritional shortfall in patients with the mitochondrial disease while highlighting the complex intra-organelle milieu that has to be accounted for: the interactions between food compounds and mitochondrial cofactors, metabolites, and signaling molecules. Section two, Essential nutrients in mitochondrial nutrition, details the effects of individual essential nutrients, including micronutrients (e.g., B vitamins, vitamin E, and vitamin C) and specific macronutrients (e.g., essential fatty acids and dietary fibers) on mitochondrial function and dynamics in various conditions and applications, along with future perspectives of research in the context of clinical applications rather than an experimental-only approach. Section three, entitled Dietary bioactive compounds and mitochondrial function, is an updated compendium of nonessential (or semiessential) bioactive compounds originating from food that are evaluated for their mitochondriamodulating potential; this includes mitochondria-targeted small-molecule antioxidants, plant-based pigments and organic compounds, nonproteogenic amino acids and derivatives, and other mitochondria-specific multifunctional nutraceuticals, administered either as monocomponent formulas or mitochondria-specific cocktails with synergetic potential. The fourth section, Whole-diet interventions and mitochondrial function, overviews the impact of several whole-diet interventions (e.g., dietary modulation and time-controlled fasting) for molecular upshots in mitochondrial signaling and DNA damage of general and disease-specific models.

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Preface

Our book provides a broad biochemical and metabolic background on nutritional deficits found in mitochondrial dysfunction, with numerous nutritional interventions for mitochondria collected in a single volume, with chapters written by top experts in the field of experimental and clinical molecular nutrition. Therefore Molecular Nutrition and Mitochondria: Metabolic Deficits, Whole-Diet Interventions, and Targeted Nutraceuticals might be a valuable resource for various audiences from both academic and industry sectors, including research and experimental medicine, neuroscience, nutrition and dietetics, biochemistry and molecular biology, food science and technology, but also for graduate and undergraduate students, physicians, and health practitioners. May 2022 Sergej M. Ostojic

Acknowledgments Being the editor of this book during the COVID-19 pandemic was a real challenge. Finding enough time and motivation to write a chapter while dealing with health issues was a difficult task for many authors while some were in a pandemic-driven limbo of changing careers and leaving academia. Sending and resending reminder emails, listening to real-life stories, encouraging people to contribute, and extending deadlines were a part of my regular job as an editor during the past 2 years, and I enjoyed it whatsoever. To acknowledge the hard work, commitment, and excellence, special thanks go to the authors of this book across the globe, from Africa and Europe to Asia, the Americas, and Australia. I would also like to sincerely thank Mrs. Samantha Allard from Elsevier, who was so swift in communicating with the authors, and professional in crafting the content and technical material for this collection. In the end, I dedicate this book to my parents Vukica and Miloljub, who set out a scene for my journey in science and medicine, and to my wife Aleksandra, son Aleksej, and daughter Nastasja, who travel along (and often navigate) this exciting voyage with me.

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Mitochondria as a target in experimental and clinical nutrition

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CHAPTER

1

Targeting mitochondrial dysfunction with nutrients: challenges and opportunities

Marie-Ce´line Frantz L’Ore´al Research & Innovation, Advanced Research, Aulnay-sous-Bois, France

1.1 Introduction For a long time, mitochondrial dysfunction was believed to be restricted to hereditary mitochondrial diseases. However, in the last couple of years, abundant scientific literature has brought a new perspective for medicine (Gueguen et al., 2021). Indeed, both in vitro and in vivo studies have demonstrated a key role of mitochondrial dysfunctions in the pathophysiology of multiple diseases, from neurodegenerative, cardiovascular, and metabolic diseases, to aging and cancer. It has been well known that oxidative stress is implicated in the pathogenesis of these diseases, and in mitochondrial function. Indeed, the mitochondrion is the organelle responsible for the energy production of the cell. Oxidative phosphorylation (OXPHOS) takes place to generate adenosine triphosphate (ATP) via oxygen consumption through the respiratory chain (RC). This process generates an oxidant local environment with a higher concentration of reactive oxygen species (ROS), making the mitochondrion the major place of oxidative stress in the cell. However, mitochondrion is also a hub for a plethora of cellular functions and signaling pathways. Thus, precisely identifying the cause of mitochondrial dysfunctions is challenging and might have to go beyond oxidative stress management. Much attention has been paid to identifying powerful antioxidants (AOX) to target mitochondrial dysfunction. Numerous nutrients and dietary supplements have a high potential as AOX, but many of them have been shown to possess extended biological activities. This chapter aims at reviewing the potential of the main classes of nutrients to target mitochondrial dysfunctions, and the limitations in their therapeutic uses.

1.2 Diseases involving mitochondrial dysfunction Historically, mitochondrial dysfunction has been targeted to treat genetic mitochondrial diseases, characterized by a deficient component of mitochondria, such Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00010-2 © 2023 Elsevier Inc. All rights reserved.

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CHAPTER 1 Targeting mitochondrial dysfunction with nutrients

as a protein, a member of the electron transport chain (ETC), mitochondrial deoxyribonucleic acid (mtDNA), etc (Rinninella et al., 2018). For instance, primary mitochondrial disorders (PMD) are associated with genetic dysfunction of the OXPHOS pathway (Camp et al., 2016). As the brain demands high energy, due to limited glycolysis capacity, its neurons require very efficacious mitochondria. Hence, any mitochondrial deficiency in neurons could be a cause, a cofactor, or a consequence and aggravating factor of neurodegenerative diseases, such as Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD), and Friedreich’s ataxia. A plethora of scientific literature is emerging in this field (Liu & Wang, 2014), especially focusing on the most widespread diseases, namely PD (Borsche et al., 2021; Jin et al., 2014; Prasuhn et al., 2021) and AD (Ke et al., 2021; Sharma et al., 2021). Beyond neurodegeneration, aging and other age-related degeneration are also investigated from the angle of mitochondrial impairment. Age-related cognitive decline was hypothesized to be linked with the age-decreased supply of oxygen and glucose, the main energy substrates of OXPHOS (Kaliszewska et al., 2021). In rheumatic and musculoskeletal diseases, oxidative stress, inflammation, cell death, and premature senescence implicated in intervertebral disk (IVD) aging and degeneration, could be a consequence of mitochondrial dysfunction (Saberi et al., 2021). Targeting age-impaired mitochondrial function in glaucoma was suggested as a treatment, retinal ganglion cells being even more affected due to the reduced oxygen delivery (Osborne et al., 2014). Similarly, mitochondrial deficiency could be implicated in age-related hearing loss, as mtDNA mutations are a major cause (Moos et al., 2018). Interestingly, some data suggest a relationship between diabetes and neurodegeneration, the common link being mitochondria and associated oxidative stress (Carvalho & Cardoso, 2020). Metabolic diseases are another group of diseases concerned by mitochondrial dysfunction. Induced by obesity, mitochondrial defects initiate inflammation in macrophages and adipocytes, and subsequent insulin resistance, a marker of type 2 diabetes (Wang et al., 2021). Hence, targeting mitochondrial dysfunction, redox status imbalance, and/or insulin dysregulation, seems to be a valuable strategy. Noteworthy, Cunarro et al. (Cunarro et al., 2018) suggested not only to focus on peripheral tissues to treat metabolic diseases, but also to target hypothalamus mitochondrial impairment, since metabolic homeostasis is centrally controlled. Importantly, mitochondrial dysfunction also contributes to the development of complications (Kaikini et al., 2017), such as diabetic cardiomyopathy (Jubaidi et al., 2020). Cardiovascular diseases are characterized by oxidative stress and toxic mitochondrial-derived aldehyde accumulation, causing inflammation and cell death. Thus, combating mitochondrial dysfunction and promoting mitochondria detoxification has been proposed to prevent or treat cardiac degeneration, such as heart failure (Kiyuna et al., 2018) or ischemic/reperfusion (I/R) injury (Carinci et al., 2021).

1.3 Targeting mitochondrial dysfunction with nutrients

1.3 Targeting mitochondrial dysfunction with nutrients Widely available through diet or food supplements and safe to use, nutrients supplementation has been envisioned to prevent, treat, or at least mitigate the outcomes of chronic pathologies. Historically, this strategy has been used for genetic mitochondrial disorders. Although nutritional intervention in PMD still requires demonstration of its efficacy (Camp et al., 2016), it can have some benefits as supportive care, when medically relevant (Rinninella et al., 2018). Similarly, adequate levels of nutrients are essential for mitochondrial function since several are implicated in cellular energy metabolism. Therefore, nutrient supplementation is required to restore mitochondrial function during fatigue syndromes or critical illness (Nicolson, 2014; Wesselink et al., 2019). Now that mitochondrial dysfunction has been associated with different diseases, dietary supplements have been studied in corresponding in vitro and in vivo models, as well as clinical trials or observational studies in some cases (Table 1.1).

1.3.1 Vitamins and cofactors 1.3.1.1 Quinone-based vitamins and coenzymes Coenzyme Q10 (CoQ10), commonly named ubiquinone, is a para-benzoquinone for which Q stands for quinone and 10 corresponds to the number of isoprene units in its side chain (Fig. 1.1). It is a lipophilic vitamin-like cofactor naturally synthesized by the human organism and considered a food supplement. It can be found in food, especially in meat, fish, canola, and soybean oils, but also in nuts and seeds. Present in almost all the eukaryotic cells, mainly in the inner mitochondrial membrane (IMM), CoQ10 is part of the substances necessary for energy production through aerobic respiration. Indeed, thanks to a redox cycle between its oxidized form (ubiquinone) and its reduced form (ubiquinol) (Fig. 1.2), CoQ10 plays the role of a shuttle by transferring electrons from nicotinamide adenine dinucleotide (NADH) deshydrogenase (complex I), succinate dehydrogenase (complex II) ETC dehydrogenase, to coenzyme Q-cytochrome c (cyt c) reductase (complex III) of the mitochondrial RC (Aaseth et al., 2021; Hidalgo-Gutie´rrez et al., 2021). The therapeutic effect of CoQ10 has been widely studied in cells, animals, and humans for several diseases (Aaseth et al., 2021; Herna´ndez-Camacho et al., 2018). For neurodegenerative diseases, CoQ10 showed promising neuroprotective properties in various in vitro and in vivo models (Jin et al., 2014; Sharma et al., 2021; Yang et al., 2016). CoQ10 was suggested to stabilize the mitochondrial membrane of neuronal cells subjected to oxidative stress. It was shown to preserve the mitochondrial membrane potential (ΔΨm) and to inhibit ROS generation (Somayajulu et al., 2005). In primary rat mesencephalic neurons, CoQ10 was able to reduce apoptosis induced by rotenone (Moon et al., 2005). This action was exerted through its prevention of mitochondrial depolarization rather than its free

5

Table 1.1 Nutrients described for their beneficial role in mitochondrial dysfunctions. Nutrient

Mode of action (demonstrated or hypothesized)

Targeted disease

Type of study/ model

Mechanistic pathways and Therapeutic effect

References

CoQ10

Stabilize mt membrane

Neurodegenerative diseases

Prevent mt depolarization

PD

Reduce total cellular ROS; maintain mt membrane potential Reduce apoptosis induced by rotenone

(Somayajulu et al., 2005)

CoQ10

CoQ10

Enhance mt function by increasing the activity of mt ETC; AOX activity Activate UCP2

PD

Neuronal cells subjected to oxidative stress Primary rat mesencephalic neurons MPTP mouse model, oral route

Protect against loss of dopamine and DA neurons

(Beal et al., 1998)

PD

MPTP primate model, oral route

(Horvath et al., 2003)

Neurodegenerative movement disorders (PD, atypical parkinsonisms, HD, Friedreich’s ataxia) PD

Clinical trials

Prevent loss of nigral dopamine cells; increase mt respiration No significant improvement

None to weak significant improvement

Reviews: (Jin et al., 2014; Negida et al., 2016; Prasuhn et al., 2021; Zhu et al., 2017)

Vitamins and cofactors

CoQ10

CoQ10

CoQ10

Clinical trials

(Moon et al., 2005)

Review: (Liu & Wang, 2014)

CoQ10




Clinical trial

Reduced CoQ10 (ubiquinol-10) CoQ10

PD

Clinical trial

Rescue CoQ10 deficiencies, energy transduction in mitochondria Rescue low endogenous CoQ10 levels, AOX

Cardiovascular diseases

Clinical trials

Cardiovascular diseases

Mostly clinical data and few in vitro data

CoQ10

Electron carrier

Clinical data

CoQ10 alone or with selenium

Cellular energy generation, AOX & antiinflammatory agent

CoQ10

Facilitate ATP production by mitochondria via ETC participation

Neuronal and muscular degenerative diseases, migraine, cancer Degenerative disorders: cardiovascular disease, diabetes, kidney disease and liver disease Heart failure

CoQ10

No impact on mt function; suppress mtROS level Improve symptoms for PD with wearing off Improvement effects but lack of a clear conclusion

Improve clinical outcome by improving mt respiration, modulate risk factors via antiatherogenic effect, increase NO levels for vasodilatation, reduce oxidative stress Encouraging results but need stronger validation

(Pham et al., 2020) (Yoritaka et al., 2015) Review: (GutierrezMariscal et al., 2021) Review: (Rabanal-Ruiz et al., 2021)

Review: (Testai et al., 2021)

Clinical trials

Reduce mortality risks

Review: (Mantle & Hargreaves, 2019)

Clinical trials

Reduction in major adverse cardiovascular events, need further trials

Review: (Sharma et al., 2016)

(Continued)

Table 1.1 Nutrients described for their beneficial role in mitochondrial dysfunctions. Continued Nutrient

Mode of action (demonstrated or hypothesized)

Targeted disease

Type of study/ model

Mechanistic pathways and Therapeutic effect

References

CoQ10

Essential component of the mt ETC and acts as a mtAOX

Age-related hearing loss (AHL)

Mice

(Someya et al., 2009)

MitoQ

Target specifically mt membrane

AD

C. elegans

MitoQ

ALS

Mouse model of ALS

MitoQ

Multiple sclerosis (MS)

Autoimmune encephalomyelitis (AE) mouse model of multiple sclerosis Cellular and mouse model

Reduce pro-apoptotic Bak expression, reduce cochlear cell death, and delay the onset of AHL Protect lifespan ETC function Decrease nitroxidative damage, increase survival, slow progression of ALS symptoms Reduce neurological disabilities and inflammation

MitoQ

Activate PGC-1α to enhance Mfn2dependent mt fusion

PD

MitoQ

Neurodegenerative diseases (PD, AD, SCA1)

Preclinical studies in mouse models

MitoQ

AD

Aged 3xTg-AD mice

Protect neurons; rescue the loss of DA neurons in the SNc of mice Increase ETC activity; decrease oxidative stress; increase neuronal function; decrease pathological markers; increase spatial memory and locomotor activity Inhibit cognitive decline and pathological markers; extend lifespan

(Ng et al., 2014) (Miquel et al., 2014)

(Mao et al., 2013)

(Xi et al., 2018)

Review: (Shinn & Lagalwar, 2021)

(Young & Franklin, 2019)

MitoQ

PD

Rotenone-treated zebrafish

MitoQ

Neurodegenerative movement disorders (PD, atypical parkinsonisms, HD, Friedreich’s ataxia) TBI

Clinical trials

Metabolic syndrome: obesity, type 2 diabetes

Animal and human data

Healthy volunteers

Clinical trial

PD

Homozygous PINK1 KO Drosophila flies CoQ10-deficient mouse cells Ubiquinonedeficient human and yeast cells gas-1(fc21) complex I mutant C. elegans

MitoQ

Nrf2/ARE pathway activation

MitoQ

Modulation of AMPK and downstream pathways (mTOR, SIRT1, Nrf2, NF-κB)

MitoQ

Vitamin K2

Electron carrier

Vitamin K2




Vitamin K2

—

Vitamin E, tocopherol

Global cellular AOX

Mt complex I disease

Preclinical and clinical trials

Improve redox balance, PD-related gene expression and mt function No significant improvement

(Ünal et al., 2020)

Neuroprotective effect in animals, clinical outcomes awaited Protect against mt damage, via regulation of mt homeostasis

Review: (Ismail et al., 2020)

No impact on mt function; suppress mtROS level Maintain normal ATP production

(Pham et al., 2020)

No effect No effect

Rescue short lifespan

Review: (Liu & Wang, 2014)

Review: (Yang et al., 2021)

(Vos et al., 2012)

(Wang & Hekimi, 2013) (Cerqua et al., 2019) (Polyak et al., 2018) (Continued)

Table 1.1 Nutrients described for their beneficial role in mitochondrial dysfunctions. Continued Nutrient

Mode of action (demonstrated or hypothesized)

Vitamin E, tocopherol

Vitamin E, tocopherol

AOX for the lipid phase of the IMM

Vitamin E, tocopherol

Inhibition of mPTP opening

Targeted disease

Type of study/ model

Mechanistic pathways and Therapeutic effect

References

Neurodegenerative diseases

Rats with memory deficit and hippocampal oxidative stress Aged rats

Improve memory impairment and mt dysfunction

(Nesari et al., 2021)

Restore mt function, prevent ROS production, restore hippocampus mt mass Reduced the blood glucose level, degree of tissue peroxidation and enzyme activity Ameliorate liver redox status and mt bioenergetics functions Contradictory results

(Navarro et al., 2010)

Brain dysfunction in aging and neurodegenerative diseases Diabetes mellitus

Streptozotocininduced diabetic rats

Vitamin E, tocopherol

Liver diseases

Mice fed a highfat diet

Vitamin E, tocopherol

PD

Vitamin E, tocopherol

AD, ALS

Preclinical, clinical and cohort studies Clinical trials, cohort studies

Vitamin E, tocopherol

Cardiovascular diseases, diabetes mellitus Diabetes

Vitamin C, ascorbic acid

Some protection

Clinical trials

No prevention, increase risk of heart failure

Diabetic rats

Long-term vitamin C supplementation ameliorate mt disturbances

(Daniel et al., 2018) Æ Sekeroglu et al., (Ë 2018) Review: (Jin et al., 2014) Review: (Hideyuki et al., 2011) Review: (Hideyuki et al., 2011) (Yusuksawad & Chaiyabutr, 2011)

Vitamin C, ascorbic acid

PD

Epidemiological and clinical studies Rats

Contradictory results

Review: (Jin et al., 2014)

Inhibit mt fission, preserve ATP levels and large-sized mitochondria Rescue OGDHC activity, restore mt respiration

(Yamada et al., 2020)

Review: (Wesselink et al., 2019) Review: (Henriques and Gomes, 2020)

Vitamin B1, thiamine

Inhibition of Drp-1 phosphorylation

Ischemic cardiac diseases

Vitamin B1, thiamine

2-oxoglutarate dehydrogenase complex (OGDHC) precursor Key metabolic precursor

Traumatic brain injury

Rats

Fatigue in critical illness

Clinical studies

Contradictory results

Precursor of flavins which act as pharmacological chaperones of flavoproteins Essential metabolic cofactor

Energy deficiency mt pathologies

Clinical cases

PMD

Clinical trials

Vitamin B3, NA or NR

NAD1 precursor

PMD

In vitro and in vivo data

Vitamin B3, niacin

NAD1 booster as NAD 1 precursor

Mitochondrial myopathy

Clinical trial

Increase cellular flavins content, promoting folding, stability and function of flavoproteins, ameliorate pathologies Some beneficial responses, improve muscle strength, but need more robust trials Restore NADH/NAD1 ratio, improve respiration, induce mt biogenesis Increase mt biogenesis and muscle strength

Vitamin B3, NAM

SIRT1 inhibitor

HD

Neurons, lymphoblasts and mice

Vitamin B1, thiamine

Vitamin B2, riboflavin

Vitamin B2, riboflavin

Increase NAD1 levels in cells, block mt-related transcription in mice, worsening motor phenotype

(Mkrtchyan et al., 2018)

Review: (Kuszak et al., 2018)

Review: (Kuszak et al., 2018) (Chini, 2020; Pirinen et al., 2020) (Naia et al., 2017)

(Continued)

Table 1.1 Nutrients described for their beneficial role in mitochondrial dysfunctions. Continued Nutrient

Mode of action (demonstrated or hypothesized)

Targeted disease

Type of study/ model

Mechanistic pathways and Therapeutic effect

References

Vitamin B3, NR

NAD1 precursor, via NRK1

PD

Improve mt function in neurons, rescue neuronal loss and motor deficit in flies

(Schöndorf et al., 2018)

Vitamin B3, NR

NAD1 precursor

ALS

Patient-derived neurons and Drosophila models of GBAPD Mice

Activate UPR mt signaling, modulate mt proteostasis, improve neurogenesis in brain Improve mt function, reduce apoptosis, reverse age-induced learning and memory impairment Improve mt health, prolong lifespan, ameliorate pathophysiology of diseases Restore youthful gene expression levels: SIRT1 activation, mt rejuvenation, antiinflammatory and antiapoptotic pathways

(Zhou et al., 2020)

Melatonin and NMN separately or in combination

Aging-induced Cognitive Impairment

Aged rats

Vitamin B3, NR

NAD1 precursor, maintenance of NAD1 levels

Aging and agerelated diseases

Preclinical and clinical trials

Vitamin B3, NMN

Key NAD1 intermediate

Age-related vascular cognitive impairment (VCI)

Aged mice

(Hosseini, Farokhi-Sisakht, et al., 2019)

Review: (Fang et al., 2017)

(Kiss et al., 2020)

Vitamin B3, NMN

Vitamin B3, niacin

immediate biosynthetic precursor to NAD1 AOX and component of NAD1

NMN preconditioning and melatonin postconditioning

Hemorrhagic shock

Rats

Promote survival, preserve mt function

(Sims et al., 2018)

Kidney I/R injury

Rats

(Tai et al., 2015)

Ischemic heart diseases

Aged rats

Reduce myocardial oxidative stress, improve mt metabolism Reduce oxidative stress and mtROS levels, improve mt membrane potential, restore NAD1/ NADH ratio No effect

Vitamin B3, NR

NAD1 precursor

Metabolic diseases (obesity, diabetes)

Clinical trial

Combination of Rα-lipoic acid, acetyl-Lcarnitine, NAM, biotin

Stimulation of PPARγc1α regulated mt biogenesis pathway

Metabolic diseases like diabetes

Type 2 diabetic rats

Vitamin B9, folic acid

Mitochondrial disorders

Clinical data

Vitamin E 1 folic acid

AD

Mice

Improve skeletal mt dysfunction and exert hypoglycemic effects, without causing weight gain Positive effects

Protect against Aβ1
40induced cognitive decline, synaptic loss, and neuronal death

(Hosseini, Vafaee, et al., 2019)

Comment: (Leduc-Gaudet et al., 2020) (Shen et al., 2008)

Reviews: (Ormazabal et al., 2015; Rinninella et al., 2018) (Figueiredo et al., 2011)

(Continued)

Table 1.1 Nutrients described for their beneficial role in mitochondrial dysfunctions. Continued Nutrient

Mode of action (demonstrated or hypothesized)

Targeted disease

Type of study/ model

Mechanistic pathways and Therapeutic effect

References

Type 2 diabetes

Myocyte/heart preparations of type 2 diabetic mice gas-1(fc21) complex I mutant C. elegans C. elegans and zebrafish

Rescue mt energetic/ redox balance

(Tocchetti et al., 2012)

Rescue short lifespan

(Polyak et al., 2018)

Improve mt membrane potential, reduce mt stress, rescue lactate, ATP and GSH levels Decrease inflammation, improve mt function, Glycine 1 NAC better than NAC alone Improve multiple key defects, including mt function, associated with premature aging in PWH Delay onset of motor deficits by ameliorating mt dysfunction Some positive effects

(Guha et al., 2021)

Endogenous antioxidants Glutathione (GSH)

N-Acetylcysteine (NAC)

Global cellular AOX

Glucose 1 NA 1 NAC

Mitochondrial complex I disease Mitochondrial complex I disease

Glycine 1 NAC

GSH precursors

Cardiovascular aging

Mice

Glycine 1 NAC

GSH precursors

Clinical trial

NAC

AOX

Geriatric comorbidities of patients with HIV (PWH) HD

Mice

Neurological disorders

Preclinical and clinical trials

NAC

(Cieslik et al., 2018)

(Kumar et al., 2020)

(Wright et al., 2015) Review: (Bavarsad Shahripour et al., 2014)

NAC

Thiolic AOX

PD

Aged mice

Enhance complex I activity

NAC

L-Cys prodrug, GSH precursor

PD

Clinical trial

NAC

Cognition

Clinical trials

NAC

Traumatic brain injury (TBI)

Preclinical and clinical trials

AOX, maintain GSH levels Maintain Cys pool, radical scavenger

Myocardial infarction Critical limb ischemia

Rats

Positive effect on dopaminergic system with positive clinical effects Statistically significant cognitive improvements but larger, targeted studies warranted Positive outcomes in animals, associated with improved mt markers, but still to demonstrate in humans Protect from mt damage

AOX, Cys donor for GSH, glutathione reductase (GR) activator

Diabetes

Insulin Resistant Rats

Bipolar depression

Clinical trial

NAC NAC

NAC

NAC

Mice

Restore limb function, improve mt respiration, reduce oxidative stress Enhance mt function, energy metabolism, prevent inflammation, apoptosis and nitrosative stress No effect at primary outcome but some positive secondary signals

(Martínez Banaclocha, 2000) (Monti et al., 2019)

Review: (Skvarc et al., 2017)

Review: (Bhatti et al., 2018)

(Basha & Priscilla, 2013) (Lejay et al., 2018) (Zalewska et al., 2020)

(Berk et al., 2019)

(Continued)

Table 1.1 Nutrients described for their beneficial role in mitochondrial dysfunctions. Continued Nutrient

Mode of action (demonstrated or hypothesized)

Targeted disease

Type of study/ model

Mechanistic pathways and Therapeutic effect

References

α-Lipoic acid

Direct (ROS scavenger, mt enzymatic cofactor) and indirect (phase 2 enzymes and mt biogenesis activation) protection ROS scavenger, Cofactor of mt enzymatic complexes Thiol compound, reduces mtROS

Age-associated cognitive dysfunction

In vitro, preclinical and preliminary clinical studies

Positive effects, further studies warranted in humans

Review: (Liu, 2008)

AD

In vitro, preclinical and clinical studies Mice

Review: (dos Santos et al., 2019) (Someya et al., 2009)

AOX

Chronic diseases, diabetes

Clinical data

Positive effects in animal, further studies warranted in humans Reduces pro-apoptotic Bak expression, reduces cochlear cell death, and delays the onset of AHL Improve neuropathic impairments

α-Lipoic acid α-Lipoic acid

α-Lipoic acid

Age-related hearing loss (AHL)

Review: (Nicolson, 2014)

Endogenous metabolites & transporters Creatine

Energy source

Mitochondrial diseases

Clinical trials

Creatine

May stabilize mt creatine kinase, and prevent activation of mPTP

Neurodegenerative diseases

Animal models

Improve physical performance and handgrip strength, reduce postexercise lactate Block neuronal death and increase lifespan

Reviews: (Kuszak et al., 2018; Rinninella et al., 2018) Review: (Beal, 2011)

Creatine

AOX, inhibitor of mPTP opening and mt iron accumulation

PD

Preclinical and clinical trials

Creatine

AOX, mt biogenesis

PD

Clinical trial

Neurodegenerative movement disorders (PD, atypical parkinsonisms, HD, Friedreich’s ataxia) Insulin resistance and diabetes

Clinical trials

No significant improvement

Preclinical and clinical trials

Ameliorate insulin sensitivity and glucose tolerance

Reviews: (Zammit et al., 2009; Marcovina et al., 2013)

Heart failure

Preclinical and clinical trials

Ameliorate pathological conditions

Idem

Hypertension

Preclinical and clinical trials

Idem

End-stage renal diseases

Clinical trials

Reduce blood pressure, improve mt function, decrease oxidative stress Controversial results

Reviews: (Marcovina et al., 2013; Zammit et al., 2009) Review: (Marcovina et al., 2013) Review: (Marcovina et al., 2013)

Creatine

L-Carnitine/Acetyl-Lcarnitine/Propionyl-Lcarnitine

L-Carnitine/Acetyl-Lcarnitine/Propionyl-Lcarnitine L-Carnitine/Acetyl-Lcarnitine/Propionyl-Lcarnitine L-Carnitine/Acetyl-Lcarnitine/Propionyl-Lcarnitine

Enhance mt protein synthesis and acetylation/ deacetylation processes Idem

Neuroprotective effect in neuronal cells, restore the MPTP-induced loss of dopamine and protected dopaminergic neurons in MPTP mouse model, improve behavioral difficulties and mood behavior in clinical trial No improvement

Review: (Jin et al., 2014)

(Investigators, 2015) Review: (Liu & Wang, 2014)

(Continued)

Table 1.1 Nutrients described for their beneficial role in mitochondrial dysfunctions. Continued Nutrient

Mode of action (demonstrated or hypothesized)

Targeted disease

Type of study/ model

Mechanistic pathways and Therapeutic effect

References

L-Carnitine

Mitochondria-specific AOX

1 patient, iv 2 g/ day for 2 weeks

Increase OXPHOS

Aged rats

L-Carnitine

Increase OXPHOS

Aging

Clinical trial

40% reduction of creatine kinase level, but no prominent recovery of muscle strength of limbs Reverse age-related decrease in L-carnitine levels, increase FAs metabolism Reduce fatigue, improve cognition

(Wang et al., 2015)

Acetyl-L-carnitine

Kennedy’s disease, spinal and bulbar muscular atrophy (SBMA) Aging

Palmitate

Type 2 diabetes

Rescue mt energetic/ redox balance

(Tocchetti et al., 2012)

Palmitate

Metabolic diseases (obesity, insulin resistance, type 2 diabetes) Metabolic diseases (obesity, insulin resistance, type 2 diabetes) Metabolic diseases (cardiac-I/R injury, obesity, type 2 diabetes, nonalcoholic fatty liver disease, cancers)

Myocyte/heart preparations of type 2 diabetic mice In vitro, in vivo, and humans

Cause insulin resistance and mt dysfunction in brain

Review: (Chudoba et al., 2019)

In vitro, in vivo, and humans

Beneficial for brain insulin action and mt function

Review: (Chudoba et al., 2019)

Animals

Fine balance between detrimental and beneficial effects

Review: (Sullivan et al., 2018)

Review: (Nicolson, 2014)

Review: (Nicolson, 2014)

Dietary Fatty Acids

MUFAs and n-3 PUFAs

n-3 PUFAs

Modulation of IMM structure-function

n-3 PUFAs

n-3 PUFAs

n-3 PUFAs

DHA

Insulin sensitivity (type 2 diabetes or insulin-resistant nondiabetic diseases) Cardiovascular diseases, neurodegenerative diseases Neurodegenerative diseases

Preclinical, clinical and observational studies

Aging and cognition

Preclinical and clinical studies

In vitro (cells) and in vivo (rodents, dogs) models Preclinical and clinical studies

Beneficial effects in animals, no effect in clinical studies, encouraging results in observational studies Beneficial effects on mt dynamic and biogenesis

Review: (Lalia & Lanza, 2016)

Positive effects on functional parameters of mitochondria in animals, equivocal results in humans Improve cognitive functions, role in synapse development

Review: (Katyare, 2016)

Review: (de Oliveira et al., 2017)

Review: (Kaliszewska et al., 2021)

Carotenoids Astaxanthin

Nrf2 and/or PGC-1α pathways activation

Glaucoma

Animal studies

lutein/zeaxanthin

AOX activities

Ocular diseases

Preclinical, clinical, and epidemiologic studies

Targeting mito-ROS, mito-apoptosis, mitobioenergetics, mitobiogenesis, mitodynamics, and mitophagy

Neurological disorders, cancer, heart disease, hyperglycemia, inflammation

Cells, ex vivo, rodents

Protect against retinal ganglion cells dysfunction and reduce ROS Protect mitochondria in primate models, inconsistent clinical data for AMD prevention

Review: (Osborne et al., 2014) Review: (Huang et al., 2020)

Influence the opening of mPTPs, balance ROS, modulate mt fission and fusion, mediate mitophagy

Review: (Huang et al., 2021)

Ginsenosides .200 different natural and transformed ginsenosides and their metabolites

(Continued)

Table 1.1 Nutrients described for their beneficial role in mitochondrial dysfunctions. Continued Nutrient

Mode of action (demonstrated or hypothesized)

Targeted disease

Type of study/ model

Mechanistic pathways and Therapeutic effect

References

Ginsenoside Rb1

AOX

Chronic kidney disease

Clinical trial

(Xu et al., 2017)

Ginsenoside Rd

Receptor-operated calcium channel antagonist

Acute ischemic stroke

Clinical trial

Significantly alleviate renal function impairments, reduce the extent of oxidative stress and inflammation Improve primary outcome

Energy regulating pathways modulation through PI3K/AKT and AMPK

Metabolic diseases

In vitro and preclinical studies

Enhance mt function in cells, reduce insulin resistance in animals

Review: (Mthembu et al., 2021)

AOX, inhibition of mPTP opening

Diabetic cardiomyopathy

In vitro and in vivo various models

Diverse mt protection

Review: (Jubaidi et al., 2020)

PI3K/Akt activation

IVD aging and degeneration Various mitochondrial dysfunctions

Neuronal cell models Various animal models

Prevent mt damage

Review: (Saberi et al., 2021) Review: (Kicinska and Jarmuszkiewicz, 2020)

(Liu et al., 2012)

Polyphenols Polyphenols: resveratrol, gingerol, quercetin, naringenin, pinosylvin, icariin, flavanols, proanthocyanidins Phenolic acids: Ferulic acid, Ellagic acid, Gallic acid, Salvianolic acid, Chlorogenic acid, Rosmarinic acid, Vanilic acid, Caffeic acid Flavonoids: naringenin/ naringin, EGCG, icariin Flavonoids

Cytoprotective effects on mt pathways

Flavonoids

PD

Cellular and animal models

Pomegranate flavonoids: kaempferol and quercetin derivatives Flavonoids: Quercetin

AD

Mouse model

Inhibition of mPTP opening

Diabetes mellitus

Diabetic rats

SIRT1 upregulation

Diabetic nephropathy Diabetic cardiomyopathy

Diabetic mice

Mitochondrial disorders

Fibroblasts from patients

Flavonoids: Fisetin Flavonoids: Bergamot polyphenols Stilbenoids: Resveratrol (RSV)

Boost OXPHOS via mt targets

Preclinical and clinical studies

Decreased DA neuronal loss and dopamine depletion, reduction of neuroinflammation, improved AOX status and mt dysfunction, activated antiapoptotic pathways, induction of neurotrophic factors, inhibition of α-synuclein aggregation Enhance synaptic plasticity, inhibit neuroinflammation, promote autophagy Reduced the blood glucose level, degree of tissue peroxidation and enzyme activity Detoxication of AGE precursor AOX effects in rats, insulin-like effects in diabetic patients Increase basal O2 consumption and ATP production, stimulate mt biogenesis, decrease ROS 5 . cell line dependent, 50/50 chance of benefits 5 . need residual OXPHOS activity

Review: (Jung and Kim, 2018)

Review: (Kaliszewska et al., 2021) (Daniel et al., 2018)

Review: (Kaikini et al., 2017) Review: (Maiuolo et al., 2021) Review: (De Paepe and Van Coster, 2017)

(Continued)

Table 1.1 Nutrients described for their beneficial role in mitochondrial dysfunctions. Continued Nutrient

Mode of action (demonstrated or hypothesized)

Targeted disease

Type of study/ model

Mechanistic pathways and Therapeutic effect

References

Stilbenoids: Resveratrol (RSV)

SIRT1 activator

HD

Neurons, lymphoblasts and mice

(Naia et al., 2017)

Stilbenoids: Resveratrol (RSV)

Diverse mechanisms

Neurodegenerative diseases

In vitro and in vivo models

Stilbenoids: Resveratrol (RSV)

AOX

Preclinical & clinical studies

Stilbenoids: polydatin

Nrf2 activation

Neurodegenerative diseases (PD, AD, SCA1) IVD aging and degeneration

Improve gene transcription associated to mt function in cells, improve motor coordination and learning in mice Cytoprotective effects via modulation of mt function and dynamics Protective effects

Stilbenoids: Resveratrol (RSV)

Numerous mt targets

Stilbenoids: Resveratrol (RSV)

Upregulate AOX capacity

Stilbenoids: Resveratrol (RSV)

SIRT1/PGC-1α activation

Cardiovascular diseases, neurodegenerative diseases, aging, cancer Cardiac diseases

Metabolic diseases

Rats

Animal models

In vitro, ex vivo, and in vivo studies Mice

Enhance cell proliferation and matrix biosynthesis, attenuate IVD aging Protective effect through mt pathways, cytotoxic for cancer cells

Positive effect via improving mt function, no conclusive available clinical study Protect from high fat diet-induced obesity and insulin resistance

Review: (Jardim et al., 2018) Review: (Shinn and Lagalwar, 2021) Review: (Saberi et al., 2021) Review: (MadreiterSokolowski et al., 2017) Review: (Arinno et al., 2021)

Review: (Kaikini et al., 2017)

Stilbenoids: Resveratrol (RSV)

AOX, activate AMPKSIRT1-PCC1α pathway

Metabolic and degenerative diseases

Animals and humans

Curcuminoids: Curcumin

NF-κB pathway inhibition

IVD aging and degeneration

Cellular models

Curcuminoids: Curcumin

Nrf2 activation

Diabetes

Cell and mouse models

Neurodegenerative diseases

In vitro and in vivo studies

SIRT1 activation

I/R injury

Neurons

AMPK/SIRT1/PGC1α pathway activation PI3/Akt/GSK3 and PI3/Akt/CREB/BDNF signaling pathways activation

I/R injury

Rats

Neurodegenerative diseases and disorders

Clinical and experimental studies

Curcuminoids: Curcumin Curcuminoids: Curcumin Curcuminoids: Curcumin Curcuminoids: Curcumin

Activate mt biogenesis, multiple beneficial effects but controversial in some studies Pro-inflammatory cytokines reduction, mitochondria protection, autophagy induction Attenuate oxidative stress, improve glucose intolerance Protection of CNS cells via numbers of mt pathways Attenuate mitochondria oxidative damage Increase number of mitochondria, upregulate biogenesis markers Neuroprotection

Review: (Uriho et al., 2021)

Review: (Saberi et al., 2021)

Review: (Kaikini et al., 2017) Review: (Bagheri et al., 2020) Review: (Kaikini et al., 2017) Review: (de Oliveira et al., 2016) Review: (Kandezi et al., 2020)

Isothiocyanates Sulforaphane (SFN)

Nrf2 activator

Diverse diseases

Rodents

Mitochondria protection

Sulforaphane (SFN)

Nrf2 activator

Diabetes

Positive effects

Sulforaphane (SFN)

Nrf2 activator and other unknown mechanisms

Brain dysfunctions

In vitro and in vivo models In vitro and in vivo models

Antiapoptotic, regulate mt fission, mt protection

Review: (DinkovaKostova & Abramov, 2015) Review: (Kaikini et al., 2017) Review: (Jardim et al., 2020) (Continued)

Table 1.1 Nutrients described for their beneficial role in mitochondrial dysfunctions. Continued Nutrient

Mode of action (demonstrated or hypothesized)

Targeted disease

Type of study/ model

Mechanistic pathways and Therapeutic effect

References

Sulforaphane (SFN)

Neurodegenerative diseases (AD, PD, multiple sclerosis)

In vitro and in vivo models

Reviews: (Kim, 2021; Schepici et al., 2020)

Sulforaphane (SFN)

Chronic inflammatory diseases

Clinical trials

Reduce oxidative stress and disease biomarkers, improve functional outcome Positive outcomes

Reviews: (Houghton, 2019; Mazarakis et al., 2020)

1.3 Targeting mitochondrial dysfunction with nutrients

FIGURE 1.1 Chemical structures of endogenous vitamins, cofactors, antioxidants, metabolites, and transporters.

FIGURE 1.2 Redox reaction between Coenzyme Q (Ubiquinone), the fully oxidized form, and its fully reduced form (Ubiquinol).

25

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CHAPTER 1 Targeting mitochondrial dysfunction with nutrients

radical scavenging properties, since no effect was observed with other AOX such as N-acetylcysteine (NAC), glutathione (GSH), or vitamin C. In a mouse model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity, CoQ10 could attenuate the loss of striatal dopamine and dopaminergic axons (Beal et al., 1998). It was presumed that CoQ10 exerted this action by enhancing the mitochondrial function, through the increase in the activity of the mitochondrial ETC. Oral administration of CoQ10 also slowed the progression of PD in a primate model of PD, by preventing the loss of nigral dopamine cells induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment (Horvath et al., 2003). An activation of the uncoupling protein UCP2 was hypothesized due to the mitochondrial respiration enhancement observed. Considerable numbers of clinical trials have been conducted to assess the therapeutic effect of CoQ10 supplementation on neurodegenerative diseases. No significant improvement was shown, except a mild symptomatic benefit in a few cases (Jin et al., 2014; Liu & Wang, 2014; Negida et al., 2016; Nicolson, 2014; Prasuhn et al., 2021; Zhu et al., 2017). Interestingly, a clinical trial using the reduced form of CoQ10 (ubiquinol-10) reported a significant improvement in the symptoms for patients with wearing off (Yoritaka et al., 2015). Of note, this has been approved in 2016 by the European Medicine Agency (EMA) for the treatment of genetic CoQ10 deficiencies (http://ec.europa.eu/health/documents/community-register/html/o1765.htm). Certainly, CoQ10 supplementation in several clinical trials appears particularly effective in PMD related with CoQ10 deficiency (Kuszak et al., 2018; Rinninella et al., 2018). However, the reduced or oxidized form of CoQ10 has no importance since both are rapidly interconverted in the organism (Pelton, 2020). In cardiovascular diseases, clinical evidence shows multiple benefits of CoQ10 supplementation, by reducing oxidative stress, enhancing mitochondrial respiration, modulating several risk factors through an antiatherogenic effect, and increasing nitric oxide (NO) levels for vasodilatation (Rabanal-Ruiz et al., 2021). Moreover, CoQ10 supplementation could be of interest to rescue deficiencies caused by statin drugs (Gutierrez-Mariscal et al., 2021). Similarly, it may reduce mortality risk in patients with type 2 diabetes, chronic kidney disease, or liver disease (Mantle & Hargreaves, 2019), as well as heart failure (Sharma et al., 2016). However, clinical data are difficult to compare making it burdensome to conclude on the most suitable dose and therapeutic usage for oral administration (Gutierrez-Mariscal et al., 2021). Beyond neurodegenerative and cardiovascular diseases, some data are encouraging with regards to benefits of CoQ10 in migraine, cancer, glaucoma, fertility, or fatigue, but warrant stronger clinical trials to conclude (Testai et al., 2021). Mitoquinone (MitoQ) is a mitochondria-targeted version of ubiquinone (Fig. 1.1). It possesses the para-benzoquinone moiety of CoQ10, grafted with a triphenyphosphonium (TPP) cation via a 10-carbon aliphatic chain spacer. TPP salts are a class of delocalized lipophilic cations designed to target mitochondria by taking advantage of the negative electrochemical potential of the IMM

1.3 Targeting mitochondrial dysfunction with nutrients

(Kelso et al., 2001). MitoQ is a synthetic compound sold as a food supplement with the argument to be up to 1000 times more effective at entering mitochondria than CoQ10 (http://www.mitoq.com). Numerous studies have been conducted to assess the potential of MitoQ in various diseases, from cellular assays to preclinical and clinical studies (Jin et al., 2014; Sharma et al., 2021). Preclinical studies in mouse models of PD, AD, or Spinocerebellar ataxia type 1 (SCA1) promote the therapeutic effects of MitoQ against neurodegenerative diseases (Shinn & Lagalwar, 2021). It was able to increase the MPTP-induced depletion of dopamine and enhance the locomotor activity of MPTP-mouse model of PD, to decrease oxidative stress and lipid peroxidation and prevent the onset of cognitive deficits in 3xTg-AD mice, and to improve mitochondrial morphology ETC activity while restoring the motor capacity of Sca1154Q/2Q mice. In another study with aged 3xTg-AD mice, MitoQ treatment improved memory retention, reduced brain oxidative stress, synapse loss, and AD pathological markers, and increased lifespan (Young & Franklin, 2019). In in vitro and in vivo PD models, MitoQ protects dopaminergic (DA) neurons by stabilizing mitochondrial morphology and function (Xi et al., 2018). It was suggested to act through the activation of proliferator-activated receptor γ coactivator 1α (PGC-1α) to enhance mitofusin-2 (Mfn-2)-dependent mitochondrial fusion. This mechanism was assessed in mice as well, whose loss of DA neurons in the substantia nigra compacta (SNc) could be rescued by MitoQ. In an AD model of Caenorhabditis elegans, MitoQ appeared to specifically target the mitochondrial membrane since it could ameliorate depletion of the mitochondrial lipid cardiolipin and protect ETC function (Ng et al., 2014). However, despite the lifespan extension, it did not reduce global oxidative stress. MitoQ also showed beneficial effects in different mouse models of sclerosis, by reducing pathological signs and improving lifespan (Miquel et al., 2014), but also by protecting neurons from oxidative stress and inflammation (Mao et al., 2013). In a PD model of zebrafish exposed to rotenone, MitoQ was shown to ameliorate the redox balance, PD¨ nal et al., 2020). related gene expression and mitochondrial function (U Despite these promising preclinical data, real therapeutic benefits remain to be demonstrated in humans. The reviewed clinical trials did not show any significant improvements of MitoQ in the motor symptoms of PD, atypical parkinsonisms, HD, or Friedreich’s ataxia patients (Liu & Wang, 2014). Neuroprotective effects of MitoQ could be demonstrated in a mouse model of traumatic brain injury (TBI), via activating the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway (Ismail et al., 2020). However clinical studies are needed to prove its efficacy in humans. Few animal and human studies show a protection of MitoQ against mitochondrial damage associated with metabolic syndrome, probably through the regulation of mitochondrial homeostasis, thanks to the modulation of 50 -adenosine monophosphate-activated protein kinase (AMPK) and downstream pathways, such as a mechanistic target of rapamycin (mTOR), sirtuin 1 (SIRT1), Nrf2, and nuclear factor kappa B (NF-κB) (Yang et al., 2021).

27

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CHAPTER 1 Targeting mitochondrial dysfunction with nutrients

While demonstration of a substantial therapeutic benefit is still required, a recent study assessed that CoQ10 and MitoQ supplementation could mildly suppress mitochondrial oxidative stress levels in healthy middle-aged men, though without impact on mitochondrial function (Pham et al., 2020). Vitamin K exists mainly in two forms: vitamin K1 (phytomenadione, phylloquinone or phytonadione) and vitamin K2 (menaquinones). It is synthetized by gut microbiota in complement to food. Vitamin K1 is present in dark green leafy vegetables such as cabbages, spinach, or salad, and in vegetable oils like olive, soya, or hemp. Vitamin K2 is found in food from animal origins: egg yolk, liver, butter, and fish. Vitamin K1 is a naphtoquinone with a 3-phytyl side chain, whereas vitamin K2 (Fig. 1.1) has an isoprene side chain, typically of 4
7 units, thus less lipophilic than CoQ10. A menaquinone of n units is noted MK-n, the most common ones being MK-4 in animals and MK-7 in some bacteria. Vitamin K is a group of liposolube vitamins required for the maturation of certain blood coagulation factors. However, in bacteria, vitamin K2 is a membrane-bound electron carrier. To assess whether it could exert this CoQ10-like function in eukaryotic cells as well, studies were conducted in Drosophila flies carrying a homozygous tensin homolog-induced kinase 1 (PINK1) knockout (Vos et al., 2012). PINK1 is a gene mutated in PD that affects mitochondrial function. Results showed that vitamin K2 was indeed able to transfer electrons in mitochondria, maintaining normal ATP production. However, other findings in CoQ10-deficient mouse cells failed to demonstrate any ability of vitamin K2 to replace CoQ10 function (Wang & Hekimi, 2013). The same observation was made in experiments with ubiquinone-deficient human and yeast cells, where vitamin K2, despite entering into mitochondria, was unable to restore neither electron flow in the RC, nor ATP synthesis (Cerqua et al., 2019). Thus, vitamin K2 (either MK-4 or MK-7) seems ineffective in mimicking CoQ10 functions in mammalian cells, probably due to its different quinone scaffold.

1.3.1.2 Vitamin E Vitamin E consists of eight chromane isomers substituted by a hydrophobic isoprenyl side chain, including four tocopherols (saturated) and four tocotrienols (unsaturated). Both tocopherols and tocotrienols exist in α, β, γ and δ forms, depending on the number and position of methyl groups on the chromanol ring. Vitamin E is present in a fatty diet of vegetable oils, cereals, meat, poultry, eggs, fruits, vegetables, and wheat germ oil. It is also sold as a food supplement. Vitamin E is well known to be an endogenous chain-breaking AOX, acting as a radical scavenger by donating a hydrogen atom to free radicals (Fig. 1.3). The resulting stabilized radical is then regenerated by a hydrogen donor like vitamin C (Traber & Stevens, 2011). Since vitamin E is fat-soluble, it is efficient to protect cells from lipid peroxidation. α-Tocopherol (Fig. 1.1) is the most biologically active. Benefits of vitamin E were shown in several preclinical models (Table 1.1). However, there are still controversies about the benefits of vitamin E

1.3 Targeting mitochondrial dysfunction with nutrients

FIGURE 1.3 Direct radical scavenging mechanism of vitamin E. α-Tocopherol reacts with a lipid hydroperoxyl (ROO•) radical. The resultant tocopheryl radical is stabilized by resonance and can be converted back to α-tocopherol by ascorbate (Traber & Stevens, 2011).

supplementation when looking at results from past clinical trials and cohort studies in different diseases. Whereas none or even detrimental effects were reported for PD (Jin et al., 2014), vascular diseases, diabetes mellitus or gastrointestinal cancers, other data suggested beneficial effects for AD and amyotrophic lateral sclerosis (ALS) patients (Hideyuki et al., 2011).

1.3.1.3 Vitamin C Vitamin C, whose chemical name is L-ascorbic acid (Fig. 1.1), is a potent watersoluble AOX with a strong reducing activity. It is found in various foods, the richest natural sources being fruits and vegetables, and is also sold as a dietary supplement, as well as a food additive to retard oxidation and browning. Humans and some other mammals depend on the diet as a source of vitamin C as they lost the ability to synthesize it. It is a required nutrient for a variety of biological functions, especially as a free radical scavenger and as a cofactor of several enzymes (Traber & Stevens, 2011). It is also involved in the biosynthesis of carnitine. As a direct AOX, ascorbic acid undergoes a one or two electron oxidation, generating the ascorbyl radical and then the dehydroascorbic acid (DHA) (Fig. 1.4). The ascorbyl radical can be regenerated to ascorbic acid through NADH phosphate NADH/NADPH systems, while the less stable DHA decomposes to 2,3-diketogulonic acid, which will further break down into oxalic acid and L-erythrulose. It is well established that ascorbic acid is part of the endogenous AOX defense system of mitochondria (Fiorani et al., 2021). Long-term vitamin C supplementation ameliorated mitochondrial disturbances in diabetic rats (Yusuksawad & Chaiyabutr, 2011). However, review of clinical data revealed contradictory results in the treatment of PD (Jin et al., 2014). Globally, metaanalysis of major clinical trials failed to demonstrate significant prevention or protection of common AOXs supplementation (vitamin E, vitamin C, carotenes, and combinations) toward cardiovascular diseases, diabetes or cancer (Houghton, 2019). This statement was also confirmed for age-related cognitive decline (Kaliszewska et al., 2021).

29

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CHAPTER 1 Targeting mitochondrial dysfunction with nutrients

e--, H+

L-ascorbate

[NAD(P)+]

e--

[NAD(P)H]

ascorbyl radical

DHA H2O

L-erythrulose

oxalic acid

2,3-diketogulonic acid

FIGURE 1.4 Direct radical scavenging mechanism of vitamin C. By reacting with a free radical, ascorbate undergoes a one or two electron oxidation, generating the ascorbyl radical and then the dehydroascorbic acid (DHA). Ascorbyl radical can be regenerated to ascorbic acid through NADH/NADPH (nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate) systems, while the less stable DHA decomposes to 2,3diketogulonic acid, which will further break down into oxalic acid and L-erythrulose.

1.3.1.4 Vitamins B The group of B vitamins (Fig. 1.1) are important micronutrients involved in numerous cellular processes and can be found in food as well as dietary supplements (Wesselink et al., 2019). Vitamin B1 (thiamine) is a metabolic precursor of thiamine pyrophosphate (TPP), an essential coenzyme for some decarboxylases and dehydrogenases. Food sources of thiamine include whole grains, fruits, vegetables, meat, and fish. In few rat models of cardiac diseases, thiamine preserved ATP levels and mitochondrial respiration, and prevented mitochondrial fission via inhibition of the dynamin-related protein Drp-1 (Mkrtchyan et al., 2018; Yamada et al., 2020). However, not much clinical data are available. Few studies looking at the effect of thiamine supplementation on mitochondrial function resulted in inconsistent conclusions (Wesselink et al., 2019). Vitamin B2 (riboflavin) is the precursor of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), two essential redox cofactors of several mitochondrial flavoproteins including membrane-bound RC complexes and β-oxidation enzymes from the mitochondrial matrix (Henriques & Gomes, 2020). It is also a key building block for complexes I and II. Riboflavin is not synthesized in mammalian cells, thus only obtained via diet. Major dietary sources are eggs, green vegetables, milk, meat, and cereals. In general, clinical data demonstrate beneficial responses of riboflavin treatment in mitochondrial diseases, although larger, more robust studies are needed (Henriques & Gomes, 2020; Kuszak et al., 2018).

1.3 Targeting mitochondrial dysfunction with nutrients

Vitamin B3 (niacin), also called vitamin PP, is composed of nicotinamide/niacinamide (NAM) and nicotinic acid (NA). Together with nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), they are precursors of NAD1, the couple NAD 1 /NADH being a key coenzyme in energy metabolism and the regulation of numerous cellular processes and pathways. In vitro and preclinical models of PMD suggested niacin supplementation could restore NADH deficiency and improve mitochondrial functions (Table 1.1). This trend was confirmed in patients suffering from mitochondrial myopathy. Similarly, NR or NMN (alone or combined with melatonin) improved mitochondrial, neuronal, and physiological parameters in neurons and animal models of age-related neurological diseases. These observations were also verified in some clinical settings, with an amelioration of the pathophysiology of the disease as well, although few in vitro and in vivo studies using NAM gave contradictory results (Table 1.1). In cardiovascular diseases, niacin or derivatives, as well as NMN associated with melatonin, also showed positive outcomes in rodent models, improving mitochondrial and cellular functions, reducing oxidative stress levels, and promoting survival. On the contrary, no effect of NR treatment was seen in clinical trials for metabolic diseases (Table 1.1). Vitamin B8 (biotin) is a coenzyme which participates in the metabolism of fatty acids (FAs), carbohydrates, and amino-acids, and in the biosynthesis of vitamins B9 and B12. It can be found in whole grains, liver, eggs, milk, soya, and nuts. Not many studies are published on biotin alone. One study reported that a combination of R-α-lipoic acid, acetyl-L-carnitine, NAM, and biotin, improved glucose tolerance and promoted mitochondrial biogenesis in type 2 diabetic rats (Shen et al., 2008). Vitamin B9 (folic acid) is necessary for transporting single-carbon groups (methyl, methylene, or formyl group). Folates are thus essential for the synthesis of DNA, methionine, and various other metabolic reactions. Folate is present in a wide range of food sources, among them peanuts, seeds, liver, lentils, or peas. A combination of folic acid and vitamin E showed protection against cognitive decline and neuronal death in a mouse model of AD, via modulation of mitochondrial complex activity (Figueiredo et al., 2011). More commonly, folic acid supplementation has been reported to be beneficial in patients with diseases characterized by folate deficiency as a determining factor, meaning not only mitochondrial diseases (Rinninella et al., 2018) but also common diseases (Ormazabal et al., 2015). Still, randomized clinical trials would be required to fully assess its therapeutic potential (Kuszak et al., 2018).

1.3.2 Endogenous antioxidants 1.3.2.1 Glutathione Glutathione (GSH, γ-glutamyl-cysteinyl-glycine) is a crucial ubiquitous cellular AOX (Fig. 1.1). It is a pseudo-tripeptidic hydrophilic molecule whose cysteine

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confers it its ability to scavenge ROS. It serves also as an essential cofactor for GSH S-transferases and peroxydases. GSH is the most important intracellular AOX, its role being to maintain the reduced state of protein thiol groups (Zalewska et al., 2020). Data suggest that it is present in multiple cell organelles, including mitochondria, probably through an active transport (Sreekumar et al., 2021). GSH declines with age. In food, GSH is particularly concentrated in certain fruits and vegetables, especially cruciferous one. It is also available as a dietary supplement, although this form encountered little success as GSH bioavailability is very poor. As it is the rate limiting substrate for GSH biosynthesis, maintaining the cysteine pool is very important. N-Acetylcycteine (NAC) is the most used precursor for this role (Mokhtari et al., 2017) (Table 1.1). Indeed, only one ex vivo study was reported, establishing a positive effect of GSH on the mitochondrial energetics of cardiac cells of type 2 diabetic mice (Tocchetti et al., 2012).

1.3.2.2 N-Acetylcycteine NAC is a nonessential thiol-containing amino-acid (Fig. 1.1). It is a cysteine donor for the biosynthesis of GSH. Hence, it demonstrates AOX properties by these direct (reductant of disulfide bonds and ROS scavenger thanks to the thiol group) and indirect (GSH concentration increase) modes of action. NAC also stimulates the activity of glutathione reductase (GR), the enzyme regenerating GSH in the reduced form (Zalewska et al., 2020). A new mechanism of action, the conversion to H2S and sulfane sulfur species, is currently emerging (Pedre et al., 2021). While NAC is only available as a drug and food supplement, studied in a wide range of diseases (Teno´rio et al., 2021), it is found naturally in food in its cysteine form. NAC (alone or in combination with NA and glucose) effectively rescued GSH levels as well as mitochondrial function and a short lifespan, in C. elegans and zebrafish models of mitochondrial complex I disease (Table 1.1). As GSH precursors, the combination of glycine 1 NAC was evaluated in mice in relation to cardiovascular aging, and in patients with human immunodeficiency virus for geriatric comorbidities. In both studies, results showed multiple key defects improvements including mitochondrial function (Table 1.1). Reviews of NAC pharmacology on neurological disorders, cognition, TBI and depression, suggested some positive effects of NAC in preclinical and clinical trials (Table 1.1), although further well-defined studies are needed. Good results were also reported in rodent models of cardiovascular diseases and diabetes, with protection of NAC from mitochondrial damage (Table 1.1).

1.3.2.3 Lipoic acid α-Lipoic-acid (or thioctic acid, Fig. 1.1) is an essential cofactor of enzymatic complexes in mitochondria, and plays also a key role in stabilizing and regulating these complexes (Solmonson & DeBerardinis, 2018). Lipoic acid is synthesized in the human body and is contained in food in a form covalently associated with lysine (lipoyllysine). It is also available as a dietary supplement. For the treatment

1.3 Targeting mitochondrial dysfunction with nutrients

of mitochondrial disorders, evidence of a lipoic acid therapeutic effect in humans is limited (Wesselink et al., 2019). Supplementation with lipoid acid and acetyl carnitine delays the mitochondrial decay of aging in rats (Ames, 2010). Lipoic acid and carnitine were also investigated for the prevention of hearing loss as AOXs, inhibitors of NF-κB, and/or activators of Nrf2/ARE pathways (Moos et al., 2018). Some promising results were obtained in animal models (Table 1.1), but limitations were seen in humans. The same trends were observed for agerelated neurological disorders, for which lipoic acid showed positive effects in vitro and in vivo, with some promising outcomes in preliminary clinical studies that need consolidation (Table 1.1). Nevertheless, lipoic acid is already used as an oral supplement in the treatment of diabetes-associated neuropathies, inflammation, and vascular complications (Nicolson, 2014).

1.3.3 Endogenous metabolites and transporters 1.3.3.1 Creatine Creatine is a guanidinium compound that forms high energy phosphate bonds (Fig. 1.1). Naturally produced in vertebrates from glycine, arginine, and methionine, it supplies energy to muscle and nerve cells. Creatine also possesses AOX properties and can be an effective inhibitor of mitochondrial permeability transition pore (mPTP) opening and mitochondrial iron accumulation (Jin et al., 2014). The main food sources are meat and fish. Creatine is also available as a food supplement, especially for athletes. As a potential energy source for defective mitochondria, creatine could improve physical performance for PMD patients in several clinical trials (Kuszak et al., 2018; Rinninella et al., 2018). Nevertheless, in combination with lipoic acid and CoQ10, no significant clinical effect was seen (Kuszak et al., 2018). Regarding neurodegenerative diseases, although creatine was neuroprotective in animal models, no clinically relevant benefices were found in all the reported studies (Table 1.1).

1.3.3.2 Carnitine L-Carnitine (Fig. 1.1) is a naturally occurring amino-acid synthesized by the human organism, but that can be provided by food as well, predominately from meat and dairy. Together with its amide derivatives, acetyl-L-carnitine and propionyl-L-carnitine, its role is to shuttle FAs across mitochondrial membranes via the carnitine/acylcarnitine transporter. Numbers of in vitro and in vivo studies reported multiple benefits of carnitine on the oxidative stress and mitochondrial function modulation (Modanloo & Mohammad, 2019). Empirically used in PMD management, there is no proven efficacy of carnitine supplementation, except for primary carnitine deficiency (Kuszak et al., 2018; Rinninella et al., 2018). Some concerns have been raised regarding potential cardiovascular risk (Kuszak et al., 2018; Rinninella et al., 2018). Carnitine supplementation showed improved clinical outcomes in hypertension, heart failure, cardiomyopathy, diabetes, and sepsis

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(Marcovina et al., 2013; Nicolson, 2014; Wesselink et al., 2019; Zammit et al., 2009), although controversial results were seen for renal disease (Table 1.1). No study was conducted on neurological diseases, except one study with only one patient having Kennedy’s disease, and without clinical amelioration (Table 1.1). In aging, old rats fed with acetyl-L-carnitine recovered their L-carnitine plasma level and had an increased FAs metabolism (Table 1.1). A randomized clinical trial on centenarians supplemented with L-carnitine showed reduced fatigue and improved cognitive function of the treated group. Other trials on diverse syndromes suggested positive effects of L-carnitine treatment (Nicolson, 2014).

1.3.4 Dietary fatty acids FAs are important as sources of energy and as structural components of cells. They can be divided into two main classes depending on their unsaturation degree: saturated FAs and unsaturated FAs. Unsaturated FAs are further divided in two subclasses: monounsaturated FAs (MUFAs) and polyunsaturated FAs (PUFAs). The degree of unsaturation and the geometric differences in PUFAs (cis vs. trans configuration of the double bonds) plays an important role in biological processes and in the construction of biological structures such as cell membranes. Among these roles, dietary FAs regulate mitochondrial membrane structurefunction, but not all of them exert the same effects on cellular energy metabolism. While saturated FAs are associated with an increased risk of cardiovascular diseases, unsaturated FAs, and among them, essential FAs, serve important cellular functions. Sources of FAs include fruits, vegetable oils, seeds, nuts, animal fats, milk, and fish oils. More than others, essential FAs must be found in the diet since there is no biochemical pathway to produce them. Omega-3 PUFAs (ω-3 or n-3 PUFAs) and omega-6 PUFAs (ω-6 or n-6 PUFAs) are the two main classes of PUFAs, characterized by the presence of a double bond three and six atoms away, respectively, from the terminal methyl group. The three types of n-3 PUFAs involved in human physiology are α-linolenic acid (ALA), eicosapentaenoic acid and DHA (Fig. 1.5). Among n-6 PUFAs, we can find for instance linoleic acid (LA), γ-linolenic acid, and ARA. Essential FAs are ALA and LA. Saturated FAs are recommended to be restricted in diet, since they are cause of cardiovascular and metabolic disorders, as demonstrated by numerous studies (Chudoba et al., 2019), though some mitochondrial benefits are sometimes reported (Tocchetti et al., 2012) (Table 1.1).

1.3.4.1 Omega-3 polyunsaturated fatty acids n-3 PUFAs are the most important PUFAs, widely studied for their health benefits, particularly due to their antiinflammatory properties. Food sources include nuts, seeds, vegetables, fruits, egg yolk, meat, and marine organisms (de Oliveira et al., 2017). In mammalian cells, differently from saturated FAs, n-3 PUFAs exert major effects on mitochondrial biogenesis, redox biology, bioenergetics, and apoptosis. They stimulate mitochondrial fusion and increase mitochondrial

1.3 Targeting mitochondrial dysfunction with nutrients

FIGURE 1.5 Chemical structures of the main omega-3 polyunsaturated fatty acids (n-3 PUFAs).

uncoupling, reducing mitochondrial ROS (mtROS) production. By attenuating endoplasmic reticulum stress, Ca21 homeostasis is preserved and inflammasome/ inflammatory pathways are therefore downregulated, improving insulin sensitivity (Lepretti et al., 2018). Hence, extensive studies have been conducted to assess the benefit of n-3 PUFAs on insulin resistance and associated diseases (Table 1.1). Animal models support the positive impact of n-3 PUFAs supplementation (Chudoba et al., 2019), though some of them revealed a fine balance between beneficial and detrimental effects (Sullivan et al., 2018). Human intervention studies fail to show benefits in type 2 diabetes and insulin resistant nondiabetic people, whereas observational studies in humans are more encouraging (Lalia & Lanza, 2016). Similarly, for neurodegenerative diseases, n-3 PUFAs ameliorated mitochondrial parameters in preclinical models (de Oliveira et al., 2017), whereas equivocal results were found in humans (Katyare, 2016). With regards to aging, DHA is an important n-3 PUFA for normal brain development thanks to its role in neurito- and synaptogenesis. DHA dietary supplementation improved learning, memory, and verbal fluency in age-related cognitive decline volunteers (Kaliszewska et al., 2021).

1.3.5 Carotenoids Carotenoids are a large group of fat-soluble pigments commonly found in our diets (Lu et al., 2020). They give the characteristic yellow, orange, or red color to certain fruits and vegetables (e.g., pumpkins, carrots, tomatoes), egg yolk, and marine products (salmon, lobster, shrimp). All of them are derivatives of tetraterpenes, meaning that they are made of eight isoprene units and thus contain 40 carbon atoms. They can be classified into two subgroups: carotenes and xanthophylls. Carotenes contain no oxygen atom and are the major sources of vitamin A, thus called pro-vitamin A carotenoids. Examples are α-carotene, β-carotene, or lycopene (Fig. 1.6). Xanthophylls, for instance zeaxanthin,

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FIGURE 1.6 Chemical structures of representative carotenoids.

astaxanthin, or lutein, carry at least one oxygen atom. Carotenoids have a high reducing potential thanks to their very high degree of unsaturation, making them reactive towards the singlet oxygen and thus, interesting lipophilic AOX (Naoi et al., 2019). Like n-3 PUFAs, other phytochemicals present in the Mediterranean diet have multiple health benefits, among them are carotenoids such as astaxanthin. Along with their AOX properties, they enhance the degradation of damaged mitochondria (mitophagy) via upregulation of mitophagy mediators and promoting new mitochondria generation (mitogenesis). Hence, they are expected to protect against premature aging and degenerative disease (Kaliszewska et al., 2021). Among other natural compounds, astaxanthin was also shown to protect against retinal ganglion cells dysfunction and reduce oxidative stress in animal models of glaucoma, presumably via activation of Nrf2 and/or PGC-1α pathways (Osborne et al., 2014). Indeed, Nrf2 activation by astaxanthin, luteolin, and lycopene, has been reported to occur through the disruption of the Nrf2/Kelch-like ECHassociated protein 1 (Keap1) complex (Naoi et al., 2019). Furthermore, astaxanthin prevents mPTP from opening, thus protecting the cell from apoptosis (Naoi et al., 2019). There are few studies and human data exploring health benefits of carotenoids, except for ocular diseases (Table 1.1). Hence, xanthophylls are pigments present at the macula, only brought via food as they cannot be biosynthesized. They were shown to upregulate carotenoid metabolic genes and to improve mitochondria biogenesis in primates (Huang et al., 2020). However, there are controversial clinical results about the capacity of lutein/zeaxanthin supplementation to prevent the development of Age-related Macular Degeneration (AMD). Hence, these inconsistencies warrant further investigations.

1.3.6 Ginsenosides Ginsenosides are the major bioactive constituents of Panax ginseng (P. ginseng), a tonifying plant traditionally used in Chinese medicine (Huang et al., 2021).

1.3 Targeting mitochondrial dysfunction with nutrients

FIGURE 1.7 Chemical structures of ginsenosides. Me, methyl; Glc, glucosyl; Rha, rhamnosyl; Ara(fur), α-L-arabinofuranosyl; Ara(pyr), α-L-arabinopyranosyl.

They are a class of triterpene saponins, divided into two groups (Fig. 1.7): the dammarane family, having a tetracyclic triterpene steroid-like core structure, and the oleanane family, composed of six-membered pentacyclic triterpenoids. The dammaranes further subdivide into two main subgroups: protopanaxadiols (2 hydroxyl groups bound at carbon-3 and -20) and protopanaxatriols (3 hydroxyl groups bound at carbon-3, -6 and -20). In protopanaxadiols, sugar groups attach to the 3-position of the carbon skeleton, for example ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rh2, and Rh3. In protopanaxatriols, sugar groups attach to the carbon-6 position, for instance ginsenosides Re, Rf, Rg1, Rg2, and Rh1. Multiple in vitro and in vivo studies demonstrate positive effects of ginsenosides on a wide range of mitochondrial functions, from mtROS control, fusion/fission, biogenesis, to mitophagy and apoptosis (Huang et al., 2021) (Table 1.1). Among the available clinical data, Ginsenoside Rb1 showed a marked amelioration in renal function for patients having chronic kidney diseases (Xu et al., 2017). Another randomized clinical trial showed the improved primary outcome of acute ischemic stroke upon treatment with ginsenoside Rd (Liu et al., 2012). Ginsenosides show great promises for multiple chronic diseases, from neurological to cardiac and metabolic diseases and even cancer, however their multitargeting capacity needs to be further explored (Huang et al., 2021).

1.3.7 Polyphenols Polyphenols are secondary metabolites of plants, where they are responsible for color, flavor, and many pharmacological activities. The very large family of polyphenols includes thousands of variants, containing at least one aromatic moiety bearing one or several hydroxyl groups, in addition to other chemical functions. They are classified according to their chemical structure into flavonoids/

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isoflavonoids and nonflavonoids (phenolic acids, monophenols, phenylethanoids, stilbenoids, lignans, flavonolignans, phytoestrogens, curcuminoids, phenolic amides, and tannins). Polyphenols are found mainly in fruits, vegetables, olive oil, nuts, seeds, roots, the leaves of different plants, herbs, whole wheat, red wine, coffee, and tea. Polyphenols demonstrate a wide range of biological activities, and are also able to act synergistically. Among these activities, they have AOX, antiinflammatory, immunomodulating, antitumor, antibacterial, and cardioprotective effects. Extensive research work has been published, exploring the pleiotropic effects of the diverse plant polyphenols. Their mitochondriotropic activities are of particular interest, though the clinical relevance of these properties remains complicated by their multifunctional and pharmacokinetics complexity (Fiorani et al., 2021). Notably, polyphenols have emerged as pharmacological mimetics of caloric restriction, which is a recognized strategy to improve mitochondrial turnover in aging phenotypes, thus promoting health and lifespan (Davinelli et al., 2020). A healthy diet (Ginkgo biloba extract, AOX) was shown to be beneficial for aging degenerative processes, by decreasing oxidative stress and increasing mitochondrial function in animals (Kaliszewska et al., 2021). Regarding metabolic diseases, the link between obesity and diet is obvious. Nutritional interventions to combat obesity and diabetes have been studied in many cellular and animal models (Mthembu et al., 2021). Phytochemicals such as polyphenols and FAs showed promising bioactivity on various mitochondrial pathways, however, further preclinical and clinical trials are warranted to assess their benefits in the treatment of diabetic complications (Kaikini et al., 2017). In addition, polyphenols are shown to protect against retinal ganglion cell dysfunction and to reduces ROS in animal models for glaucoma (Osborne et al., 2014). They are studied as possible therapeutics for ocular diseases (Huang et al., 2020).

1.3.7.1 Phenolic acids Phenolic acids can be divided into two major groups: hydroxybenzoic acids and hydroxycinnamic acids (Fig. 1.8). Hydroxybenzoic acids are derived from hydroxylation of benzoic acid, whereas hydroxycinnamic acids are derived from hydroxylation of cinnamic acid. Phenolic acids are widely present in plants and food such as nuts and fruits, like raspberries, grapes, strawberries, walnuts, cranberries, and blackcurrants. Phenolic acids prevent oxidative damage through their antioxidative action but also by preventing mPTP from opening, resulting in decreased mtROS production (Jubaidi et al., 2020) (Table 1.1). More particularly, ferulic acid activates Nrf2 pathway, vanillic acid activates AMPK pathway, and salvianolic acids and caffeic acid are potent free radical scavengers. Salicylates are allosteric activators of AMPK and inhibitors of pAMPK dephosphorylation, causing a decrease in lipids levels as observed in animals and humans (Kaikini et al., 2017). Phenolic acids showed cardioprotection in in vitro and in vivo studies, although clinical data would be needed to conclude their therapeutic outcome.

1.3 Targeting mitochondrial dysfunction with nutrients

FIGURE 1.8 General chemical structures of selected polyphenols. Me, methyl; Glc, glucosyl.

1.3.7.2 Flavonoids Flavonoids have a core skeleton made of two phenyl rings (A and B) connected by a heterocyclic ring (C, containing the embedded oxygen atom) (Fig. 1.8). They can be classified into flavones, flavonols, flavanones, flavanols, flavanonols, isoflavonoids, neoflavonoids, chalconoids, aurones, anthocyanins and their aglycones anthocyanidins, and the oligomeric proanthocyanidins. Dietary sources are mainly fruits and vegetables. There are multiple mitochondrial targets of flavonoids (Kaikini et al., 2017; Kicinska & Jarmuszkiewicz, 2020; Naoi et al., 2019): they can act as direct mtROS scavengers, inhibitors of mitochondrial complex I thus suppressing ROS production, inhibitors of the apoptotic pathway through maintaining mitochondrial membrane potential, and modulating B-cell lymphoma 2 (Bcl-2)/Bcl-2 associated X protein (Bax) signaling pathways. They can stimulate mitochondrial biogenesis via activation or upregulation of nuclear transcription factors such as proliferatoractivated receptor gamma coactivators (PGC-1α and PGC-1β) and nuclear respiratory factors (NRF1 and NRF2), as well as upstream regulators including SIRT1, AMPK, cAMP-response element binding protein (CREB), and forkhead

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transcription factor (FOXO1). They were found to be regulators of mitochondrial autophagy (mitophagy) via the PINK1/Parkin pathway. They are implicated in the control of mitochondrial fission and fusion, possibly through the regulation of the expression of related proteins such as Drp-1, dynamin 2 (Dnm-2), or mitofusins 1 and 2 (Mfn-1, Mfn-2). Interestingly, flavonoids also behave as mitochondrial ion channels openers, notably the potassium channels mitoBKCa and mitoKATP. Certain flavonoids were reported to activate the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) signaling pathway, triggering mitochondrial protective effects in neuronal cell models (Naoi et al., 2019; Saberi et al., 2021) (Table 1.1). This is the case for the flavanones naringenin and naringin, the catechin epigallocatechin-3-gallate (EGCG), or the peroxylated flavonol icariin. This protection could be further demonstrated in an AD mouse model, where a pomegranate diet, containing kaempferol- and quercetin-related flavonols, enhanced synaptic plasticity and inhibited neuroinflammation (Kaliszewska et al., 2021). Jung & Kim (Jung & Kim, 2018) reviewed the cellular and animal studies proving the potential of multiple flavonoids in prevention and treatment of PD, however lacking long-term clinical studies. With regards to metabolic diseases, the well-known flavonol quercetin was shown to be very effective at protecting hepatic tissue from oxidative stress in diabetic rats, via prevention of the mPTP opening (Daniel et al., 2018). Another flavonol, fisetin, prevented Advanced Glycation End-products (AGE)-related diabetic complications in a mouse model of Type 1 diabetes, supposedly through SIRT1 upregulation (Kaikini et al., 2017). With a high content of flavonoids, naringin being the main constituent, Bergamot polyphenols supplementation was stressed as a promising therapeutic intervention for diabetic patients, according to clinical and epidemiological studies (Maiuolo et al., 2021).

1.3.7.3 Stilbenoids Stilbenoids are metabolites produced by plants to defend against pathogens, or sometimes as growth regulators. They are formed from two benzene moieties linked by a 2-carbon chain. They divide into three classes according to the bridge between the two benzene rings: bibenzyls (ethane bridge), stilbenes (ethene bridge, Fig. 1.8), and phenanthrenes (cyclized). Among the stilbene class, resveratrol (RSV) is the most studied (Fig. 1.8). It can be found in a variety of plants and foods such as grapes, peanuts, berries, cocoa, dates, groundnuts, tomatoes, pines (Uriho et al., 2021). RSV is known for the so-called “French paradox,” which describes the low incidence of coronary heart diseases in French people despite their saturated FAs-rich diet. This protection was attributed to the consumption of red wine, which contains RSV (Ferrie`res, 2004). In addition to its direct ROS scavenging ability, RSV exerts AOX activity and mitochondrial protection through activation of the Nrf2/ARE and AMPK/SIRT1/PGC-1α signaling pathways (Naoi et al., 2019). Like flavonoids, RSV modulates many mitochondrial functions, from fission/fusion, biogenesis, to membrane permeabilization and apoptosis (Table 1.1). RSV protects

1.3 Targeting mitochondrial dysfunction with nutrients

complexes I and IV, thus correcting OXPHOS defect (Kuszak et al., 2018). This could be beneficial for PMD pathologies, although in vitro data suggest the requirement of a residual OXPHOS activity for RSV efficacy (De Paepe & Van Coster, 2017). RSV protects neurons in vitro and in vivo and promotes survival via PI3K/Akt pathway activation (Naoi et al., 2019). It improves cognitive function in some clinical trials and shows potential for neurodegenerative disease treatments (Table 1.1), however clinical data should be completed. The RSV glucoside polydatin was shown to attenuate the progression of IVD degeneration and to enhance matrix biosynthesis and cell proliferation in rats (Saberi et al., 2021). RSV also exhibits beneficial effects in cardiac and metabolic diseases according to several animal and human studies, nevertheless some data are controversial (Table 1.1).

1.3.7.4 Curcuminoids Curcumin (diferuloylmethane) is the most abundant turmeric component extracted from the rhizomes of the dietary spice Curcuma longa, widely used in Asian food and traditional medicine. It is a symmetric polyphenol subjected to keto-enol tautomerism and able to directly scavenge free radicals, generating a stabilized radical intermediate thanks to its electron resonance properties (Fig. 1.9). It can then be regenerated by a proton donor such as ascorbic acid (Rainey et al., 2020). Furthermore, the α,β-unsaturated β-diketo moiety of curcumin behaves as a metal chelating agent for Fe21, Fe31 and Cu21. This gives it interesting capacity to reduce hydroxyl radicals (HO•) production from H2O2, by inhibiting the Fenton reaction. Curcumin also possesses hormetic properties (Rainey et al., 2020), mainly through the activation of Nrf2 pathway (Trujillo et al., 2014). Hormesis refers to an adaptive response activated by repeated low doses of a natural product. Curcumin is active as an AOX, antiinflammatory, antiapoptotic, antitumor, and antiviral agent (de Oliveira et al., 2016). In vitro and in vivo data (Table 1.1) suggest curcumin may modulate various mitochondria parameters such as ETC complexes activity, mitophagy, fusion/fission and biogenesis, oxidative stress and apoptotic pathways (Bagheri et al., 2020; Kaliszewska et al., 2021), although its impact on key regulators needs to be further assessed (Rainey et al., 2020). In particular, inhibition of apoptosis was explained by the prevention of oxidation of the critical thiol residues of mPTP components (Naoi et al., 2019). In addition, it could activate the AMPK/SIRT1/ PGC-1α signaling pathway in neurons (Kaikini et al., 2017) and rats, but the triggering of mitochondria biogenesis was not verified in the experiment (de Oliveira et al., 2016). Curcumin was shown to prevent the incidence of neurodegeneration via two essential signaling pathways: PI3K/Akt/glycogen synthase kinase-3 (GSK3) and PI3K/Akt CREB/brain-derived neurotrophic factor (BDNF) (Kandezi et al., 2020; Naoi et al., 2019). Curcumin was also proposed to protect from IVD degeneration (Saberi et al., 2021) thanks to its action on mitochondria and antiinflammatory activity through NF-κB pathway inhibition (Naoi et al., 2019). Finally, as Nrf2 activator, curcumin was shown to attenuate high glucose-induced

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FIGURE 1.9 Chemical structure of curcumin, its keto-enol tautomerism, and its free radical scavenging mechanism. Upon reaction with a radical R•, the resultant radical is stabilized by resonance and can be converted back to curcumin by a hydrogen donor such as ascorbate (Rainey et al., 2020).

oxidative stress in cells while improving glucose intolerance in a high-fat diet-fed mice (Kaikini et al., 2017). Numerous other cellular pathways are suggested to be modulated by curcumin beyond mitochondrial targets (Naoi et al., 2019). Of note, concerns are raised about the PAINS (Pan Assay Interference Compounds) nature of curcumin (Rainey et al., 2020), which should be considered when interpreting results of biological assays.

1.3.8 Isothiocyanates 1.3.8.1 Sulforaphane Sulforaphane (SFN) belongs to the group of isothiocyanates (ITCs), obtained from cruciferous vegetables such as broccoli, brussel sprouts, cauliflower, and cabbage. Very reactive and unstable, it is produced when its glucosinolate precursor glucoraphanin is hydrolyzed by the enzyme myrosinase, upon damage of the plant (such as from chewing or boiling), allowing the two components to mix and react (Fig. 1.10). It is a potent cytoprotective agent with AOX, antiinflammatory, antitumor, and antimicrobial activities. SFN is a reference compound as Nrf2 activator, by reacting with the cysteine residues (C151, C489, C583) of its repressor Keap1, which allows Nrf2 nuclear translocation and activation of ARE-responsive genes. Beyond the most studied Nrf2 pathway, SFN exhibits a range of other effects (Table 1.1). Among them, another transcription factor, NF-κB, associated with inflammatory pathways, is downregulated by SFN (Houghton, 2019). In cellular and rat models of diabetes, SFN prevented cell and tissue damage, and mitigated the manifestations of diabetic neuropathy (Kaikini et al., 2017). Treatment of rodents with SFN protected mitochondria from damage and respiration impairment induced by toxic or oxidant compounds (Dinkova-Kostova & Abramov, 2015). Mitochondrial integrity was preserved via resistance to mPTP opening, and endogenous AOX defenses

1.4 Challenges and limitations of using nutrients

FIGURE 1.10 Chemical structure of sulforaphane and its generation from the hydrolysis of its precursor glucoraphanin by the enzyme myrosinase.

were increased. SFN exhibits antiapoptotic properties by regulation of mitochondrial biomarkers such as Bax and cyt c. It also modulates fission through downregulation of Drp-1, independently of Nrf2 (Jardim et al., 2020). Hence, its mechanism of action is still not fully unraveled. In vitro and in vivo studies showed reduction of oxidative stress and disease biomarkers upon SFN administration, while ameliorating the physiopathology of neurodegenerative diseases (Kim, 2021; Schepici et al., 2020). Moreover, review of clinical data suggests an interesting potential of SFN to treat chronic inflammatory diseases (Houghton, 2019; Mazarakis et al., 2020). By contrast to other phytochemicals, SFN shows great promises as a therapeutic intervention, thanks to its favorable physicochemical profile, with an absolute bioavailability around 80% (Houghton, 2019). However, mastering its sourcing and stabilization remains challenging (Mazarakis et al., 2020).

1.4 Challenges and limitations of using nutrients to target mitochondrial dysfunction Several important issues remain to be addressed when attempting dietary supplementation to target mitochondrial dysfunction. First, the exact molecular mechanism of action and degree of selectivity of the compounds need to be determined. Notably, dietary phytochemicals affect almost all cell signaling routes and are not restricted to mitochondria (Kaikini et al., 2017; Naoi et al., 2019). Experimentation can hardly identify pathways directly targeted vs. those in which phytonutrients have no effect. Though it is of primary importance to identify the main targets of chemicals, allowing to distinguish between direct and indirect effects. Furthermore, the observed effects and underlying pathways engaged are model-dependent. For instance, flavonoids can exert pro-oxidative and cytotoxic activity in cancer cells (Kyselova, 2012). Second, there are clear limitations in translating results from animal to human studies. In the example of n-3 PUFAs, studies in rodents were mostly preventive, whereas human studies were curative (Lalia & Lanza, 2016). This fact could also

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explain why MitoQ, while active in animal models, may not be efficient for PD clinical remission, as 50% of DA neurons and 80% of striatal dopamine were lost before PD was diagnosed (Snow et al., 2010). In addition, variations in dosage and duration of n-3 PUFAs administered in both preclinical and clinical trials preclude clear conclusions (Lalia & Lanza, 2016). Third, is the importance of the clinical assay design. A lot of human studies have been observational. Clinical trials suffer from a lack of randomization and of control groups, insufficient group size, heterogeneity in dose, duration and route of administration, nonstandard measures of symptom improvement, different laboratory or clinical endpoints, variations in patient population, and patient response (Aaseth et al., 2021; Herna´ndez-Camacho et al., 2018; Hideyuki et al., 2011; Lalia & Lanza, 2016; Turton et al., 2021). Therefore, well-designed, randomized, double-blinded, placebo-controlled trials are clearly needed, with adequate sample size, outcome measures and endpoints, trial duration, dosage, way of administration, and disease stage of patients. Whatever the nutrient evaluated, finding the optimal dose remains a challenge. In the case of RSV (Uriho et al., 2021), this is particularly important as it induces cytotoxicity depending on its dosage (Jardim et al., 2018; Madreiter-Sokolowski et al., 2017). Thus, the nonreproducible clinical results with dietary polyphenols might by due to a narrow therapeutic window (Naoi et al., 2019). Similarly, Bakalova et al. draw the hypothesis that vitamin C could function in a “protective mode” or a “destructive mode” depending on the intracellular concentration of the cytotoxic ascorbyl radical, thus proposing a potential anticancer effect from high doses of vitamin C (Bakalova et al., 2020). This antioxidant vs. pro-oxidant action may explain in part the reported contradictory results (Gonza´lez et al., 2010). Although nutrients are relatively safe, their low efficacy and/or bioavailability can require high doses in treatments, increasing safety risks. Hence, a phase I clinical trial revealed tolerance concerns for high doses (800
1200 mg) of ALA (Moos et al., 2018). Furthermore, the L-carnitine case illustrates the importance of the route of administration. In order to attain the necessary supraphysiological concentrations of L-carnitine in plasma and target organs, intravenous administration would be required to overcome its very low oral bioavailability (Zammit et al., 2009). Of note as well, the nutrient active concentration may not be reached in humans during normal consumption of food (de Oliveira et al., 2016). Hence, nutritional supplementation may not be sufficient in certain pathologies. Another concern is the capacity of nutraceutics to effectively target mitochondria. Intriguingly, vitamin E reaches mitochondria in the flight muscle of birds only when combined with regular exercise (Cooper-Mullin et al., 2021). Vitamin C was shown to be present in high concentrations in mitochondria, possibly through an active transport (Fiorani et al., 2021). Interestingly, Fiorani et al. also reported a similar observation for the polyphenol quercetin, explained by its affinity for heme proteins. The authors suggest a poor efficacy should be expected for untargeted AOX, which would not be able to accumulate in sufficient amounts in

1.5 Topical use of nutrients for dermo-cosmetic applications

critical sites of mtROS production. This was the purpose, notably, of MitoQ design. Nevertheless, this statement should be balanced with the fact that entering mitochondria might not be mandatory, as multiple sites of action are possible depending on the targeted pathway. The last, but certainly the most critical issue, is the poor Adsorption Distribution Metabolism Excretion (ADME) profile of most nutrients. Globally, oral absorption is limited, distribution could be challenging, especially to cross the blood-brain barrier (BBB) for neuroprotection therapy, lipophilic nutrients and most phytochemicals are poorly soluble in water, metabolization and excretion are usually rapid. For instance, CoQ10 is very poorly absorbed by the intestine (less than 40% of the ingested dose), which limits its usage (Arenas-Jal et al., 2020; Mantle & Dybring, 2020). In addition, its limited ability to cross BBB might explain its lack of clinical efficacy in neurological disorders (Turton et al., 2021). Similarly, L-carnitine is poorly absorbed, it has a very high renal clearance, and an active uptake into tissues by a high-affinity transporter. Therefore, even with high oral doses (e.g., more than 2 grams per day), its plasma concentration remains modest (Zammit et al., 2009). Phytonutrients encounter the same drawbacks. Absorption rate of ginsenosides in the intestines is about 1% to 3.7% (Huang et al., 2021). Polyphenols exhibit low oral bioavailability due to poor absorption, low solubility, and rapid excretion associated with extensive in situ biotransformation and conjugation (Fiorani et al., 2021). One possible strategy to overcome these limitations is a specific formulation and delivery system. For example, complexation of dietary plant bioactives such as tocotrienols, pentacyclic triterpenoids, or curcumin, into cyclodextrins, was shown to improve their stability, bioavailability and bioactivity in animal models and humans (Wu¨pper et al., 2021). Similarly, the poor availability of CoQ10 could perhaps be overcome by innovative formulations (Pastor-Maldonado et al., 2020; Pelton, 2020) like the new Ubisol-Q10 nanomicellar technology (Wear et al., 2021) or microencapsulation using the ionic gelation vibrational jet flow technique (Jones et al., 2020). Interestingly, CoQ10 analogs with a shorter side chain, were suggested as an alternative to treat patients with CoQ10 deficiencies. For instance, the less lipophilic CoQ4 was shown to effectively mimic the CoQ10 function of electron transport into mitochondria of mammalian cells (Cerqua et al., 2019). Other CoQ10 analogs with putative better physicochemical properties are investigated to this end (Sua´rez-Rivero et al., 2021).

1.5 Topical use of nutrients for dermo-cosmetic applications Skin, and by extension the scalp (Tru¨eb, 2015), is an organ subjected to high oxidative stress due to its direct interaction with environment, such as ultraviolet radiation (UVR) or pollution (Rinnerthaler et al., 2015). In addition to being the

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first in line, epidermal keratinocytes need a high-energy production to assure the continuous epidermal renewal. Hence, mitochondria play an important role in skin and their dysfunction can have serious consequences, especially skin cancers (Gupta & Abdullah, 2021). Notably, mtDNA damage (mutation, deletion, thymine-dimers, etc.) has been proposed as a relevant marker of UVR exposure (Birch-Machin, 2000). Pollutants were also demonstrated to exacerbate sun damage in keratinocytes and reconstructed skin, including in mitochondria, with mtROS generation, mitochondrial membrane depolarization and reduced ATP production (Soeur et al., 2017). Decline in mitochondrial functions is linked to skin and hair aging clinical signs such as wrinkles, pigmentation disorders, hair graying, and hair loss (Stout & Birch-Machin, 2019). This was further supported by a mtDNA-depleter mouse model (Singh et al., 2018; Villavicencio et al., 2021). Interestingly, there is increasing evidence showing that some skin manifestations are signs of mitochondrial disorders (Birch-Machin, 2000). Certainly, a part of patients with PMD and those with genetic diseases of premature aging exhibit hair and skin abnormalities (Hussain et al., 2021). For all these reasons, targeting mitochondria has been proposed to combat skin (photo)aging and to promote skin regeneration (Sreedhar et al., 2020) as well as wound healing (Cano Sanchez et al., 2018). Specific nutrients are studied or already used for such endpoints (Table 1.2), most of the time via topical applications but nutritional supplements are not excluded (nutricosmetics). CoQ10 is a widely used ingredient in antiaging creams. Indeed, aged skin was shown to shift to an anaerobic pathway most likely linked to deficient mitochondrial activity (Prahl et al., 2008). Treatment of irradiated fibroblasts with CoQ10 (Schniertshauer et al., 2016), as well as topical application of CoQ10 formulas on human volunteers, resulted in a restoration of mitochondrial function, with a skin AOX capacity improvement, and a stimulation of energy metabolism (Knott et al., 2015). A study using UVB-irradiated hairless mice further showed that CoQ10 could protect skin from oxidative stress through increasing the AOX enzymes manganese superoxide dismutase (SOD2) and glutathione peroxidase (GPx) (Kim et al., 2007). Of note, a recent study on rabbits and rats demonstrated the capacity of MitoQ, incorporated in a chitosan/hyaluronan topical membrane, to accelerate wound healing probably through the mitigation of inflammation (Tamer et al., 2018). In skin, vitamin E is known to exert important photoprotective and antiaging activities, as an AOX but also as modulator of different signaling pathways (Rinnerthaler et al., 2015). Notably, the isomer γ-tocotrienol delayed cellular aging in stress-induced senescent skin fibroblasts, through regulation of the apoptotic cascade (Makpol et al., 2012). α-Tocopherol also showed protection of human dermal fibroblasts against oxidative stress, via improvement of mitochondrial function and redox status, and inhibition of NO release and inflammation (Camillo et al., 2022). In addition, in the field of environmental pollution, a proteomic study on keratinocytes exposed to diesel particles showed vitamin E

Table 1.2 Nutrients described for their beneficial role in dermo-cosmetic applications, linked with skin mitochondrial dysfunctions. Nutrient

Mode of action (demonstrated or hypothesized)

Targeted application

Type of study/ model

Mechanistic pathways and Therapeutic effect

References

Restore ATP level, increase mt function, preserve mt membrane potential Increase SOD2 and GPx

(Schniertshauer et al., 2016)

Vitamins and cofactors CoQ10

AOX function

Skin UVR protection

UV irradiated fibroblasts

CoQ10

Naturally occurring AOX and component of mt ETC Part of mt respiratory chain, decline with age Only endogenously produced lipidsoluble AOX

Photoaging protection

UVB-irradiated hairless mice

Skin antiaging and UVR protection

Clinical trial, 0.01% topical (no placebo)

Restore mt membrane potential after UVR

(Prahl et al., 2008)

Skin antiaging and UVR protection

Increase CoQ10 levels, stimulate energy metabolism, increase AOX capacity of skin

(Knott et al., 2015)

Mt-targeted AOX

Wound healing

Human keratinocytes and clinical trial, topical (348 μM & 870 μM, no placebo) Rabbit and rat models Senescent skin fibroblasts

Accelerate healing process through inflammation control Inhibit H2O2-induced apoptosis, inhibit cyt c release and caspase-9/-3 activation Improve mt membrane potential and redox status, inhibit iNOSdependent NO release and proinflammatory cytokine gene expression

(Tamer et al., 2018) (Makpol et al., 2012)

CoQ10

CoQ10

MitoQ Vitamin E, γ-tocotrienol

Skin aging

Vitamin E, α-tocopherol

Photoaging-related skin cancers

Human dermal fibroblasts subjected to oxidative stress

(Kim et al., 2007)

(Camillo et al., 2022)

(Continued)

Table 1.2 Nutrients described for their beneficial role in dermo-cosmetic applications, linked with skin mitochondrial dysfunctions. Continued Nutrient

Mode of action (demonstrated or hypothesized)

Targeted application

Type of study/ model

Mechanistic pathways and Therapeutic effect

References

Vitamin E

AOX

Environmental pollution protection

Partially restore the altered proteins

(Rajagopalan et al., 2019)

Vitamin C

AOX

Photoaged skin protection

AOX and cofactor

Skin care

Vitamin B3, NAM Vitamin B3, NAM

NAD1 primary precursor Used in the synthesis of NAD1 coenzymes

Skin aging and photoaging protection Skin aging and pigmentation control

Dermal fibroblasts

Increase density of skin microrelief, decrease deep furrows Photoprotection, antiaging, depigmentation, antiinflammatory action Protect against oxidative stress, increase mt efficiency Protect against oxidative stress, antiinflammatory effect, modulate epidermal differentiation, enhance ECM and skin barrier, modulate melanosome transfer

(Humbert et al., 2003)

Vitamin C

Primary skin keratinocytes exposed to diesel particles and vapor Clinical trial, 5% topical on photoaged skin Clinical trials, topical

Skin protection from photodamage and aging

Ex vivo and clinical data, topical

Cells, reconstructed skin models, clinical trials, topical

Review: (Telang, 2013) Review: (Oblong, 2014) Review: (Boo, 2021)

Endogenous antioxidants α-Lipoic acid

Essential cofactor of the mt enzymatic complex, potent AOX

Improve skin quality (wrinkles, roughness). Controversial data for photoprotection.

Review: (Matsugo et al., 2011)

Carotenoids β-Carotene

Singlet oxygen quencher

Lycopene

AOX

Photosensitive skin diseases, phototoxic drugs protection, acute UV damage protection, skin carcinogenesis prevention Photoprotection

Phytoene and phytofluene

UV absorber, ROS scavenger

Photoprotection, skin coloration, skin lightening

In vitro, in vivo and human data, oral & topical

Skin lightening effect, skin quality improvement (aging signs)

Flavonoids

Senostatic and senolytic

Skin aging prevention

In vitro and in vivo data, systemic and topical

Plant polyphenols

Various biological activity

Skin protection from UV damage

Growing evidence in senescence-associated secretory phenotype (SASP) modulation, but need solid preclinical and clinical data Promising efficacy for skin disorders management

Plant polyphenols

AOX and antiinflammatory agents Activation of Wnt-α-catenin signaling pathway

Skin protection from air pollution

In vitro, in vivo models & human data, mostly oral

Clinical studies

Effective for photosensitive diseases and drug phototoxicity protection, but no clear evidence for aging/photoaging protection Protect against skin photodamage

Review: (Bayerl, 2008)

Review: (Dilokthornsakul et al., 2018) Review: (MeléndezMartínez et al., 2019)

Polyphenols

Plant flavonoids

Anti-hair loss

In vitro, ex vivo, in vivo and clinical studies In vitro, ex vivo and in vivo studies

Review: (DomaszewskaSzostek et al., 2021)

Decrease levels of ROS and inflammation mediators

Review: (Farjadmand et al., 2021) Review: (Boo, 2019)

Reduce rate of hair loss or stimulate hair growth, need clinical confirmation

Review: (Bassino et al., 2020) (Continued)

Table 1.2 Nutrients described for their beneficial role in dermo-cosmetic applications, linked with skin mitochondrial dysfunctions. Continued Nutrient

Mode of action (demonstrated or hypothesized)

Targeted application

Stilbenoids: Resveratrol (RSV) Stilbenoids: Resveratrol (RSV) Stilbenoids: Resveratrol (RSV)

Multiple mechanisms

Skin aging treatment

AMPK-FOXO3 cascade activation Nrf2 pathway activation

Skin aging prevention

Primary human keratinocytes

Skin protection from oxidative stress

Primary normal human keratinocytes and full-thickness reconstructed skin

Type of study/ model

Mechanistic pathways and Therapeutic effect

References

Unique AOX activity including effect on mt function

Review: (Farris et al., 2013)

Prevent oxidative stressinduced senescence and proliferative dysfunction Prevent oxidative stressinduced cellular alterations and increase GSH content via Nrf2 pathway

(Ido et al., 2015)

(Soeur et al., 2015)

1.5 Topical use of nutrients for dermo-cosmetic applications

partially restored altered proteins implicated in skin integrity maintenance, mitochondrial OXPHOS, or cellular migration (Rajagopalan et al., 2019). Together with vitamin E, vitamin C is a major cosmetic AOX for skin care. Indeed, ascorbate skin levels decrease with age, either chronological aging or photoaging (Rinnerthaler et al., 2015). Topical application of 5% vitamin C led to the clinical improvement of photodamaged skin (Humbert et al., 2003). In addition to protect skin from sun damage, topical vitamin C is also used in antiwrinkle products via increasing collagen synthesis, as a depigmenting agent, and possessing some antiinflammatory activity (Telang, 2013). NAM, whose deficiency causes pellagra, is very commonly used in a plethora of skincare products due to its important role in skin homeostasis (Oblong, 2014). Cellular and reconstructed skin models, as well as clinical data, demonstrate the implication of NAM and its metabolites in skin aging and pigmentation control (Boo, 2021). Different mechanisms are involved, mainly direct or indirect AOX capacity, antiinflammatory effects, modulation of cell senescence and epidermal differentiation, enhancement of extracellular matrix (ECM) and skin barrier, and modulation of melanosome transfer leading to skin lightening. α-Lipoic acid was reported to improve skin photoaging clinical signs in a human study, but controversial results were noticed between an ex vivo model and human for protection against UV-induced oxidative damage (Matsugo et al., 2011). β-Carotene is a provitamin for the famous skin antiaging product retinol (Rinnerthaler et al., 2015). Carotenoids are potent singlet oxygen quenchers described mainly for their skin photoprotective action (Table 1.2). However additional clinical data and mechanistical studies are needed to fully understand their potential skin beneficial effects. Noteworthy, their color and instability make them difficult to be formulated in topical creams. Hence, most of the carotenoids data are from diet. Finally, plant polyphenols are an important phytochemical family exhibiting high potential in skin aging prevention, skin protection from UV damage and pollution, as well as hair loss treatment (Table 1.2). Among them, RSV possesses a unique mechanistic profile which makes it an important antiaging topical ingredient (Table 1.2). Notably, it was shown to prevent oxidative stress-induced damage in keratinocytes and/or reconstructed skin via AMPK-FOXO3 and Nrf2 pathways activation (Table 1.2). Importantly, even if efficacy is established, specific ADME issues must be addressed for topical administration of those nutrients. Skin penetration is limited especially for highly lipophilic compounds like CoQ10 or vitamin E, which will stay in the stratum corneum. Some chemicals might be photosensitizers, such as riboflavin. Strong color of phytochemical dyes like carotenoids or anthocyanins, makes them difficult to incorporate into face creams. Chemical stability, among them oxidability and photostability, is a major drawback in many cases. For instance, vitamin C is oxidized to DHA (Fig. 1.4) when exposed to light and further degradation generates a yellow color. Stable derivatives are presented as

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FIGURE 1.11 Schematic representation of the main mitochondrial targets and pathways of nutrients. Endogenous cofactors, vitamins, and antioxidants support OXPHOS (vitamins B2 and B3, CoQ10 and MitoQ) and metabolic pathways (vitamins B1, B2, B3, B8 and B9, L-carnitine, lipoic acid, creatine), and enhance antioxidant defense (CoQ10 and MitoQ, vitamins C and E, lipoic acid, GSH and NAC). Phytonutrients intervene in different mitochondrial functions and pathways, such as direct mtROS inhibition (carotenoids, ginsenosides, phenolic acids, flavonoids, stilbenoids), or possibly through UCP2 opening (n-3 FAs, RSV), antioxidant defense induction via Nrf2 pathway (lipoic acid, carotenoids, ginsenosides, ferulic acid, quercetin, RSV and polydatin, curcumin, SFN), metabolic pathways increase such as FAs β-oxidation (n-3 FAs), regulation of mitochondrial dynamics and mitophagy via Mfn-1/Mfn-2, Drp-1/Dnm-2, and/or PINK1/Parkin (n-3 FAs, ginsenosides, flavonoids) as well as mitochondrial biogenesis promotion via AMPK/SIRT1/PCG-1α pathway (n-3 FAs, carotenoids, ginsenosides, polyphenols such as salicylates, vanillic acid, fisetin, quercetin, RSV, curcumin), reduction of mitochondrial swelling and apoptosis via Bax inhibition and/or Bcl-2 activation (ginsenosides, flavonoids, curcumin, SFN) or potassium channels opening (flavonoids) or mPTP inhibition, preventing ΔΨm to decrease (carotenoids, ginsenosides, phenolic acids, quercetin, curcumin, SFN), and also antiinflammation action through inhibition of NF-κB signaling (carotenoids, curcumin, SFN). Purple (endogenous) and black (nutrients) arrows indicate metabolic support/ activation/upregulation or inhibition/downregulation. Acetyl-CoA, acetyl-coenzyme A; ADP, adenosine diphosphate; AMPK, 5-adenosine monophosphate-activated protein kinase; AOX, antioxidant; ATP, adenosine triphosphate; Bax, Bcl-2 associated X protein; Bcl-2, B-cell lymphoma 2; CACT, carnitine/acylcarnitine transporter; CoQ10, Coenzyme Q10; ΔΨ m, mitochondrial membrane potential; Drp-1, dynamin-related protein 1; Dnm-2, (Continued)

1.6 Conclusion and perspectives

alternatives (Telang, 2013). Lipoic acid presents a poor thermal stability and is prone to photodegradation due to its dithiolane ring (Matsugo et al., 2011). β-Carotene is readily oxidized due to its multiple conjugated double bonds, hence barely used in cosmetic products (Bayerl, 2008). Photoinstable due to cis-trans isomerization, RSV must be formulated with UV filters or in night products only (Farris et al., 2013). Flavonoids in general are poorly soluble, and specific topical delivery systems have been developed such as encapsulation or microemulsions (Nagula & Wairkar, 2019). Hence, when topical formulation is a major hurdle, the oral route through nutricosmetics could represent an interesting opportunity.

1.6 Conclusion and perspectives

L

Targeting mitochondria dysfunction can be understood in two ways: either mitochondria dysfunction is the cause of the disease, or it is a consequence. Therefore, targeting mitochondria dysfunction will only treat the symptoms, though it could also mitigate the disease progression. Consequently, targeting only the mitochondria would be a reductionist therapeutic approach, oversimplifying their complex role in cellular homeostasis (Prasuhn et al., 2021). However, this complexity could also represent an opportunity for drug development, as many cellular alterations in various pathologies can be treated by restoring mitochondrial impairment. Potential therapeutic targets have multiple mitochondria functions, including mtROS production, mitochondrial dynamics and trafficking, mitochondrial biogenesis, mitophagy, and mitochondrial swelling and apoptosis (Fig. 1.11). Nutrients can be classified into two groups: (1) endogenous cofactors, vitamins, and AOXs, and (2) dietary phytochemicals. Endogenous cofactors and vitamins are implicated in energy metabolism and ATP production through OXPHOS, while the AOXs’ role is to maintain cellular redox homeostasis. When some diseases or dysfunctions are due to a deficiency in these nutrients, external supplementation could restore them to the normal level. This is mainly the case for CoQ10 and vitamins B, as important constituents of the mitochondrial ETC. Similarly, consequently to mutations or age, enzymes can lose binding affinity dynamin 2; ETC, electron transport chain; FAs; fatty acids; GSH, glutathione; IMM, inner mitochondrial membrane; Mfn, mitofusin; MitoQ, mitoquinone; mPTP, mitochondrial permeability transition pore; mt, mitochondrial; mtROS, mitochondrial reactive oxygen species; NAC, N-acetylcysteine; NAD 1 /NADH, nicotinamide adenine dinucleotide; NF-κB, nuclear factor kappa B; Nrf2, nuclear factor erythroid 2-related factor 2; OMM, outer mitochondrial membrane; ROS, reactive oxygen species; RSV, resveratrol; SFN, sulforaphane; SIRT1, sirtuin 1; TCA, tricarboxylic acid; OXPHOS, oxidative phosphorylation; PGC-1α, proliferator-activated receptor γ coactivator 1α; PINK1, tensin homolog-induced kinase 1; UCP2, uncoupling protein 2; Vit, vitamin.

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(increased Km) for coenzymes and substrates: the use of high doses of vitamins B could be one strategy to overcome this loss in binding (Ames, 2010). Phytochemicals are multitargeted: they impact many pathways, not only related to mitochondria. The difficulty lies in the identification of the primary target and the determination of the effective dose. Of note, beyond oral supplementation, a lot of nutrients are also used topically for dermatological or cosmetic applications, mainly antiaging/antiwrinkle (e.g., CoQ10, vitamin C), skin protection from sun damage (e.g., vitamin E, vitamin C, polyphenols), skin tone and pigmentation disorders management (e.g., niacinamide), or antiinflammation related to skin diseases (e.g., niacinamide, various polyphenols). However, again, these usages are not necessarily linked to mitochondrial dysfunction. The general trend is a mitochondrial, cellular and/or tissular protective effect of nutrients demonstrated in most in vitro and in vivo assays, but barely confirmed in a clinical setting. In addition to the need for better pharmacological characterization and robust clinical trials, both classes of nutrients suffer from poor pharmacokinetics, hampering their therapeutic use. Nevertheless, there are some proven benefits of long-term fruit and vegetable supplementation in aging processes. Mediterranean diet and related are good illustrations of the mitigative effect of dietary phytochemicals, although the exact mechanisms are still to be understood (Kaliszewska et al., 2021). With a known mode of action, the role of nutrients in preventing oxidative damage and mitochondria protection should be considered beyond foods and could be investigated as lead compounds for the discovery and development of new drugs having improved pharmacological activity, selectivity, ADMET properties, solubility and stability (Naoi et al., 2019).

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Mitochondrion at the crossroads between nutrients and the epigenome

2

Laura Bordoni1 and Domenico Sergi2 1

Unit of Molecular Biology and Nutrigenomics, School of Pharmacy, University of Camerino, Camerino, MC, Italy 2 Department of Translational Medicine, University of Ferrara, Ferrara, FE, Italy

2.1 Introduction The modulation of gene expression is a pivotal regulatory mechanism by which cells adapt to endogenous and exogenous stimuli, including nutrition. In this context, the epigenome represents a crucial modulator of gene expression, with epigenetic modifications being finely tuned by extracellular and intracellular signals. At the same time, cellular responses to environmental cues are mediated, at least in part, by the mitochondria, that regulate both energetic and redox homeostasis. Recently, a tight link between epigenetic and mitochondrial metabolism has been described. This relationship is further supported by the fact that mitochondria contain their own DNA (mtDNA), which is a circular double-stranded molecule, present in multiple copies in each mitochondrion and that also undergo epigenetic modifications. Moreover, a pivotal role of nutrition in affecting both epigenetic and mitochondrial dynamics and function has been highlighted. While the role of nutrition in modulating mitochondrial functions is extensively discussed in other sections of this book, this chapter will focus on the impact of nutrition in boosting epigenetic regulations and on the role of the mitochondria in mediating this complex relationship. In this chapter, first, a brief introduction on the epigenome will be provided, explaining why nutrition is essential for its homeostasis and regulation. Then, the bidirectional crosstalk between the nuclear and the mtDNA will be described, with a particular focus on its implications on the epigenome. Finally, the current evidence on the impact of the diet on mitochondrial epigenetics is summarized, describing both limits and future prospectives in this research field.

Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00015-1 © 2023 Elsevier Inc. All rights reserved.

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2.2 Epigenetic modifications The word “epigenetics,” first coined by Conrad Waddington (Waddington, 1942), defines stably heritable phenotypes resulting from changes in a chromosome without changes in the gene sequence (Berger et al., 2009). Epigenetics is currently defined as the study of inherited changes in phenotype or gene expression that are caused by mechanisms other than changes in the underlying DNA sequence (Bird, 2007). Epigenetic mechanisms of gene regulation, which collectively make up the epigenome, include: DNA methylation, histone posttranslational modifications, chromatin remodeling and noncoding RNAs (ncRNAs). Epigenetic modifications are first established during the developmental stages to ensure proper differentiation, but they can change during the life course, in both physiological (i.e., aging) and pathological (i.e., cancer) conditions, and in response to numerous environmental factors (Bordoni & Gabbianelli, 2019). Epigenetic mechanisms also allow organisms to adapt to environmental changes, orchestrating cellular responses to a stimulus also in the long term. Due to their plasticity and reversibility, epigenetic modifications have attracted increasing attention for their potential roles as biomarkers and/or therapeutic targets.

2.2.1 DNA methylation DNA methylation is the covalent addition of a methyl group at the 5-carbon of a cytosine ring, resulting in 5-methylcytosine (5mC). In mammals, DNA methylation occurs primarily on cytosines within a CpG dinucleotide even though nonCpG methylation also exists (Jang et al., 2017). Depending on the location where DNA methylation occurs (e.g., gene promoter or gene bodies), methylation represses or enhances gene repression. This reaction is catalyzed by specific enzymes, the DNA methyltransferases (DNMTs). DNMT1, also called “the maintenance” DNMT, copies methylation marks from the parental strand to the newly synthesized DNA strand. It is essential for the transmission of DNA methylation patterns from cell-to-cell during replication, allowing the conservation of the cellular epigenetic memory (D’Urso & Brickner, 2014). Conversely, DNMT3A and DNMT3B establish de novo methylation patterns. On the contrary, DNA demethylation can occur through both passive and active mechanisms. Passive DNA demethylation is mediated by suppression of the DNMT1 activity, which is reflected in a progressive loss of methyl marks during the cells’ replication. Active DNA demethylation is mediated by specific enzymes that remove methyl groups through a multistep process (Kohli & Zhang, 2013). Among the known DNA demethylases, Ten-Eleven Translocation (TET) proteins are enzymes that promote DNA demethylation by oxidizing 5mC into 5-hydroxymethylcytosine (5-hmc), as a first step. Of note, this intermediate of the DNA demethylation process has been demonstrated to have its own role as an epigenetic mark (Guibert et al., 2013), particularly in specific cell types (i.e., neurons) (Szulwach et al., 2011).

2.2 Epigenetic modifications

DNA methylation is not only involved in gene expression regulation, but also plays a pivotal role in the silencing of transposable elements along the genome, in X chromosome inactivation, in genomic imprinting, and in other major cellular physiological processes. Since an in-depth description of all the roles of DNA methylation is outside of the scope of this chapter, for a detailed overview of this topic, refer to this comprehensive review focusing on the molecular hallmarks of epigenetic control (Allis & Jenuwein, 2016).

2.2.2 Histone modifications and chromatin remodeling In the nucleus, DNA is wrapped around histone proteins and packaged into nucleosomes. Posttranslational modifications of histone tails, which include methylation, phosphorylation, acetylation, ubiquitylation, and sumoylation (Lawrence et al., 2016) play a key role in the regulation of gene expression. Certainly, such modifications, affects gene expression by modulating the accessibility of the DNA to the transcription machinery, by altering chromatin structure or recruiting histone modifiers. Besides their role in regulating transcription, these posttranslational modifications also affect chromosome packaging, DNA damage, and DNA repair. Histone acetylation encompasses the enzymatic addition of an acetyl group from acetyl-coenzyme A (acetyl-CoA) and is catalyzed by histone acetyltransferases (HATs). Contrarily, the hydrolytic removal of acetyl groups from histone residues is mediated by histone deacetylases (HDACs). The addition of the aforementioned functional groups on the histones tails is mediated by other specific enzymes including, among others, histone methyltransferases and histone phosphoriylases referred to as “writers.” Enzymes catalyzing the removal of the functional group (e.g., HDACs, demethylases, or phosphatases) are named “erasers.” While histone acetylation is mainly associated with gene expression (since it increases accessibility to DNA by reducing the electrostatic interaction between DNA and histones), the other histone modifications exert different effects depending on which proteins they recruit. Indeed, the combination of these posttranslational modifications along the chromatin generates a histone code which is “read” by specific regulatory proteins (“readers”), able to recognize the histone modifications and modulate the structure of each specific chromatin domain (Zhang et al., 2015).

2.2.3 Noncoding RNA ncRNAs are a cluster of RNAs that do not encode proteins. A diverse catalog of ncRNAs are transcribed from the genome. They include micro-RNA (miRNAs), endogenous small interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs), and long noncoding RNAs (lncRNAs). They are important regulators of the gene expression networks by controlling nuclear architecture, gene transcription, and by modulating mRNA stability, translation, and posttranslational modifications in the cytoplasm. Although first recognized as regulators of gene expression at the

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posttranscriptional level, they can also act as transcriptional regulators. They have been shown to have a role in heterochromatin formation, histone modification, DNA methylation targeting, and gene silencing (Beermann et al., 2016; Yao et al., 2019). siRNAs and miRNAs are broadly distributed in both physiological and phylogenetic terms, while piRNAs are primarily found in animals and exert their functions most clearly in the germline. In all cases, the small RNAs act as specificity factors that direct bound effector proteins (members of the Argonaute protein superfamily) to target nucleic acid molecules via base-pairing interactions. siRNAs and miRNAs bind to members of the Ago clade of Argonaute proteins, whereas piRNAs bind to members of the Piwi clade. miRNAs mainly act as regulators of endogenous genes, siRNAs act as defenders of genome integrity in response to foreign or invasive nucleic acids such as viruses, transposons, and transgenes, while piRNAs have a major role in transposon control and genome defense (Carthew & Sontheimer, 2009).

2.3 Mitochondrial epigenetics and mito-epigenetics Many of the pathways described in the previous sections are closely related to mitochondrial metabolism. Mitochondrial metabolism can regulate epigenetic dynamics in the nucleus, thus exerting pleiotropic effects on the entire cellular metabolism (Castegna et al., 2015). Epigenetic mechanisms, in turn, regulate the expression of proteins directed to the mitochondria, thus modulating their functions. This generates a vicious cycle, with mitochondrial metabolism influencing nuclear epigenetic modifications which, in turn, regulate the expression of genes involved in mitochondrial function. Moreover, mitochondria are unique organelles containing their own DNA, and it has been shown that epigenetic regulations can also occur directly in the mitochondrial genome, opening new possibilities for an additional level of regulation of mitochondrial dynamics. A bidirectional crosstalk between the nuclear and the mtDNA exists (Cagin & Enriquez, 2015), to generate a coordinated gene response to various environmental cues. Much of this bidirectional crosstalk relies on epigenetic processes, including DNA, RNA, and histone modification pathways (Wiese & Bannister, 2020).

2.3.1 Mitochondrial epigenetics: how mitochondria affect epigenetic pathways 2.3.1.1 Epigenetic regulations in the nucleus affect mitochondrial functions The mitochondrial proteome comprises an estimated 1158 proteins (Calvo et al., 2016) that are encoded by nuclear genes and imported into the organelle via various import pathways (Dudek et al., 2013). For this reason, epigenetic regulation occurring in the nuclear genome can affect mitochondrial functions by modulating

2.3 Mitochondrial epigenetics and mito-epigenetics

gene expression of proteins directed to the mitochondria. For instance, the expression of the nuclear-encoded DNA polymerase γA, a mitochondrial enzyme that controls the mtDNA copy number, is regulated by DNA methylation in a specific area of its sequence (exon 2) (Kelly et al., 2012). Also, mtDNA copy numbers are strictly regulated by nuclear-encoded mtDNA-specific replication factors, the expression of which is under the control of cell-specific DNA methylation (Sun & John, 2018). Not only nuclear DNA methylation, but also nuclear-encoded sncRNAs affect mitochondrial functions. Short sncRNAs transcribed from the nuclear genome can target mitochondria, thus contributing to gene silencing of both ncDNA- and mtDNA-encoded genes. Similarly, nuclear-encoded lncRNAs can be transported into the mitochondria to influence mitochondrial metabolism, biosynthesis, and apoptosis (Noh et al., 2016).

2.3.1.2 Mitochondrial functions impact the nuclear epigenome It has been demonstrated that mtDNA and mitochondrial activity can regulate methylation patterns of nuclear genes. For instance, in vitro experiments showed that the removal of mtDNA causes significant alterations in ncDNA methylation (both hypomethylation and hypermethylation); these alterations could be reversed by the reintroduction of mtDNA (Smiraglia et al., 2008). This might be due a mtDNA copy number-dependent expression of DNMT1 (Xie et al., 2007). Not only the abundance of mtDNA copy number but also the presence of specific mitochondrial haplotypes in mtDNA appear to regulate nDNA methylation. For example, carriers of the J haplogroup show higher global DNA methylation levels relative to non-J carriers in European populations (Bellizzi et al., 2012). Furthermore, it has been shown that mtDNA mutations can impact epigenome homeostasis. For example, the mtDNA transfer RNAs (tRNA)Leu(UUR) nucleotide (nt) 3243 A . G mutation, which has been associated with diabetes, neuromuscular degenerative disease, and perinatal lethality, can induce profound changes in the nuclear epigenome. In particular, mitochondrially-derived acetyl-CoA levels decreased at high heteroplasmy, thus causing decreased histone H4 acetylation; at midlevel heteroplasmy, α-ketoglutarate levels increased and were inversely correlated with histone H3 methylation; inhibition of mitochondrial protein synthesis induced acetylation and methylation changes, while restoration of mitochondrial function reverses these effects; mtDNA heteroplasmy also affects mitochondrial NAD 1 /NADH ratio, which correlates with nuclear histone acetylation, whereas nuclear NAD 1 /NADH ratio correlates with changes in nDNA and mtDNA transcription (Kopinski et al., 2019). Not only mtDNA, but also metabolic regulations of the mitochondria can indirectly affect nuclear epigenetic dynamics. In this context, serine biosynthesis, the folate cycle, the methionine cycle, the transsulfuration pathway, and the Krebs cycle (Lopes, 2020; Martı´nez-Reyes & Chandel, 2020) are among the crucial finely tuned metabolic pathways affecting DNA methylation. Particularly interesting is the correlation of these pathways with the one-carbon cycle (Iacobazzi et al., 2013), which provides the cell with S-adenosylmethionine (SAM), the

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universal methyl-donor. Once having donated its methyl group, SAM is transformed into S-adenosyl-homocysteine and then into homocysteine, which can later be recycled back to methionine or feed into the transsulfuration pathway (leading to the production of cysteine). Since folate cycle reactions are duplicated in the cytosol and mitochondria, mitochondria contribute to the production of SAM. The mitochondrial and cytosolic cycles are linked through the exchange of serine and glycine, which are interconverted by the serine hydroxymethyltransferase through methylenetetrahydrofolate. Based on the central role of one-carbon pathway in methylation reactions, it is likely that impairment of both cytosolic and mitochondrial one-carbon metabolism affect both nDNA and mtDNA methylation (Lopes, 2020). Moreover, the production of the cysteine through the transsulfuration pathway influences the synthesis of glutathione (GSH), a major antioxidant affecting the cell redox homeostasis (Lu, 2009). In addition, mitochondria contribute to the regulation of the acetyl-CoA levels. Acetyl-CoA can be produced in the cytosol but also in the mitochondria, where it can be generated from the oxidation of pyruvate and fatty acid β-oxidation (Martı´nez-Reyes & Chandel, 2020). As previously mentioned, the maintenance of an acetyl-CoA pool is crucial to sustain the Krebs cycle as well as for the acetylation of histones, leading to the activation of transcriptional programs through the alteration of chromatin dynamics. Moreover, the Krebs cycle uses acetyl-CoA to produce intermediates like α-ketoglutarate, succinate, and fumarate, that are able to influence the activity of TET enzymes (Martı´nez-Reyes & Chandel, 2020), thus affecting 5-hmc production and active demethylation processes (Tahiliani et al., 2009). In particular, α-ketoglutarate is a regulator of both the Jumonji Cdomain-containing protein (JMJDs) families (which are histone demethylases) and of TET proteins (which are DNA demethylases). Thus, deregulation of the TCA cycle or dysfunction of the mitochondria can affect a-KG levels and consequently JMJD and TET activity (Kaelin & McKnight, 2013). Finally, mitochondrial metabolism mediates the epigenetic regulations occurring in the nucleus through trafficking of metabolic enzymes and metabolites able to influence the activity of epigenetic enzymes, ultimately affecting gene regulation in response to metabolic cues (for an in-depth review, see Wiese & Bannister, 2020).

2.3.2 Mito-epigenetics: epigenetic regulations in the mitochondrial genome The mtDNA is a circular double-stranded molecule, comprising 16,569 bp, where one strand is purine rich (i.e., the heavy strand) and the complementary strand is rich in pyrimidines (i.e., the light strand). Each mitochondrion contains multiple copies of mtDNA. Although most of the mitochondrial proteome is encoded by nuclear genes, mtDNA encodes for 13 polypeptides that are crucial components of the electron transport chain (ETC) and therefore are required for the generation of ATP via oxidative phosphorylation (OXPHOS). Moreover, it encodes for 22 tRNAs and 2

2.3 Mitochondrial epigenetics and mito-epigenetics

ribosomal RNAs (rRNAs) (Calvo & Mootha, 2010; Yasukawa & Kang, 2018). Thus, mtDNA needs to be dependably replicated, transcribed, and translated to maintain the proper mitochondrial function. Remarkably, it has been shown that epigenetic regulations might occur in the mtDNA (Ghosh et al., 2015). However, since the structure and the origin of the mtDNA is different from the nuclear DNA, epigenetic regulations of the mitochondrial genome have peculiar features.

2.3.2.1 mtDNA methylation The most extensively studied epigenetic modification in the mitochondrial genome is DNA methylation. The assessment of methylation in the mtDNA has been a challenge for a long time, with a lack of consensus about the existence of this epigenetic mark in the mtDNA (Iacobazzi et al., 2013; Mechta et al., 2017; Stimpfel et al., 2018). Despite some uncertainties, Shock et al. demonstrated the presence of a specific isoform of DNMT1 (the mtDNMT1) inside mitochondria. This isoform possesses a mitochondrial targeting sequence that facilitates the translocation of the DNMT1 prepeptide into mitochondria (Shock et al., 2011). Chestnut et al. found that also the DNMT3A was present in mitochondrial fractions of mouse and human CNS (Chestnut et al., 2011). It has been demonstrated that DNMT3B can also modify the frequency and quantity of mtDNA methylation in healthy breast cells, albeit in a strand specific manner, affecting the Lstrand more significantly. Therefore, the discovery of DNMTs within the mitochondria corroborated the hypothesis that DNA methylation might occur within this organelle. However, the extent to which mtDNA is methylated is still debated. Uncertainties about the level of methylation in mtDNA are mainly related to technical issues. Firstly, it seems that methylation in the mtDNA is frequently also outside of CpGs (Liu et al., 2016; Shock et al., 2011). This might explain why bisulfite-related techniques aimed at measuring methylation have revealed such low levels of this mark in mtDNA. Moreover, mtDNA is particularly abundant in a specific area of the mitochondrial genome: the D-loop. De-methylation of the displacement loop of mitochondrial DNA is associated with increased mitochondria (Stoccoro et al., 2018; Zheng et al., 2015). The human mtDNA is extensively methylated on a non-CpG context (Patil et al., 2019) and the two mtDNA strains (heavy and light) can be differentially methylated (Vos et al., 2021). Moreover, mtDNA is particularly rich in hydroxymethylation marks (Shock et al., 2011). Finally, mtDNA methylation showed a remarkable plasticity to environmental factors (Sharma et al., 2019) and has been suggested as an interesting emerging biomarker for the detection and diagnosis of diseases and the understanding of cellular behavior in particular conditions (Iacobazzi et al., 2013).

2.3.2.2 Mitochondrial transcription factor A and the mitochromosome structure According to the current dogma, mtDNA is not protected by histone proteins and is not arranged into nucleosome structures (despite some histone proteins having

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been identified in mitochondria (Choi et al., 2011)). Instead, it appears to be organized into nucleoids, which are nucleoprotein complexes whose primary structural constituent is the mitochondrial transcription factor A (TFAM). TFAM is an abundant protein involved in packaging mtDNA into nucleoids and regulating mtDNA copy number (Ekstrand et al., 2004). Furthermore, as its name suggests, TFAM (Wang & Bogenhagen, 2006) initiates the transcription of the genes encoded by mtDNA, by binding to the regulatory D-loop (Scarpulla, 2008). Although we cannot talk about histone modifications in the mitochondrial genome, posttranslational modifications of the TFAM also occur (King et al., 2018). TFAM is modified by acetylation, phosphorylation, and O-linked glycosylation. Despite evidence about a role of these modifications in the regulation of both compaction and transcription of mtDNA emerging, the specific sites of these modifications and their respective downstream consequences are poorly understood.

2.3.2.3 mitoMIRs As previously described, microRNAs (miRNAs) are small, single-stranded ncRNAs molecules involved in posttranscriptional control of gene expression. miRNAs align and bind especially to 30 UTR sequences of their target genes and initiate either mRNA degradation or translational repression, resulting in reduced protein levels. miRNAs derived from the nuclear genome can be imported into the mitochondria where they interact with some mtDNA-derived mRNA molecules, thereby modulating gene expression (Das et al., 2012; Tomasetti et al., 2014). miRNAs identified in the mitochondria are collectively termed mitochondrial miRNAs or mito-miR (Sripada et al., 2012). Moreover, human mtDNA also seems to harbor mito-miR sequences (namely, miR-1974, miR-1977, and miR1978), but it remains to be established if they are actually transcribed inside the mitochondria or derived from mitochondrial genes integrated into the nuclear genome (i.e., NUMTs) (Bandiera et al., 2011). miRNAs are recognized as major players in virtually every biological process, including the activity of mitochondria (i.e., mitochondrial OXPHOS, ETC components, lipid metabolism) (Shepherd et al., 2017).

2.4 Impact of diet on the epigenome: the mediation of mitochondria 2.4.1 How diet modulates the epigenome As already described, the epigenome is a plastic level of gene expression regulation and mediates the effects of environmental factors on the genome. Diet represents a key exogenous, modifiable factor that shapes the epigenome. Epigenetic modifications, in turn, may represent a pivotal mechanism by which nutrient availability, quality, as well as food bioactive derivatives impact human health.

2.4 Impact of diet on the epigenome: the mediation of mitochondria

Indeed, while nutrients for many years have been described as a mere source of energy, they now represent crucial signals able to influence cellular biochemistry and physiology at the gene expression level. Besides the already established role of dietary constituents to directly modulate gene expression (Andreescu, 2018), dietary patterns as well as nutrients also impact gene expression by modulating epigenetic modifications (Ideraabdullah & Zeisel, 2018; Mehedint et al., 2010; Waterland et al., 2006). First, nutrients may provide the substrates that comprise the epigenetic marks such as methyl groups for DNA methylation; second, nutrients modulate the activity of epigenetic regulatory enzymes. In terms of the first mechanism, a pivotal role is played by folic acid, vitamins B12, and B6 which participated in one-carbon metabolism alongside dietary methyl donors (choline, betaine, or methionine) to form SAM, which in turn provides the methyl groups for DNA methylation (Ideraabdullah & Zeisel, 2018). In agreement with this notion, a diet low in methyl donors leads to a decrease in DNA methylation as reported in the hepatic nuclear DNA of rodents (Wilson et al., 1984), further supporting the link between diet and epigenetic modifications. Besides the aforementioned B vitamins and methyl donor availability, other dietary factors also affect DNA methylation which include protein restriction which promotes hypermethylation (Lillycrop et al., 2008) as well as food bioactive derivatives such as sulforaphane and genistein which have also been shown to modulate methylation patterns. As already described, epigenetic modifications not only directly affect the DNA, but can also target histones, with diet modulating these processes. These modifications are keys to adjusting nutrient absorption and metabolism to dietary intake to allow the body metabolic machinery to adapt to switch in nutrient quality, such as the ratio of carbohydrates-to-fat intake. In this regard, in rodents a high-starch/low-fat diet promotes the methylation of histone H3K4 on the transcribed regions and promoter of genes involved in carbohydrates digestion and absorption like the sucrase-isomaltase and sodium/glucose cotransporter protein 1, thereby promoting their expression (Inoue et al., 2015). Histone acetylation which relies on acetyl group donors availability such as acetyl-CoA, malonyl-CoA, glutaryl-CoA, and succinyl-CoA is also influenced by dietary components. In this context, the NAD1-dependent deacetylases sirtuins are at the interface between nutritional status and histone acetylation. From a nutritional prospective, sirtuin activity is modulated by caloric restriction (Schwer & Verdin, 2008). As a consequence, calories influence histone acetylation status to modulate the expression of genes involved in fatty acid oxidation, endogenous glucose production, and insulin secretion, providing a key example of how nutritional status adjusts energy and substrate metabolism to changes in nutrient availability. Besides energy availability, food bioactive derivatives have also been proven to modulate histone deacetylation as in the case of resveratrol, which promotes histone deacetylation by enhancing the affinity of sirtuin 1 for acylated histones (Borra et al., 2005). Finally, diet can influence gene expression by up- or downregulating miRNAs as demonstrated in monkeys where caloric restriction was able to counteract the

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changes in skeletal muscle miRNAs induced by aging (Mercken et al., 2013). The impact of diet on micro-RNA expression has also been demonstrated in humans, with vitamin D (Jorde et al., 2012), zinc (Ryu et al., 2011), vitamin E, and sodium (Ferrero et al., 2021) correlating with the expression of several miRNAs whose function are strictly related to the impact of these nutrients on human health. For instance, sodium intake has been reported to affect the expression of miRNAs (Ferrero et al., 2021) whose levels have been reported to be altered in cardiovascular diseases, which is in line with the close relationship between high intake of sodium cardiovascular health (O’Donnell et al., 2010). Similarly, vitamin D intake negatively correlated with microRNA-144
3p whose expression, not surprisingly, is altered in individuals with osteoporosis (Wang et al., 2018). Thus, epigenetic modifications represent one of the factors mediating the impact of diet on human health, with the epigenome being modulated in response to nutrient quality and quantity. It must not be overlooked that energy availability, micronutrients, and macronutrients are not the only dietary factors shaping the epigenome. Certainly, food bioactive derivatives, which are also part of the diet, and are particularly abundant in healthy dietary patterns, are also responsible for fostering epigenetic modifications, extensively reviewed elsewhere (Kumari et al., 2020).

2.4.2 Focus on diet-related metabolic connections between mitochondria and cytoplasm able to affect the epigenome 2.4.2.1 Methyl donors, the one-carbon cycle and methylation reactions As previously mentioned, the one-carbon cycle (1CC) regulates numerous biochemical pathways (including epigenetic reactions that require the SAM, the universal methyl donor) and is duplicated in the cytosol and in mitochondria (Stoccoro et al., 2018; Zheng et al., 2015). A central role in this pathway is played by folates, a group of water soluble compounds within the vitamin B9 family that are acquired through the diet (e.g., leafy green vegetables) and as reduced forms or as folic acid in fortified foods (Dekhne et al., 2020). Folates are compartmentalized in the cytosol and mitochondria, with a smaller pool in the nucleus (Tibbetts & Appling, 2010). Cytosolic and mitochondrial 1CCs are connected by an exchange of serine, glycine, and formate, with uptake of folates from the cytosol into mitochondria occurring through a specific transporter (Lawrence et al., 2016). In the mitochondria, folates are required for the 1C metabolism originating from serine, whose catabolism serves as the principal source of 1C units, whereas glycine is employed for cellular biosynthesis (i.e., de novo synthesis of purine nucleotides and thymidylate in the cytosol). Moreover, mitochondrial 1CC is an important source of NADPH and glycine which, in turn, contributes to GSH synthesis and ATP production (Ducker & Rabinowitz, 2017). Finally, the methylated form of folate (5,10-methylene tetrahydrofolate) is then metabolized in the cytosol to produce 5-methyl tetrahydrofolate used as a methyl

2.4 Impact of diet on the epigenome: the mediation of mitochondria

donor for the conversion of homocysteine to methionine and then into SAM. Histone methyltransferases and DNA methyltransferases use SAM as a methyl donor to methylate histones at lysine or arginine residues, or DNA, respectively. In summary (Dekhne et al., 2020), serine metabolism in the mitochondria is related to epigenetic regulations supporting cellular methylation reactions via 5methyl tetrahydrofolate
dependent methylation of homocysteine (which also needs vitamin B12 as a cofactor) and de novo synthesis of ATP (that is an essential cofactor for ATP-dependent remodeling activities that use the energy derived from ATP hydrolysis to move/slide nucleosomes) (Hargreaves & Crabtree, 2011). Of note, alterations of these pathways have been reported to be essential in cancer development (Possemato et al., 2011; Yang & Vousden, 2016). The relevance of both mitochondrial and cytosolic 1CC to epigenetic regulations highlights the role of all the diet-derived micronutrients involved in this pathway (i.e., vitamins B12, B2, B6, B9, zinc, betaine, choline).

2.4.2.2 Acetyl-coA and acetylation reactions As previously described, acetylation reactions are essential regulators of the chromatin structure. HATs utilize the acetyl-coenzyme A as a cofactor, which is mainly generated in the mitochondria through the fatty acid β-oxidation and the Krebs cycle. Intra-mitochondrially-derived acetyl groups from pyruvate can exit mitochondria in the presence of L-carnitine and become an important source of acetyl groups for nuclear histone acetylation (Madiraju et al., 2009). Indeed, numerous GWAS have shown that histone acetylation is sensitive to the availability of acetyl-CoA, which responds to nutrient availability or metabolic reprogramming (Choudhary et al., 2014; Pietrocola et al., 2015; Sivanand et al., 2018). Moreover, a family of HDAC, the sirtuins, uses NAD 1 as a cofactor, which is generated in the mitochondria, cytoplasm, and nucleus. Sirtuins (e.g., human Sirt1
7) catalyze the removal of acyl groups from lysine residues in an NAD 1 dependent manner, and loss of sirtuin deacylase activity correlates with the development of aging-related diseases. Sirtuins have been found in the nucleus (SIRT1, 2, 3, and 7), cytoplasm (SIRT1, 2), and mitochondria (SIRT3, 4, and 5). Since they depend on NAD 1 as a cofactor, their activity is primarily influenced by fluctuations in NAD 1 levels, and therefore they are sensitive to nutrient availability, in consideration of the fact that low nutrient availability induces a drop in the NADH/NAD 1 ratio. Through their epigenetic actions, sirtuins regulate cellular metabolism by affecting the functions of key transcription factors regulating metabolic genes. For an in-depth description of these metabolic pathways, please refer to (Wiese & Bannister, 2020). Several dietary factors have been shown to affect sirtuin functions (Howitz et al., 2003), thus mediating these effects on the epigenome. Not only bioactive compounds, such as resveratrol, but also a dietary regimen of caloric restriction has been shown to modulate the activity of these proteins (Guarente, 2012). Finally, not only histone deacetylation, but also histone demethylation is affected by the availability of a metabolic intermediate generated

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in the mitochondria: the flavin adenine dinucleotide, which is a cofactor of lysine-specific histone demethylase (Hino et al., 2012).

2.4.2.3 Antioxidants Reactive oxygen species (ROS) are natural byproducts of oxidative metabolism, with mitochondria representing the major endogenous source of ROS (Turrens, 2003). Despite normal conditions, ROS act as cellular messengers in redox signaling, an excess of free radicals not readily neutralized by antioxidants can react with DNA or cross-link with proteins or lipids (Cutler, 2005). The oxidative balance of a cell is also correlated to the epigenetic homeostasis through numerous pathways. ROS can indirectly modulate the activity of the epigenetic machinery since histone-modifying enzymes depend on intracellular levels of essential metabolites, such as Acetyl-CoA, Fe, ketoglutarate, NAD1, and SAM (Simpson et al., 2012). Moreover, increased oxidative stress promotes oxidized DNA lesions formed by hydroxylation of pyrimidines and 5-methylcytosine (5mC), which can interfere, due to structural similarities, with epigenetic signals related to 5-hmC (Lewandowska & Bartoszek, 2011). The 1CC provides precursors for the synthesis of GSH that can be generated from cysteine through the transsulfuration pathway. Therefore, oxidative stress can globally influence the cell on multiple levels, from DNA and histones to histone modifiers, which will directly affect the epigenetic landscape of the cell. Also, ROS also affects DNA demethylation by DNA oxidation and TET-mediated hydroxymethylation (Chia et al., 2011). This evidence indicates that epigenetic changes are tightly linked to both global cellular metabolism and energy levels of the cell, with mitochondria acting as mediators in this network.

2.4.3 Effects of nutrients and diet on mitochondrial epigenetics and mito-epigenetics Given the strict integration between metabolism, epigenetic and epitranscriptomic processes, it is intuitive to assume that mitochondria, due to their essential metabolic role, have a major role in this network. In this regard, nutrients and food bioactive derivatives shape the epigenome by modulating mitochondrial metabolism which is particularly susceptible to nutrient availability as well as quality (Sergi et al., 2019). Mitochondria are perfectly placed to translate nutritional signals into epigenetic modifications. They are a crucial source of metabolites which provide functional groups for epigenetic modifications as well as the organelles responsible for the conversion of nutrients into the energy currency of the cell: ATP. As a consequence, nutritional intervention which alters mitochondrial metabolome will affect the availability of mark donors and downstream epigenetic modifications. For this reason, mitochondrial health is essential not only for the bioenergetic but also for the epigenetic homeostasis (Wiese & Bannister, 2020).

2.4 Impact of diet on the epigenome: the mediation of mitochondria

A key representative molecule in this context is represented by acetyl-CoA produced in the mitochondria as part of fatty acid β-oxidation and the Krebs cycle. A decrease in acetyl-CoA levels as in response to ketogenic diets, caloric restrictions, or fasting, is concomitant with a decrease in acetyl mark availability for histone acetylation. Similarly, an increase in NADH/NAD1 in response to low energy supply results in a drop in S-adenosyl-L-methionine produced in the mitochondria and the subsequent decline in a pivotal methyl donor affecting both DNA and histone methylation (Taormina et al., 2019). It is plausible, therefore, that nutrients and food bioactives are able to boost mitochondrial metabolism, and as molecules able to dampen mitochondrial ROS production by interfering with the mitochondrial metabolome and the availability of epigenetic mark donors. A key example of this paradigm is resveratrol, a polyphenol present in red wine, grapes, and peanuts and that has been shown to exploit a variety of health promoting effects which are dependent of its antiaging, antiinflammatory and antidiabetic properties. In terms of it role on mitochondrial health, resveratrol promotes mitochondrial biogenesis with a mechanism which depends upon SIRT1-mediated deacetylation and activation of the master regulator of mitochondrial function, PGC1α. Moreover, due to the tight link between oxidative stress and the epigenome, diet can have a role in modulating this relationship also, by serving as a supply of exogenous antioxidants that can contribute to a proper redox homeostasis. Indeed, both endogenous and exogenous (diet-derived) antioxidants play a major role in keeping free radicals in check and preventing them from causing cellular damage (Valko et al., 2007). Micronutrients such as vitamin C, E (tocopherols and tocotrienols), A (β-carotene with other carotenoids, retinol), selenium and many others are common dietary antioxidants that can decrease oxidative damage. The same paradigm holds true for bioactive molecules (e.g., epigallocatechin-3-gallate, curcumin, sulforaphane, genistein, stilbenoids, and others), which by dampening oxidative stressm modulate the cellular redox status. However, it remains to be elucidated, whether the effects of these molecules are a direct consequence of their activity as ROS quencher or depend on the induction of intracellular pathways aimed at protecting the cells against oxidative stress (Munialo et al., 2019). Independently of their mechanism(s) of action, these bioactive molecules can also modulate epigenetic marks through their antioxidant effects (Beetch et al., 2020). Finally, mtDNA can also undergo diet-induced epigenetic modifications as described in the case of the Mediterranean diet which has been shown to increase mtDNA methylation (Corsi et al., 2020). Additionally, mtDNA epigenetics appear to be responsive to l-carnitine supplementation as marked by an increase in Dloop methylation in platelets (Bordoni et al., 2020). However, despite the wellknown role of diet and food bioactive derivatives in modulating mitochondrial function, their impact on mitochondrial epigenetics remains to be elucidated and may open novel perspectives in the field of nutri-epigenetics. This represents a novel field of research which, by teasing out the relationship between nutrients, food bioactives, as well as dietary patterns and mitochondrial epigenetics, will

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further shed light on the role of mitochondria in the pathogenesis of diseases, in which the dysfunction of these organelles has been shown to represent a crucial pathogenetic feature, including type 2 diabetes mellitus and neurodegenerative diseases (Reddy, 2009; Sergi et al., 2019).

2.5 Conclusions Since epigenetic modifications are reversible and tightly connected with the energetic metabolism of the cell, dietary modulations of the epigenome appear to be major determinants of human health. Metabolic changes induced by the diet alter the relative abundances of various metabolites, thus directly affecting the epigenetic machinery. Mitochondria, which are the primary regulators of the cellular energetic and redox metabolism, also contribute to epigenome homeostasis. Epigenetic communication between nuclear and mitochondrial genomes occurs at multiple levels, ensuring a coordinated gene expression response to differential environmental stimuli. A better understanding of this crosstalk and the effects of diet on it will open the way to new prevention strategies using nutrition to prevent complex and environmentally-driven noncommunicable diseases.

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60.

CHAPTER

Nutritional assessment and malnutrition in adult patients with mitochondrial disease

3 Heidi Zweers

Department of Gastroenterology and Hepatology-Dietetics, Radboud University Medical Centre, Nijmegen, The Netherlands

3.1 Introduction According to the definition by Stratton (Stratton et al., 2003), the term “disease related malnutrition” coins a shortage and/or imbalance of nutrient intake that leads to negative changes in body composition, functioning, and clinical outcome (Fig. 3.1). By this definition, the energy shortage in MD is malnutrition on a cellular level. Malnutrition is a complex problem and according to the definition of Stratton, both undernutrition and obesity could be present, and both are known to occur in MD patients (Apabhai et al., 2011; Aubry et al., 2018). The combination of low muscle mass and low muscle strength seen in malnourished MD-patients may also be defined as sarcopenia (Cruz-Jentoft et al., 2019; Hiona & Leeuwenburgh, 2008).

FIGURE 3.1 Malnutrition according to Stratton. Malnutrition according to Stratton.

Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00005-9 © 2023 Elsevier Inc. All rights reserved.

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Body composition is known to be important for physical functioning in the general population and even more in neuromuscular disorders (Nau et al., 1997; Palmieri et al., 1996; Pruna et al., 2011; Skalsky et al., 2008). Altered body composition in MD patients has been reported (Aubry et al., 2018; Hou et al., 2019; Zweers et al., 2021b) and a higher skeletal muscle mass index in these patients was correlated with higher muscle strength (Hou et al., 2019; Zweers et al., 2021b). The low physical functioning in MD patients may also be related to alterations in both nutritional intake and nutritional status. Recent observational studies suggested that MD patients have inadequate protein intake and are at risk of malnutrition (Cruz-Jentoft et al., 2019; Rinninella et al., 2018; Zweers et al., 2021b). This could affect both body composition and physical functioning. The DYNAMO study (Zweers et al., 2021b) demonstrates that muscle strength is related to body composition and protein intake in MD patients. The physical functioning of MD patients appears to be determined by the availability of energy (ATP) on the one hand and the nutritional status on the other. The “assumed” relationship between these is summarized in Fig. 3.2. It is suggested that malnutrition may worsen symptoms in MD, which was shown in a study in young MD patients in which an association between nutritional status and mitochondrial functioning was observed (Wortmann et al., 2009). Vice versa, it is known from other diseases such as anorexia nervosa (Chinnery & Turnbull, 1998) or cancer cachexia (Fearon et al., 2013; Ushmorov et al., 1999) that malnutrition causes secondary mitochondrial dysfunction. From data of children with mitochondrial disorders, it was suggested that by improving the nutritional status in MD patients, ATP production may be improved (Morava et al., 2006).

FIGURE 3.2 Assumed relationship between body composition, nutritional intake, and physical functioning in patients with MD. Assumed relationship between body composition, nutritional intake and physical functioning in patients with MD.

3.1 Introduction

3.1.1 Gastro intestinal problems and BMI de Laat et al. (2015) published an observational study that explores and characterizes the frequency and severity of gastrointestinal complaints including dysphagia in 92 adult patients carrying the m.3243 A . G mutation in mitochondrial DNA using a validated questionnaire. Data were compared with those obtained in healthy controls. Gastrointestinal problems (86%), especially constipation (69%), bloating (51%), flatulence (40%), and mild dysphagia (33%), are very common in carriers of the m.3243 A . G mutation and are not related to heteroplasmy levels in urinary epithelial cells, nor to disease severity. The severity of gastrointestinal problems as well as overall disease severity was associated with an increased risk for malnutrition in the form of a low BMI (de Laat et al., 2015). There was however, no relevant correlation between gastrointestinal problems and BMI, indicating that perhaps other factors contribute to the risk of malnutrition. The results support the hypothesis that severely affected patients have a higher risk for malnutrition. Apabhai et al. (2011) studied a similar MD population in the UK (Newcastle) and in contrast these authors found that almost half of the patients were overweight, similar to healthy controls. On the other hand, de Laat et al. (2015) showed a significantly lower BMI in patients compared to controls, while around one-third of these patients had a BMI . 25. Therefore, both under- and overweight seems to be a problem in this population. It should be realized here that patients with a normal or high BMI may still be at risk for malnutrition because of an inadequate diet, with altered body composition (low muscle mass and high fat mass), and low functioning.

3.1.2 Food intake Zweers et al. (2018) performed an observational, cross-sectional, retrospective study, 60 three-day nutrition diaries of adult MD patients were analyzed and compared with the Dutch recommended daily allowance and the Dutch National Food Consumption Survey. This study demonstrated that most MD patients had an inadequate diet. Specifically, intake of protein, calcium, dairy products, and fluids were low although interindividual differences were high. Overall, maintaining a healthy diet seems as difficult for MD patients as it is for the general population: recommendations for fiber, sugars, saturated fat, and vitamin D intake were not met in either group.

3.1.3 Prevalence of malnutrition in mitochondrial diseases In the DYNAMO study, Zweers et al. (2021b) found a high prevalence of malnutrition in MD patients (73%) and this confirms previous work (Apabhai et al., 2011; Aubry et al., 2018; Hou et al., 2019). According to the GLIM criteria, 46% of MD patients were malnourished, whereas 43% were according to the PG-SGA criteria. This seems to be a consistent result, however, there was a low overlap in

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malnutrition between the two methods. A better comparability was seen between sarcopenia and malnutrition since all patients diagnosed with sarcopenia were also classified as malnourished. This low comparability underlines the challenges of diagnosing malnutrition and underlines the conclusions of Aubry et al. (2018), that a nutritional assessment should be part of patient care in all adult MD patients.

3.1.4 The optimal method for nutritional assessment in adult mitochondrial diseases patients 3.1.4.1 Nutritional assessment Nutritional assessment entails the systematic assessment of nutritional status and nutritional needs. Measurements are performed in a structured (subjective and objective) manner that can be categorized into three domains: • • •

Food intake and requirements Body composition Functional parameters.

These three domains correspond with the definition of malnutrition (Stratton et al., 2003) (Fig. 3.1). Nutritional assessment is part of every dietary consultation and is essential in dietary diagnostics and research. The individual dietary treatment plan is based on data from the nutritional assessment. Nutritional assessment as such can be performed on many levels, ranging from very basic to more sophisticated (and accurate) assessments. A basic one for example, is comprised of an assessment of dietary history regarding food intake, anthropometry (height and weight measurements) for body composition, and interview data for functioning. “More accurate” also implies more time-consuming, expensive, and invasive tests for patients. In striving for the best possible individual dietary treatment options, dietitians search for the optimal balance between intensive assessment methods and obtaining reliable data to design the individual treatment plan.

3.1.4.2 Energy requirements The optimal method to estimate total energy expenditure in MD patients was studied by Zweers et al. (2021c). Resting energy expenditure was measured in adult MD patients carrying the m.3243 A . G mutation using indirect calorimetry and was compared with results of 21 predictive equations for resting energy expenditure, as well as with indirect calorimetry data in healthy controls. Physical activity level (PAL) was assessed using accelerometery (SenseWear) and compared with a fixed average PAL as well as with patients’ self-estimated activity levels. Total energy expenditure was lower in MD patients than the recommendations for healthy adults because of their lower physical activity. In MD patients, six prediction equations for resting energy expenditure provided a reliable alternative for indirect calorimetry, with an accuracy of 71%
76%. As PAL are highly

3.1 Introduction

variable and are not reliably estimated by the patients themselves, measurement of PAL using accelerometery is recommended in this population.

3.1.4.3 Body composition In the DYNAMO study (Zweers et al., 2021b), the second domain (i.e., body composition) was evaluated and the diagnostic accuracy of bioelectrical impedance analysis (BIA) versus dual xray absorptiometry (DXA) measurements in adult MD was tested. The conclusion was that for measuring body composition, DXA should be preferably used instead of BIA because of the higher accuracy. If DXA is not available, waist circumference is the preferred additional method besides BIA to assess the risk for obesity and metabolic syndrome in this population because BIA has a low sensitivity and specificity for diagnosing a high-fat percentage.

3.1.4.4 Functional parameters In the DYNAMO study (Zweers et al., 2021b) multiple functional tests from the third domain were executed: handgrip strength, 30 s sit to stand test, 6-min walking test, and the 6-min mastication test. The choice for these tests was based on the expertise of the physiotherapist of the multidisciplinary mitochondrial expertise team from the Radboudumc. In both the GLIM consensus for diagnoses of malnutrition (Cederholm et al., 2019), and in the sarcopenia consensus (CruzJentoft et al., 2019), handgrip strength is included which is easy to perform by patients, and dietitians can include this in their consultation with minimal additional effort or costs. The handgrip strength test provides valuable data on functioning, however reference values and cut off points are unclear. For instance, in the Sarcopenia consensus, cut off points are used based on data provided by Dodds et al. (2014) for 70-year-old patients. This makes sense because sarcopenia is mainly a muscle disorder in the elderly. However, chronically ill patients, including the MD population, frequently suffer from sarcopenia as well (Hiona & Leeuwenburgh, 2008; Hou et al., 2019; Shah et al., 2009). Since the mean age of the MD population is between 40 and 45 years, it is incorrect to use the cut off point for 70-year-old patients without further evidence. The DYNAMO study demonstrated that sarcopenia does get underdiagnosed when the mentioned consensus cut off point are used. Therefore, the recommendation is to use the agespecific cut off point of the Dodds reference (Dodds et al., 2014).

3.1.4.5 PG SGA The PG SGA has been validated in oncology patients and it is suggested to be suitable for other patient categories (Kosters et al., 2016). The MD patient, however, in contrast to cancer patients in general, does not suffer from inflammation. Another drawback of the PG SGA is that as found in the DYNAMITE study [23], the improved nutritional status after diet intervention is not reflected in an improved PG SGA score. This could be explained by the fact that gastrointestinal symptoms are frequently seen in MD patients (de Laat et al., 2015), which

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comprise a substantial part of the PG-SGA score. For example, the individual diet intervention could decrease the severity of constipation, but the patient still received one point for constipation in the PG SGA because severity of symptoms is not reflected by a lower PG SGA score. Moreover, the “functioning section” of the PG SGA contains a question about the lowering of daily activity, which is not suitable for a chronic patient population. Apart from these drawbacks, the use of the PG SGA is supported by the positive fact that it is a patient-generated instrument that includes all three domains of nutritional assessment and takes minimal effort to measure.

3.1.4.6 GLIM criteria The GLIM criteria (Cederholm et al., 2019; Jensen et al., 2019) for malnutrition are categorized into two domains. One is the phenotypic criterion on body composition. The other domain comprises the etiologic criterion. To meet this criterion, patients need to have either a low nutritional intake and/or inflammation should be present. Unfortunately, a “functioning domain” is not part of this consensus. The GLIM criteria have not specifically been designed for the analysis of a chronic patient category.

3.1.4.7 Sarcopenia The sarcopenia consensus (Cruz-Jentoft et al., 2019) on the other hand, is more suitable for chronic patients, yet sarcopenia shows overlap with malnutrition. Also, while both nutrition and mitochondrial dysfunction can play a role here (Shah et al., 2009), sarcopenia is a muscle disorder that mainly occurs in elderly. Therefore, the consensus only contains two domains: body composition and functioning. For sarcopenia diagnosis, body composition (muscle mass) has to be measured (different from the PG SGA or GLIM criteria). Although this measurement is more invasive, body composition is crucial for the MD patient population because many patients with normal or even high BMI still have a low muscle mass, which may easily remain undetected if not actually measured (Aubry et al., 2018). In the sarcopenia consensus, functioning also has to be measured, using either hand grip strength or the sit-to-stand tests. The sit-to-stand test has additional advantages in MD patients because of the endurance component, which is highly relevant in patients with a muscular energy deficit.

3.1.4.8 NRS_2002 screening tool Aubry et al. (2018) performed nutritional assessment on all domains in 11 MD patients and 15 controls. These authors concluded that all patients were malnourished according to the definition by the European Society of Clinical Nutrition and Metabolism (ESPEN), but none were malnourished according to the screening tool (nutritional risk score NRS-2002). Thus, the latter tool seems less sensitive in chronically ill outpatients.

3.2 Nutritional assessment and dietary interventions

3.1.5 Sex differences The MD population is a highly heterogeneous group of patients and this heterogeneity is based on both genotype and phenotype. Sex differences could also play a role here. References used in nutritional assessment are mostly age and gender-specific, and some nutritional recommendations are as well (e.g., the iron recommendation is higher in females because of menstrual losses). In addition, formulas to calculate energy requirements and body composition use gender and age and also height and weight as variables to correct for these differences (Zweers et al., 2021b).

3.2 Nutritional assessment and dietary interventions The DINAMITE study (Zweers et al., 2020), is a randomized controlled trial to explore the effect of an individually tailored dietary intervention on personalized goals, body composition, functioning, and quality of life in 39 adult MD patients due to the m.3243 A . G mutation. The intervention group (n 5 20) received an individually tailored dietary intervention over a 6-month period, whereas controls (n 5 19) received standard care during 6-months (control period), followed by an individually tailored dietary intervention for the next 6 months (intervention period). Nutritional assessment and QoL measurements were performed at 3month intervals. After 3 months of dietary intervention, 57% of the personalized goals were achieved. The most successfully realized goals were: improved body composition, handgrip strength, and diminished gastrointestinal complaints. Consistent effects on functioning included improved handgrip strength, vitality, and fatigue score. These effects, however, did not seem to last after 3 months. The DINAMITE study concludes that because of the heterogenicity of MD patients, nutritional interventions should be tailored in a personalized manner and based on the nutritional assessment outcomes. The nutritional implications and recommendations based on the nutritional assessment are summarized in Fig. 3.3. (1) low energy intake , 90% individual needs 5 calculated REE 1 measured PAL (with actometre); (2) low protein intake ,1.2 g/kg ideal bodyweight; (3) low handgrip according to (Dodds et al., 2014); (4) low muscle mass; preferably measured by DXA 5 SMI , 7 kg/m2 for men and , 5,5 kg/m2 for women or FFMI measured by BIA , 15 kg/m2 for women and ,17 kg/m2 for men (Cederholm et al., 2019; Jensen et al., 2019); (5) weight loss 5 . 5% in 6 months or . 10% . 6 months (Cederholm et al., 2019); (6) low BMI 5 , 20 kg/m2 (Cederholm et al., 2019); (7) high waist circumference 5 men $ 94 cm women $ 80 cm (WHO, 2008); (8) high BMI 5 . 30 kg/m2 (Weir and Jan, 2022); (9) high fat percentage 5 . 30% for women and .25% for men (Okorodudu et al., 2010); (10) malnutrition 5 according to GLIM 2018 (Cederholm et al., 2019) 5 low muscle mass4, low BMI6 or weight loss5 1 low nutritional intake1,2 or malnutrition according to PG SGA; (11) sarcopenia 5 consensus 2018 (Cruz-Jentoft et al., 2019) 5 low Handgrip Strenght3 1 low muscle mass.4

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FIGURE 3.3 Summary of nutritional implications and recommendations based on nutritional assessment.

3.3 Conclusion Malnutrition is very common in MD, even in patients with a normal or high BMI. Unfortunately, typical screening tools lack sensitivity to detect malnutrition in this chronically ill patient group. All adult patients with MD should receive a full nutritional assessment of all three domains (1) food intake and requirements; (2) body composition; (3) functional parameters. This nutritional assessment should

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152. Available from https://doi.org/ 10.1097/GIM.0b013e31819307a2. Skalsky, A. J., Abresch, R. T., Han, J. J., Shin, C. S., & McDonald, C. M. (2008). The relationship between regional body composition and quantitative strength in facioscapulohumeral muscular dystrophy (FSHD). Neuromuscular Disorders, 18(11), 873
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Stratton, R. J., Green, C. J., & Elia, M. (2003). Disease related malnutrition: An evidence based approach to treatment. Ushmorov, A., Hack, V., & Dro¨ge, W. (1999). Differential reconstitution of mitochondrial respiratory chain activity and plasma redox state by cysteine and ornithine in a model of cancer cachexia. Cancer Research, 59(14), 3527
3534. Weir, C.B., & Jan, A. (2022). BMI classification percentile and cut off points. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; Available from: https://www.ncbi.nlm.nih.gov/books/NBK541070/. WHO. (2008). Waist circumference and waist-hip ratio. Wortmann, S. B., Van Essen, H. Z., Rodenburg, R. J. T., Van Den Heuvel, L. P., De Vries, M. C., Rasmussen-Conrad, E., Smeitink, J. A. M., & Morava, E. (2009). Mitochondrial energy production correlates with the age-related BMI. Pediatric Research, 65(1), 103
108. Available from https://doi.org/10.1203/PDR.0b013e31818d1c8a. Zweers, H., Janssen, M. C. H., Leij, S., & Wanten, G. (2018). Patients with mitochondrial disease have an inadequate nutritional intake. Journal of Parenteral and Enteral Nutrition, 42(3), 581
586. Zweers, H., Smit, D., Leij, S., Wanten, G., & Janssen, M. C. H. (2020). Individual dietary intervention in adult patients with mitochondrial disease due to the m.3243 A . G mutation. Nutrition (Burbank, Los Angeles County, Calif.), 69, 110544. Available from https://doi.org/10.1016/j.nut.2019.06.025. Zweers, H. E. E., Janssen, M. C. H., & Wanten, G. J. A. (2021a). Response to energy requirements in m.3243A . G carriers depend on multiple factors. Journal of Parenteral and Enteral Nutrition, 45(2), 229. Available from https://doi.org/10.1002/ jpen.2023. Zweers, H. E. E., Bordier, V., in ‘t Hulst, J., Janssen, M. C. H., Wanten, G. J. A., & LeijHalfwerk, S. (2021b). Association of body composition, physical functioning, and protein intake in adult patients with mitochondrial diseases. Journal of Parenteral and Enteral Nutrition, 45(1), 165
174. Available from https://doi.org/10.1002/jpen.1826. Zweers, H. E. E., Janssen, M. C. H., & Wanten, G. J. A. (2021c). Optimal estimate for energy requirements in adult patients with the m.3243A . G mutation in mitochondrial DNA. Journal of Parenteral and Enteral Nutrition, 45(1), 158
164. Available from https://doi.org/10.1002/jpen.1965.

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CHAPTER

Therapeutic potential and metabolic impact of alternative respiratory chain enzymes

4 Sina Saari1,2

1

Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland Obesity Research Unit, Research Program for Clinical and Molecular Metabolism, Faculty of Medicine, University of Helsinki, Helsinki, Finland

2

4.1 Introduction The human core oxidative phosphorylation system (OXPHOS) is widely conserved throughout all kingdoms of life. Yet, many organisms, including plants and fungi, possess additional enzymes that enable alternative respiratory pathways. The core OXPHOS system, composed of large enzyme complexes, is a rather rigid machinery that is efficient in ATP production but poorly suited for sudden changes in the environment. The prevalent theory behind the appearance and preservation of additional OXPHOS branches in many organisms and species is based on their ability to improve metabolic flexibility. Still, the question remains why vertebrates have lost these respiratory pathways. The selection pressures underlying the evolution of alternative respiratory enzymes remain to be established. For a long time, these enzymes were thought to be only present in plants, bacteria, protozoans, and fungi (McDonald & Gospodaryov, 2019). Recently, the sequencing of several animal genomes has shown that genes that have strong similarities with alternative respiratory enzymes are present in several animal phyla. Whether these genes provide functional alternative respiratory enzymes, and, if so, under which circumstances are these enzymes active, is under investigation. In any case, alternative respiratory pathways remain absent from vertebrates and from most of the arthropods. The two main alternative respiratory chain (RC) enzyme families, the type 2 alternative NADH dehydrogenases (NDH2s) and the alternative oxidases (AOXs), are small, nontransmembrane complexes located at the inner mitochondrial membrane, where they provide the RC with branching points for ubiquinone reduction (NADH:ubiquinone) and ubiquinol oxidation (ubioquinol:O2), respectively (McDonald & Gospodaryov, 2019) (Fig. 4.1). Unlike the RC complex that they

Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00021-7 © 2023 Elsevier Inc. All rights reserved.

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FIGURE 4.1 Alternative respiratory chain pathways. (A) Core OXPHOS system in mammalians (e.g., Homo sapiens). (B
D) Examples of core OXPHOS systems with one or both alternative respiratory chain enzymes, as observed in some animals (e.g., Ciona intestinalis), in various fungi (e.g., Saccharomyces cerevisiae) and in plants (e.g., Arabidopsis thaliana). AOX, Alternative oxidase; C, cytochrome c; NDE, external alternative NADH dehydrogenase; NDI, internal alternative NADH dehydrogenase; NDX, alternative NADH dehydrogenase; Q, ubiquinol/ubiquinone.

can replace, the alternative respiratory enzymes do not pump protons, but instead dissipate the energy of the electron transfer reaction as heat. AOXs and NDH2s have been found to contribute to the general metabolic state of the cell and to respond to changes in the environment via for example, redox homeostasis and thermogenesis (McDonald & Gospodaryov, 2019). The alternative respiratory pathways have been extensively studied in plants where their main role seems to be in helping the organism adapt to sudden changes in the environment (Florez-Sarasa et al., 2020; van Dongen et al., 2011). The majority of animals that still have active alternative respiratory pathways are marine animals (Saari, Garcia, et al., 2019; Tward et al., 2019). It is therefore plausible, that in vertebrates and arthropods that have lost these pathways, the metabolic adaptability is provided by other, more convenient mechanisms suitable for their habitat or “way of life”. Another theory is that the alternative respiratory pathways were counter-selected in the course of evolution due to their harmful or disadvantageous properties. In fact, evidence exist that the NDH2 and AOX genes that have been preserved in vertebrates have altered functions. In humans, for example the FSP1 gene, a member of the NDH2 protein family, is transcribed and translated into a protein (Bersuker et al., 2019). This protein is involved in

4.2 Alternative oxidase

ferroptosis and mitochondrial oxidative stress signaling but cannot provide alternative respiration. Due to their unique features, and the absence of alternative respiratory activity in humans, these enzymes have created exciting new perspectives for the treatment of mitochondrial diseases (Rajendran et al., 2019; Seo et al., 1998).

4.2 Alternative oxidase AOXs are localized on the matrix side of the inner mitochondrial membrane where they provide an alternative route that directly transfers electrons from ubiquinol to molecular oxygen, bypassing Complex III (CIII) and Complex IV (CIV) of the classical RC (Fig. 4.1). Because of its potential as a therapeutic target, the AOXs of the parasitic protist Trypanosoma brucei is one of the most studied AOXs. Its crystal structure reveals a homodimeric protein with a nonheme diiron at the active site (Shiba et al., 2013). The diiron core is responsible, in association with a tyrosine residue at 220 (Tyr220), for the reduction of O2. It is located in the vicinity of a hydrophobic cavity proposed to serve as an ubiquinol binding site. The structure of the active site appears highly conserved across species (May et al., 2017). However, species differences in ubiquinol oxidase activity of AOXs have been found. These differences appear linked to the variability in the residues lining the ubiquinol-binding cavity (May et al., 2017). Respirometry studies in cells expressing AOXs have systematically shown that AOXs possess lower affinity for ubiquinol than CIII, supporting the idea that AOXs only participate in quinol reduction when CIII or CIV are inactive, dysfunctional, or saturated (Hakkaart et al., 2006). However, it is now recognized that “classical” respirometry studies using inhibitors may underestimate AOX activity. Oxygen isotope fractionation studies offer a better view at the partitioning of the electrons between the alternative and classic oxidases, showing that the alternative respiratory pathways are involved in 10%
50% of the basal respiration in the absence of stress (Del-Saz et al., 2018; Florez-Sarasa et al., 2016). The regulation of AOXs and the metabolic consequences of their activity also vary amongst isoforms and organisms where it is expressed. For example, in plants two subfamilies of AOXs are present. AOX2 family members are constitutively expressed isoforms while oxidases of AOX1 family are stress induced. Most studies from plants, fungi, and protists point towards a rapid transcriptional activation of enzymes in the AOX1 family in response to various stress conditions such as intense light, low temperature, and changes in pH (McDonald & Gospodaryov, 2019). In addition, posttranslational modifications have been observed (Day et al., 1995). Both isoforms possess two highly-conserved cysteine residues in the N-terminal domain of the protein. The intermolecular disulfide bond between the first cysteine residues of each monomer causes the inactivation of AOX1 enzymes while the noncovalently linked dimer is the active form. The

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balance between oxidized (inactive) and reduced (active) forms is controlled by the redox state of the NAD(P)H pool and therefore, by mitochondrial metabolism (Juszczuk & Rychter, 2003; Vanlerberghe et al., 1995). It is important to note, however, that AOXs should rarely be in their oxidized/inactive forms, in vivo. Independent of their ability to undergo oxidative inactivation, AOX1 and AOX2 subfamilies can be activated by the association of 2-oxo acids such as pyruvate and glyoxylate to the cysteine residues. The physiological role of AOX remains poorly understood in animals that naturally possess this gene. Most of the existing information comes from in silico analysis of genomes and transcriptomes, and little is known about the posttranslational regulation of AOX in animals (McDonald et al., 2009). Tward et al. (2019) recently demonstrated that AOX is naturally transcribed and translated in the marine copepod Tigriopus californicus, introducing this organism as a model to study the role of AOX in animals (Tward et al., 2019). The discovery of AOX in animals has solidified the perspective of its use in human medicine, and particularly in the treatment of currently incurable mitochondrial diseases. Animal AOXs may provide a more suitable protein than AOXs of plant or fungal origin, as it is more likely to be compatible with human metabolism (Hakkaart et al., 2006).

4.3 Alternative NADH dehydrogenase Yeast and plants express alternative NADH dehydrogenases (NDH2s) that localize either on the external (NDEs) or on the internal (NDIs) side of the inner mitochondrial membrane (Fig. 4.1). NDH2s provide a branching point for NADH: ubiquinone oxidoreduction and are therefore capable of replacing the OXPHOS reaction that is catalyzed by Complex I (CI). Like AOXs, NDH2s do not contribute to proton pumping or to the generation of a ΔΨ (Matus-Ortega et al., 2011; Saladi et al., 2020; Sweetman et al., 2019). NDEs transfer electrons from cytosolic NADH to the OXPHOS and are therefore involved in the cytosolic metabolism. In yeast, NDEs have been found to direct cells with mitochondrial dysfunction for apoptosis (Saladi et al., 2020). In contrast, NDIs provide the RC with resistance to inhibitors targeting CI, such as rotenone, allowing for the preservation of the tricarboxylic acid (TCA) cycle metabolism. This is why the internal alternative NADH dehydrogenases, NDIs, are presented as an alternative for the treatment of CI-related diseases. Phylogenetic studies show that NDH2s are present in metazoans, although with a more limited distribution than AOX. In addition, and in contrast with AOXs, they are also found in archaea. Crystal structure of the NDI1 from Saccharomyces cerevisiae revealed a homodimer with FAD and NAD1-binding domains as well as an amphiphilic domain that anchors the enzyme to the inner mitochondrial membrane (Iwata et al., 2012). The structure shows an overlap between the binding

4.4 Transgenic models of alternative respiratory chain enzymes

sites for NAD1 and ubiquinol suggesting that their binding is mutually exclusive. Similar to the AOX, NDIs have been implicated in cold adaptation and photosynthetic metabolism in plants (Staffan Svensson & Rasmusson, 2001; Svensson et al., 2002). NDIs also function as an “overflow system,” keeping reducing equivalents at physiological levels and thereby preventing oxidative stress. In Aspergillus niger, which possesses an alternative NDH2 in addition to CI but has no AOX, inhibition of NDH2 leads to increased growth and protein production but also increased susceptibility to oxidative stress (Voulgaris et al., 2012), supporting a general role of NDIs in antioxidant defense and metabolic regulation. Expression of the yeast (S. cerevisiae) NDI1 in mammalian cells has provided clear respirometry-based evidence that NDI1 affinity for ubiquinol may equal that of the human CI (Seo et al., 1999).

4.4 Transgenic models of alternative respiratory chain enzymes Although the reasons for the loss of alternative respiratory enzymes in vertebrates and in many arthropods remains a mystery, their potential as a therapeutic treatment has given birth to several transgenic models including mammalian cells, fruit flies, and rodents. In these models, the aptitude and limits of alternative respiratory pathways were tested by combining them with mitochondrial dysfunction as well as metabolic stressors such as elevated temperature, cold, and malnutrition.

4.4.1 Mammalian cell models Since the alternative RC enzymes have been lost during evolution in most animals, including mammals, the viability of their reintroduction into mammalian cells was a strong concern. Originating from one of the closest related species to humans with a functional AOXs, the AOX gene from the sea squirt Ciona intestinalis was considered more likely to be innocuous than for example, AOXs from plants or fungi. Introduction of Ciona AOX into human HEK293T cells (Hakkaart et al., 2006) showed no detectable effect on proliferation under standard culture conditions, while providing resistance to antimycin, an inhibitor of CIII, and to cyanide, an inhibitor of CIV (Fig. 4.2). In later studies (Dassa et al., 2009), inhibition of AOX with n-propyl gallate (nPG, Fig. 4.2) confirmed that the alternative enzyme was the source of antimycin- and cyanide-resistant respiration. Importantly, the expression as well as the inhibition of AOX barely affected the overall oxygen consumption of the RC in respirometry studies, indicating that in the absence of defects in CIII, CIV or Complex V (CV), AOX does not interfere with the activity of the human OXPHOS complexes (Dassa et al., 2009; Hakkaart et al., 2006). Similar results were obtained using salicylic hydroxamic acid,

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FIGURE 4.2 The OXPHOS and OXPHOS inhibitors. Inhibitors are presented in red with the blunted arrow indicating the RC complex they target. Only a subset of inhibitors that are commonly used to estimate the activity of the RC pathways are presented. AOX, Alternative oxidase; C, cytochrome c; NDH2, alternative NADH dehydrogenase; Q, ubiquinol/ubiquinone.

another AOX inhibitor. This lack of interference was attributed to the low affinity of AOX for ubiquinol compared to the mammalian CIII. Kinetic and respirometry studies confirmed that, like in plants, Ciona AOX is engaged in circumstances of RC dysfunction, and more precisely, when the RC is oversaturated and the ubiquinol pool is consequently builtup (Dassa et al., 2009; Hakkaart et al., 2006). However, respirometry has been shown to underestimate the activity of AOX in plants (Del-Saz et al., 2018; Florez-Sarasa et al., 2016). In HEK293T cells, AOX decreased superoxide dismutase (SOD) expression, even in conditions where its respiratory role was undetectable (Hakkaart et al., 2006). This is an interesting effect since SOD2 expression is known to be tightly regulated by oxidative stress. It is plausible that AOX is active, maybe in a small subset of mitochondria, which would decrease ROS level and, in turn, negatively influence SOD2 expression. Oxygen isotope fractionation studies could clarify the in vivo partitioning of electrons between AOX and CIV. On HEK293T cells, the antioxidant effect of AOX was later demonstrated and, unexpectedly, found to be dependent on the metabolic state of the cells (Cannino et al., 2012). In the presence of a fermentable source of carbon (glucose), ROS levels were maintained low independently of AOX expression, even when cells were treated with antimycin. On the contrary, nonfermentable culture conditions, where glucose was replaced by galactose, led to an increase in basal ROS levels as well as in ROS level after antimycin (Cannino et al., 2012). This increase was blunted in AOX-expressing cells, supporting the strong antioxidant effect of AOX (Cannino et al., 2012).

4.4 Transgenic models of alternative respiratory chain enzymes

Since then, Ciona AOX has been successfully used to clarify the role of mitochondria in epigenetic signaling (Lozoya et al., 2019), cancer (Hollinshead et al., 2020; Martı´nez-Reyes et al., 2020), and immunity (Billingham & Chandel, 2019; Mills et al., 2016). Interestingly, the AOX from the thermogenic skunk cabbage Symplocarpus renifolius was also capable of driving antimycin- and cyanideresistant respiration in HeLa cells (Matsukawa et al., 2009). Plant AOXs are known to be posttranslationally regulated by pyruvate and ketoglutarate (Gray et al., 2004). HeLa cells expressing Symplocarpus AOX showed enhanced cyanide-resistant respiration when supplemented with pyruvate in nonfermentable culture conditions but not when pyruvate supplementation was combined with a fermentable substrate (Matsukawa et al., 2009). The seemingly negative impact of fermentable metabolism on AOX activity could suggest that higher organisms have developed alternative metabolic pathways to modulate RC activity in the absence of alternative RC enzymes for example, rerouting the metabolism towards fermentation (Warburg-like effect). In support of this idea, when the ability of AOX to alleviate mitochondrial dysfunction was tested in HEK293T cells with CIV defects, whether due to the knockdown of COX10 by RNAi or to a mutation in COX15 (Antonicka et al., 2003), only in nonfermentable conditions was AOX capable to restore cell growth (Dassa et al., 2009). Furthermore, in AOX-expressing HEK293T cells, the coexpression of NDI1 suppressed the effect of the substrates on AOX activity (Cannino et al., 2012). In both fermentable and nonfermentable conditions, when coexpressed with NDI1, AOX provided strong respiration and antioxidant capacity after antimycin or cyanide inhibition of the RC. This led the authors to conclude that the metabolism-driven adaptation was linked to a posttranslational regulation of CI, bypassed by NDI1. The yeast alternative NADH dehydrogenase, NDI1, was first introduced into Chinese hamster cells and later into human HEK293 cells. Both NDI1-expressing models demonstrated unaltered viability associated with resistance to CI inhibition (Seo et al., 1998). However, in HEK293 cells, NDI1 expression decreased the ADP/O ratio of NADH oxidase-driven OXPHOS activity, as expected by the lack of H1 pumping ability of alternative respiratory pathways, while succinate dehydrogenase-driven OXPHOS activity, which does not require NDI1 or CI, remained unchanged (Seo et al., 1999). Therefore, NDI1 competes with CI for NADH. NDI1 has nevertheless been considered for complementation therapy of pathologies associated with CI dysfunction. NDI1 was found to improve NADH oxidation in studies using Chinese hamster cells, human osteosarcoma cells (Bai et al., 2001), as well as in a cell model of CI deficiency associated with Leber hereditary optic neuropathy (Park et al., 2007; Seo et al., 1998). Furthermore, in SH-SY5Y neuroblastoma cells, that are widely used as a model for neuronal differentiation, metabolism and neurodegeneration, the symptoms of Parkinson’s disease caused by mitochondrial replacement with mitochondria from patients, were clearly attenuated by the expression of NDI1 (Cronin-Furman et al., 2019). The improvements included enhanced RC activity, better mitochondrial biogenesis as

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well as lowered levels of Lewy bodies, which are protein aggregates associated with Parkinson’s disease. The alternative NADH dehydrogenases from Arabidopsis thaliana, AtNDA2 and AtNDB4, have also been introduced into CI-defective human fibroblasts (Catania et al., 2019). Unlike the NDI1 from yeast, AtNDA2 and AtNDB4 naturally coexist with CI in Arabidopsis (Fig. 4.1). This led to the expectation that AtNDA2 and AtNDB4 would be less likely to compete with CI. Unfortunately, while promoting rotenone-resistant respiration and being able to compensate for CI dysfunction in human cells, the alternative NADH dehydrogenases from Arabidopsis also competed with CI (Catania et al., 2019). The study nevertheless provided further evidence of the potential of xenotropic gene expression for therapeutic treatments in human.

4.4.2 Drosophila melanogaster Ciona AOX was first introduced into Drosophila melanogaster by FernandezAyala et al., in 2009. Under nonstress conditions, ubiquitous AOX-expression had no significant detrimental effects on lifespan, viability, and fecundity of the flies, while it slightly yet significantly delayed development. AOX-expressing flies were cyanide- and antimycin-resistant demonstrating the functionality of the enzyme. In Drosophila, AOX complemented the defects induced by a pathological mutation of DJ1 (Fernandez-Ayala et al., 2009), which, in humans, causes autosomal recessive Parkinson’s disease. AOX-activity did also alleviate most of the pathological phenotypes associated with the knockdown of CIV-subunits and assembly factors such as Cox5a, Cox5b, Cox6c, and Surf1, limiting locomotor dysfunction and early developmental lethality (Fernandez-Ayala et al., 2009; Kemppainen et al., 2014). AOX-expressing flies also offered the first clear evidence of a detrimental effect of the transgene. When the flies were tested in mating competition assays, AOX males were unable to compete with wild-type (WT) males, leading to a decrease in the proportion of AOX progeny. This defect was linked to modest spermatogenesis defects in flies expressing AOX (Saari et al., 2017), with no detectable consequences in the absence of reproductive competition. Adaptation analysis also revealed some serious limitations caused by AOX expression in flies. AOX flies were more sensitive to high environmental temperatures than the nontransgenic controls (Saari, Garcia, et al., 2019), potentially because of a thermogenic burden brought on by AOX activation, though this hypothesis remains to be tested. AOX-expressing flies were also hypersensitive to limited nutritional conditions, being rarely able to develop beyond the pupal stage (Saari, Garcia, et al., 2019). The developmental defect was associated with a complex failure of nutrient utilization, linked to the inefficient turnover of TCA cycle intermediates (Saari, Garcia, et al., 2019; Saari, Kemppainen, et al., 2019). These studies show unequivocally that xenotropic expression of AOX can have deleterious

4.4 Transgenic models of alternative respiratory chain enzymes

consequences, especially during stages of cellular development that require considerable metabolic reorganization, such as spermatogenesis and metamorphosis. A transgenic Drosophila model of NDI1 was first introduced and characterized in 2010 (Sanz et al., 2010). NDI1-expression did not decrease the fitness of the flies, fertility, viability, and developmental time of the flies were unchanged. In fact, NDI1 increased the lifespan of the flies. Unlike in human cells, and despite compensating for the knockdown of CI activity, NDI1 expression did not perturb the NAD/NADH balance in Drosophila (Sanz et al., 2010). This would suggest that the enzyme is poorly or not at all competing with CI in Drosophila in controlled environmental conditions. The NDH2 protein of Ciona intestinalis, NDX, also increased the lifespan of Drosophila, although the effect was weaker compared to the enzyme of yeast origin (Gospodaryov et al., 2014). NDX was also found to provide resistance to both heat and cold stress, a feature likely connected to the role of NDH2s in thermoregulation (Gospodaryov et al., 2014; Saari, Garcia, et al., 2019). Interestingly, unlike its yeast counterpart, in conditions of low nutrient availability, NDX expression decreased the flies’ viability (Gospodaryov et al., 2014). The difference between NDX and NDI1 could be due to a threshold effect, leading to a state of starvation instead of caloric restriction in NDX flies. It would be interesting to compare the metabolic signatures of these transgenic flies in conditions of caloric restriction.

4.4.3 Rodent models Two mouse models ubiquitously expressing Ciona AOX have already been published. In 2013, El-Khoury et al. generated mice lines in which AOX was introduced by viral transduction. Each line harbored multiple copies of the transgene inserted at multiple loci (El-Khoury et al., 2013). In the transgenic animals, the AOX protein was detected in all eleven tissues that were tested, including the brain. The animals exhibited clear resistance to cyanide. Isolated mitochondria, as well as intact tissues, presented cyanide-resistant respiration that was sensitive to AOX-inhibitor nPG (Fig. 4.2). Analysis of the ADP/O ratio in brains of the mice showed no significant difference between AOX and control mice. Therefore, as in the insect and cell models, AOX does not seem to participate in the RC activity in the absence OXPHOS dysfunction or environmental stress. A second AOX expressing mouse model was created in 2016, where the gene was introduced at the Rosa26 locus, as a single copy, under the control of a strong and ubiquitous CAG enhancer-promoter (Szibor et al., 2017). The AOX mice expressed a functional enzyme, which provided resistance to respiratory toxins like cyanide. The transgenic animals displayed unaltered physiology and a metabolomics profile under normal animal husbandry (Szibor et al., 2017). Unexpectedly, however, in this mouse model, AOX activity was undetectable in the brain, despite the fact that the CAG promoter and the Rosa26 locus are commonly used for targeted gene expression in the brain.

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Since then, the use of the transgenic AOX-mouse model has advanced our understanding of mitochondria and associated diseases. It revealed new mechanisms of ROS-induced inflammation in a broader study of the role of Complex II (CII) in sepsis (Mills et al., 2016). AOX has also been shown to protect from cigarette smoke-induced damages in mouse lungs and in isolated primary mouse embryonic fibroblasts (Giordano et al., 2019). One promising example of the therapeutic potential of alternative pathways is highlighted by the studies of AOX-expressing GRACILE mice. The GRACILE mutation leads to fetal growth restriction, aminoaciduria, cholestasis, liver iron overload, lactic acidosis, and death during early infancy in humans (Rajendran et al., 2019). This severe phenotype is caused by a mutation in the BCS1L gene, which encodes for a translocase of the inner mitochondrial membrane, required for the assembly of CIII. Expression of AOX in mice with a homozygous knockin of the GRACILE mutation in BCS1L increased lifespan and prevented lethal cardiomyopathy (Rajendran et al., 2019). AOX also fully prevented astrogliosis of the somatosensory cortex, a pathology not found in GRACILE patients but commonly found in GRACILE mice. In the kidney, AOX maintained the mass and tubular volume, but failed to prevent fibrosis associated with the GRACILE mutation. AOX also failed to restore the increased levels of the liver enzymes alanine aminotransferase and alkaline phosphatase in plasma. These promising, although contrasted effects, suggest a context-dependent therapeutic potential for AOX, possibly linked to tissue- and cell type-specific variations in metabolism. In a mouse model for cardiac ischemia-reperfusion injury, AOX was able to improve mitochondrial function postinjury but failed to protect from the injury during anoxia and failed to improve tissue remodeling (Szibor et al., 2020). Proteome analyses of a postischemic heart showed an increase in proteins involved in extracellular matrix reorganization in AOX-expressing heart, which could have increased the stiffness of the muscle and hampered heart contractility. ROS signaling is known to be involved in activating muscle tissue repair in both cardiac and skeletal muscle and therefore, it is possible that the antioxidant properties of AOX blunted these signals and impaired tissue regeneration. On the opposite side of the therapeutic spectrum, heart-specific knockout of COX15 in mice, which causes mitochondrial cardiomyopathy, was not rescued but instead worsened by ubiquitous AOX expression, leading to a shortened lifespan (Dogan et al., 2018). Mouse studies using AOX as a potential treatment for RC-related disorders have shown promising results but also more limitations than what could be expected with the in vitro and Drosophila systems. These results point strongly towards the fact that AOX therapeutic use will depend on “contextual” factors like tissue type, developmental stage, environmental conditions, and metabolic state. On the other hand, physiological characterization of AOX-expressing mice demonstrated no obvious differences in body composition and cardiac performance, nor did they show any abnormality in their response to a high fat or ketogenic diet (Dhandapani et al., 2019). It is important to study more environmental

4.5 Metabolic impact of alternative enzymes

conditions, like cold exposure or maternal malnutrition, in adults or in the context of development, to better understand the applicability of AOX-therapy. In any case, a better understanding of the metabolic consequences of AOX activity in mammals is needed. A murine model of ubiquitous NDH2 expression has yet to be presented to the scientific community. Transient expression of NDI1 through local intracerebral injection of recombinant-associated adenovirus (AAVs) has nonetheless been successful in rodents. In the first study, rats were injected with rotenone, which induces the formation of the Lewy bodies, characteristic for Parkinson’s disease (Marella et al., 2008). In another independent study, Parkinsonism was induced by MPTP (methyl-4-phenyl-1,2,3,6-tetrahydropyridine) treatment (Barber-Singh et al., 2011). In both studies, local expression of NDI1-protein was observed in dopaminergic neurons following the injection, and this expression was associated with improved locomotor abilities. NDI1-transduction therapy was also applied to the treatment of experimental autoimmune encephalomyelitis in mice, which is a model for multiple sclerosis. Local, AAVs-mediated, transduction of NDI1 improved visual function, cell viability, and mitochondrial cristae organization, suggesting that the visual improvements were due to enhanced mitochondrial function which prevented retinal ganglion cell loss and axonal degeneration (Talla et al., 2020). In 2020, a mouse model with conditional expression of NDI1 was created (McElroy et al., 2020). The expression of NDI1 was induced in the brains of mice suffering from the knockout of NDUFS4, a CI subunit. NDUFS4-deficient mice have a pathological presentation similar to that of patients suffering from Leigh syndrome. Expression of NDI1, increased the lifespan of the NDUFS42/2 animals and prevented neuro-inflammation but failed to improve motor functions. NDI1 expression also restored normal metabolite and transcript levels in the cerebellum of the knockout mice. Interestingly, in astrocytes, U13C-glucose tracing showed an increase in TCA intermediates like citrate, malate, and succinate, but a decrease in pyruvate in the NDI1 expressing NDUFS42/2 -mice, suggesting that NDI1 expression enhanced the flux of metabolites in the TCA cycle.

4.5 Metabolic impact of alternative enzymes 4.5.1 Nutrition The therapeutic use of the alternative RC enzymes depends heavily on their metabolic impact and the consequences thereof, whether beneficial or harmful. Lessons from other kingdoms may help us to predict these consequences. In plants, alternative respiratory pathways have been suggested to improve the modulation of ATP synthesis by providing pathways for the activation of the TCA metabolism in situations where ATP production is not required or not possible, such as when the ATP/ADP ratio is very high due to active photosynthesis. In

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fact, the combined activities of NDH2 and AOX can recycle the NADH generated by the TCA independently of the ATP synthase since their participation would lead to electron transfer without proton pumping (Fig. 4.3). Consequently, alternative pathways can preserve the availability of carbon skeletons of the TCA cycle intermediates in conditions where, in mammals, they would be depleted (Martı´nez-Reyes et al., 2016). Maintaining TCA intermediates allows for the continuation of other metabolic processes (e.g., heme, fatty acid synthesis, and amino acid synthesis). Importantly, the activity of alternative RC enzymes would enhance the recycling of NADH without causing oxidative stress (Martı´nez-Reyes et al., 2016) (Fig. 4.3).

FIGURE 4.3 Alternative respiratory chain enzymes and their role in mitochondrial metabolism. Alternative respiratory chain enzymes can maintain mitochondrial metabolism when one or several of the core OXPHOS complexes are defective, with the exception of Complex II (insert). The insert (dashed box) presents the electron flow and theoretical H 1 production of the traditional respiratory pathway (on top), in comparison to when one or both of the alternative respiratory pathways are actively participating in respiration. 1C, One carbon cycle; A, AOX; I, Complex I, II, Complex II, III, Complex IV, N, NDH2; TCA, tricarboxylic acid cycle.

4.5 Metabolic impact of alternative enzymes

In plants, the activity of the AOX-protein and the expression of AOXencoding genes are induced by some TCA cycle intermediates and by mitochondrial ROS (mtROS), suggesting that AOX has a role in enhancing TCA cycle flux while restraining phosphorylating electron transfer (Fernie et al., 2004; Gray et al., 2004). The different subtypes of AOX proteins are activated by various TCA metabolites in plants, with α-ketoglutarate and oxaloacetate being the most common posttranslational activators. In contrast, citrate and malate, while they increase AOX transcription, they have no direct effects on the activity of the enzyme (Gray et al., 2004). Whether these regulatory qualities are conserved in animal AOXs, remains to be studied. Also, how these regulations will impact the effect of AOX in mammalian organisms is an open and critical question. The regulation of internal NADH dehydrogenases in plants and lower eukaryotes is less clear (Antos-Krzeminska & Jarmuszkiewicz, 2019; Rasmusson et al., 2004). Unlike many NDEs, their activity seems to be Ca21 independent. Nevertheless, they are suggested to participate in NAD1 recycling during photorespiration and to be activated by elevated NADH levels in the mitochondrial matrix (Fernie et al., 2004; Wallstro¨m et al., 2014). Dietary studies in Drosophila expressing AOX show that although it is able to partially restore OXPHOS function, the enzyme compromises efficient nutrient utilization under conditions of restricted diet (Saari, Garcia, et al., 2019). This could mean that, as in plants, Ciona AOX is regulated by intermediates of the carbon metabolism, or that it is perturbing the metabolic adaptation to fasting. In contrast, NDI1 flies were not hypersensitive to caloric restriction (Sanz et al., 2010). The increased longevity of NDI1 flies may nevertheless be the signature of a constitutively altered metabolism; for example NDI1 flies could be in a permanent state of “caloric restriction,” which is known to prolong lifespan in many model organisms including Drosophila. This would explain why NDI1 flies live longer, and caloric restriction has no additive effect. AOX is expected to have lower affinity for ubiquinol than CIII and therefore, even if active, it is expected to cause minimal disturbances on OXPHOS function and ATP production. The developmental delay in Drosophila and the slight weight loss in the adult flies (Fernandez-Ayala et al., 2009) potentially contradicts these expectations. Since weight loss is a signature of energy wasting or decreased energy production, this would imply interference by AOX in OXPHOS. Considering that these effects are measurable in flies, in standard conditions, we suspect that AOX is active in a subset of tissues, physiological conditions, cells and/or life cycle phases. The deleterious consequences of this activity are likely masked by the combination of a nutritionally rich diet and optimal environment (controlled temperature, humidity, light cycle, husbandry, etc.). Metabolite-targeted analysis showed no significant difference between Drosophila larvae with AOX and control larvae in the steady-state levels of triglycerides, a major form of energy storage in Drosophila, nor in lactate and pyruvate levels (Saari, Kemppainen, et al., 2019). When nutrition was limited, these metabolites were decreased to the same extent in both genotypes of larvae. In

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addition, the developmental defect caused by AOX on a low-nutrient diet affects the entire process of metamorphosis while a very small but significant number of transgenic animals reach adult stage. This points towards a systemic effect, rather than a tissue-specific or metabolite-specific phenomenon (Saari, Kemppainen, et al., 2019). Metabolomics studies could help us to understand the mechanisms of this developmental defect. Whether AOX-expressing mice would be affected by limited nutritional conditions, remains to be studied.

4.5.2 Reactive oxygen species During recent years, mtROS have gained attention, not only as a damaging byproduct of RC activity, but also as a central signaling molecule that regulates the nutrient-sensing pathways of the cell as well as the metabolic state of mitochondria. Under normal physiological conditions, ROS influences cell proliferation, growth, and differentiation (Brunelle et al., 2005; Chen et al., 2009). It is therefore expected that the expression of AOX and NDH2, which are known to alter ROS production (Fernandez-Ayala et al., 2009; Mills et al., 2016), will alter ROS signaling and oxidative stress in mammals. While there are no circumstances in which AOX would be expected to act as a pro-oxidant, the connection between NDH2 and ROS is more ambiguous. On one hand, NDH2 has been shown to decrease ROS production in conditions of CI dysfunction (Seo et al., 2006). On the other hand, NDH2 has the ability to promote reverse electron transfer (RET) at CI and thereby to dramatically enhance ROS production (Scialo` et al., 2016). Activation of the AMP-activated protein kinase, a master regulator of cellular energy homeostasis in conditions of metabolic stress, has recently been shown to depend on increased mtROS (Rabinovitch et al., 2017). Reactive oxygen species are usually produced in cascades that are catalyzed by either spontaneous chemical reaction (e.g., H2O2 and iron) or by enzymatic reaction (e.g., O22 and SOD). Several recent and excellent reviews presented these reactions in great detail (Schieber & Chandel, 2014; Zhang et al., 2019). In general, the first ROS species generated at the RC is the superoxide anion (O22). In normal conditions, it is mainly produced in the mitochondria, as a spontaneous and accidental reaction between an electron in transit through the RC and an oxygen molecule. Mitochondrial superoxide is rapidly converted into H2O2 by the SODs that are present in the intermembrane space (SOD1) and in the mitochondrial matrix (SOD2). While still not fully elucidated, the regulatory functions of these two ROS species (O22 and H2O2) appear largely different. For example, starvationrelated autophagy is normally associated with the accumulation of O22, whereas amino acid deprivation is connected to H2O2 accumulation (Chen et al., 2009; Scherz-Shouval et al., 2007). Mitochondrial O22 remains a major threat for the organism as demonstrated by the severe phenotypes of SOD22/2 (Li et al., 1995) and SOD12/2 mice (Meissner et al., 2008). Conversion of O22 into H2O2 is therefore essential for survival, in addition to being crucial for ROS signaling.

4.5 Metabolic impact of alternative enzymes

Understanding ROS-dependent signaling hence requires elucidating the mechanisms that balance antioxidant defense with ROS signals. In disease states, AOX may provide protection from damage caused by excessive ROS, whereas under normal physiological conditions, AOX may dilute or perturb signals relayed by these ROS species. This may lead to disconnected cellular responses to environmental changes and metabolic maladaptation, potentially escalating a pathological state. Such abrogation of metabolic flexibility by AOX was observed in the COX15 knockdown mice (Dogan et al., 2018). Furthermore, disruption of spermatogenesis and metamorphosis by AOX, two highly coordinated processes of cell differentiation and tissue reorganization in Drosophila, could be the consequences of defective ROS signaling or redox homeostasis (Saari et al., 2017; Saari, Garcia, et al., 2019). NDI1 has also emerged as a valuable tool in understanding ROS signaling. In Drosophila, NDI1 improved the locomotive activity, time spent flying, and lifespan of PINK1 knockdown flies (Scialo` et al., 2016). Interestingly, it also increased lifespan of WT Drosophila. This increase was found to be due to the overreduction of the ubiquinol pool that caused RET and an increase in ROS levels (Scialo` et al., 2016). RET describes the flow of electrons through CI from quinones to NADH. For a long time, RET was suspected to occur only in vitro in very specific conditions, such as in isolated mitochondria fed with succinate in conditions of low oxygen. In this case, the succinate dehydrogenase feeds electrons to the quinones, which cannot transfer them to the CIII
CIV segment of the RC. When the buildup of ubiquinone becomes very high, the quinones will transfer their electrons back to CI, which will convey them to NAD1. However, in most instances, the electrons will not reach NAD1, because the pool of NAD1 will rapidly become depleted and because RET is so inefficient that many of the electrons will end up interacting directly with O2. By providing a third entry point for the electrons into the RC, NDI1 potentially enhances the quinone reduction. It is important to note that in physiological conditions, RET should not be enhanced in cells expressing NDI1 since NDI1 and CI have very similar affinity for NADH, unless the NADH dehydrogenase activity of CI can be downregulated or its RET activity upregulated. The results obtained by Cannino et al. (2012), provide evidence for a metabolism-dependent regulation of CI activity in conditions of RC dysfunction (Cannino et al., 2012). A logical reason for such downregulation would be to protect the cell from harmful ROS production caused by the stalling of electrons in the RC. Not only would the activity of NDI1 directly increase electron stalling and ROS production, but it could lead, as observed by Scialo` et al. (2016), to secondary ROS production because of RET (Scialo` et al., 2016). In contrast, AOX should alleviate these effects by providing an escape path for the electrons, if electron transfer through AOX is more efficient than through RET. This was confirmed in the same publication, by showing that AOX nullifies the increase in lifespan caused by NDI1 expression.

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4.6 Therapeutic potential of alternative enzymes in mitochondria-related diseases The use of alternative enzymes as a therapeutic tool has heavily focused on limiting ROS production and its deleterious consequences, which are presumed to be an underlying cause for mitochondrial disorders but also for a variety of conditions such as Alzheimer’s and inflammation. However, it is now clearly recognized that mitochondrial dysfunction is not systematically associated with ROS production, and the causes of the diseases should be searched for in the alterations in substrate flux and distortions in metabolism (Nikkanen et al., 2016). Limitations in carbon skeleton flux may lead to deprivation of essential nutrients and excess of others while disturbances in, for example calcium homeostasis, may disturb efficient transport across membranes. Metabolic characterization of the consequences of alternative enzymes may provide new ways to understand and influence the crosstalk between OXPHOS enzyme complexes and nutrient balance in the cell. A recent review summarizing the metabolomics studies of various mitochondrial diseases (Esterhuizen et al., 2017) clearly underlines the complex, and unpredictable metabolic consequences of mitochondrial dysfunction, which could limit the applications of alternative RC enzymes in therapeutic and research use. This complexity appears inherent to the metabolism. The metabolism of cells carrying mitochondrial dysfunction should be seen as an intertwined combination of tissue-specific metabolism, dysfunction-dependant immediate responses, secondary metabolic responses, and dysfunction-independent (environmental, genetic, or stochastic) metabolic and regulatory differences. Currently, we are not capable of elucidating such complex settings, however, we can, using the alternative respiratory pathways, replace the activity of the dysfunctional RC enzyme and verify the metabolic consequences of this replacement therapy. Not only could this help elucidate the mechanisms of the metabolic adaptation to mitochondrial diseases, but it could allow for their treatment. The growing body of evidence underlining the ability of AOX and NDH2 to alleviate even the most severe consequences of mitochondrial diseases (Mills et al., 2016; Rajendran et al., 2019), supports the idea that these enzymes could be utilized for therapeutic purposes. Unfortunately, in-depth studies in conditions where the adaptive abilities of the metabolism were challenged, led to the conclusion that alternative respiratory pathways can have harmful effects (Saari, Garcia, et al., 2019). Despite the therapeutic use of AOX and NDH2 in treating diseases remaining a distant perspective, the two enzymes appear to be a critical tool in understanding and possibly reversing the metabolic defects caused by mitochondrial diseases but also by more common pathologies such as Parkinson’s disease (Cronin-Furman et al., 2019). Considering the current difficulties associated with the treatments of these disorders and their well-known high socio-economic impact, studies of alternative respiratory pathways, whether aiming to elucidate the mechanisms or to treat diseases, are strongly welcomed.

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9. Available from https://doi.org/10.1016/j.celrep.2017.09.026. Rajendran, J., Purhonen, J., Tegelberg, S., Smolander, O. P., Mo¨rgelin, M., Rozman, J., Gailus-Durner, V., Fuchs, H., Hrabe de Angelis, M., Auvinen, P., Mervaala, E., Jacobs, H. T., Szibor, M., Fellman, V., & Kallija¨rvi, J. (2019). Alternative oxidase-mediated respiration prevents lethal mitochondrial cardiomyopathy. EMBO Molecular Medicine, 11(1). Available from https://doi.org/10.15252/emmm.201809456. Rasmusson, A. G., Soole, K. L., & Elthon, T. E. (2004). Alternative NAD(P)H dehydrogenases of plant mitochondria. Annual Review of Plant Biology, 55, 23
39. Available from https://doi.org/10.1146/annurev.arplant.55.031903.141720. Saari, S., Andjelkovi´c, A., Garcia, G. S., Jacobs, H. T., & Oliveira, M. T. (2017). Expression of Ciona intestinalis AOX causes male reproductive defects in Drosophila melanogaster. BMC Developmental Biology, 17(1). Available from https://doi.org/ 10.1186/s12861-017-0151-3. Saari, S., Garcia, G. S., Bremer, K., Chioda, M. M., Andjelkovi´c, A., Debes, P. V., Nikinmaa, M., Szibor, M., Dufour, E., Rustin, P., Oliveira, M. T., & Jacobs, H. T. (2019). Alternative respiratory chain enzymes: Therapeutic potential and possible pitfalls. Biochimica et Biophysica Acta—Molecular Basis of Disease, 1865(4), 854
866. Available from https://doi.org/10.1016/j.bbadis.2018.10.012. Saari, S., Kemppainen, E., Tuomela, T., Oliveira, M. T., Dufour, E., & Jacobs, H. T. (2019). Alternative oxidase confers nutritional limitation on Drosophila development. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology, 331 (6), 341
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9171. Available from https://doi.org/10.1073/pnas.95.16.9167. Seo, B. B., Marella, M., Yagi, T., & Matsuno-Yagi, A. (2006). The single subunit NADH dehydrogenase reduces generation of reactive oxygen species from complex I. FEBS Letters, 580(26), 6105
6108. Available from https://doi.org/10.1016/j. febslet.2006.10.008. Seo, B. B., Matsuno-Yagi, A., & Yagi, T. (1999). Modulation of oxidative phosphorylation of human kidney 293 cells by transfection with the internal rotenone-insensitive NADH-quinone oxidoreductase (NDI1) gene of Saccharomyces cerevisiae. Biochimica et Biophysica Acta—Bioenergetics, 1412(1), 56
65. Available from https://doi.org/ 10.1016/S0005-2728(99)00051-1. Shiba, T., Kido, Y., Sakamoto, K., Inaoka, D. K., Tsuge, C., Tatsumi, R., Takahashi, G., Balogun, E. O., Nara, T., Aoki, T., Honma, T., Tanaka, A., Inoue, M., Matsuoka, S., Saimoto, H., Moore, A. L., Harada, S., & Kita, K. (2013). Structure of the trypanosome cyanide-insensitive alternative oxidase. Proceedings of the National Academy of Sciences of the United States of America, 110(12), 4580
4585. Available from https:// doi.org/10.1073/pnas.1218386110. Staffan Svensson, A., & Rasmusson, A. G. (2001). Light-dependent gene expression for proteins in the respiratory chain of potato leaves. Plant Journal, 28(1), 73
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361. Available from https://doi.org/10.1104/pp.109.2.353. Voulgaris, I., O’Donnell, A., Harvey, L. M., & McNeil, B. (2012). Inactivating alternative NADH dehydrogenases: Enhancing fungal bioprocesses by improving growth and biomass yield? Scientific Reports, 2. Available from https://doi.org/10.1038/srep00322. Wallstro¨m, S. V., Florez-Sarasa, I., Arau´jo, W. L., Escobar, M. A., Geisler, D. A., Aidemark, M., Lager, I., Fernie, A. R., Ribas-Carbo´, M., & Rasmusson, A. G. (2014). Suppression of NDA-type alternative mitochondrial NAD(P)H dehydrogenases in arabidopsis thaliana modifies growth and metabolism, but not high light stimulation of mitochondrial electron transport. Plant and Cell Physiology, 55(5), 881
896. Available from https://doi.org/10.1093/pcp/pcu021. Zhang, L., Wang, X., Cueto, R., Effi, C., Zhang, Y., Tan, H., Qin, X., Ji, Y., Yang, X., & Wang, H. (2019). Biochemical basis and metabolic interplay of redox regulation. Redox Biology, 26, 101284. Available from https://doi.org/10.1016/j. redox.2019.101284.

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Essential nutrients in mitochondrial nutrition

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Aging, mitochondrial dysfunctions, and vitamin E

5

Gaetana Napolitano1, Gianluca Fasciolo2 and Paola Venditti2 1

Department of Science and Technology, University of Naples “Parthenope”, Naples, Italy Department of Biology, University of Naples “Napoli Federico II,” Complesso Universitario di Monte Sant’Angelo, Naples, Italy

2

5.1 Introduction Aging is a progressive degenerative state accompanied by an increasing loss of physiological integrity and function. These lead to an increase in the risk factors for major human diseases, including cancer, diabetes, cardiovascular disorders, and neurodegenerative diseases (Lo´pez-Otı´n et al., 2013). Several genetic pathways and biochemical processes preserved in evolution control the aging process. Recent studies identify several factors simultaneously involved in the aging process. They include impaired intercellular communication, stem cell exhaustion, deregulated nutrient sensing, epigenetic alterations, loss of proteostasis, genomic instability, and mitochondrial dysfunctions (Lo´pez-Otı´n et al., 2013). Mitochondria are relevant players in the aging process since they are involved in critical metabolic roles in all organs (Harman, 1956). They regulate intracellular homeostasis, calcium balance, metabolism of dietary substrates in the fed and fasted state, and are involved in the cellular signaling pathways in response to stress conditions. In addition, mitochondria are subject to fusion and fission processes, the balance of which is essential for cell health. They are also involved in apoptosis and are fundamental for innate immunity. Mitochondrial functionality declines with age in several tissues and organisms because of the accumulation of alterations (Lo´pezOtı´n et al., 2013). The relationship between mitochondrial dysfunctions and aging has been investigated for a long time but, even today, complex underlying mechanisms are not fully understood and represent a challenge for aging research. Based on the observation that metabolic rates appeared to inversely correlate with lifespan, in the 1970s Harman (Harman, 1972) proposed “The mitochondrial free radical theory of aging” (MFRTA). According to MFRTA, the accumulation of reactive oxygen species (ROS) produced by mitochondria during life generates oxidative damage to proteins, lipids, and DNA. The oxidative damage, in turn, contributes to aging and age-related diseases in an inevitable but stochastic process. The MFRTA is an extension of the previous “Free radical theory of aging” proposed in the 1950s by Harman (Harman, 1956), based on the fact that respiratory enzymes, Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00025-4 © 2023 Elsevier Inc. All rights reserved.

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which utilize oxygen to generate readily available energy, are the main generators of ROS. Because of molecular disfigurement and functional decay, it is possible that antioxidant molecules which are capable of efficiently quenching a variety of ROS or reduce their production, especially in mitochondria, can slow down the rate of aging and reduce the incidence of age-related diseases and prolong longevity (Speakman, 2005). On this assumption, multiple studies were established and aimed at estimating the effect of the level of endogenous antioxidants and/or of the administration of exogenous antioxidants on aging and age-related processes and diseases (Mock et al., 2017; Prasad et al., 2017). A more recent investigation relies on the fact that mitochondria are not only production sites for metabolic energy and macromolecules but also regulatory hubs. They communicate and coordinate many vital physiological processes at the cellular and organismal levels. Information concerning proteotoxic and metabolic stress and inflammatory signals is sent to the nucleus from mitochondria to maintain cellular homeostasis (Son & Lee, 2021). Several molecular compounds are involved in mitochondria communication, including proteostasis signaling molecules, mitochondrial metabolites, and mitokines such as mitochondrial-derived peptides (MDPs). Multiple age-related mitochondrial dysfunctions affect the communication processes, induce maladaptive metabolic shifts, and reduce organismal fitness. Thus, they contribute to aging phenotypes and age-related disabilities (Son & Lee, 2021). However, MFRTA has been questioned recently for several reasons. First, the administration of antioxidants has been unsuccessful in extending lifespan (Scialo et al., 2013). Additionally, the naked rat experimental model lives seven times longer than the domestic rat despite generating the same amount of ROS, higher levels of oxidative damage, and lower content of antioxidants (Scialo et al., 2013). On the other hand, supplementation with antioxidants has been linked to an increased incidence of several diseases with adverse effects on human longevity (Shields et al., 2021). Moreover, the literature reports inconsistent results between increased ROS levels and longevity, putting in evidence that ROS can have both beneficial or detrimental effects on longevity depending on the species and conditions (Shields et al., 2021). These last aspects are linked to the Mitochondrial Hormesis (Mitohormesis) theory (Ristow & Schmeisser, 2014). The Free Radical Theory of Aging suggests a linear dose-response relationship between increasing amounts of ROS and oxidative stress on the one hand and mortality events on the other. The concept of mitohormesis indicates a nonlinear dose-response relationship where low doses of ROS exposure decrease mortality and higher doses promote mortality (Ristow & Schmeisser, 2014) (Fig. 5.1). In this context, antioxidants appear to have a negative effect, presumably by preventing the hormetic response. Antioxidant supplementation with vitamin C, vitamin E, or C and E complexes knocked out the whole cell adaptive response to environmental changes by reducing the activation of factors involved in such adaptations (Gomez-Cabrera et al., 2008; Morrison et al., 2015; Paulsen et al., 2014; Venditti et al., 2014).

5.1 Introduction

FIGURE 5.1 “Mitohormesis process.” Lower mitochondrial ROS concentrations produced upon cellular stress engage a nonlinear response (blue) that results in health span-promoting effects. When ROS production is higher, it may cause cellular and systemic damage culminating in increased mortality.

This chapter aims to describe an overview of the mechanisms involved in the aging process with particular attention to the mitochondria. The chapter will be articulated in the first part, focused on several aspects of the involvement of mitochondria in the aging process. In the second part, we will report knowledge about the functions of antioxidant molecules in the prevention of age-linked mitochondrial dysfunctions, with particular attention to vitamin E. Finally, we will discuss the experimental evidence which challenges MFRTA. We will focus the attention on the recent role of ROS as signaling molecules, pointing out the importance of the maintenance of adequate ROS levels for redox signaling by ROS-sensitive components which activate specific cell survival pathways.

5.1.1 Mitochondria, reactive oxygen species and the free radical theory of aging Mitochondria represent the powerhouse of the cells as they are involved in the production of most cellular ATP by the oxidative phosphorylation process

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(OXPHOS), which relies on the inner membrane electron transfer chain (etc). The etc includes protein complexes organized into five multisubunit enzymes embedded in the mitochondrial inner membrane. They are known as complexes I, II, III, IV, and V. The complexes are also referred to as NADH dehydrogenase, succinate dehydrogenase, ubiquinol-cytochrome c reductase, cytochrome c oxidase, and F1F0-ATP synthase, respectively. In addition, two diffusible factors that function as electron shuttles within the mitochondrial intermembrane space take part in the system: coenzyme Q, a lipophilic quinone, and cytochrome c (Cyt c), a hydrophilic heme protein localized on the external surface of the inner membrane (Venditti et al., 2015). The electrons flow through the respiratory complexes from reduced equivalents (NADH and FADH2), obtained from the oxidation of the energy molecules of the food to the oxygen driven by the redox gradient. Mitochondria use about 95% of the oxygen to convert the chemical energy from food into an energetic molecule usable by the cell (ATP). The electron transfer along the inner mitochondrial membrane carriers is associated with radicals and other ROS production. These molecules derive from the incomplete reduction of molecular oxygen in the cell (Venditti et al., 2015). The ROS can be free radicals, atoms, and molecules with an unpaired electron in their outer shell, or molecules that can generate free radicals. The univalent autooxidation of mitochondrial carriers leads to anion superoxide (O2• 2 ) production (Turrens & Boveris, 1980). Then, mitochondrial superoxide dismutase (SOD) converts superoxide to hydrogen peroxide (H2O2), which can be turned into a hydroxyl radical (•OH) via the Fenton reaction (Halliwell & Gutteridge, 1990). ROS are considered a normal by-product of aerobic metabolism, and they play a crucial role in redox signaling. Low ROS levels are involved in redox signaling by addressing specific targets, whereas higher levels result in the disruption of redox signaling and/or damage to biomolecules (Sies, 2018). In this view, a condition of oxidative stress onset begins when an imbalance between oxidants and antioxidants in favor of the oxidants occurs, and disruption of redox signaling and control and/or molecular damage happens (Sies & Jones, 2007). Because of their chemical structure, ROS are unstable and highly reactive species. Therefore, they are generally short-lived and often leave the subcellular production site after undergoing a reduction process (Venditti et al., 2015). Thus, ROS may cause oxidative damage to macromolecules such as DNA, proteins, and lipids (Pham-Huy et al., 2008). The oxidation of DNA modifies the structure of the nucleotides favoring DNA double-stranded breaks that lead to genomic instability (Kregel & Zhang, 2007). ROS can affect protein activity directly or indirectly. They are responsible for nitrosylation, carbonylation, disulfide bond formation, and glutathionylation of proteins (Sharma et al., 2012). Alternatively, proteins can be conjugated with breakdown products of fatty acid peroxidation (Sharma et al., 2012). Thus, site-specific amino acid modification, fragmentation of the peptide chain, aggregation of cross-linked reaction products, altered electric charge, and increased susceptibility of proteins to proteolysis, occur (Sharma et al., 2012). Finally, the lipid peroxidation following the exposure of lipids to ROS, prompts

5.1 Introduction

cell membrane damage and generates reactive by-products that can further damage the cell (Mylonas & Kouretas, 1999). Since the amount of all oxidized macromolecules increases with age, they are regarded as biomarkers of the aging process (Liguori et al., 2018) and, according to the MFRTA, aging represents the result of the accumulation of molecular damage caused by mitochondrial ROS generated by normal metabolism (Harman, 1956). The theory fits with the idea that the mechanism of aging is intrinsic since ROS production is continuous as they represent the by-products of a process essential to all known aerobic organisms. In addition, MFRTA highlights that the mitochondrial ROS, not cellular ones, are responsible for aging. This aspect is crucial since it underlies the dependence of the lifespan of an organism on its rate of oxygen consumption by the mitochondrial respiratory chain (believed to be the primary process that produces ROS). This allows us to explain why the rate of aging and the maximum lifespan varies so significantly among species (Barja, 2013). MFRTA has been supported for a long time by several observations linking ROS, mitochondria, and longevity. These observations sustain that the aging process is accompanied by the increase of ROS production (Capel et al., 2005), oxidative damage (Muller et al., 2007), mitochondrial dysfunctions (Sun et al., 2016), and oxidative stress in several agerelated diseases (Pham-Huy et al., 2008). All these markers of aging have as the common denominator the physiological alterations of mitochondria and, therefore, mitochondrial dysfunctions implicating increased oxidative stress, in turn, are associated with the aging process (Bratic & Larsson, 2013; Fukui & Moraes, 2008; Tatsuta & Langer, 2008).

5.1.2 Mitocondrial DNA and aging Mitochondria are eukaryotic organelles of proteobacterial origin characterized by their DNA (mtDNA; also referred to as the nucleoid) encoding only a few of the mitochondrial proteins (13 in humans and 8 in yeast) (Chen & Butow, 2005). The mitochondrially-encoded proteins interact with other components encoded by the nuclear cell genome constituting the OXPHOS complexes, which produce ATP as a source of cellular chemical energy. MtDNA point mutations and deletions are known to accumulate with age (Park & Larsson, 2011), even if the debate is still ongoing to define whether the accumulation of mtDNA mutations is a causal or just correlation with aging. MtDNA has been considered the primary target for aging-associated somatic mutations for several reasons. These include the lack of protective histones, the limited efficiency of the mtDNA repair mechanisms compared to nuclear DNA (Linnane et al., 1989), and replication errors early in life undergoing polyclonal expansion and respiratory chain dysfunction in different tissues (Ameur et al., 2011). In experiments performed in homozygous knock-in mice that expressed a proof-reading-deficient version of PolgA, the nucleusencoded catalytic subunit of mtDNA polymerase, showed for the first time the link between mtDNA mutations and aging phenotype in a mammalian model. This model developed an mtDNA mutator phenotype load (B2500-fold in the

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homozygous and B500-fold higher in the heterozygous) associated with reduced lifespan and premature onset of aging-related phenotypes (Trifunovic et al., 2004). However, the observation that only in homozygous mice the lifespan was shorter, suggested that mtDNA mutation load alone does not determine lifespan (Ng et al., 2019; Szczepanowska & Trifunovic, 2020) and that a more complex manifestation of mitochondrial genomic instability is likely involved (Ha¨ma¨la¨inen et al., 2019). The accumulation of mitochondrial DNA mutations is responsible for increased ROS production that causes oxidative damage to cellular macromolecules that, in turn, leads to reduced respiratory chain activity and ATP generation.

5.1.3 Mitochondrial dynamics, mitophagy and aging To preserve mitochondrial function and quality, mitochondria undergo a continuous turnover. Alterations in the fine balance of the processes involved in the mitochondrial turnover (mitophagy, mitochondrial biogenesis, and mitochondrial dynamics) play a crucial role in the aging process (Srivastava, 2017). Dysfunctional mitophagy, a process of removing damaged mitochondria, may contribute to the accumulation of nonfunctional mitochondria during the aging process (Palikaras et al., 2015). One of the most characterized mitophagy pathways is phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1)/Parkin-dependent pathway, although mitophagy can also occur in a Parkin-independent manner (Srivastava, 2017). The canonical PINK1/Parkindependent mitophagy pathway is activated in response to mitochondrial damage (e.g., loss of mitochondrial membrane potential or accumulation of misfolded proteins) and involves stabilization of PINK1 on the outer mitochondrial membrane where it phosphorylates ubiquitin (Narendra et al., 2010). The ubiquitin phosphorylation recruits cytosolic E3 ubiquitin-protein ligase
Parkin to the mitochondrial outer membrane which polyubiquitinates mitochondrial proteins to facilitate their association with the autophagy receptors, thereby leading to the formation of autophagosome (Kane et al., 2014; Koyano et al., 2014). The autophagosome subsequently fuses with the lysosome to promote mitochondrial degradation (Lazarou et al., 2015). Ubiquitination of mitochondrial proteins thus identifies or tags damaged mitochondria for Parkin-mediated mitophagy. Alternatively, PINK1 can also recruit autophagy receptors directly to mitochondria in a Parkin-independent manner to mediate mitophagy (Lazarou et al., 2015). Mitochondrial fusion and fission events also referred to as “mitochondrial dynamics” tightly regulate the mitophagy process (Chan, 2012) (Fig. 5.2). Mitochondrial fusion allows two adjacent mitochondria to fuse in a more elongated mitochondrion. It is regulated in mammals by three GTPases: mitofusin 1 (MFN1) and mitofusin 2 (MFN2), located in the outer mitochondrial membrane and optic atrophy gene 1 (OPA1), located in the inner mitochondrial membrane and the intermembrane space (Sebastia´n et al., 2017). Fusion allows the exchange of matrix contents and mtDNA molecules between mitochondria. This process

5.1 Introduction

FIGURE 5.2 Mitochondrial dynamics. Mitochondrial fission separates one mitochondrion into two, while fusion joins two mitochondria together. Fission begins when the endoplasmic reticulum is recruited to the constriction site, marked by mtDNA. Next, multiple outer-mitochondrial membrane-bound proteins (FIS1, MFF, MiD49, and MiD51) recruit DRP1 to the surface of the mitochondria, aiding in endoplasmic reticulum-mediated constriction (Liu et al., 2020). Mitofusins (MFN1 and MFN2) coordinate fusion on the outer-mitochondrial membrane, while optic atrophy 1 (OPA1) works on the inner-mitochondrial membrane (Liu et al., 2020).

favors optimal mitochondrial physiology by diluting mutated mtDNA and rescuing damaged mitochondria by key components acquisition from healthy mitochondria (Zorzano & Claret, 2015). Mitochondrial fission generates two distinct types of mitochondria: one with high mitochondrial membrane potential, and the other with a low mitochondrial membrane potential which can recover its membrane potential and undergo fusion with another mitochondrion or remain depolarized and then eliminated by mitophagy (Twig et al., 2008). Mitochondrial fission undergoes the regulation by two classes of protein, namely a soluble cytosolic protein, dynamin-related protein 1 (DRP1), and mitochondria-bound proteins, including fission 1 homolog protein (FIS1), mitochondrial fission factor (MFF), mitochondrial dynamics protein of 49 kDa (MiD49), and mitochondrial dynamics protein of 51 kDa/mitochondrial elongation factor 1 (MiD51/MIEF1) (Sebastia´n et al., 2017). Mitochondrial dynamics regulate mitophagy so they contribute to mitochondrial quality control. Twig and coworkers (Twig et al., 2008) report that mitochondrial

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fission in mouse pancreatic β cells allows the segregation of damaged mitochondria from the mitochondrial network and the subsequent removal by mitophagy. Therefore, alterations in mitochondrial fusion or fission processes may induce intrinsic mitochondrial defects followed by an impaired mitochondrial activity. The unbalanced mitochondrial dynamics can affect mitophagy and prevent the elimination of damaged mitochondria. These alterations are associated with several agerelated diseases (Manczak et al., 2011; Sebastia´n et al., 2016; Wang et al., 2015). Several proteins involved in mitochondrial fission are dysregulated in aging. Aged mice show reduced DRP1 activity, and the mitochondria of neurons, skeletal muscle, and oocytes present altered morphology (Kageyama et al., 2012; Udagawa et al., 2014). An increased mitofusin 2-DRP1 ratio is associated with longer intermyofibrillar mitochondria in the skeletal muscle of aged mice (LeducGaudet et al., 2015). Elongated mitochondrial networks are found also in aged human endothelial cells (HUVECs) associated with downregulation of both DRP1 and FIS1 expression (Mai et al., 2010). Reduced MFN2 expression has been observed with age, indicating a correlation between decreased fusion and agerelated dysfunction in mitochondrial dynamics (Sebastia´n et al., 2016). There is accumulating evidence that even alterations in the mitophagy process are involved in health span and lifespan in different model organisms. Sun et al. (2015) reported a decreased mitophagy level in the hippocampal dentate gyrus in 21-month-old mice compared to 3-month-old mice. Satellite cells isolated from skeletal muscle of aged humans or mice showed defective mitophagy (Garcı´aPrat et al., 2016). Even in hearts from aged mice, the mitophagy was defective (Hoshino et al., 2013). Decreased expression of mitophagy genes was also observed in the skeletal muscle of physically inactive elderly women (Drummond et al., 2014). The alteration of mitochondrial dynamics, and/or defective mitophagy with chronological age can lead to the accumulation of damaged or dysfunctional mitochondria in cells. Moreover, the decline in mitophagy with increasing age prevents clearance of dysfunctional mitochondria leading to further mitochondrial damage accrual and deterioration of cellular function (Fig. 5.3). Maintenance of mitochondrial function and cellular homeostasis requires selective elimination of defective mitochondria and the generation of newly synthesized mitochondria by stimulation of a mitochondrial biogenesis program to maintain adequate mitochondrial mass and quality (Palikaras et al., 2015). Mitochondrial biogenesis is a tightly regulated process. It involves transcription factors such as nuclear respiratory factors that is, NRF1 and NRF2, and transcriptional coactivators, such as members of the PPARγ coactivator-1 family that is, PGC1α, PGC1β, and PGC1 related coactivator (Scarpulla et al., 2012). Both NRF1 and NRF2 are involved in the expression of proteins of the mitochondrial respiratory chain, import machinery, and heme biosynthesis, as well as several nuclear-encoded mitochondrial proteins that include mtDNA transcription factors A, B1, and B2 (i.e., TFAM, TFB1M, and TFB2M) (Scarpulla, 2008; Scarpulla et al., 2012). PGC1α plays a crucial regulator role since it coactivates the

5.1 Introduction

FIGURE 5.3 Mitochondrial dysfunctions during aging and age-related disorders. Mitochondrial dysfunctions are strictly associated with aging and oxidative damage due to increased ROS production with aging. Oxidative damage plays a crucial role in the generation of dysfunctional mitochondria. In addition, the alterations of mitochondrial fission and fusion processes are relevant since they contribute to defective mitophagy generating other dysfunctional mitochondria and triggering age-related disorders onset.

expression of multiple transcription factors that include NRF1, NRF2, and estrogen-related receptor α (Lin et al., 2005). Several pieces of evidence report the involvement of reduced biogenesis of mitochondria in the aging process. Telomeres play a critical role in this context. Telomeres consist of repeating nucleotide sequences and a set of particular proteins that interact with DNA to form a nucleoprotein complex (De Lange, 2006). The aging theory that links mitochondria and telomeres mainly relies on a stem cell defect caused by the activation of p53 and the induction of growth arrest, senescence, and apoptosis (Sharpless & DePinho, 2004). Indeed, telomere shortening goes along with increased p53 activity and, consequently, high levels of apoptosis (Flores & Blasco, 2009). Mice lacking p53 or its downstream targets show functional rescue of stem and progenitor cells in the hematopoietic system, skin, and gastrointestinal tract, and the concomitant rescue of tissue pathologies (Choudhury et al., 2007; Flores & Blasco, 2009). P53 binds to the promoters of PPARγ coactivator 1α (PGC1α) and PGC1β and represses the expression of PGC1A and PGC1B. The repression of both coactivators impairs overall

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mitochondrial biogenesis and function, resulting in the functional decline in tissue stem cells and postmitotic tissues and aging (Sahin & DePinho, 2012). This mitochondrial decline also occurs during physiological aging in wild-type mice and can be partially reversed by telomerase activation (Bernardes de Jesus et al., 2012). Mitochondria biogenesis also involves a class of NAD1 -dependent deacetylases, the sirtuins (van de Ven et al., 2017). Mammals contain seven sirtuin enzymes (SIRT1
7) and three sirtuins (SIRT3, SIRT4, and SIRT5) localized into the mitochondrial matrix (van de Ven et al., 2017). Among these, SIRT3 deacetylates and activates several enzymes involved in the cellular response to oxidative stress, and enzymes that regulate mitochondrial metabolism (isocitrate dehydrogenase or complexes of the mitochondrial respiratory chain) (Boland et al., 2013). SIRT3 may regulate mitochondrial fusion by targeting Opa1 (Samant et al., 2014). A more recent study demonstrated that SIRT3 silencing in SW620 cancer cells is associated with decreased mitochondrial biogenesis and dysfunctions as evidenced by the decrease in proteins such as PGC-1α and TFAM and lower levels of OXPHOS complexes (Torrens-Mas et al., 2019). Therefore, SIRT3 may regulate mitochondrial homeostasis at several levels, including mitochondrial biogenesis and function. Moreover, the use of genetic mouse models evidenced that the loss of SIRT3
5 is involved in age-related disease development (van de Ven et al., 2017). In addition, decreased NAD1 levels observed in aging can reduce sirtuin activity, which, in turn, may contribute to the aging process. Emerging evidence indicates that sirtuins also regulate another relevant cellular process, autophagy. Autophagy is a process by which damaged proteins and organelles, including damaged mitochondria, endoplasmic reticulum, and peroxisomes, are targeted for lysosomal-mediated degradation. Both autophagy and sirtuins appear to protect cells from environmental stresses. The deacetylation of p53 by SIRT1 suppresses the activity of p53 in essential physiological roles such as apoptosis and induces autophagy (Liu et al., 2018). The activation of autophagy by sirtuins also attenuates cellular senescence. SIRT6 overexpression inhibits the senescence of human bronchial epithelial cells induced by cigarette smoke extract (Takasaka et al., 2014). In addition, SIRT6 knockdown and mutant SIRT6 (H133Y) without histone deacetylase activity enhances the senescence process (Takasaka et al., 2014). Furthermore, negative regulation of SIRT1dependent autophagy by miR-212 augments cellular senescence (Ramalinga et al., 2015). Therefore, the modulation of sirtuins, which are involved in mitochondrial biogenesis (Rodgers et al., 2005) and removal of damaged mitochondria by autophagy (Lee et al., 2008), may control mitochondrial function, thus playing a protective role against age-associated diseases.

5.1.4 Retrograde signaling: from mitochondria to nucleus The mitochondrial destruction by lysosomes (mitophagy) is a thermodynamically expensive process. Therefore, it seems more useful for the cells to remove only a

5.1 Introduction

small region of the mitochondrion, presumably the most heavily damaged (Jang et al., 2018). This process is known as retrograde signaling and it may occur through the creation of mitochondrial-derived vesicles, a parkin-dependent process (Sugiura et al., 2014), by which mitochondria send information to the nucleus to activate a nuclear transcriptional response to minimize mitochondrial stress and subsequent damage (Jang et al., 2018). Recent studies report that the pathway of the mitochondrial unfolded protein response (UPRmt) is involved in retrograde signaling (Melber & Haynes, 2018). Mitochondrial molecular chaperones maintain the mitochondrial protein-folding environment. Their expression levels are coupled to the state of mitochondrial protein homeostasis by a mitochondria-to-nuclear signaling pathway termed the UPRmt (Nargund et al., 2012). The transcription factor ATFS-1 in Caenorhabditis elegans and potentially orthologous transcription factors in mammals (ATF4, ATF5, CHOP) regulate UPRmt. In addition to a nuclear localization sequence, ATFS-1 has an amino-terminal mitochondrial targeting sequence essential for UPRmt repression. Generally, ATFS-1 is imported into mitochondria and degraded. When mitochondria are stressed and dysfunctional, import efficiency decreases, allowing a percentage of ATFS-1 to accumulate in the cytosol and traffic to the nucleus (Nargund et al., 2012). Here, it promotes the expression of genes responsible for the survival and recovery of the mitochondrial network (Melber & Haynes, 2018). It seems that the cells can perceive the severity of mitochondrial dysfunction and define the nuclear response to an SOS sent from this critical organelle. This response might be the elimination of the whole-cell organelle, part of it, or, the activation of genes coding for proteases, antioxidant enzymes, and genes involved in mitochondrial protein import, mitochondrial dynamics, and cellular metabolism (Melber & Haynes, 2018) (Fig. 5.4).

FIGURE 5.4 Quality control mechanisms to counteract mitochondrial stress. The mitochondrial response to a stress condition changes with the magnitude of perceived stress. It includes (i) the activation of UPRmt to trigger a transcriptional program to potentially reduce the stress; (ii) the removal of mitochondrial damaged part into a mitochondrial-derived vesicle (MDV) to rescue the healthy portion; (iii) mitophagy activation to remove stressed mitochondrion; (iv) induction of cell death to remove the entire damaged cell.

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UPRmt can play a role in the aging process and age-related diseases. Indeed, the augment of activity of UPRmt permits the preservation of mitochondrial functionality and promotes longevity. It has been found that an aggregation-prone protein expressed in the neurons of C. elegans, comparable to protein aggregate seen in Huntington’s disease, binds to mitochondria, eliciting a global induction of UPRmt and affecting whole-animal physiology (Berendzen et al., 2016). Similarly, Aβ accumulation induces both UPRmt and mitophagy in a manner conserved from C. elegans to humans. The alteration of the mitochondrial import process is disrupted during Aβ proteotoxic stress (Cenini et al., 2016). Moreover, it is downregulated in patients with Alzheimer’s disease (Sorrentino et al., 2017). Further support for these findings comes from studies reporting that pharmacological interventions (metformin, resveratrol, rapamycin, NAD1 supplementation) seem to activate retrograde signaling from the mitochondria, thus increasing lifespan (De Haes et al., 2014; Houtkooper et al., 2013; Zhang et al., 2016).

5.1.5 Mitochondria and the “inflammaging” Aging is accompanied by a chronic state of low-grade inflammation referred to as “inflammaging” interconnected with other relevant mechanisms of aging and agerelated diseases (Franceschi et al., 2018). The inflammatory process has beneficial effects of allowing to neutralize dangerous or harmful agents in adulthood, and it becomes detrimental in old age with the onset of the inflammaging process (Franceschi et al., 2017). A recent study reports that the inflammaging process does not simply reflect increases in pro-inflammatory markers, but also antiinflammatory markers are involved in the process. This observation suggests that “inflammaging players” are more numerous and not restricted to classical cytokines (Morrisette-Thomas et al., 2014). It is thought that alterations in the complex machine involved in scavenging cellular debris and misfolded and/or misplaced self-molecules, which develop with age, are the basis for the inflammaging process (Franceschi et al., 2017). These molecules, often termed damageassociated molecular patterns (DAMPs) (Jang et al., 2018), are recognized by a specific class of receptors, pattern recognition receptors, that lead to unwanted and/or unnecessary inflammatory responses (Franceschi et al., 2017). In this context, the mitochondria represent a source of potential immune-stimulating DAMPs, including mtDNA, N-formyl peptides generated by translation of mitochondrial-encoded protein, and the phospholipid cardiolipin, a lipid almost exclusively found in mitochondrial membranes (De la Cruz & Kang, 2018; Dudek, 2017). The circulating mtDNA triggers an immune response activating the Toll-like receptor 9 (TLR9), while mitochondrial proteins interact with the formyl peptide receptor-1 (Jang et al., 2018). It is to point out that circulating mtDNA levels increase gradually with age after the fifth decade of life. The subjects with the highest mtDNA plasma levels have the highest amounts of inflammatory cytokines, and the ones with the lowest mtDNA levels have the lowest levels of the same cytokines (Pinti et al., 2014). In the same work, the authors

5.1 Introduction

report that in vitro stimulation of monocytes with mtDNA concentrations similar to the highest levels observed in vivo results in an increased production of TNFα, suggesting that mtDNA can modulate the production of pro-inflammatory cytokines (Pinti et al., 2014). The involvement of mitochondria in inflammatory pathways activation is very tight and complex. DAMPs may also trigger the assembly of the inflammasome, a high
molecular weight protein complex, in particular the NLRP3 inflammasome, that activates caspase-1. This then triggers the proteolytic activation of potent cytokines such as IL-1β and IL-18 (Broz & Dixit, 2016). Interestingly, the caspase-1 activation may also induce mitochondrial damage, further increasing inflammaging (Yu et al., 2014). The inflammasome activation plays a role also for the mitochondria-associated adaptor molecules on the outer mitochondrial membrane (Subramanian et al., 2013). There is also evidence that the mitochondrial phospholipid cardiolipin can directly bind and activate NLRP3 (Iyer et al., 2013). Finally, the release of mtDNA into the cytosol can also activate the inflammasome (Shimada et al., 2012). However, mtDNA release would be associated with both inflammasome and mitophagy activation. A recent study suggests that mitophagy may restrain inflammasome activation by removing damaged mitochondria (Zhong et al., 2016). These last could act as NLRP3 activators via the release of mtDNA (Zhong et al., 2016). The observation that in elders, there is evidence for chronic and persistent viral infections (Brunner et al., 2010) but, at the same time, it has been observed an involution of the thymus with a reduction of T-cells, got the researchers to hypothesize an alternative mechanism to the TLR9 activation by mtDNA. Given the endosymbiotic origin of mitochondria, it may be possible that mtDNA is recognized foreign, like bacterial and viral DNA, by cyclic GMP-AMP synthase (cGAS), a cytosolic sensor of doublestranded DNA (dsDNA). Once bound to dsDNA, cGAS induces signaling through the adaptor protein STING, leading to activation of the transcription factor IRF3. This finally activates the transcription of genes involved in response to viral infection (Jang et al., 2018). This hypothesis is supported by research showing a constitutive activation of the cGAS/STING/IRF3 pathway in mouse embryonic fibroblasts haploinsufficient for the mitochondrial transcription factor TFAM (West et al., 2015). This is involved in inflammation in microglial cells (Little et al., 2014). This mitochondrial-initiated virally triggered pathway might play a significant role in the inflammation observed in the elderly (Fig. 5.5). Even the mitochondrial succinate, a tricarboxylic acid (TCA) metabolite, may induce an inflammatory pathway in aging. When the Gram-negative bacterial product lipopolysaccharide activates the macrophages, they switch their metabolism from oxidative phosphorylation to glycolysis with a subsequent increase of the TCA metabolite succinate (Tannahill, 2013). Interestingly, this increase in succinate can stabilize HIF-1α that regulates IL-1β production (Tannahill, 2013). In addition, mitochondrial-produced succinate can interact with specific G proteincoupled succinate receptors localized on both immune and nonimmune cells, thus acting as a chemokine (Rubic et al., 2008). In this way, TCA metabolite succinate may contribute to the age-dependent alteration in immunity, and this is not

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FIGURE 5.5 “Inflammaging” pathways activated by mtDNA. MtDNA may activate immune response through an extracellular pathway, by the interaction between TRL9 and extracellular mtDNA, or an intracellular pathway by the NLRP3 inflammasome and the cGAS/STING pathway for intracellular mtDNA release.

surprising considering that in model organisms, such as yeast and worms, levels of TCA metabolites appear to regulate overall lifespan (Edwards et al., 2013). In conclusion, impaired mitochondrial function and increased oxidative stress represent the primary mediators of the aging process and subsequent age-related diseases (Szentesi et al., 2019). Whether the reactions of free radicals and other ROS contribute to the accumulation of molecular damages and give rise to aging and age-related diseases, antioxidants should prevent or slow down these processes and prolong longevity. Based on this assumption, many studies were established and aimed to estimate the effect of the level of endogenous antioxidants and/or of the administration of exogenous antioxidants on aging and age-related processes and diseases. Among molecules with antioxidant properties, vitamin E has gained attention since its supplementation can increase the vitamin content in mitochondria, protecting them from the dysfunction due to increased oxidative damage (Napolitano et al., 2019).

5.2 Vitamin E Tocopherols and tocotrienols are two subgroups with a shared chromanol ring and a saturated or tively (Schneider, 2005). Each subgroup has four plants, the α-isoform of tocopherols (α-T) is the

of vitamin E antioxidant class unsaturated side chain, respecisomers (α, β, γ, and δ). In the major isoform found in leaves,

5.2 Vitamin E

the -isoform (γ-T) is the primary form in seeds, whereas β- and δ-tocopherols (β-T and δ-T) are much less abundant (Napolitano et al., 2019). Tocotrienols (α, β, γ, and δ-T3) occur mainly in cereals and are less widespread (Napolitano et al., 2019). All plant species contain tocopherols, whereas tocotrienols are only present in certain plants such as annatto, palm, grains, nuts, and rubber (Sookwong et al., 2010).

5.2.1 Vitamin E and antioxidant capacity Vitamin E is a potent antioxidant molecule and, according to its antioxidant activity, it reduces the inflammatory processes by limiting the generation of ROS and their damage (Calder et al., 2009). The antioxidant property of vitamin E is due first to its capacity to deactivate the oxygen singlet (1O2) by quenching (Napolitano et al., 2019). Tocopherols can also act as potent chain-breaking antioxidants chemically scavenging 1O2 and lipid peroxyl radicals (Munne´-Bosch, 2005). Again, tocopherols may stabilize membrane structures by interacting with polyunsaturated fatty acyl chains (nonantioxidant functions) (Blokhina et al., 2003; Sattler et al., 2003) and are involved in carbohydrate metabolism (Maeda & Della Penna, 2007) and cell proliferation (Blokhina et al., 2003) in plants.

5.2.2 Uptake and cellular distribution of vitamin E The most common form of Vitamin E in human tissues is tocopherol, and the dietary intake of fruit and vegetables represents the primary source (Napolitano et al., 2019). Vitamin E from natural sources forms micelles with bile acids, cholesterol, phospholipid, and triacylglycerol to reach the intestinal lumen (Schmolz et al., 2016). In the next step, the micelles are absorbed into the intestinal cells. When vitamin E accesses the lacteal vessel, it forms chylomicrons with cholesterol, phospholipid, and triacylglycerol. Chylomicrons circulate via the lymphatic system to the blood flow via the thoracic duct. When they reach the liver, they are taken up by very-low-density lipoprotein and released into the bloodstream. Upon transition from the liver to the bloodstream, α-T is preferentially released into the bloodstream due to the presence of α-tocopherol transfer protein (α-TTP) in the liver (Miyazawa et al., 2019). The mechanism of selective release of α-T from the liver via α-TTP is not fully understood. The current opinion suggests that the interaction between α-T and α-TTP facilitates the transport of α-T to the plasma membrane. Here, it binds the resident phosphatidylinositol 4,5-bisphosphate and induces a conformational change that results in the release of α-T and its incorporation into the membrane (Chung et al., 2016). The next bond with ATP-binding cassettes A1 receptor allows α-T to exit the cells, be incorporated into lipoproteins, and move to extrahepatic tissues. α-TTP is largely affine to α-T (100%) and has a much lower affinity to β-T, γ-T, and δ-T (50%, 10%
30%, or 1%, respectively) (Jiang et al., 2014). The vitamin E isomer unbound to α-TTP undergoes metabolization by phase I metabolism (catabolism and sidechain shortening) and phase II metabolism (sulfation and glucuronidation). The resulting

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metabolites are excreted from the body via feces and urine (Miyazawa et al., 2019). Unlike tocopherols, α-TTP shows a lower affinity for tocotrienols (Miyazawa et al., 2019). Because of its poor affinity with α-TTP, tocotrienol is unlikely to be liberated from the liver into the bloodstream. Peh et al. (2016) reported that the accumulation of tocotrienol in the brain and adipose tissue does not occur via the bloodstream, but via the lymphatic system. Moreover, tissue distribution of tocotrienol is not only affected by α-TTP but also other factors, such as cytochrome P450 (CYP) (Abe et al., 2007). Abe et al. (2007) reported that α and γ-tocotrienol concentrations in the plasma and various tissues were enhanced by ketoconazole, a potent inhibitor of CYP, in a vitamin E deficient rat. Vitamin E localizes in the membrane of organelles such as the endoplasmic reticulum, mitochondria, and peroxisomes (Saito et al., 2004). The problem concerning vitamin E distribution among the membrane of cellular organelles has only been recently investigated by Irı´as-Mata and colleagues (2018). The authors proposed diffusion as the main driving force of the distribution of vitamin E isoforms within the cell (Irı´as-Mata et al., 2018). Moreover, they found that the metabolites of γ-T, α-T3, and γ-T3, rather than the parent compounds, localize in mitochondria, and the metabolites of β-T, γ-T, and δ-T, localize in peroxisomes (Irı´as-Mata et al., 2018).

5.2.3 Vitamin E functions in mitochondria The localization of α-tocopherol within the highly unsaturated phospholipid bilayer of cell membranes provides a means of controlling lipid oxidation at the initiation site. Therefore, the incorporation of α-tocopherol and other antioxidants into mitochondria is relevant to maintain oxidative stability of the membranebound lipids. Many studies regarding mitochondrial disease and dysfunction focused on vitamin E (Rafique et al., 2004) and other antioxidant deficiencies, and relatively sparse information is available regarding the eventual beneficial effects of antioxidant-enriched mitochondria in terms of health and function. Experiments performed on subcellular membranes of microsome and mitochondria from porcine and chicken muscle evidenced an increase in α-tocopherol content with a α-tocopherol-supplemented diet for the animals (Lauridsen et al., 2012). In addition, other dietary components may increase the subcellular concentration of α-tocopherol. In the study by Lauridsen et al. (2000), an additive effect of dietary all-rac-α-tocopheryl acetate and copper affected the concentration of α-tocopherol in mitochondria and microsomes. Although the mechanism of vitamin E delivery to cellular organelles is not fully understood, its preferential distribution to mitochondria concurs with the presence of a cytosolic binding protein able to transfer the vitamin from liposomes to mitochondria (Mowri et al., 1981). The accumulation of vitamin E in membranes may counteract oxidative stress due to enhanced ROS production. In the experiments reported by Lauridsen et al. (2012), membrane enrichment with vitamin E is responsible for oxidative stability.

5.2 Vitamin E

The reduced rates of Fe21-catalyzed lipid peroxidation, lower peroxidation rates of subcellular fractions in the presence of metmyoglobin/hydrogen peroxide, and lower rate of formation of free radicals, sustain the conclusion of the work of Lauridsen et al. (2012). Vitamin E treatment reduces the thyroid hormone-induced increase in lipid oxidative damage and prevents the reduction in total antioxidant capacities in mitochondria isolated from liver rats (Venditti et al., 1999). In the same study, the authors demonstrate that vitamin E treatment prevents the thyroid hormone-induced changes in the distribution of the mitochondrial population among its fractions (Venditti et al., 1999), thanks to the capacity to reduce the degradation of the M1 fraction (the most abundant and susceptible to oxidants) by increasing antioxidant defenses. Vitamin E supplementation to cold-exposed animals reduces the levels of oxidative damage in both liver (Venditti et al., 2007) and muscle (Venditti et al., 2009) mitochondria. In addition, the reduced mitochondrial oxidative damage by the vitamin E administration ameliorates the heart functional recovery after ischemia-reperfusion in both experimental (Venditti et al., 2000) and functional (Venditti et al., 2011) hyperthyroidism.

5.2.4 Vitamin E, mitochondria, and aging Vitamin E supplementation prevents or ameliorates chronic and age-associated diseases such as cardiovascular disease, chronic inflammation, and neurological disorders (Azzi, 2004). The first experiment that demonstrated a protective effect on mtDNA, was performed by administering a vitamin E and C supplemented diet (De la Asuncion et al., 1996). Antioxidant supplementation preserved mtDNA from damage associated with aging in the brain, liver, and kidney of aged rats (De la Asuncion et al., 1996). Subsequently, although vitamin E has been extensively studied, there has been no consistent information about the effects of vitamin E on mitochondrial dysfunction in aging. Moreover, studies performed on vitamin E supplementation led to controversial conclusions. Sumien et al. (2003) shows that the supplementation with α-tocopheryl acetate increased α-tocopherol concentrations 3
5-fold in plasma and tissue homogenates, and 2
3-fold in mitochondria isolated from the liver, skeletal muscle, and heart of aged mice. However, supplementation affected neither the rate of heart mitochondrial H2O2 generation nor products of lipid peroxidation and protein oxidation (protein carbonyls) (Sumien et al., 2003). The same authors report that the vitamin E supplemented diet in relatively old mice did not reverse preexisting agerelated disorders of cognitive or motor function (Sumien et al., 2004). In addition, the supplementation provides little effect on the oxidative damage to lipid and protein (Sumien et al., 2004). Conversely, Shetty et al. (2014) suggest that tocopherol supplementation could ameliorate age-related impairment and reduce protein oxidation in the liver, brain, heart, and skeletal muscle of male aged C57BL/6J mice. Probably, the administration of different isoforms of α-tocopherol and initiated in different ages of the animals may explain the diversity of the results obtained. Indeed, a diet supplemented with vitamin E can

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improve cognitive and motor performance and reduce oxidative stress when the administration is initiated in younger rats and is maintained into old age (Joseph et al., 1998). Subsequently, Navarro et al. (2005) showed that the beneficial effects of high doses of vitamin E on the average and maximum lifespan of male mice, parallel with the beneficial effect on the decline in neurological performance and mitochondrial function associated with aging. Another study by the same research group reports that dietary supplementation with vitamin E from nine to 12 months of age in rats restores, in a dose-dependent manner, the decreases in tissue and mitochondrial respiration, the activities of complexes I and IV, and mtNOS activities almost to the values of 4-month-old rats (Navarro et al., 2011). Moreover, vitamin E prevents the accumulation of oxidized products of lipids, proteins, and DNA and the increase in superoxide anion and hydrogen peroxide production (Navarro et al., 2011). Finally, vitamin E supplementation seems to sustain mitochondrial biogenesis in synaptic areas, thus reverting the loss of hippocampus mitochondrial mass (Navarro et al., 2011). The effect of vitamin E supplementation does not only concern mitochondrial ROS production associated with the aging process. Cosmas et al. (1994) reported that in rat myocardium, the size of mitochondria increases with aging. Indeed, senescence is associated with a shift in the mitochondrial population distribution towards a higher percentage of wider organelles, consistent with a decline in physiological function (Bertoni-Freddari et al., 1993; Cosmas et al., 1994). Vitamin E supplementation restores the size of hepatic mitochondria in “old” mice supplemented with vitamin E, as suggested by significantly smaller mitochondria than age-matched controls (Agostinucci et al., 2002). Continuous vitamin E supplementation maintains mitochondrial size with age (Agostinucci et al., 2002). Referring to randomized clinical trials, the difficulty in performing specific and uniform human studies is widely responsible for the inconsistent outcomes reported in the literature. Therefore, the benefit of vitamin E as a treatment for aging and neurodegenerative disorders is still under debate. However, epidemiological studies demonstrate that consumption of specific micronutrients, including vitamin E, is linked to improved cognitive performance in humans (Fata et al., 2014). Morris et al. (2002) show that four different cognitive tests revealed a reduced cognitive decline in individuals (aged 65
102 years) with higher vitamin E intake (users) (obtained by diet and from supplements) when compared to nonusers (low vitamin E intake). In a similar study, vitamin E intake improved cognitive performance and decreases the risk of developing Alzheimer’s disease (Morris et al., 2005). In sharp contrast, randomized clinical trials with vitamin E do not fully support this evidence (Joshi & Pratico`, 2012). Most likely, many clinic epidemiological and interventional studies do not consider new emerging roles for vitamin E, among which the modulation of specific signaling pathways and genes involved in metabolic and inflammatory events (Joshi & Pratico`, 2012), thus with inconclusive results. While the experiments with vitamin E alone result in controversial outcomes, supplementation involving multiple antioxidants or antioxidant-rich food has

5.3 The necessity for an alternative theory

furnishes positive results (Joseph et al., 2009). In rodents, the combination of vitamins C (ascorbate) and E reduced age-associated impairments in cognitive function (Arzi et al., 2004). It decreased the oxidative stress in the brain of old diabetic rats (Naziroglu et al., 2011) and protected against intermittent cold exposure-induced oxidative stress in the hypothalamus and cortex of aged rats (Devi & Manjula, 2014). The combination is successful because vitamin E (tocopherol) is involved in a chain-breaking mechanism to prevent lipid peroxidation by scavenging peroxyl radicals. In this process, it becomes oxidized to α-tocopheroxyl radical. The ascorbate, a lipophilic coantioxidant, can reduce back the tocopheroxyl radical to tocopherol. Ascorbate is then oxidized and is recycled by enzyme systems using NADH or NADPH (Buettner, 1993). Patients with mild cognitive impairments and Alzheimer’s disease show a reduction in plasma concentration of vitamins C and E, along with other antioxidants (Rinaldi et al., 2003). However, these studies do not compare every single supplementation combination. Therefore, more studies are needed to determine the nature of the interactions between antioxidants on cellular redox state during aging.

5.3 The necessity for an alternative theory Even if the MFRTA furnishes valuable bases for aging research, its validity was questioned (Pomatto & Davies, 2018). Growing evidence suggests that short-term low concentrations of free radicals and ROS render them advantageous, whereas high concentrations make them deleterious (Pomatto & Davies, 2018). That means that different concentrations of ROS may exert a nonlinear response, defined as “hormesis” or, more precisely, “mitochondrial hormesis” (mitohormesis), being that ROS is predominantly produced by the mitochondria in the physiological state (Yun & Finkel, 2014). In this view, ROS become essential, health-engendering signaling molecules. Some evidence suggested that a transient ROS increase induces antioxidant defense enzymes that detoxify the cell by increased ROS production (Venditti et al., 2016). Wood et al. (2003) show that lower concentrations of peroxiredoxins can remove both cytosolic and mitochondrial H2O2. When H2O2 concentration increases, peroxiredoxins become inactive, and H2O2 accumulation triggers a cascade of cellular signaling events involving the Nrf2 stress-protective pathway (Zhang et al., 2015). Thus, oxidant levels are highly responsible for initiating cellular signaling responses. In this view, even if the supplementation with antioxidants triggers ROS detoxification, it may cover multiple other pathways that are ROS-induced, compromising cell survival. In an appealing review, Hekimi et al. (2011) reported that MFRTA does not fully explain the aging process for different reasons. First, there is no correlation between the produced ROS and longevity in some species. In addition, the administration of antioxidants has detrimental rather than beneficial effects on lifespan, and the inactivation or overexpression of antioxidant activities fails to produce

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outcomes that support the MFRTA. Finally, long-lived mutants and species with high ROS production and oxidative damage exist. Several scientific pieces of evidence sustain these items. A study on C. elegans shows that knockout mutations affecting type 2 mitochondrial SOD activity increase oxidative stress. These mutations surprisingly prolong lifespan (Doonan et al., 2008). Again, mice deficient in both Mn-SOD and glutathione peroxidase-1 show oxidative damage and a greater incidence of pathology but no reduction in longevity Zhang et al., 2009). Mice that lack one copy of Sod2 (Sod21/2) display enhanced mitochondrial oxidative stress, but they live as long as the wild-type (Van Remmen et al., 2003). A more recent model for the aging process suggests that the properties of ROS as signaling molecules may provide an alternative way to explain a series of phenomena related to ROS biology (Hekimi et al., 2011). More specifically, ROS are signaling molecules that function as stress signals in response to agedependent damage. In fact, under physiological conditions, the transient generation of ROS appears to be essential to maintain cellular homeostasis by activating multiple pathways. They include ERK, JNK, and MAPK cascades, as well as the PI3-K/Akt, PLC-γ1, and JAK/STAT pathways (Hekimi et al., 2011). These pathways, in turn, modulate the activities of central transcription factors, among them NF-kB, AP-1, Nrf2, FoxOs, HIF-1α, and p53 (Hekimi et al., 2011). Furthermore, kinases and phosphatases susceptible to oxidative modifications can regulate the activities of enzymes such as catalase, GPxs, and Prdx.

5.3.1 ROS signaling, aging, and lifespan Mounting evidence in several systems reports that ROS-activated signaling pathways can stimulate beneficial responses to the cellular stresses produced during aging. Two mutations in two mitochondrial respiratory complexes of a mutant young C. elegans are responsible for the increased superoxide generation (Yang & Siegfried, 2010). This in turn, activates signals to trigger changes of gene expression to prevent or attenuate the effects of subsequent aging (Yang & Siegfried, 2010). The increase in superoxide generation seems necessary and sufficient to increase longevity since the antioxidants NAC and vitamin C suppressed it (Lee et al., 2010; Yang & Siegfried, 2010). Liu et al. (2005) demonstrated that lack of one copy of the enzyme necessary for the synthesis of the antioxidant and redox cofactor ubiquinone (Mclk1) is associated with an increased mitochondrial oxidative stress, but also with extended lifespan with respect to the wild-type siblings (Liu et al., 2005), and with slower development of biomarkers of aging, including the loss of mitochondrial function (Lapointe et al., 2009). It is assumed that produced ROS activate the transcription of the protective factor hypoxiainducible factor 1α (HIF-1α) which, in turn, stimulates the expression of genes that are important for the effector function of macrophages (Wang et al., 2010). These findings demonstrate that changes in mitochondrial function within physiologically tolerable limits modulate the immune response. Moreover, they suggest

5.3 The necessity for an alternative theory

that altered immune function through a limited increase in HIF-1α expression can positively impact animal longevity. In another study, a mice mutant for the glutathione transferase mGSTA4
4, an enzyme implicated in the detoxification of 4-hydroxynonenal (4-HNE), have an extended lifespan (Singh et al., 2010). 4-HNE, the product of lipid oxidation, can modulate ligand-independent signaling by membrane receptors and interact with a range of kinases (Dwivedi et al., 2007). Similarly, deacetylases of the sirtuin family produce NAD1 metabolites that generate a ROS signal, whereas antioxidants hinder the lifespan-prolonging properties of sirtuin activation (Schmeisser et al., 2013).

5.3.2 “The gradual ROS response hypothesis” The scientific evidence reported below led Hekimi et al. (2011) to formulate “The gradual ROS response hypothesis.” According to the theory: . . .. cellular constituents sustain a variety of age-dependent damage that trigger ROS-dependent, protective, stress-response pathways. The ROS generation that is triggered by these mechanisms is well handled by the cellular detoxification systems and is therefore not deleterious. These protective mechanisms appear unable to fully prevent the gradual accumulation of age-related damage. Thus, the gradual increase in damage induces an ever-greater stimulation of ROS production as the cell attempts to enhance its stress response. As aging progresses, ROS generation partially escapes control by the antioxidant systems and ROS toxicity starts to participate in causing the very damage that the ROS-dependent stress pathways are meant to neutralize. This could trigger a toxic runaway process that might form the basis of the involvement of ROS in age-dependent diseases, which tend to develop only in the second half of lifespan (Hekimi et al., 2011).

In this view, the hormetic response would share common signaling pathways with the theory of gradual ROS response even if the normal aging process for living organisms is based on this theory. The new aging theory could provide an elucidation regarding the increased lifespan of younger animals with an extensive production of ROS. The increase in ROS production independent from age and induced by several mutations in younger animals can turn on protective signaling pathways. These are, in turn, responsible for an extension of lifespan. The theory can explain the capacity of antioxidant systems endogenously activated to extend lifespan while exogenous supplementations do not induce the same effect. The gradual ROS response hypothesis is interesting since it furnishes alternative explanations for the well-established correlation between ROS production and aging. However, further studies concerning the new interpretation of ROS biology are necessary to identify mechanisms potentially relevant to initiate the aging process.

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5.4 Concluding remarks The literature analysis reported here highlights that, although frequently attacked, the “Mitochondrial Free Radical Theory of Aging” continues to be widely investigated and sustained. Therefore, mitochondrial ROS production plays a crucial role in the cellular damage associated with the aging process, and antioxidant molecules supplementation may represent a valid strategy to contain oxidative damage and aging progression. Nevertheless, the new point of view considering mitochondria not merely like the cellular powerhouses, and new models for the relationship between ROS, redox signaling, oxidative damage, and lifespan, suggest that simplistic interpretation or application of the Theory led to inconclusive solutions. Perturbations in mitochondrial function, biogenesis, and dynamics impair cellular homeostasis and trigger mitochondrial quality control mechanisms. Altered mitochondrial dynamics and quality control promote the accumulation of damaged mitochondria that contributes to aging and several age-related pathologies. Thus, strategies that effectively improve or rescue the defect in mitochondrial dynamics and quality control may be beneficial in counteracting aging and ageassociated diseases. In addition, the “Mithormesis” perspective opposes the traditional view of ROS as being invariably harmful and unwanted by-products of mitochondrial metabolism. The theory suggests that ROS plays a crucial role in cell survival by turning on stress signaling pathways responsible for triggering desired cellular survival pathways. In addition, the mithormetic theory reconsiders the protective effects of antioxidant molecules such as vitamin E: supplementation of several antioxidants could interfere with “adaptive homeostasis” linked to transient ROS production. Mithormesis relies on the recent new role for the oxidants not merely as inducers of macromolecular damage, but also as regulators of signaling pathways. Growing recent research concerning the aging mechanisms focuses attention on redox signaling and suggests alternative roles for ROS, such as mitonuclear redox signaling, a regulatory process involved in the development of cellular senescence. In this overview, it would be more accurate to modify the free radical theory of aging from “Organisms age because cells accumulate reactive oxygen speciesdependent damage overtime” to: “Organisms age because cells accumulate oxidants”-dependent damage and oxidants’-dependent senescent characteristics over time’ as recently suggested by Clement and Luo (2020).

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CHAPTER

The role of B vitamins in protecting mitochondrial function

6

Sandip Mukherjee, Oly Banerjee and Siddhartha Singh Department of Physiology, Serampore College, Hooghly, West Bengal, India

6.1 Introduction Vitamins are essential micronutrients that are synthesized by bacteria, yeasts, and plants, but not by mammals (Yoshii et al., 2019). Therefore, mammals must obtain vitamins from the diet or rely on their synthesis by commensal bacteria in the gastrointestinal tract. Some vitamins are water-soluble (e.g., vitazmin B family and vitamin C), whereas others are fat-soluble (e.g., vitamins A, D, E, and K). Water-soluble vitamins are not stored by the body and any excess is excreted in the urine; therefore, it is important to consume a diet containing the necessary amounts of these vitamins. Vitamin deficiency associated with insufficient dietary intake occurs not only in developing countries but also in developed countries as a result of an increasingly unbalanced diet for individuals (Whatham et al., 2008). In addition to the diet, commensal bacteria are recognized as important players in the control of host health (Lee & Hase, 2014; Rooks & Garrett, 2016). From the point of view of vitamins, commensal bacteria are both providers and consumers of B vitamins and vitamin K. Although dietary B vitamins are generally absorbed through the small intestine, bacterial B vitamins are produced and absorbed mainly through the colon (Magnu´sdo´ttir et al., 2015; Yoshii et al., 2019), indicating that dietary and gut microbiota-derived B vitamins are possibly handled differently by the human body. B vitamins are important cofactors and coenzymes in several metabolic pathways, and it has been reported recently that B vitamins also play significant roles in the maintenance of immune homeostasis (Hosomi & Kunisawa, 2017; Suzuki & Kunisawa, 2015). Thus, both dietary components and the gut microbiota modulate host immune function via B vitamins. There are eight members in the B vitamin family: B1 (thiamin), B2 (riboflavin), B3 (niacin), B5 (pantothenic acid), B6 (pyridoxine), B7 (biotin), B9 [folate (folic acid)], and B12 (cobalamin). Choline, also known as B4, which was officially recognized as an essential nutrient by the Institute of Medicine in 1998 (Zeisel & da Costa, 2009). Vitamin B deficiencies are highly prevalent in many developing countries, especially where diets are low in animal products, fruit, and vegetables, and Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00001-1 © 2023 Elsevier Inc. All rights reserved.

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FIGURE 6.1 Molecular structure of eight B vitamins (Janssen et al., 2019).

where cereals are milled prior to consumption. Pregnant and lactating women, infants and children are mostly at risk of Vitamin B deficiencies (Ashley, 2016) (Fig. 6.1).

6.2 B vitamins and mitochondrial metabolism 6.2.1 Vitamin B1 (thiamine) Mitochondria are the major holder (more than 90%) of all cellular thiamine as they are highly endowed with vitamin B1 or thiamine (Bettendorff et al., 1994). Thiamine diphosphate is the active form of thiamine and is essential cofactor of several mitochondrial dehydrogenase complexes as essential cofactor. These complexes are alpha-ketoglutarate (α-KG) dehydrogenase (OGDH) complex, pyruvate dehydrogenase (PDH) complex, and branched-chain keto-acid dehydrogenase complex. Later research established the role of thiamine as allosteric regulator of two components of malate-aspartate shuttle,—glutamate dehydrogenase and malate dehydrogenage (Mkrtchyan et al., 2015). Thus, regulation of enzymes related to malate-aspartate shuttle by thiamine may impact on malate/citrate exchange and responsible for decline in the efflux of citrate from the mitochondria and escalation in citrate flux through the TCA cycle (Mkrtchyan et al., 2015). Further, in addition to its role as coenzyme for acetyl-CoA production, thiamine also acts as an allosteric regulator of acetyl-CoA metabolism including regulatory acetylation of proteins and acetylcholine biosynthesis (Mkrtchyan et al., 2015).

6.2.2 Vitamin B2 (riboflavin) The biologically active forms of vitamin B2 or riboflavin are the oxidized and reduced forms of flavin adenine dinucleotide (FAD and FADH2, respectively)

6.2 B vitamins and mitochondrial metabolism

FIGURE 6.2 Structure of (A) Flavin monophosphate (FMN), and (B) Flavin adenine dinucleotide (FAD). (Suwannasom et al., 2020).

and flavin mononucleotide (FMN and FMNH2, respectively) (McCormick, 2000; Thurnham, 2000) (Fig. 6.2). Riboflavin is first phosphorylated intracellularly to FMN by riboflavin kinase and further metabolized to FAD by FAD synthase (FADS) that subsequently transfers an AMP unit from ATP to FMN (Janssen et al., 2019). Both FMN and FAD act as electron carriers and comprise the essential prosthetic group of flavoenzymes. About 90 flavoproteins are encoded in the human genome and utilize either FAD (84%) or FMN (16%) while five human flavoenzymes require both FMN and FAD (Lienhart et al., 2013). There are five acyl-CoA dehydrogenases present in the mitochondria that require FAD and catalyze the first step in each cycle of β-oxidation (Depeint et al., 2006). They are isovaleryl CoA dehydrogenase, branched-chain acyl CoA dehydrogenase, 2-methyl-branched-chain acyl CoA dehydrogenase, isobutyryl CoA dehydrogenase and short-chain acyl CoA dehydrogenase (Depeint et al., 2006). Being located in mitochondria, riboflavin catalyzes several redox reactions:— oxidation, reduction and dehydrogenase reactions. Riboflavin reinforces the redox reaction catalyzed by succinate dehydrogenase (SDH) and glutathione reductase (GR) by providing FAD (Depeint et al., 2006). SDH catalyzes the oxidation of succinate to fumarate in which reduction of FAD to FADH2 is an intermediary step. This intermediary step also utilizes FAD-dependent GR to cause reduction of oxidized glutathione (GSSG) to two molecules of glutathione (GSH), which is also coupled to the reduction of NADPH to NADP 1 . In addition to its supportive role in antioxidant defense mechanisms, riboflavin is also proposed to act as an antioxidant by its own oxidation (Janssen et al., 2019).

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6.2.3 Vitamin B3 (niacin) Vitamin B3 encompasses several dietary forms—nicotinic acid (NA), nicotinamide (NAM), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN) (Yoshino et al., 2018). These are the precursors for the formation of NAD1 in the salvage pathway. Furthermore, NAD 1 synthesis can also occur through de novo synthesis from tryptophan (Mehmel et al., 2020). NAD1 plays a pivotal role in hydrogen transfer in redox reaction and also receives hydride from several metabolic processes including glycolysis, the TCA cycle, and fatty acid oxidation to form NADH (Xie et al., 2020). NAD1 can be converted into its phosphorylated form NADP1, with a principal role in lipid metabolism and redox balance. Additionally, NADP1 can also receive the electrons from NADH to form NADPH by nicotinamide nucleotide transhydrogenase which is located in the mitochondrial inner membrane (Xie et al., 2020). Alternatively, NADPH can also be generated from the pentose phosphate pathway, the serine synthesis pathway, and glutamate dehydrogenase (Ronchi et al., 2016; Lewis et al., 2014). Through mitochondrial oxidative phosphorylation (OXPHOS), NADH serves as a central hydride donor to ATP synthesis, whereas, NADPH mostly regulates cellular redox status with the generation of reactive oxygen species (ROS). Besides mediating mitochondrial metabolic signals via the electron transport chain, NAD1 is also a direct regulator of protein post-translational acylation and ADP-ribosylation modifications (Houtkooper et al., 2010). The important role of NAD 1 has also been expanded as a cosubstrate for various enzymes including sirtuins, poly-ADP ribose polymerase (PARPs), CD157, CD73, CD38 and SARM1 (Xie et al., 2020). In addition, NAD1 is also utilized as a nucleotide analog in DNA ligation and RNA capping (Chen & Yu, 2019; Bird et al., 2016). In cellular biology, NAD 1 has a fundamental importance due to its essential role as a cofactor of various metabolic redox reactions, as well as an obligate cosubstrate for NAD 1 -consuming enzymes, which are involved in many fundamental cellular processes including aging/longevity.

6.2.4 Vitamin B5 (pantothenic acid) Vitamin B5 (pantothenic acid) is the precursor of Coenzyme A (CoA) and is involved in the oxidation of lipids and carbohydrates. CoA, being the essential cofactor in metabolic processes, transfers acyl group and functions as an acyl moiety transporter (Janssen et al., 2019). CoA also serves as carbonyl-activating group which is essential for the mitochondrial enzymes PDH and alpha-keto glutarate dehydrogenase of the TCA cycle as well as for the beta oxidation pathway (Atamna, 2004). Apart from its role in acetyl transferase reaction, CoA regulates glucose oxidation in the TCA cycle and fatty acid oxidation and thus maintains a balance in between carbohydrate and lipid metabolism (Leonardi et al., 2005). CoA also serves as the required cofactor for the biosynthesis of ketone bodies (Puchalska &

6.2 B vitamins and mitochondrial metabolism

Crawford, 2017). Thus, CoA is an essential cofactor in cellular metabolism and has pivotal importance in metabolic functions, which explain its predominace in mitochondria (2.2 mM), with less presence in peroxisomes (20
140 μM) and the least presence in the cytoplasm (less than 15 μM) (Williamson & Corkey, 1979).

6.2.5 Vitamin B6 (pyridoxal phosphate) The biologically active form of vitamin B6 is pyridoxal 50 phosphate (PLP). In contrary to microorganisms, mammals are not able to synthesize PLP, but they recycle it from B6 vitamers such as pyridoxal (PL), pyridoxamine, and pyridoxine contained in food through a salvage pathway (McCormick & Chen, 1999). PLP acts as coenzyme in about 160 distinct enzymatic activities and serves many essential metabolic functions in amino acid biosynthesis and catabolism, 1-carbon metabolism, membrane lipid biosynthesis, production of neurotransmitters and biogenic polyamines, as well as glycogen cycling and iron metabolism (iron-sulfur cluster and heme biosynthesis) (Whittaker, 2016). PLP is specifically involved in amino acid metabolism, de novo synthesis of NAD1 and iron-sulfur (Fe-S) cluster biosynthesis, and functioning as a cofactor for several aminotransferases and decarboxylases (Depeint et al., 2006; Braymer & Lill, 2017). The FeS cluster is an essential component of many metabolic protein complexes, including aconitase and ETS complexes and other cellular protein complexes like DNA polymerases and helicases (Braymer & Lill, 2017). Additionally, PLP is also the essential cofactor for mitochondrial aminolevulinate synthase which catalyzes the first step of the heme biosynthetic pathway (Hunter & Ferreira, 2009). In addition to its role in GSH synthesis from homocysteine, vitamin B6 participates in protecting mitochondria from oxidative stress by maintaining a balance between GSH and GSSG (Depeint et al., 2006). A mechanism that is independent of energy was found to give PLP easy access to different compartments of the mitochondria (Depeint et al., 2006).

6.2.6 Vitamin B8/B7 (biotin) Biologically, vitamin B8/B7 (biotin) is utilized by organisms without further chemical or enzymatic modifications. Biotin resides mainly in mitochondria and cytosol, and its cellular location is consistent with its function. However, biotin can enhance mitochondrial biogenesis thorough guanylate cyclase activation (Nisoli et al., 2003). As a coenzyme, biotin is required by five carboxylases, four of which are found within mitochondria. These carboxylases encompass carboxyl transferases and catalyze the addition of a carboxylic acid group to an organic compound, a reaction involving carbon dioxide (CO2). Pyruvate carboxylase catalyzes the carboxylation of pyruvate to oxaloacetate and thus functions to replace the oxaloacetate in the TCA cycle. Furthermore, this enzyme is crucial for both gluconeogenesis (in liver and kidney) and fatty acid synthesis (in adipose tissue, liver, brain) since it provides oxaloacetate, a precursor to malate and citrate,

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which are both citric acid cycle intermediates and leave the mitochondria during biosynthetic processes. Another enzyme, Propionyl-CoA carboxylase plays a pivotal role in the catabolism of odd chain fatty acids and amino acids (isoleucine, methionine, threonine and valine) as they convert propionyl-CoA to methylmalonyl-CoA. Methylcrotonyl-CoA carboxylase (MCCC) catalyzes carboxylation of 3-methylcrotonyl-CoA to generate 3-methylglutaconyl-CoA and this is an essential step for the catabolism of leucine and isovalerate (Tomassetti et al., 2018). The other enzyme acetyl-CoA carboxylase (ACACB) is a biotin carboxyl carrier protein and can act as both biotin carboxylase and carboxyltransferase. In its carboxyltransferase role, ACACB catalyzes the carboxylation of acetyl-CoA to malonyl-CoA and this reaction is ATP-dependent. Being localized in the mitochondrial outer membrane, ACACB associates with carnitine palmitoyltransferase 1 and regulates the channeling of acetyl CoA toward either lipid synthesis in the cytosol or mitochondrial beta-oxidation (Zu et al., 2013). AcetylCoA carboxylase (ACACA), a cytoplasmic biotin containing enzyme, plays a pivotal role in the biosynthesis of long-chain fatty acid (Janssen et al., 2019).

6.2.7 Vitamin B11/B9 (folate) Folate is the generic term used for several forms of this B vitamin but is mostly referred to as vitamin B11 or B9. Mitochondria contain more than half of the cellular folates and therefore several important steps of folate metabolism occur here (Tibbetts & Appling, 2010). The folate metabolism is highly complex, particularly because folate is involved in numerous metabolic reactions (Depeint et al., 2006; Nijhout et al., 2008). The network of folate metabolism is compartmentalized within mammalian cells in the nucleus, the cytosol, and in the mitochondria. The various forms of folate are necessary for the synthesis of ADP and GDP, purines and thymidylate synthesis, metabolism of cellular GSH, amino acid metabolism, and for providing S-adenosylmethionine (SAM) for methylation reaction. Additionally, folate is also essential for methionine recycling that occurs in the cytoplasm and interconversion of serine-glycine occurring in mitochondria (Ulrich et al., 2008; Reed et al., 2008). The integrity of the mitochondrial and nuclear genomes is preserved through maintaining an adequate and balanced cellular deoxythymidine (dT) monophosphate (dTMP) pool. Both depletion and expansion of dTMP pools can impact genomic (both nuclear and mitochondrial) DNA integrity. In order to sustain a continuous supply of dTMP to mtDNA replication, the salvage pathway is not sufficient, so the de novo pathway is also required. The folate-dependent enzymes serine hydroxymethyltransferase 2, thymidylate synthase, and dihydrofolate reductase 2 are required by the de novo dTMP synthesis pathway to catalyze the conversion of dUMP to dTMP (Zhou et al., 2008). Further, folate deficiency also results in the accumulation of mtDNA deletions in model systems, which may lead to reduced expression of mitochondrial genes that are important for mitochondrial function and energy production (Fenech, 2012; Chou et al., 2009).

6.3 Oxidative stress and mitochondrial toxicity: role of B vitamins

6.2.8 Vitamin B12 (cobalamin) The largest and most complex B-vitamin is vitamin B12 (cobalamin). It functions as a coenzyme in mitochondria and cytosol. In addition to cyanocobalamin, other forms of B12 found naturally in biological systems include methylcobalamin, cob (I)alamin, 50 -deoxy adenosylcobalamin, and hydroxycobalamin. As the cofactor for methylmalonyl-CoA mutase (converts methylmalonyl-CoA into succinylCoA) in mitochondria, deoxyadenosylcobalamin plays a role in the degradation of amino acids and odd-chain fatty acids. Branched-chain amino acids (BCAAs) are utilized for fatty acid synthesis in differentiating adipocytes and the catabolism of BCAAs are assisted by adenosylcobalamin (Green et al., 2016). Cobalamin is essential for methylmalonyl-CoA mutase as cobalamin deficiency leads to accumulation of methyl-malonic acid (MMA), methylmalonyl-CoA, and odd-chain fatty acids (Green et al., 2016). MMA is reported to impair mitochondrial function by acting as a mitochondrial toxin that inhibits SDH (Narasimhan et al., 1996). Another form of vitamin B12, methylcobalamin serves as a cofactor for 5methyltetrahydrofolate-homocysteine methyltransferase (MTR), which is also known as methionine synthase. MTR catalyzes the transmethylation of homocysteine via methyltetrahydrofolate (MTHF) to methionine (Matthews et al., 1998). Methylcobalamin also functions in the synthesis of GSH by acting as a cofactor for methionine synthase. Tetrahydrofolate is the fully reduced form of folate and its synthesis is dependent on methyl synthase activity (Scott, 1999). Thus adequate availability of methylcobalamin is also very crucial for folate metabolism (Fig. 6.3).

6.3 Oxidative stress and mitochondrial toxicity: role of B vitamins Thiamine (vitamin B1) serves as a cofactor for most mitochondrial localized enzymes. Besides its involvement in cellular metabolism, thiamine serves a pivotal role in oxidative stress (Depeint et al., 2006). Thiamine pyrophosphate, which is synthesized from thiamine, is an essential cofactor of various enzymes during cell metabolism, protecting against tissue oxidative damage by maintaining the reduced NADP1 concentration (Gioda et al., 2010). A major function of thiamine is to increase the activity of a major component of cellular antioxidant enzymes, —glutathione peroxidase (GPx). It is a selenoprotein enzyme that catalyzes the breakdown of hydrogen peroxide and organic peroxides through GSH metabolism (Cominetti et al., 2011). Furthermore, thiamine dependent enzymes, —pyruvate dehydrogenase complex and α-ketoglutarate dehydrogenase complex, can be major sources of ROS in mitochondria (Fig. 6.4). The matrix soluble dihydrolipoyl containing dehydrogenases was also found to generate superoxide radicals,

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FIGURE 6.3 Role of B vitamins in the mitochondrial metabolism (Janssen et al., 2019). ACAC, acetylCoA carboxylase; ACAD, acyl-CoA dehydrogenase; ACO, aconitase; a-KG, alphaketoglutarate; BCAA, branched-chain amino acids; BCAT, branched-chain amino acid transaminase; BCKA, branched-chain ketoacids; BCKDH, branched-chain ketoacid dehydrogenase; 1,3-BPG, 1,3-bisphosphoglyceric acid; C, cytochrome C;CBS, cystathionine-beta-synthase; CoA-SH, Coenzyme A; CS, citrate synthase; FAD, flavin adenine dinucleotide; FADH2, hydroquinone form of FAD; FH, fumarate hydratase; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; G3P, glyceraldehydes 3-phosphate; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, glutathione; GSSG, glutathione disulfide; HCY, homocysteine; IDH, isocitrate dehydrogenase; IMM, inner mitochondrial membrane; IRG1, immunoresponsive gene 1; MAT, methionine adenosyltransferase; MDH, malate dehydrogenase; Ms, methionine synthase; 5-MTHF, 5methyltetrahydrofolate; 5,10-MTHF, 5,10-methylenetetrahydrofolate; MTHFR, methylene tetrahydrofolate reductase; MUT, methylmalonyl-CoA mutase; NADC, nicotinamide adenine dinucleotide; NADH, reduced form of NADC; NNT, nicotinamide nucleotide transhydrogenase; OAA, oxaloacetate; OGDH, 2-oxoglutarate dehydrogenase complex; OMM, outer mitochondrial membrane; PCC, propionyl-CoA carboxylase; PDH, pyruvate dehydrogenase complex; Q, coenzyme Q; SAM, S-adenosylmethionine; SCS, succinyl coenzyme A synthetase; SDH, succinate dehydrogenase; SHMT, serine hydroxymethyltransferase; suc-CoA, succinyl-CoA; THF, tetrahydrofolate.

6.3 Oxidative stress and mitochondrial toxicity: role of B vitamins

FIGURE 6.4 The ketoglutarate dehydrogenase complex (KGDHC). There are three subunits in the KGDHC. E1k subunit catalyzes the reaction where thiamine is a critical component. The sulfhydryl groups of the E2 subunit can also act as antioxidants. E•Lip(SH)(SH), E•Lip (SCOR)(SH) and E•Lip(S)2 are intermediates of E2k subunit during catalysis.

production of which is stimulated by low NAD1 availability or by high NADH/ NAD1 ratios (Gibson & Blass, 2007). Additionally, thiamine deprivation reduces activities of oxidant-scavenging enzymes for example, superoxide dismutase, GSH peroxydase and catalases, inducing oxidative stress in mitochondria and increasing lipid peroxidation (Sharma et al., 2013). Dysregulation of these oxidant-scavenging enzymes at both cellular and mitochondrial levels straightway causes the opening of the mitochondrial permeability transition pore, followed by induction of apoptotic makers, namely chromatin margination and condensation, vacuolization of the cytosol and damage of the plasma membrane (Deryabina et al., 2013). Riboflavin (vitamin B2) is a key component of the mitochondrial respiratory chain and thus its deficiency can lead to the disruption of the mitochondrial respiratory chain and accordingly cause mitochondrial dysfunction (Barile et al., 2016). As a consequence, excess ROS was produced dismantling cellular antioxidant balance. Excessive ROS production including superoxide O22, hydrogen peroxide, and hydroxyl radical generation favors oxidative stress resulting into the alteration of Ca21 homeostasis in mitochondria. All these events triggers membrane lipid peroxidation and potentially induces nuclear and mtDNA damage (Van Houten et al., 2006). Riboflavin also supports NADPH-dependent biliverdin reductase B, which is involved in the protection against I/R oxidative injuries (Sanches et al., 2014). Due to its involvement within the GSH redox cycle and its conversion from reduced to oxidized forms, riboflavin exhibits some antioxidant

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properties. Riboflavin functions as coenzyme for redox enzymes in its FAD and FMN forms (Saedisomeolia & Ashoori, 2018). Oxidized GSH is converted to its reduced form by GR, which needs FAD as coenzyme. Then, in the intracellular milieu, reduced GSH scavenges ROS and is converted to its oxidized form (Dringen et al., 2000). FAD is essential to re-convert the oxidized GSH to its reduced form, thereby regaining its antioxidant potential. NAD and NADP, two distinct forms of vitamin B3 (niacin) along with niacincontaining enzymes, scavenge free radicals and protect tissues from oxidative stress-mediated damage. NADPH (a niacin dependent biomolecule) serves as an important coenzyme of the GR, which converts oxidized glutathione (GSSG) to its reduced form (GSH). GSH acts as a cosubstrate for GPx (Ambrosio et al., 1991). NADPH also provides sufficient substrate for GSH regeneration via GR reaction. In this way, niacin deficiency promotes oxidative stress. Nicotinamide being the precursor for NAD and NADH, can also protect against oxidative stress-induced apoptosis. Nicotinamide can also prevent the release of cytochrome C and inhibits apoptosis because they inhibit caspase 1, caspase 3, and caspase 8 during cellular injury (Maiese & Chong, 2003). Nicotinamide supplementation during global ischemia characterized by depletion of NAD 1 and ATP levels causes increase in NAD 1 supply and PARP inhibition (Klaidman et al., 2003). Pantothenic acid also protects mitochondrial constituents from oxidative damage by increasing both CoA and GSH levels and enhancing GPx activity (Wojtczak & Slyshenkov, 2003). Pantothenic acid or pantothenol are reported to exert partial protection against ROS-mediated lipid peroxidation (Wojtczak & Slyshenkov, 2003). However, pantothenic acid or pantothenol are not free radical scavengers but, rather, they exerted a metabolic effect. Being the precursor of CoA, pantothenic acid exerts its beneficial effect in various kinds of cell damage mediated by ROS and it is thought to be related to the increased content or stimulated biosynthesis of this coenzyme. Pantothenol also protects the activities of some enzymes such as catalase, GPx, GR to prevent oxidative stress and also the activity of malic enzyme that is involved in keeping NADP in the reduced state (Wojtczak & Slyshenkov, 2003). Thus, pantothenic acid and its reduced derivative, pantothenol promotes the synthesis of GSH or preventing its degradation and/or efflux from the cell and make use of their potential against oxidative stress because GSH is an essential element controlling apoptosis and other kinds of ROS-induced cytotoxicity (Umansky et al., 2000; Davis et al., 2001). Pyridoxin (vitamin B6), not being classified as classical antioxidant compound, has been shown to have thoroughly effectual antioxidant properties (Stocker et al., 2003) as Vitamin B6 is an effective quencher of a singlet oxygen. Both pyridoxine and PL are reported to be the efficient superoxide (or •OOH) scavengers and to prevent lipid peroxidation (Matxain et al., 2006). Pyridoxine also reported to serve as a highly efficient hydroxyl radical (•OH) quencher with a capacity of scavenging up to eight •OH molecules (Stocker et al., 2003; Matxain et al., 2006). Moreover, vitamin B6-dependent enzymes cystathionine

6.3 Oxidative stress and mitochondrial toxicity: role of B vitamins

13-syntase and cystathionine γ-lyase are associated with conversion of methionine to cysteine, which is pivotal for the synthesis of GSH. Thus vitamin B6 deficiency leads to decreased GSH synthesis (Cabrini et al., 1998). Additionally, pyridoxine deficiency was found to influence long-chain polyunsaturated fatty acids biosynthesis, potentiates lipid peroxidation, and perturbs antioxidant defense (Cabrini et al., 1998; Keles et al., 2010). Paraoxonase is a group of hydrolytic enzymes with a wide range of substrates that have the ability to protect against lipid oxidation. Paraoxonase protein is localized to the inner mitochondrial membrane, and is associated with respiratory complex III (Devarajan et al., 2011). Vitamin B6 supplementation enhanced serum paraoxonase and arylesterase activities which might be related to a possible direct effect of this vitamin on the enzyme and/or related to its ability to reduce oxidative stress (Ta¸s et al., 2014). Vitamin B7 (biotin) attenuates 7b-hydroxycholesterol (a lipid peroxidation product) induced cytotoxicity and reported to increase antioxidant activities in neurodegenerative diseases (Sghaier et al., 2019). In the same neurodegenerative model, biotin also restricted the overproduction of O2•2 and H2O2 and consequently, lipid peroxidation and protein carbonylation. Moreover, biotin also improves the mitochondrial function through the upregulation of the activity of SDH and counteracts the loss of transmembrane mitochondrial potential and reduces the overproduction of mitochondrial O2•2 (Sghaier et al., 2019). Biotin was reported to induce the endogenous antioxidant response of nuclear factor erythroid 2
related factor 2 (Nrf-2) and limit the cellular ROS as well as mitochondrial ROS production (Fourcade et al., 2020). A different mechanism was also reported in favor of the antioxidant role of biotin which is dependent on the biotinylation of heat shock protein, 60 (HSP60) at its lysines, a mitochondrial heatshock protein that ameliorates oxidative stress in cells (Li et al., 2014). Another key vitamin is folic acid, which cooperates with vitamin B12 to promote the regeneration of methionine from homocysteine. Homocysteine is toxic to mitochondrial function (Coppen & Bolander-Gouaille, 2005). Folic acid has immense impact on mitochondrial oxidative stress and serves as a direct antioxidant and scavenger molecule. Folate deficiency was reported to promote oxidative
nitrosative stress resulting in endoplasmic reticulum stress which could trigger cellular GSH depletion (Lai et al., 2017). This is accomplished by two mechanisms. Firstly, there was huge depletion of mitochondrial GSH pool caused by folate deficiency mediated reduced Bcl-2 expression. Secondly, folate deficiency was associated with two pivotal enzymes of cellular GSH redox regulation,—γ-glutamylcysteinyl synthetase heavy chain (a catalytic enzyme for GSH biosynthesis) and mitochondrial isocitrate dehydrogenase 2. The latter enzyme provides nicotinamide adenine dinucleotide phosphate and is responsible for regeneration of GSH from oxidized GSH disulfide via mitochondrial GR (Lai et al., 2017). Besides its ability to interact with nitric oxide synthase, folate also improves quinonoid BH4 availability and causes a reduction in superoxide production (Doshi et al., 2001). Furthermore, in folate-depleted mitochondria, increased oxidative stress was

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involved in the accumulation of mitochondrial DNA (mtDNA) and a large deletion which has been found to be correlated with increased 8-hydroxydeoxyguanosine (8-OHdG) levels (Lezza et al., 1999). This 8-OHdG was further reported to cause strand breakage and recombination between homologous sequences flanking deletion site (Lezza et al., 1999). Mitochondria contain vitamin B12 (cobalamin) and B80% of it is in its cobalamin (II) form. Cobalamin-dependent L-methylmalonyl-CoA mutase is a mitochondrial enzyme (Padovani & Banerjee, 2006). Several studies have reported the effectiveness of physiologically relevant concentration of vitamin B12 against mitochondrial oxidative stress (Moreira et al., 2011; Green et al., 2017; Karamshetty et al., 2016). Vitamin B12 is one of the important cofactor in homocysteine metabolism and homocysteine is believed to mediate ROS accumulation by multiple mechanisms (Green et al., 2017). Homocysteine mediated generation of mitochondrial O2•2 was effectively inhibited by cobalamin (Moreira et al., 2011). Vitamin B12 also maintains GSH levels, and thus antioxidant status, as well as modulates the effects of nitrosylation in cell signal transduction (Wheatley, 2007).

6.4 Role of B vitamins as mitochondrial nutrients Thiamin is very crucial for maintaining function/structure of mitochondria. It acts as cofactor in critical metabolic reactions related to ATP generation, oxidative energy metabolism and oxidative stress (Subramanian et al., 2013). Free thiamin or the phosphorylated form of thiamin plays pivotal role in the mitochondrial energy production. Three mitochondrial enzymes, PDH, alpha ketoglutarate dehydrogenase, and branched-chain ketoacid dehydrogenase play indispensable role in the generation of energy by the mitochondria and they all need thiamin pyrophosphate (TPP) as coenzyme (Lapointe, 2014). Most of the TPP is generated from the thiamin in cytoplasm and then transported into the mitochondria by mitochondrial thiamin pyrophosphate transporter via a carrier-mediated process (Subramanian et al., 2013). Mitochondria serves as host for a wide array of metabolites essential for normal cellular growth and development involving key regulatory pathways of mitochondria, such as metabolism of amino acids, fatty acids, and purines, and the oxidation-reduction reaction (Barja & Herrero, 2000). All these processes depend significantly on flavoenzymes, such as oxidases, reductases, and dehydrogenases. Flavoenzymes are functionally dependent on biologically active FAD or FMN, for which riboflavin (vitamin B2) is the precursor (Udhayabanu et al., 2017). Both FAD and FMN act as redox cofactors in both complexes I and II of the respiratory chain. Thus, riboflavin has a crucial impact on mitochondrial energy production mediated by the electron transport chain (etc.) (Powers, 2003).

6.4 Role of B vitamins as mitochondrial nutrients

Furthermore, flavoenzymes, along with cytochrome P450, are also involved in drug and toxin metabolism. Moreover, GR needs FAD as coenzyme for recycling of oxidized glutathione (GSSG) to its reduced form GSH (Depeint et al., 2006). The amide form of niacin or nicotinic acid (vitamin B3), nicotinamide is a precursor for both nicotinamide adenine dinucleotide (NAD/NADH) and nicotinamide adenine dinucleotide phosphate (NADP/NADPH) (Lapointe, 2014). NADH is involved in electron transfer from metabolite intermediates to the respiratory chain and NAD is implied in the activity of PDH and alpha-ketoglutarate dehydrogenase, two key enzymes of the TCA cycle (Lapointe, 2014). NAD is further implicated in the mitochondrial DNA repair mechanism by serving as substrate for the crucial DNA repair enzyme, poly (ADP-ribose) polymerase (Kirsch & De Groot, 2001). NAD’s natural precursor, NR, also boosts the PGC1α-dependent mitochondrial biogenesis pathway and thereby potentiates the transcription of genes of OXPHOS (Amjad et al., 2021). Additionally, manipulation of mitochondrial NAD levels by NMN leads to metabolic changes that protect mitochondria from ROS and excessive fragmentation, and provides treatments for pathophysiological stress conditions (Klimova et al., 2019). High doses of nicotinic acid are demonstrated to elevate NAD/NADP levels in both mitochondria and cytoplasm (Ames et al., 2002). Vitamin B5 (pantothenic acid) is implicated in fatty acid and carbohydrate oxidation. Pantothenic acid is a precursor to CoA, a molecule that is required for a variety of mitochondrial enzymatic activities. In general, CoA is a carbonylactivating group that serves as an acyl group carrier in the mitochondria, where it is required for the TCA cycle enzyme, PDH, and alpha-ketoglutarate dehydrogenase, as well as the β-fatty acid oxidation pathway (Atamna, 2004). Pantothenic acid also serves to defend mitochondrial constituents from oxidative damage which is accomplished by increasing CoA and GSH as well as potentiating GPx activity (Slyshenkov et al., 2004). By elevating the content of mitochondrial CoA, pantothenic acid or pantothenol, enhanced the production of ATP and thereby increases glutathione to anticipate mitochondrial oxidative stress (Slyshenkov et al., 2004) (Fig. 6.5). Pyridoxal 50 -phosphate (PLP) being the biologically active form of vitamin B6, acting as a cofactor for many essential enzymes, the majority of which are involved in amino acid metabolism and fatty acid metabolism. Besides these roles, PLP also represents an important cofactor for the degradation of storage carbohydrates, such as glycogen (Wu & Lu, 2012). Deficiency of vitamin B6 can cause damage and toxicity to mitochondria, which depends more on PLP for its functions (Nan et al., 2021). PLP-dependent transaminases mediate NADH oxidation in the mitochondria and are involved in the malate-aspartate shuttle. Moreover, vitamin B6 is also indispensible for the decarboxylation reaction in the mitochondrial matrix, which is crucial for the synthesis of heme (a component of hemoglobin). Lack of vitamin B6 leads to a decrease in heme/cytochrome resulting in the increase in ROS formation (Ames et al., 2005). Also vitamin B6 deficiency causes excessive increments in lipid peroxidation, which in turn is

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FIGURE 6.5 Mode of action of mitochondrial nutrients: role of B vitamins (Lapointe, 2014). a-KGDH, alpha-ketoglutarate dehydrogenase; ALCAR, acetyl-L-carnitine; alpha-LA, alpha-lipoic acid; B1
B12, B vitamins; BC-KADH, branched-chain ketoacid dehydrogenase; (c), cytochrome C; C, vitamin C; CoA, coenzyme A; Citrate S., citrate synthase; CoQ, coenzyme Q or ubiquinone; Cu, copper; E, vitamin E; FAD, flavin adenine dinucleotide oxidized or reduced (FADH2); GPxs, glutathione peroxidases; GSH, glutathione; I
V, mitochondrial complexes I
V; Mn, manganese; NAC, N-acetylcysteine; NAD, nicotinamide adenine dinucleotide oxidized or reduced (NADH); PDH, pyruvate dehydrogenase; PUFAs, polyunsaturated fatty acids; ROS, reactive oxygen species; Se, selenium; SODs, superoxide dismutases; TCA cycle, tricarboxylic acid cycle.

responsible for mitochondrial damage (Kannan & Jain, 2004). Vitamin B6 is also involved in the manufacture of glutathione (GSH) from homocysteine and the maintenance of an acceptable GSH/GSSG ratio, and is thus linked to mitochondrial oxidative stress management (Kannan & Jain, 2004). Biotin (vitamin B7) acts as coenzyme in five different biotin-dependent carboxylases (BDCs), among which four BDCs are located in mitochondria. Biotin is known to maintain the biochemical integrity of the TCA cycle and biotin deficiency exerts detrimental effects on the level of TCA cycle intermediates (Atamna et al., 2007). Biotin is also implicated in the maintenance of mitochondrial complex IV and heme metabolism, and thus helps to prevent oxidative damage to DNA (Atamna et al., 2007). Furthermore, biotin is associated with mitochondrial biogenesis, which involves guanylate cyclase activation (Lapointe, 2014).

6.5 Mitochondrial signaling metabolites: impact of B vitamins

Within human cells, folate-mediated one-carbon metabolism is compartmentalized into the cytosol, nucleus, and mitochondria. Folates help purines, thymidine, and methionine biosynthesis by activating and transferring one-carbon units (Morscher et al., 2018). Folate-bound one-carbon units are used by mammalian mitochondria to methylate tRNA, which is essential for mitochondrial translation and consequently OXPHOS (Morscher et al., 2018). The transport and metabolism of folate in cells is highly compartmentalized and mitochondrial folate transport and metabolism is comparatively different from cytosolic folate metabolism. Impaired folate metabolism is reported to be potentially involved in the pathophysiology of mitochondrial disorders (Ormazabal et al., 2015). Further, supplementation of high-dose folates can lower oxidative stress levels, but a lack of folates promotes mitochondrial oxidative damage (Huang et al., 2004; Chang et al., 2007). Vitamin B12 (cobalamin) is essential for the function of methylmalonyl-CoA mutase in mitochondria, which catalyzes the conversion of L-methyl-malonyl-CoA to succinyl-CoA (an important intermediate of the TCA Cycle) and uses adenosylcobalamin as a cofactor (Moras et al., 2007). In human cells, the majority of cellular cobalamin has been found to be bound to methylmalonyl-CoA mutase in the mitochondria.

6.5 Mitochondrial signaling metabolites: impact of B vitamins Different mitochondrial metabolites produced in B vitamin-supported reactions (TCA cycle, ETS, oxidation of fatty acids and amino acids) perform a crucial role in mitochondria-nuclear signaling.

6.5.1 B vitamins and HIF1 signaling B vitamins are known to impacts level of TCA cycle intermediates such as α-KG, succinate, and fumarate. Specifically, α-KG dependent Egl nine (EGLNs, also called prolyl hydroxylase enzymes) when influenced by B vitamins, affects signaling of Hypoxia-Inducible Factor 1 (HIF1) (Strowitzki et al., 2019). Moreover, α-KG accumulation because of the uncontrolled metabolism of mitochondrial NAD 1 , turn down the stability as well as target gene expression of HIF1A (Ho et al., 2017). During the course of aging-induced normoxia, supplementation of NMN (form of vitamin B3) stabilizes HIF1A in muscle and thus anticipates pseudo-hypoxic states (Gomes et al., 2013). However, this effect of NMN was abolished in animal models when EGLNs and SIRT1 (sirtuins) were knocked down and thus highlighted the role of TCA cycle metabolites. Furthermore, nicotinamide and NMN have also been shown to impart protection against ischemiareperfusion (I/R) injury in the brain via attenuating ROS signaling and providing HIF1 stability by modulating TCA cycle intermediates. However, long-term activation of HIF1 has been shown to be deleterious in the I/R state (Shoji et al., 2014).

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FIGURE 6.6 Regulatory role of B vitamins on HIF 1 signaling (Janssen et al., 2019). a-KG, alphaketoglutarate; ARNT, aryl hydrocarbon receptor nuclear translocator; EGLNs, egg-layingdefective nine family or HIF prolyl hydroxylases; HIF1A, hypoxia-inducible factor 1 alpha; HRE, hypoxia response element; IMM, inner mitochondrial membrane; OGDH, 2oxoglutarate dehydrogenase complex; OMM, outer mitochondrial membrane; PDH, pyruvate dehydrogenase complex; pVHL, von Hippel
Lindau protein; SLC25A10, mitochondrial dicarboxylate carrier; SLC25A11, mitochondrial 2-oxoglutarate/malate carrier protein; Ub, ubiquitin E3 ligase.

Increased HIF1 expression in fibroblast of a clinically CII (complex II: SDH) deficient patients was the outcome of accumulated succinate, which competes with EGLNs to destabilize HIF1A. Vitamin B2 brings about necessary FAD for complex II or SDH complex to act in the mitochondrial etc and therefore, in these patients, supplementation of vitamin B2 checked succinate level and stabilized SDH complex to reduce HIF1A expression (Maio et al., 2016). Another B vitamin, thiamin is critical for the activity of PDH and alpha ketoglutarate (α-KG) dehydrogenase and deficiency of thiamin curtailed activity of these enzymes, resulting in a raised level of lactate and pyruvate in plasma. Pyruvate is thought to interact with the catalytic site of EGLN to suppress its activity and thereby stabilize HIF1A. Alternatively, α-KG is associated with HIF1A degradation but its detailed mechanism is still pending. Taken together, B vitamins have a profound role on HIF1 signaling by regulating the interplay between TCA cycle intermediate, ROS, other metabolites, and EGLNs but the critical impact of B vitamins on HIF1 signaling still needs extensive elucidation and exploration (Fig. 6.6).

6.5.2 Impacts of B vitamin on methylation of histone and DNA Methylation of histones is very pivotal in health, disease, and inheritance. Methyl donors are mandatory for both histone and DNA methylation. Several B vitamins including vitamin B2, B6, B11 (folate), and B12 (cobalamin) are required to generate methyl donor, SAM, via one carbon metabolism in both the cytosol and mitochondria. Mitochondria-derived serine is the most potent precursor of

6.5 Mitochondrial signaling metabolites: impact of B vitamins

methionine to form SAM (Ducker & Rabinowitz, 2017). Neural tube defects caused by folate deficiency have also been linked to changes in the methylation landscape during embryonic development of the brain (Chang et al., 2011). Folate deficiency is also associated with hypomethylation of DNA in lymphocytes. All basic residues, such as arginines, lysines, and histidines are the target of histone methylation and largely explored histone methylation sites include histone H3, lysine 4 (H3K4), H3K9, H3K27, H3K36, H3K79, and H4K20 (Greer & Shi, 2012). FAD (a form of vitamin B2) is known to be a cofactor of KDM1 histone lysine demethylases (α-KG independent histone demethylases), which are involved in the demethylation of H3K4 (involved in the transcriptional repression) and H3K9 (involved in the transcriptional activation) (Maes et al., 2015). Inhibition of activity of KDM1 histone lysine demethylases via dampening of FADs and silencing of riboflavin kinase, resulted in escalated histone methylation. This causes loss of repression of energy expenditure related genes leading to raised mitochondrial respiration and initiation of lipolysis (Hino et al., 2012). Furthermore, deficiency of vitamin B2 impacts immune signaling because it heightened methylation of histones on proinflammatory cytokine genes including TNF-α and IL-1 beta (Liu & Zempleni, 2014). Moreover, TCA cycle intermediates regulate ten-eleven translocation (TET) and KDM demethylases to impact the status of DNA and histone methylation (Janssen et al., 2019). Thus, vitamin B1, B2, and B3 maintain TCA cycle function and regulate DNA and histone methylation in the nucleus (Fig. 6.7).

FIGURE 6.7 Regulatory role of B vitamins on methylation of histones and DNA (Janssen et al., 2019). α-KG, alpha-ketoglutarate; HCY, homocysteine; IMM, inner mitochondrial membrane; KDMs, histone lysine demethylase family; KDM1A, histone lysine demethylase family 1A or lysine-specific histone demethylase 1A (LSD1); Met, methyl-group (CH3); OMM, outer mitochondrial membrane; SAM, S-adenosylmethionine; SLC25A10, mitochondrial dicarboxylate carrier; SLC25A11, mitochondrial 2-oxoglutarate/malate carrier protein; TETs, ten-eleven translocation family of DNA demethylases; THF, tetrahydrofolate.

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6.5.3 B vitamin: as regulator of histone acetylation According to growing body of research, protein acetylation appears to have a vital role in key biological functions (Choudhary et al., 2009). The interaction between histone acetyltransferases (HATS) and histone deacetylases are well known to play a role in protein acetylation homeostasis (Siudeja et al., 2011). CoA is essential for the synthesis of acetyl CoA from acetate or citrate and CoA is a promising candidate metabolite with the ability to alter protein acetylation (Siudeja et al., 2011). Thus, inhibition of enzymes of the acetyl-CoA biosynthesis pathway reduces the acetylation of specific proteins. Chemical inhibition or genetic knockdown of pantothenate kinase (as shown in Drosophila or in mammalian cell model) interferes with de novo synthesis of CoA, which is necessary to aid acetylation of histone and tubulin (Siudeja et al., 2011). Vitamin B3 dependent sirutins (SIRTs) (among which sirutin 3, sirutin 4, and sirutin 5 are predominantly located in the mitochondria) are other critical regulators of lysine acetylation (Iwahara et al., 2012). Among the sirutins, SIRT3
5 are predominantly located in mitochondria and SIRT1, 2, 6, and 7 are in the nucleocytoplasmic compartment. In the NAD-dependent pathway, SIRT 1 causes deacetylation of several histones and this is pivotal in the regulation of chromatin and mapping of the epigenetic code (Zhang & Kraus, 2010). Additionally, EP300acetyltransferase, a general HATS, is also regulated by SIRT1. In addition to SIRT1, SIRT6, and SIRT7 are also reported to deacetylate multiple histone targets which are critical regulators of several cellular activities like metabolic homeostasis, biogenesis of ribosomes, DNA damage repair, and cell proliferation (Tasselli et al., 2017; Blank & Grummt, 2017). Further, B vitamins also regulate the oxidation of fatty acids where several acyl-CoAs required for acetylation are produced. Thus, B vitamins concerned with fatty acid oxidation also play critical roles in histone acetylation. Mitochondrial acyl-CoA dehydrogenases, that executes the first step of fatty acid oxidation, are essentially flavoproteins which need FAD as cofactor and therefore are dependent on riboflavin (vitamin B2). Moreover, vitamin B2 deficiency affects several acyl CoA dehydrogenases, resulting in alteration in the mitochondrial CoA pool and fatty acid derivatives (Veitch et al., 1988). Therefore, it is noteworthy to mention that FAD deprivation or vitamin B2 deficiency associated changes on the activity of these metabolic enzymes, not only affect mitochondrial lipid metabolism but also influence signaling pathways modulated by acyl-CoA. Another B vitamin, biotin (vitamin B7/B8), expedites the generation of methylmalonyl CoA from propionyl CoA, utilizing propionyl CoA caboxylase. Therefore, biotin deficiency leads to an elevated level of several mitochondrial CoA intermediates including propionyl CoA (Herna´ndez-Va´zquez et al., 2013; Schwab et al., 2006), and this elevated level of propionyl CoA is known to induce mitochondrial toxicity via impairment of CIII activity within ETS and inhibition of activity of both PDH and OGDH. Further, vitamin B12 is pivotal for methylmalonyl-CoA mutase (MUT) function (Green et al., 2016). Vitamin B12

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FIGURE 6.8 Regulatory role of B vitamins on acetylation of histone (Janssen et al., 2019). Acyl-CoA transport from the mitochondria to the nucleus, here depicted as dashed arrow, follows multiple different routes as described in the text. ACAD, acyl-CoA dehydrogenase; AceCoA, acetyl-CoA; ACLY, ATP-citrate lyase; But-CoA, butyryl-CoA; CACT, carnitine/ acylcarnitine translocase; CoA, Coenzyme A; CPT1, carnitine palmitoyltransferase 1; CPT2, carnitine palmitoyltransferase 2; Crot-CoA, crotonyl-CoA; CS, citrate synthase; ETS, electron transfer system; FAD, flavin adenine dinucleotide; FADH2, hydroquinone form of FAD; IMM, inner mitochondrial membrane; KAT, lysine acetyltransferase; KDAC, lysine deacetylase; MMA-CoA, methylmalonyl-CoA; MUT, methylmalonyl-CoA mutase; NADC, nicotinamide adenine dinucleotide; NADH, reduced form of NADC; NAM, nicotinamide; OAA, oxaloacetate; OGDH, 2-oxoglutarate dehydrogenase complex; OMM, outer mitochondrial membrane; PDH, pyruvate dehydrogenase complex; Prop-CoA, propionylCoA; SLC25A1, mitochondrial tricarboxylate transport protein; Suc-CoA, succinyl-CoAPCC, propionyl-CoA carboxylase; VDAC, voltage-dependent anion channels.

deficiency increases methylmalonyl-CoA level and causes accumulation of methyl-malonic acid. The latter is reported to act as a mitochondrial toxic agent that inhibits SDH and impairs mitochondrial activity (Toyoshima et al., 1995; Narasimhan et al., 1996). Thus vitamin B12 deficiency could affect histone acetylation impacting specific acyl CoA species (Fig. 6.8).

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Analysis of the mitochondrial status of murine neuronal N2a cells treated with resveratrol and synthetic isomeric resveratrol analogs: aza-stilbenes

7

Mohamed Ksila1,2, Imen Ghzaiel1,3,4, Aline Yammine1, Thomas Nury1, Anne Vejux1, Dominique Vervandier-Fasseur5, Norbert Latruffe1, Emmanuelle Prost-Camus6, Smail Meziane7, Olfa Masmoudi-Kouki2, Amira Zarrouk3,8, Taoufik Ghrairi2 and Ge´rard Lizard1 1

Team “Biochemistry of the Peroxisome, Inflammation and Lipid Metabolism”, University Bourgogne Franche-Comte´, Dijon, France 2 Laboratory of Neurophysiology, Department of Biologie, Faculty of Sciences, Cellular Physiopathology and Valorisation of BioMolecules, University Tunis-El Manar, Tunis, Tunisia 3 Lab-NAFS “Nutrition—Functional Food & Vascular Health,” Faculty of Medicine, University of Monastir, Monastir, Tunisia 4 Faculty of Sciences of Tunis, University Tunis-El Manar, Tunis, Tunisia 5 Team OCS, Institute of Molecular Chemistry of University of Burgundy (ICMUB UMR CNRS 6302), University of Bourgogne Franche-Comte´, Dijon, France 6 LARA-Spiral Laboratories, Couternon, France 7 Institut Europe´en des Antioxydants, Neuves-Maisons, France 8 University of Sousse, Faculty of Medicine, Sousse, Tunisia

7.1 Introduction Polyphenols are a family of organic molecules widely present in the plant kingdom. They are characterized by the presence of phenolic groups (Bertelli et al., 2021). Polyphenols are subdivided into two classes of molecules: flavonoids and nonflavonoids (Jimenez-Del-Rio & Velez-Pardo, 2015) (Fig. 7.1). Among the flavonoids, two subclasses are distinguished: anthocyanins and anthoxanthins. The latter includes flavonols (quercetin, myricetin, kaempferol), flavanones (maringenin, hesperetin), flavanols (catechin), flavones (apigenin, luteolin) and isoflavones (genistein, daidzein). The nonflavonoids include phenolic acids (p-coumaric acid, ferulic acid, sinapic acid, and gallic acid), stilbenes, and lignans. Among the Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00023-0 © 2023 Elsevier Inc. All rights reserved.

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POLYPHENOLS Flavonoids

Non-Flavonoids Phenolic acids Stilbenes resveratrol

Lignans secoisolariciresinol

Red wine

Anthocyanins

Hydrobenzoic acids

gallic acid

Hydroxycinnamic acids

coumaric acid

Anthoxantins

cyanidin

Grapes Pistachios Strawberries

Apples Green and black tea

Tomatoes

Flavonols

Flavones

Flavanols

Isoflavones

Flavanones

quercetin

apigenin

catechin

daidzein

naringenin

Honey

Rosemary

Parsley

FIGURE 7.1 Classification of polyphenols.

stilbenes, resveratrol is one of the most studied molecules because of its physicochemical characteristics and its different biological activities, which vary according to the concentration used. Thus, resveratrol (under its trans configuration) is a powerful antioxidant capable of neutralizing reactive oxygen and nitrogen species, but it is also a metal chelator, which contributes to its antioxidant activities (Rodrigo & Bosco, 2006; Rodrigo et al., 2011). Resveratrol also attenuates the cytotoxic activities of cholesterol oxide derivatives such as 7-ketocholesterol which is increased in patients with age related diseases (Dugas et al., 2010; Yammine et al., 2020). In vitro and in vivo, the trans form of resveratrol, which is biologically active, has several biological activities: antiaging activities, protective activities against some age-related diseases (e.g., cardiovascular diseases, age-related macular degeneration), and antitumor activities (Silva et al., 2019; Vervandier-Fasseur & Latruffe, 2019). Furthermore, trans-resveratrol also has neurotrophic properties, defined as antioxidant and pro-differentiating activities on nerve cells (Uddin et al., 2020, 2021), which make it possible to envisage its use in the treatment of certain neurodegenerative diseases where oxidative stress is increased in association with a decrease in functional mature neurons as is the case in Parkinson’s and Alzheimer’s disease (Fukutomi et al., 2021; Namsi et al., 2018). Trans-resveratrol’s differentiating activities have also been described on C2C12 murine myoblasts, suggesting that trans-resveratrol can also be of interest in the prevention and/or treatment of sarcopenia, which is a highly disabling muscle weakness in the elderly due to the loss of autonomy it causes (Kaminski et al., 2012). Despite its attractive physicochemical characteristics and biological activities, trans-resveratrol has the disadvantage of being rapidly metabolized both at the intestinal level under the influence of

7.1 Introduction

the microbiota and at the blood level (Yammine et al., 2021). Consequently, the moderate bioavailability for trans-resveratrol prompted the investigation of innovative and relevant synthetic trans-resveratrol derivatives. In this context, isosteric analogs of trans-resveratrol have been synthesized with the aim of obtaining compounds whose catabolism is less important than that of trans-resveratrol. These are isosteric analogs of trans-resveratrol, aza-stilbenes, and azo-stilbenes in which the C 5 C bond between both aromatic rings was replaced with C 5 N or N 5 N bonds, respectively (Lizard et al., 2020). Similar to trans-resveratrol, aza- and azo-stilbenes may have a therapeutic interest for Alzheimer’s disease. These molecules oppose the aggregation of β-amyloid proteins considered as determining factors in neurodegeneration (Rana et al., 2018) and also inhibit cholinesterases (Biscussi et al., 2020). However, in Alzheimer’s disease, the increase in the activity of these enzymes leads to decreases in acetylcholine associated with cognitive decline and behavioral disorders (Giacobini et al., 2022). As it has been reported with trans-RSV, iron chelating activities have also been described with azo-stilbenes suggesting that these molecules have cytoprotective activities against ferroptosis (Rana et al., 2018), an iron-induced type of cell death considered to play a major role in neuronal loss in Alzheimer’s patients (Jakaria et al., 2021). In addition, like trans-resveratrol, antitumor activities of aza- and azo-stilbenes have been described (Siddiqui et al., 2013) as well as differentiating activities of azo-stilbenes on human HL-60 promyelocytic leukemia cells (Kagechika et al., 1985). On the other hand, studies comparing the effects of aza- and azo-stilbenes vs. those of trans-resveratrol at the mitochondrial level have not yet been carried out. A better understanding of the effects of aza- and azo-stilbenes at the mitochondrial level is an important point since this organelle is involved both in the control of RedOx equilibrium and in cell metabolism, which play major roles in neurodegeneration and cell death induction (Fransen et al., 2020; Lismont et al., 2015). In the present study, aza-stilbenes were synthetized (AZA ST I to VII) and their cytotoxic effects were compared to those of resveratrol, under its trans configuration, using murine neuronal N2a cells. The effects on cell viability were quantified by two complementary assays: the fluorescein diacetate (FDA) assay, which is a cell viability assay based on measuring esterase activity (Namsi et al., 2018), and the crystal violet assay which measures the density of adherent cells (Namsi et al., 2019; Vega-Avila & Pugsley, 2011). In these conditions, the concentrations decreasing cell viability and density (50% Inhibiting Concentration: IC50) were determined for AZA ST I to VII comparatively to resveratrol and the effects of azo-stilbenes at the mitochondrial level were measured by taking into account the mitochondrial transmembrane potential (ΔΨm) per cell after staining with 3,30 -dihexyloxacarbocyanine iodide (DiOC6(3)), and the production of reactive oxygen species, mainly superoxide anion, at the mitochondrial level by staining with MitoSOX. The data obtained on N2a cells cultured for 48 h with and without resveratrol, or aza-stilbenes (in a concentration range from 1.5 to 100 μM) are presented.

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7.2 Material and methods 7.2.1 Synthesis of aza-stilbenes I to VII The procedure used to synthetize the aza-stilbenes is summarized in Scheme 7.1. Following the Kotora’s procedure (Kotora et al., 2016), an equimolar mixture of 4-aminophenol and aromatic aldehyde in distillated water was stirred at room temperature for 4.5 h. After filtration and drying in the air, the aza-stilbenes were recrystallized from EtOH (II and V), from acetone (I and III), from ethyl acetate (IV), or from acetonitrile (VI and VII). The main chemical characteristics of resveratrol (trans-resveratrol) and AZA-ST I to VII are presented in Table 7.1. In addition, while the resveratrol solution (50 mM, EtOH) is colorless, AZA-ST solutions are most often colored (yellow, orange, brown) with the exception of AZA-ST V, which is slightly whitish (Fig. 7.2).

7.2.2 Cell culture and treatments The mouse neuro-2a (N2a) neuroblastoma cell line (Ref: CCL-131, American Type Culture Collection, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle medium (DMEM, Lonza, Amboise, France) containing 10% (v/v) of heat-inactivated fetal bovine serum (FBS) (Pan Biotech, Aidenbach, Germany) (30 min, 56 C) and 1% (v/v) of penicillin (100 U/mL)/streptomycin (100 mg/mL) (Pan Biotech). The cells were maintained at 37 C in a humidified atmosphere (5% CO2, 95% air), trypsinized (0.05% trypsin
0.02% EDTA solution) and passaged twice a week. The cells were seeded at 60,000 cells per well containing 1 mL of DMEM supplemented with 5% (v/v) heat-inactivated FBS, 1% antibiotics (penicillin, streptomycin), and 10% FBS in 24-well plates (FALCON, Becton Dickinson, Franklin Lakes, NJ, USA). The stock solutions of resveratrol (trans-resveratrol) and aza-stilbenes were prepared as follows: resveratrol was prepared at 50 mM in absolute ethanol (EtOH; Carlo Erba Reagents, Val de Reuil, France) and aza-stilbenes (AZA-ST) were prepared at 50 mM in dimethyl sulfoxide (DMSO; Sigma-Aldrich). In order to evaluate the effects of aza-stilbenes on N2a cells comparatively to resveratrol, the growth medium was removed after 24 h of culture and the N2a cells were incubated either with resveratrol, or aza-stilbenes used at various concentrations ranging from 1.5 to 100 μM for 48 h.

SCHEME 7.1 General procedure of synthesis of aza-stilbenes (AZA-ST) I to VII (Kotora et al., 2016).

7.2 Material and methods

Table 7.1 Main chemical characteristics of resveratrol and aza-stilbenes (AZA-ST). Molecular formula

Molecular weight (g/mol)

Solubility (50 mM, RT)

C14H12O3

228.24

DMSO

C13H11NO2

213.228

DMSO, absolute ethanol

C13H11NO2

213.228

DMSO

C13H11NO2

213.228

DMSO

C13H11NO3

229.228

DMSO, absolute ethanol

C14H13NO2

227.254

DMSO

C13H10NOBr

276.11

DMSO

C13H10NOBr

276.11

DMSO, absolute ethanol

Resveratrol

Aza-Stilbenes

RT, room temperature.

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FIGURE 7.2 Aspect of resveratrol (trans RSV: 50 mM) and aza-stilbenes (AZA-ST: 50 mM) diluted in absolute ethanol (EtOH) and DMSO, respectively.

7.2.3 Measurement of cell viability with the fluorescein diacetate assay Cell viability was measured with the FDA (Sigma-Aldrich) assay which takes into account esterase activity (Namsi et al., 2018, 2019). The N2a cells previously cultured for 24 h into 24-well plates in DMEM containing 10% FBS, were further incubated for 48 h with and without resveratrol, or aza-stilbenes used at different concentrations (1.5
100 μM). At the end of treatment, cells were incubated in the dark with 15 μg/mL FDA for 5 minutes at 37 C, rinsed twice with phosphate buffered saline (PBS), then lysed with 10 mM Tris
HCl solution containing 1% sodium dodecyl containing 1% sodium dodecyl sulfate (SDS) for 10 minutes. Using a TECAN fluorescence microplate reader (Sunrise spectrophotometer, TECAN, Lyon, France), the fluorescence intensity was measured with an excitation at 485 nm and an emission at 528 nm. All assays were performed in three independent experiments and performed in triplicate. Data were expressed as a percentage of untreated cells (control).

7.2.4 Evaluation of adherent cells with crystal violet staining assay The crystal violet viability test is used to mark living cells that have remained adherent (Vega-Avila & Pugsley, 2011). Cells were seeded in 96-well plates at an initial seeding density of 30,000 cellscm22, and incubated for 24 h at 37 C, 5%

7.2 Material and methods

CO2. After exposure to resveratrol (1.5
100 μM) and AZA-ST (1.5
100 μM) for 48 h as described previously, the supernatants containing the dead cells are eliminated and the cell layer is rinsed once with PBS. A solution of crystal violet (Sigma Aldrich) is then added on adherent cells for 5 minutes. This dye contains ethanol which will fix the cells and mark them at the level of the nuclei. Three washes are carried out with distilled water to eliminate excess crystal violet. The cells are then dried and incubated in a 0.1 M sodium citrate solution in 50% ethanol. The crystal violet present in the cells is thus dissolved and the absorbance was read at 570 nm (Sassi et al., 2019). Data obtained with crystal violet staining (optical density per well) were expressed as % control (untreated cells).

7.2.5 Flow cytometric quantification of cells with depolarized mitochondria with DiOC6(3) The variation of the mitochondrial transmembrane potential (ΔΨm) was measured using 3,30 -dihexyloxacarbocyanine iodide (DiOC6(3)) (Invitrogen/Thermo Fisher Scientific, Montigny le Bretonneux, France). This fluorochrome accumulates in the mitochondria proportionally to the ΔΨm value (Ragot et al., 2013); the higher the ΔΨm, the more the probe accumulates. After 48 h of treatment, adherent cells collected by trypsinization were pooled with nonadherent cells, and stained with a solution of DiOC6(3) at 40 nM (15 min; 37 C). The cells were immediately analyzed on a BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). The loss of ΔΨm is indicated by a decrease in the intensity of the green fluorescence collected through a band pass filter of 520 6 10 nm. For each sample, 10,000 cells were acquired and the data were analyzed with FlowJo (Tree Star Inc., Carrboro, NC, USA) software. All assays were performed in triplicate.

7.2.6 Flow cytometric measurement of mitochondrial reactive oxygen species production with MitoSOX-Red The MitoSOX-Red is a selective fluorochrome used for the detection of superoxide anion (O2•2) at the mitochondrial level (Zarrouk et al., 2012). In the mitochondria, this fluorochrome is oxidized by O2•2, and exhibits an orange/red fluorescence (λEx 5 510 nm; λEm 5 580 nm). Mitochondrial production of O2• 2 was quantified by flow cytometry after staining with MitoSOX-Red (Thermo Fisher Scientific), initially prepared at 5 mM in PBS (Ghzaiel et al., 2021). Briefly, after 48 h of treatment, adherent and nonadherent N2a cells were pooled, stained with a 5 μM MitoSOX-Red solution and incubated for 15 min at 37 C. At the end of the incubation time, the cells were immediately analyzed by flow cytometry. The fluorescent signals were detected through a 580 6 20 nm band pass filter using a BD Accuri C6 flow cytometer (BD Biosciences). For each sample, 10,000 cells were acquired. Data were analyzed with FlowJo software (Tree Star Inc.). All assays were performed in triplicate.

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CHAPTER 7 Analysis of the mitochondrial status of murine

7.2.7 Statistical analysis The experimental results were statistically analyzed with GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA). Data were expressed as the mean 6 standard deviation (SD) and compared with a Student’s t-test. A p-value less than 0.05 was considered statistically significant.

7.3 Results The impact of resveratrol and AZA-ST I to VII on cell viability and cell growth were evaluated by two complementary assays: the FDA assay, which is frequently used to evaluate esterase activity correlated with cell viability (Namsi et al., 2018) and the crystal violet assay, which permits to quantify adherent cells (Namsi et al., 2018). The quantity of adherent cells permits to evaluate cell growth and also cell death since cytotoxic agents induce a loss of cell adhesion. Noteworthy, under treatment with resveratrol and AZA-ST, the decrease of cell viability was associated with a more or less pronounced decrease of FDA positive cells as shown in Fig. 7.3. Based on the IC50 values determined with the FDA assay, as well as taking into account the effects of concentrations above IC50 on cell viability, the cytotoxicity of the different molecules studied was in the following range of order: AZA-ST IV (IC50 B 6.25 μM) . resveratrol (6.25 , IC50 , 12.5 μM) . AZA-ST VI (IC50 B 12.5 μM) . AZA-ST II (IC50 B 12.5 μM) . AZA-ST VII (12.5 , IC50 , 25 μM) . AZA-ST V (12.5 , IC50 , 25 μM) . AZA-ST III (12.5 , IC50 , 25 μM) . AZA-ST I (12.5 , IC50 , 50 μM). Noteworthy, with the crystal violet assay (Fig. 7.4), the IC50 values were in the same range of order than with the FDA assay. This demonstrates that the loss of cell viability under treatment with resveratrol and AZA-ST is associated with a decreased number of adherent cells. As it is well established on different cell types that resveratrol triggers mitochondrial dysfunctions in a concentration dependent manner, the effects of AZA-ST have been studied at the mitochondrial level and compared with those of resveratrol. With the use of DiOC6(3), which allows for quantifying the ΔΨm, a more or less pronounced loss of ΔΨm is observed with the different AZA-ST since 12.5 μM (Fig. 7.5). This loss of ΔΨm is associated with an overproduction of superoxide anions at the mitochondrial level measured with MitoSOX (Fig. 7.6). Altogether, the data obtained show various cytotoxic effects with the different AZA-ST studied. All the AZA-ST studied induce cytotoxic effects in a concentration-dependent manner, characterized by a decrease of esterase activity and a reduced number of adherent cells which are associated with mitochondrial dysfunctions: loss of ΔΨm and increased production of superoxide anions at the mitochondrial level.

AZA-ST I (µM)

*

100

*

80 60

*

* *

40

*

20

Viable cells (%control)-FDA

120

*

100

*

80

*

* 20

*

*

* *

60

*

40 20

*

Viable cells (%control)-FDA

*

80

50

10 0

25

12 .5

6. 25

AZA-ST III (µM)

120 100

3. 12 5

rl Ct

10 0

25

50

12 .5

6. 25

3. 12 5

rl

1. 5

Ct

0

120 100 80

*

60

*

40

* *

20

*

100 80

*

60

* 40

*

10 0

25

50

12 .5

6. 25

AZA-ST V (µM)

120

*

*

20

Viable cells (%control)-FDA

AZA-ST IV (µM)

3. 12 5

Ct rl

10 0

50

25

12 .5

6. 25

3. 12 5

rl Ct

1. 5

0 1. 5

*

100 80 60

*

*

40

*

20

*

* *

60 40

*

20

*

Viable cells (%control)-FDA

*

80

10 0

25

50

12 .5

6. 25

AZA-ST VII (µM)

AZA-ST VI (µM) 120 100

3. 12 5

1. 5

rl Ct

10 0

25

50

12 .5

6. 25

3. 12 5

1. 5

0 Ct rl

0

120

120

*

100 80

*

60

*

40

*

20

* 10 0

50

25

12 .5

6. 25

1. 5

10 0

25

50

12 .5

6. 25

3. 12 5

rl

1. 5

Ct

Ct rl

0

0

3. 12 5

Viable cells (%control)-FDA

*

40

AZA-ST II (µM)

Viable cells (%control)-FDA

*

0

0

Viable cells (%control)-FDA

*

60

1. 5

Viable cells (%control)-FDA

Resveratrol (µM) 120

FIGURE 7.3 Evaluation with the fluorescein diacetate (FDA) assay of the effects of resveratrol and AZA-ST I to VII on cell viability. N2a cells were incubated for 48 h with or without resveratrol or AZAST I to VII in a range of concentrations from 1.5 to 100 μM. The dotted red line makes it possible to evaluate the value of the concentration (or the range of concentrations) reducing cell viability by 50% (IC50). Data are the mean 6 SD of two independent experiments performed in triplicate. Significance of the differences between control (untreated cells), resveratrol or AZA-ST I to VII—treated cells; Student’s t-test:  P ,.05 or less.

100

*

80

*

60

*

40

*

20

50

10 0

25

12 .5

6. 25

3. 12 5

Ct

r

0

50

10 0

25

12 .5

6. 25

1. 5 3. 12 5

Ct rl

50

10 0

25

12 .5

6. 25

3. 12 5

* *

20 0 50

50

AZA-ST VI (µM) 120

*

40

10 0

25

12 .5

6. 25

3. 12 5

Ct

rl

0

*

60

10 0

20

80

AZA-ST VII (µM) 120 100

*

*

80

*

60

*

40

*

*

20 0 50

*

100

10 0

*

AZA-ST V (µM) 120

25

40

*

0

25

60

*

*

20

6. 25

*

*

40

12 .5

*

*

12 .5

*

*

6. 25

*

80

*

60

Ct

50

AZA-ST IV (µM) 120 100

* 80

10 0

25

12 .5

6. 25

3. 12 5

rl Ct

1. 5

0

100

3. 12 5

20

AZA-ST III (µM) 120

rl

40

*

0

3. 12 5

*

*

*

rl

*

*

20

1. 5

*

60

*

40

1. 5

*

80

*

60

Ct

50

AZA-ST II (µM) 120 100

* 80

10 0

25

12 .5

6. 25

3. 12 5

rl Ct

1. 5

0

1. 5

Adherent cells (%control)-Crystal Violet Adherent cells (%control)-Crystal Violet Adherent cells (%control)-Crystal Violet

*

Adherent cells (%control)-Crystal Violet

*

20

100

1. 5

*

40

Adherent cells (%control)-Crystal Violet

60

Adherent cells (%control)-Crystal Violet

80

AZA-ST I (µM) 120

Ct rl

*

Adherent cells (%control)-Crystal Violet

100

1. 5

Adherent cells (%control)-Crystal Violet

Resveratrol (µM) 120

FIGURE 7.4 Evaluation with the crystal violet assay of the effects of resveratrol and AZA-ST I to VII on cell density. N2a cells were incubated for 48 h with or without resveratrol or AZA-ST I to VII in a range of concentrations from 1.5 to 100 μM. The dotted red line makes it possible to evaluate the value of the concentration (or the range of concentrations) reducing the number of adherent cells by 50% (IC50). Data are the mean 6 SD of two independent experiments performed in triplicate. Significance of the differences between control (untreated cells), resveratrol or AZA-ST I to VII—treated cells; Student’s t-test:  P ,.05 or less.

Resveratrol (µM)

*

50

*

40

* 30

*

20 10

*

*

*

60

*

AZA-ST I (µM)

50

*

40

*

30

*

20 10

10 0

M SO Ct r Et (0. l 1 O H %) (0 .1 % ) 1. 5 3. 12 5 6. 25 12 .5

D

D

50 10 0

25

M SO Ct r Et (0. l 1 O H %) (0 .1 % 1. ) 5 µM 3. 12 5 6. 25 12 .5

25

0

0

50

% DiOC6(3) negative cells

60

% DiOC6(3) negative cells

70

70

*

40

* *

*

*

*

*

10 0

50 40 30

*

*

20

*

*

*

30 20

*

*

10

*

*

*

*

60

AZA-ST V (µM)

10 0

*

50 40 30 20

*

* *

10

*

*

*

40

*

30

*

*

20

*

% DiOC6(3) negative cells

AZA-ST VI (µM)

50

10

10 0

25

M SO Ct r Et (0. l 1 O H %) (0 .1 % ) 1. 5 3. 12 5 6. 25 12 .5

D

10 0

50

25

M SO Ct r Et (0. l 1 O H %) (0 .1 % ) 1. 5 3. 12 5 6. 25 12 .5

D

70

70 60

50

0

0

60

AZA-ST VII (µM)

*

50 40

*

30 20 10

* *

*

*

*

10 0

50

10 0

50

25

M SO Ct r Et (0. l 1 O H %) (0 .1 % ) 1. 5 3. 12 5 6. 25 12 .5

D

25

0

0

M SO Ct r Et (0. l 1 O H %) (0 .1 % ) 1. 5 3. 12 5 6. 25 12 .5

% DiOC6(3) negative cells

40

70 % DiOC6(3) negative cells

% DiOC6(3) negative cells

10 0

50

25

D M SO Ct r Et (0. l 1 O H %) (0 .1 % ) 1. 5 3. 12 5 6. 25 12 .5

* AZA-ST IV (µM)

10

*

0

70 60

AZA-ST III (µM)

50

50

20

60

25

% DiOC6(3) negative cells

50

30

70

D M SO Ct r Et (0. l 1 O H %) (0 .1 % ) 1. 5 3. 12 5 6. 25 12 .5

AZA-ST II (µM) 60

D

% DiOC6(3) negative cells

70

FIGURE 7.5 Flow cytometric evaluation of transmembrane mitochondrial potential (ΔΨm) with DiOC6(3) under treatment with resveratrol and AZA-ST I to VII. N2a cells were incubated for 48 h with or without resveratrol or AZA-ST I to VII in a range of concentrations from 1.5 to 100 μM. Data are the mean 6 SD of two independent experiments performed in triplicate. Significance of the differences between control (untreated cells), resveratrol or AZA-ST I to VII—treated cells; Student’s t-test:  P ,.05 or less.

%MitoSOX positive cells

80 60

* 40

*

*

20

100

*

Resveratrol (µM)

*

0

%MitoSOX positive cells

40

* *

*

*

*

*

25

50 10 0

*

AZA-ST III (µM)

80

*

60 40 20

* 60

*

40

* 20

* *

*

*

*

50 10 0

25

M SO Ct r Et (0. l 1 O H %) (0 .1 % ) 1. 5 3. 12 5 6. 25 12 .5

100

AZA-ST V (µM)

*

80 60 40

* 20

*

*

*

*

*

80 60

*

40

* *

25

60

*

40 20

*

*

*

10 0

50

25

10 0

0

0

D M SO Ct r Et (0. l 1 O H %) (0 .1 % ) 1. 5 3. 12 5 6. 25 12 .5

80

25

*

AZA-ST VII (µM)

50

*

*

100

%MitoSOX positive cells

AZA-ST VI (µM)

50 10 0

D M SO Ct r Et (0. l 1 O H %) (0 .1 % ) 1. 5 3. 12 5 6. 25 12 .5

50 10 0

D

25

0

M SO Ct r Et (0. l 1 O H %) (0 .1 % ) 1. 5 3. 12 5 6. 25 12 .5

0

%MitoSOX positive cells

*

D

25

50 10 0

*

%MitoSOX positive cells

%MitoSOX positive cells

AZA-ST IV (µM)

80

20

20

0

D M SO Ct r Et (0. l 1 O H %) (0 .1 % ) 1. 5 3. 12 5 6. 25 12 .5

0

100

*

*

D M SO Ct r Et (0. l 1 O H %) (0 .1 % ) 1. 5 3. 12 5 6. 25 12 .5

50 10 0

25

*

60

100

*

40

100

*

AZA-ST II (µM)

80

20

60

0

D M SO Ct r Et (0. l 1 O H %) (0 .1 % ) 1. 5 3. 12 5 6. 25 12 .5

%MitoSOX positive cells

100

*

AZA-ST I (µM) 80

D M SO Ct r Et (0. l 1 O H %) (0 .1 % ) 1. 5 3. 12 5 6. 25 12 .5

%MitoSOX positive cells

100

FIGURE 7.6 Flow cytometric evaluation of superoxide anions production at the mitochondrial level with MitoSOX under treatment with resveratrol and AZA-ST I to VII. N2a cells were incubated for 48 h with or without resveratrol or AZA-ST I to VII in a range of concentrations from 1.5 to 100 μM. Data are the mean 6 SD of two independent experiments performed in triplicate. Significance of the differences between control (untreated cells), resveratrol or AZA-ST I to VII—treated cells; Student’s t-test:  P ,.05 or less.

7.4 Discussion and conclusion

7.4 Discussion and conclusion Resveratrol, under its trans configuration, is one of the most studied polyphenols both in vitro and in vivo (Zhang et al., 2021). It is a major compound of the Mediterranean diet with antiaging (Li et al., 2021), antioxidant (Silva et al., 2019), antiinflammatory (Alesci et al., 2022) and antitumoral (Yang et al., 2022) activities. In vivo, the major problem of resveratrol is its rapid catabolism that attenuates its biological activity (Yammine et al., 2021). Resveratrol derivatives, aza-stilbenes (Lizard et al., 2020), have therefore been synthesized with the aim of having a biological activity close to or more efficient than resveratrol with a lower catabolism. In terms of toxicity, taking into account cell viability evaluated with esterase activity (FDA assay) and the quantity of adherent cells (crystal violet assay), the results obtained with aza-stilbenes (AZA-ST I to VII) show variable toxicity, either lower or stronger than resveratrol. Thus, with the different AZA-ST which have been synthetized, the IC50 values are in the range of 6.25
25 μM. Thus, as observed with resveratrol, AZA-ST—induced cell death is associated with a decrease of esterase activity. This decrease, which corresponds to a loss of cell viability, is associated with a lower number of adherent cells. Truly, as previously reported with RSV (Namsi et al., 2018), cell death induction, not only reduces cell growth but also favors cell detachment associated with an increase number of nonadherent cells floating in the culture medium. In addition, as observed with RSV, the cytotoxic effects of AZA-ST are associated with marked mitochondrial dysfunctions characterized by a loss of ΔΨm occurring since 12.5
25 μM and associated with an overproduction of superoxide anions. Therefore, from 12.5 μM and higher, it is suggested that AZA-ST could have antitumoral activities, as reported with resveratrol (Vervandier-Fasseur & Latruffe, 2019; Yammine et al., 2021). At lower concentrations, in the range of 1.5
6.25 μM, it will be interesting to evaluate the cytoprotective and neurotrophic activities of AZA-ST. Indeed, at low concentrations, some polyphenols (resveratrol, quercetin, apigenin) are known to induce cell differentiation on different cell types (N2a cells which are murine neuronal cells) (Namsi et al., 2018); C2C12 cells which are murine skeletal muscle cells (Kaminski et al., 2012)) and also to prevent 7-ketocholesterol-induced cell death by oxiapoptophagy involving OXIdative stress, APOPTOsis and autoPHAGY on N2a cells (Nury et al., 2021; Yammine et al., 2020). Altogether, the data obtained support the hypothesis that AZA-ST could act on the cell signaling involved in the control of the RedOx status, mitochondrial activity, and cell death (apoptosis and autophagy). In addition, as immunostimulant activities of polyphenols have been reported on the innate and adaptive immune response in cancer (Ghiringhelli et al., 2012), it will be important to study the effects of AZA-ST in the context of cancer immunotherapy. In conclusion, on N2a cells, although the cytotoxic activities of the AZA-ST studied are more or less pronounced depending on the molecule considered, all

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CHAPTER 7 Analysis of the mitochondrial status of murine

the AZA-ST analyzed induce (as observed with resveratrol) a decrease in esterase activity associated with a decrease in the number of adherent cells and induce significant dysfunctions at the mitochondrial level: loss of ΔΨm and overproduction of superoxide anions. These biological activities, which evocate those of resveratrol, encourage us to specify the activities of AZA-ST in vivo and to simultaneously evaluate their catabolism.

Acknowledgments The authors thank the University de Bourgogne and the University of Tunis El Manar for their administrative assistance (collaboration France—Tunisia) and their financial support. This work was also supported by the Regional Council of Bourgogne Franche-Comte´ (MEDICTA Research Project; Dominique Vervandier-Fasseur, Anne Vejux, and Ge´rard Lizard) and is also a part of a Hubert Curien project (PHC Utique 2021
2022; Code CMCU: 22G0809/Code Campus France: 47608VJ; title: potentiels neurotrophiques d’huiles essentielles, de stilbe`nes et oxyste´rols sur des mode`les cellulaires de neurode´ge´nerescence; University of Bourgogne and University of Tunis El Manar; Taoufik Ghrairi/Olfa Masmoudi-Kouki and Ge´rard Lizard).

Conflict of interest The authors declare no conflict of interest.

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Dietary eicosapentaenoic acid and docosahexaenoic acid for mitochondrial biogenesis and dynamics

8

Sebastian Jannas-Vela1 and Mauricio Castro-Sepulveda2 1

Instituto de Ciencias de la Salud, Universidad de O’Higgins, Rancagua, Chile Laboratorio Ciencias del Ejercicio, Escuela de Kinesiologı´a, Universidad Finis Terrae, Santiago, Chile

2

8.1 Introduction Fatty acids (FAs) are lipids that have diverse functions of major physiological importance as they are important sources of energy, major components of cellular membranes, precursors for the synthesis of bioactive lipids, and regulators of diverse molecular signaling pathways (Calder, 2015; Mozaffarian & Wu, 2011). They have a generic structure characterized by hydrocarbon chains containing a carboxylic acid at one end and a methyl group at the other end. The most abundant FAs in human diet and thus in cellular membranes are saturated fatty acids (SFAs) which contain no double bonds. Conversely, unsaturated FAs have one or more double bonds in their hydrocarbon chain; those with one double bond are classified as monounsaturated FAs, and those with two or more are classified as polyunsaturated fatty acids (PUFAs). Omega-6 (n-6) and omega-3 (n-3) are the two main families of PUFAs. The shortest members of each family are the essential FAs linoleic acid (LA—n-6) and α-linolenic acid (ALA—n-3) which are 18 hydrocarbon acyl chains in length. Dietary sources of LA include margarine, eggs, poultry and sunflower, while ALA is high in canola, flaxseed, walnuts and leafy green vegetables. By a set of reactions catalyzed by the same enzymes in the liver, LA and ALA can be further metabolized by desaturation (insertion of additional double bonds) and elongation (insertion of two carbons) to arachidonic acid (AA—n-6), and to eicosapentaenoic acid (EPA—n-3) and docosahexaenoic acid (DHA—n-3). As both n-6 and n-3 PUFAs compete for the same set of enzymes, and the present-day diet is deficient in n-3 PUFAs with a ratio of n-6 to n-3 of 15/1 (Simopoulos, 2002), this ultimately results in a higher relative conversion of LA to AA, at the expense of EPA and DHA production from ALA (Calder, 2011). As a result, ALA, EPA and DHA should be consumed regularly. Dietary sources of EPA and DHA mostly Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00028-X © 2023 Elsevier Inc. All rights reserved.

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occur in seafood and algae, including fatty fish such as salmon, herring, mackerel, trout, tuna, oysters, and scallops, among others. Interestingly, many of the biological effects of n-3 and n-6 PUFAs coincide, including their incorporation into cell membranes (Gerling et al., 2019; Herbst et al., 2014), the expression of genes related to FA metabolism (Muhlhausler et al., 2010), and the production of bioactive lipids, including the oxylipins -PUFA oxidation products formed via oxygen-dependent reactions - (Zulyniak et al., 2013; Gabbs et al. 2015), specialized proresolving mediators (Gabbs et al., 2015) and endocannabinoids (McDougle et al., 2017). While n-6 PUFAs and their derived metabolites have been associated with proinflammatory effects (Egan & Kuehl, 1980), the n-3 PUFAs and their resulting metabolites are known for their proresolving and lower inflammatory action (Kuda, 2017). Consequently, the overconsumption of n-6 PUFAs in parallel with a lower intake of n-3 PUFAs, a common characteristic of western diets, promotes a proinflammatory environment linked to poor metabolic health, affecting key biological processes such as inflammation, glucose and fatty acid transport, muscle protein synthesis, and mitochondrial function. Skeletal muscle is a key metabolic tissue that has been purported to be affected by n-3 PUFAs. There is growing evidence showing that consumption and incorporation of EPA and DHA into skeletal muscle membranes may positively affect muscle metabolism and function (Gerling et al., 2019; Smith et al., 2011). Although the mechanisms remain unclear, recent data has shown that these benefits may in part be mediated by the capacity of n-3 PUFAs to be incorporated into tissue membranes including mitochondrial membranes, thereby potentially altering mitochondrial biosynthesis and dynamics. The following sections will address current evidence from in vitro, animal, and human studies regarding the effects of n-3 PUFAs on skeletal muscle mitochondrial biogenesis and dynamics.

8.2 Mitochondrial biogenesis and dynamics Mitochondria are double-membraned (inner and outer), highly dynamic organelles involved in a large variety of functions including but not limited to: ATP synthesis, reactive oxygen species production, and calcium handling. As such, they are highly susceptible to different stressors affecting their number, activity, and shape. In the following sections, we will give an introduction on mitochondrial biogenesis and dynamics and later discuss how these processes are affected by dietary EPA and DHA.

8.2.1 Mitochondrial biogenesis The pioneering work of Holloszy (1967) showed that in response to disruptions in cellular homeostasis (i.e., habitual contractile activity), rodent skeletal muscle

8.2 Mitochondrial biogenesis and dynamics

displays a remarkable plasticity and capacity to alter both the type and amount of protein. This study paved the way for subsequent work showing that after several weeks of exercise training, the increases in rodent muscle mitochondrial enzyme activity are related to a greater mitochondrial number and size, and to more densely packed cristae (Arribat et al., 2019; Menshikova et al., 2006; Nielsen et al., 2017). A crucial finding was that these adaptations were not accompanied by increases in the cytosolic enzymes, creatine kinase, and adenylate kinase, providing evidence that improved ATP synthesis via oxygen-dependent pathways is a defining feature of the exercise-trained muscle. Further studies in humans, revealed that the higher maximal mitochondrial enzyme activities and greater mitochondrial volume based on molecular biology techniques from vastus lateralis samples of trained subjects were positively associated with training-induced improvements in aerobic capacity or whole-body maximal oxygen uptake (VO2max). The results of these seminal studies provided evidence that whole-body aerobic capacity could be limited not only by “central” cardiorespiratory factors (i.e., oxygen delivery/transport system) but also by “local” factors, including skeletal muscle mitochondrial content. The increase in mitochondrial content is known as mitochondrial biogenesis (Perry & Hawley, 2018). It is now recognized that mitochondrial biogenesis in part is accomplished through the recruitment of newly synthesized proteins. Mitochondrial biogenesis involves the transcription of proteins encoded by both nuclear and mitochondrial genes. The cotranscription factor peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) is considered one of the major regulators of mitochondrial biogenesis (Olesen et al., 2010). This coactivator enhances the transcription of other transcription factors including the nuclear respiratory factors, which activate mitochondrial transcription factor A (TFAM), promoting the transcription and replication of mitochondrial DNA (mtDNA) (Safdar et al., 2018). A recent explosion of new information indicates that the PGC-1 family of coactivators serves key functions in the dynamic transcriptional control of mitochondrial biogenesis and energy metabolic pathways in a variety of mammalian tissues. The importance of PGC-1α has provided exciting new avenues for understanding the fundamental connections between alterations in the external environment and adaptive metabolic responses of striated muscle. For instance, decreased mitochondrial content in skeletal muscle has been associated with various pathologies and conditions, such as muscular dystrophy (e.g., Duchenne dystrophy), disuse atrophy, sarcopenia, and insulin resistance (Gan et al., 2018; Ryan & Hoogenraad, 2007). In this context, it is important to develop strategies that enhance mitochondrial biogenesis.

8.2.2 Mitochondrial dynamics Mitochondria are organelles that are constantly remodeling through two highly conserved processes, namely fission and fusion. Together, these processes are known as mitochondrial dynamics. In the 1990s, time-lapse analysis using

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mitochondrial-targeted fluorescent proteins and dyes allowed for a detailed and dynamic visualization of changes in mitochondrial shape and structure, which were compatible with fusion events in living cells (from yeast to hepatocytes) (Bereiter-Hahn & Vo¨th, 1994; Cortese, 1998). Moreover, electron tomography analysis unraveled additional mitochondrial inner membrane structures, such as cristae junctions, and their dynamic regulation, and the first genes that participate in the fusion of mitochondria were also identified. These findings, among others, led to the discovery of a group of genes that control and execute mitochondrial fusion and fission. It is now accepted that the protein mitofusins 1/2 (Mfn1 and Mfn2), located in the outer mitochondrial membrane, and optic atrophy protein 1 (Opa1), located in the inner mitochondrial membrane, regulate mitochondrial fusion (elongated mitochondrial phenotype), while dynamin-related protein 1 (Drp1) and mitochondrial fission 1 (FIS1) protein, located in the cytosol and outer mitochondrial membrane respectively, regulate mitochondrial fission (Fig. 8.1) (Mishra & Chan, 2016). Fusion-mediated mitochondrial-component-sharing supports multiple elements of mitochondrial biology including mtDNA integrity, mitochondrial respiration, mitochondrial membrane potential equilibration, apoptosis, and signaling events such as [Ca21] oscillations. Not surprisingly, these proteins also have several nonusogenic functions. For instance, Mfn2 modulates mitochondria-endoplasmic/

FIGURE 8.1 Mitochondrial fusion and fission dynamic in skeletal muscle.

8.2 Mitochondrial biogenesis and dynamics

sarcoplasmic reticulum (ER/SR) tethering to regulate mitochondrial activity through the flux of calcium from the ER to the mitochondria. Meanwhile, mitochondrial fission, among other adaptor proteins, depends on the recruitment of Drp1, a cytosolic GTPase, located in the outer mitochondrial membrane, and the mitochondrial protein FIS1. Fission can facilitate motility and is required for the segregation of damaged mitochondria for mitophagy, mtDNA replication, and mitochondrial redistribution during cell division (circular mitochondrial phenotype) (Eisner et al., 2018). Thus, mitochondrial fusion/fission dynamics is central to organelle quality control and the regulation of a variety of cellular functions. As such, whole-body knockouts for each protein (fusion/fission proteins, e.g., Opa1) interfere with early development and are embryonically lethal (Eisner et al., 2018), revealing the importance of these proteins. Furthermore, dysregulation of the mitochondrial fusion/fission process has been associated with several pathologies such as type 2 diabetes and myopathies like Duchenne muscle dystrophy (Bi et al., 2016; Fealy et al., 2018; Rovira-Llopis et al., 2017). Mitochondrial dynamics are also often associated with the energetic states of the cells. For example, nonfermentable culture conditions that force increased oxidative phosphorylation (OXPHOS) activity in yeast are accompanied by the elongation of the mitochondrial network (increased fusion) (Mishra & Chan, 2016). An analogous study done in fibroblast human cells incubated in galactose media, found that mitochondria are further elongated and rely more heavily on OXPHOS for ATP production (Mishra et al., 2014). In other conditions that promote mitochondrial fusion, mitochondria have also been observed to be associated with increased aerobic ATP production (Castro-Sepu´lveda et al., 2021). Conversely, mitochondrial fission has been associated with decreased mitochondrial respiration, lower mitochondrial membrane potential, lower OXPHOS mitochondrial ATP production, and thus increased production of ATP via glycolysis (CastroSepu´lveda et al., 2021). These observations suggest that high OXPHOS activity correlates with mitochondrial fusion and is consistent with the proposal that elongated mitochondrial networks are more efficient at energy generation and capable of distributing energy through long distances, potentially increasing cell health and viability (Chan, 2012). By using fluorescent photoactivated protein technology, mitochondrial fusion events have also been observed in living skeletal muscle cells (myoblast) and in isolated mouse muscle fibers. Indeed, in skeletal muscle, mitochondrial fusion is considered a quality control for mitochondrial function; its prolonged loss endangers bioenergetics and excitation-contraction coupling (Eisner et al., 2014). In mice, skeletal muscle-specific depletion of Mfn1 and Mfn2 results in increased mtDNA mutations, decreased muscle mass, exercise intolerance, and lactic acidosis (Chen et al., 2010). Moreover, low levels of Mfn2 in myotubes reduces oxygen consumption and mitochondrial membrane potential, and in human skeletal muscle, a lower Mfn2 protein amount is associated with a reduced mitochondriaSR interaction, lower mitochondrial cristae density, reduced maximal oxygen uptake, and lower fatty acid oxidation capacity (Castro-Sepulveda et al., 2020).

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Finally, and also in humans, Opa1 protein mass is directly correlated with myofiber diameter (Castro-Sepulveda et al., 2021). Altogether, the evidence provided shows that mitochondrial biogenesis and fusion/fission dynamics are interrelated and central to maintain muscle function via efficient energy production and organelle quality control through the regulation of gene expression, reactive species production, and calcium handling, among other processes.

8.3 Effect of n-3 polyunsaturated fatty acids on mitochondrial biogenesis and dynamics Different sources of dietary fats have been suggested to have different effects on mitochondrial function and dynamic behavior. In fact, in contrast to the effect of SFAs, n-3 PUFAs have been reported to improve mitochondrial function, reduce ROS production, and promote mitochondrial fusion for both in vitro and in vivo experiments. N-3 PUFA supplementation, particularly with fish oil enriched in EPA and DHA, results in significant incorporation of these FAs into numerous membrane phospholipid species, within skeletal muscle including the sarcolemma, SR, and mitochondria in rodents and in humans. Although the content of n-3 PUFAs in membrane phospholipids is relatively low (,10%), their incorporation into skeletal muscle membranes has been suggested to alter the organization and function of membrane proteins and subsequently, energy metabolism (Gerling et al., 2019; Turner et al., 2003). This may be particularly important in mitochondria, where electron transfer is tightly coupled between the complexes embedded within the electron transport chain. The following paragraphs will describe how the incorporation of n-3 PUFAs within skeletal muscle membranes potentially affects mitochondrial biogenesis and dynamics. In human muscle cells, incubation of 50 μM of the n-3 PUFAs EPA and DHA for 24 and 48 h resulted in increased mitochondrial content (Vaughan et al., 2012). The mechanism for this adaptation was likely a result of an upregulation of PGC-1α expression. A recent study confirmed these observations, as skeletal muscle cells incubated with 10 μM DHA resulted in mitochondrial biogenesis and muscle fiber type conversion from fast-twitch to slow-twitch, which was mediated by the PGC1-α pathway (Chen et al., 2022). Similarly, in rodents, n-3 PUFA supplementation has been shown to increase the activity of the mitochondrial protein CPT-I (Power & Newsholme, 1997) and enhance mitochondrial biogenesis and CPT-1 expression in red muscle (Totland et al., 2000). However, it is important to consider that the relative dosages given in rodent and some cell studies were extremely high, which is unrealistic for human trials. Moreover, several studies have shown that high-fat feeding may induce mitochondrial biogenesis, independent of the type of fatty acid consumed (Hancock et al., 2008 Jun; Jain et al., 2014; Turner et al., 2007). This could potentially explain the increases in mitochondrial content observed with high doses of n-3 PUFAs in muscle cells and

8.3 Effect of n-3 polyunsaturated fatty acids

rodent skeletal muscle. Moreover, it is important to point out, that not all studies have observed a positive link between n-3 PUFAs and mitochondrial biogenesis. For example, Hsue and coworkers (Hsueh et al., 2018) observed that after 72 h of myogenic differentiation with 50 μM EPA and DHA, there were fewer myotubes formed along with a reduction in mitochondrial content and oxygen consumption rate. Similarly, studies in aged or obese rodent models, as well as in healthy young and insulin-resistant humans have observed no change in mitochondrial content after long-term supplementation with n-3 PUFAs (Chorner et al., 2016; Herbst et al., 2014; Johnson et al., 2015; Lalia et al., 2015; Matravadia et al., 2014). Altogether, these findings suggest a variable and small response with n-3 PUFAs on mitochondrial biogenesis in muscle cells and rodent skeletal muscle. Furthermore, in human skeletal muscle it is unlikely that n-3 PUFA supplementation at normal doses (0,5
5 g/d) leads to increased mitochondrial biogenesis. That being said, n-3 PUFAs have been shown to alter mitochondrial function and dynamics in the absence of changes in mitochondrial content. For instance, in aged mice, a 10-week supplementation period with EPA partially restored skeletal muscle mitochondrial oxidative capacity via increased coupling efficiency of the mitochondrial electron transport chain leading to improved intrinsic function of mitochondria (Johnson et al., 2015). In humans, the incorporation of EPA and DHA into mitochondrial skeletal muscle membranes after 12-weeks of n-3 PUFA supplementation resulted in increased sub-maximal ADP-stimulated respiration (i.e. ADP sensitivity), independent of changes in the protein content of ATP synthase or ADP transporters (Herbst et al., 2014). These findings provide evidence that the changes in mitochondrial respiratory function after n-3 PUFA supplementation are independent of changes in mitochondrial content. Furthermore, these results also suggest that other intrinsic mitochondrial factors could be mediating the beneficial effects of n-3 PUFAs on mitochondrial respiratory function. A recent study provided evidence that changes in mitochondrial bioenergetics could be associated with changes in mitochondrial dynamics after n-3 PUFA supplementation. After a 4-week supplementation period with fish oil or a placebo, healthy young women were assigned to a 2-week single-limb immobilization period while maintaining their supplementation regimen (Miotto et al., 2019). Muscle biopsy samples taken after immobilization showed that n-3 PUFA supplementation prevented skeletal muscle mitochondrial derangements,—a 20% reduction was observed in the placebo-control group,—by maintaining mitochondrial content, ADP-stimulated respiration, and the ratio between the mitochondrial dynamic proteins Mfn2 (fusion) and Drp1 (fission). In support of these findings, skeletal muscles of rats given a high fish oil diet exhibited greater levels of Mfn2 and OPA1 proteins as well as lower levels of Drp1 and FIS1 compared to highlard fed rats (Lionetti et al., 2013). In addition, electron microscopy observations showed a prominent presence of fusion events in rats fed fish oil. Recent evidence from skeletal muscle cells has reported differential effects of SFAs and PUFAs on mitochondrial morphology and dynamics, where treatment with SFAs induced a mitochondrial fission phenotype, whereas unsaturated and PUFAs protected against palmitate-induced mitochondrial fission (Jheng et al.,

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2012). In support of these findings, skeletal muscle cells incubated with DHA reported a higher proportion of large and elongated mitochondria with a concomitant downregulation of the fission genes Drp1 and FIS1 (Casanova et al., 2014). Altogether, these results suggest that the changes in skeletal muscle mitochondrial bioenergetics observed after n-3 PUFA supplementation could be mediated via changes in mitochondrial dynamics; however, further evidence is needed to support a casual role of n-3 PUFAs on mitochondrial dynamics and mitochondrial bioenergetics.

8.4 Conclusion In conclusion, it appears that n-3 PUFA supplementation has a minimal effect on mitochondrial biogenesis in humans. However, recent studies suggest a positive effect of n-3 PUFAs on mitochondrial function and dynamics. Further studies are needed to fully understand the mechanism behind these adaptations, mainly focusing on the association between mitochondrial dynamics and mitochondrial function (e.g., bioenergetics). We hypothesize that incorporation of n-3 PUFAs into the mitochondrial membrane favors mitochondrial fusion, leading to improved mitochondrial function and enhanced skeletal muscle health (Fig. 8.2).

FIGURE 8.2 Hypothesis: Omega-3 (n-3) polyunsaturated fatty acids (PUFAs) are incorporated into mitochondrial phospholipids potentially inducing mitochondrial fusion and leading to enhanced mitochondrial function and skeletal muscle health.

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CHAPTER

Vitamin C and mitochondrial function in health and exercise

9

Michael J. Gonzalez1,2, Jorge R. Miranda-Massari3 and Jose Olalde4 1

School of Public Health, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico 2 Universidad Central del Caribe, School of Chiropractic Medicine, Bayamon, Puerto Rico 3 School of Pharmacy, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico 4 Centro Me´dico Regenerativo (CMR), Bayamon, and Caguas, Puerto Rico

9.1 Vitamin C (ascorbic acid, ascorbate) Vitamin C (L-ascorbic acid, AA) is a water-soluble vitamin with multiple physiological functions. The best food sources are fruits and vegetables, which are available as a dietary supplements. Unlike most species, humans cannot synthesize vitamin C endogenously, so it is an essential dietary component (Roy & Guha, 1958). Vitamin C is required for the biosynthesis of collagen, L-carnitine, and certain neurotransmitters. Vitamin C is also involved in protein metabolism (Diliberto et al., 1991; Jeffrey & Martin, 1966), and it is an important physiological antioxidant (Padayatty et al., 2003) that can regenerate other antioxidants within the body, including alpha-tocopherol (vitamin E) (Niki, 1987). In addition to its function as a cofactor and antioxidant, vitamin C facilitates the intestinal absorption of non-heme iron (Lynch & Cook, 1980). Over 90% of vitamin C is absorbed in the moderate intake of 60
200 mg/day. However, at higher doses, the efficiency of absorption decreases. We must mention that changes in absorption have been observed when utilizing a bowel tolerance regime when the body is responding to physiological stress such as disease, trauma, or toxin exposure which increases the physiological need for ascorbate. This is discussed by Cathcart (Cathcart, 1981) and Hickey (Hickey et al., 2005, 2008). The absorbed, unmetabolized ascorbic acid is excreted in the urine (Lykkesfeldt & Tveden-Nyborg, 2019). Supplements typically contain vitamin C in the form of ascorbic acid, which has the equivalent bioavailability to that of naturally occurring ascorbic acid in foods, such as citrus fruits and broccoli. Other forms of vitamin C supplements include sodium ascorbate, calcium ascorbate, other mineral ascorbates, ascorbic Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00016-3 © 2023 Elsevier Inc. All rights reserved.

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acid with bioflavonoids, and combination products include whole foods (Johnston & Luo, 1994).

9.2 Mitochondria Mitochondria serve several important cellular roles, but first, let’s discuss background history, structure, and the roles mitochondria play in cellular health. It is generally recognized and agreed that mitochondria originated from an aerobic bacterium approximately 1
3 billion years ago, which merged with a preexisting unicellular organism. Both organisms developed a symbiotic relationship which provided a way to create aerobic cellular respiration and produce much more energy. This additional energy supported the development of complex multicellular aerobic organisms. Mitochondria (and chloroplasts) are the only subcellular (and bioenergetic) organelles with its own DNA (mtDNA) (Allen, 2015). The mtDNA is maternally transmitted by the ovum at conception (inherited from one’s mother) and its genetic defects or variants, deficiencies (if present) are limited to the mitochondria. The cellular-nuclear DNA (nDNA) is governed by Mendelian inheritance principles. In contrast to nDNA, which is made up of 3.3 billion base pairs (bp) of genes, mtDNA is circular and composed of 16,569 bp. These bp include 37 genes, of which 24 encode for mitochondrial translation and 13 encode for the cellular respiratory chain (Chinnery & Hudson, 2013). The nDNA is protected by histones which shield nDNA from free radical damage. However, mtDNA is not protected by histones, so they are more susceptible to oxidative damage (Chinnery & Hudson, 2013). mtDNA may generate up to ten times the number of nDNA mutations for two reasons
mtDNA resides close to the ETS inside the inner mitochondrial membrane and mtDNA lacks repair mechanisms, so once damaged, the mitochondria may be slated for apoptosis (Chial & Craig, 2008).

9.3 Mitochondria structure and roles The number of mitochondria per cell is energy/function dependent, i.e., those cells that require and spend the most energy contain the highest number of mitochondria. Most cells have between a few hundred to over 20,000 mitochondria; they are concentrated most heavily in cells of the heart, brain, liver, muscles, gastrointestinal tract, and kidneys (Johnson et al., 2019). Mitochondria are composed of two membranes. The more porous outer membrane contains porin and allows molecules up to approximately 10 kDa to freely diffuse across the membrane. The inner, more tightly constructed (less permeable) membrane contains cardiolipin, a phospholipid which has both a higher affinity for inner membrane proteins and, having two unsaturated bonds, is more susceptible to oxidative damage.

9.4 Vitamin C and the mitochondria

Components of the ETS are found along the inner membrane. The space between the two membranes is the intermembrane space where cytochrome c is found. Inside the inner membrane is the mitochondrial matrix which contains many of the enzymes necessary for adenosine triphosphate (ATP) production (enzymes associated with the Kreb’s Cycle), as well as the mitochondrial genome. Mitochondria play many important roles in human biology, including synthesis of heme, lipids, amino acids, and nucleotides. As mentioned above, they are involved in initiating cellular apoptosis. Their most important role, however, is the production of ATP. Mitochondria generate 95% of the ATP in the cell and rely on ATP for its own functions (Chinopoulos & Adam-Vizi, 2010; Tzameli, 2012). Due to the location of the ETS adjacent to the inner mitochondrial membrane, the generation of free radicals as a normal part of oxidative phosphorylation (production of ATP), as well as the lack of histone protection for mtDNA, much oxidative damage can occur to mitochondria, in normal physiological reactions as well as in chronic disease.

9.4 Vitamin C and the mitochondria The decrease in mitochondrial membrane fluidity caused by mitochondrial membrane injury may reduce the mitochondrial function and the activity of enzymes in its membrane. Vitamin C is a free radical scavenger and has been successfully used to prevent mitochondrial injury in ischemic reperfused myocardium. A rat model of acute in chronic hypoxia, used large doses of vitamin C to determine the effects on mitochondrial function, ATP production, and myocardial structure. The use of vitamin C significantly improved myocardial energy metabolism and protected myocardial structure from injury in acute and chronic hypoxic rats (Luo et al., 1998) (see Fig. 9.1). A common feature in the very early stages of Alzheimer’s disease is mitochondrial dysfunction which is also accompanied by exacerbation of oxidative stress, which contributes to disease pathology. Two different mitochondrial mutations were studied in mice. Although the mutations differed in the amount of oxygen consumption, and membrane potential, they had a lower ATP/ADP ratio than the wild type and produced greater amounts of reactive oxygen species (ROS) than the wild type. Acute administration of ascorbate to mitochondria isolated from wild-type mice increased oxygen consumption compared with untreated mitochondria suggesting ascorbate may support energy production. This study suggests that the presence of ascorbate deficiency can contribute to mitochondrial dysfunction, even at an early stage of Alzheimer’s disease. Ascorbate may, therefore, provide a useful preventative strategy against neurodegenerative disease (Dixit et al., 2017). Vitamin C has been used to treat primary mitochondrial disease as an antioxidant (Eleff et al., 1984; Enns, 2014) and in combination with vitamin K as an

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FIGURE 9.1 Vitamin C is effective in improving myocardial and skeletal energy metabolism, protecting against myocardial structural injury, mitochondrial & inflammation recovery.

electron transfer mediator to bypass complex III deficiency in the electron transport system (Eleff et al., 1984; Mowat et al., 1987). Vitamin C in its oxidized form, DHA, is transported into the mitochondria via facilitative glucose transporter 1 and reduced to mitochondrial AA. Mitochondrial AA quenches ROS and protects the mitochondrial genome. Mitochondrial vitamin C serves to maintain healthy mitochondrial membrane potentials and inhibits mitochondrial membrane depolarization. Moreover, vitamin C as a reduction molecule facilitates electron movement which favors energy production (Gonzalez et al., 2005).

9.5 Mitochondriopathies Mitochondriopathies are a group of conditions characterized by the inability of the cell mitochondria to produce adequate amounts of ATP, by a faulty element in the biochemical pathway such as defective enzymes and/or insufficient essential micronutrients. Mitochondrial dysfunction can be caused by mutations (acquired or inherited) of the mtDNA or in nuclear genes that encode for mitochondrial components. However, mitochondrial dysfunction can also be a result of adverse drugs effects, environmental toxins, and infections. A study conducted among 60 patients with mitochondrial diseases showed that many patients have an inadequate diet to support adequate mitochondrial function. It was found that the intake of protein, calcium, and fluids was low, and

9.6 Role of vitamin C in mitochondrial disease

FIGURE 9.2 Acquired mitochondrial dysfunction can be caused by various injury factors. These can be caused by deficiencies in Vitamin C and other cofactors, toxins, drugs, infections. These can cause gradual mitochondrial damage that starts with mitochondrial membrane damage, Krebs cycle damage, etc damage, inflammation, and mtDNA mutation.

therefore individualized nutritional counseling is recommended in patients with mitochondrial dysfunction (Zweers et al., 2018). Other reports proposed that carnitine insufficiency might contribute to mitochondrial dysfunction and obesityrelated impairments in glucose tolerance (Noland et al., 2009). Mitochondrial dysfunctions are associated with a variety of chronic disease conditions such as cancer, diseases of the brain, peripheral nerves, endocrine system, skeletal muscles, heart, and other major organs (Finsterer, 2004; Kamp et al., 2011; Vanotti et al., 2007) (See Fig. 9.2).

9.6 Role of vitamin C in mitochondrial disease No studies have assessed the effect of vitamin C alone in patients with Primary Mitochondrial Diseases. Two case reports of patients with mitochondrial myopathy associated with complex III deficiency using a combination vitamin C with vitamin K3 are reviewed herein (Argov et al., 1986; Eleff et al., 1984). A 17-year-old patient with a severe defect in mitochondrial complex III of the electron transport chain was examined by nuclear magnetic resonance by measuring the molar ratio of the phosphocreatine to inorganic phosphate. The quantitative evaluation of the oxidative metabolism determined that the rate of recovery from exercise was 2.5% of normal. After administration of menadione

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(vitamin K) and ascorbate (vitamin C), they found a 21-fold increase of the recovery rate relative to the pretherapy value, which was about 56% of the recovery rate of the young female control. The patient also demonstrated significant improvements in functional ability. In the year prior to the intervention, the patient’s tolerance to exercise was severely compromised. She could walk only half a block or climb five to ten steps without resting, and she frequently needed a wheelchair. She received 10 mg of menadione and 1 gram of ascorbate every 6 h. Within 24 h of starting redox therapy, there was a marked contrast, the patient claimed to have “more energy.” Within two days, she ceased using her wheelchair and was able to walk several blocks without stopping or complaining of fatigue. The reported improvement was maintained for months as reported in a followup report (Eleff et al., 1984). The followup one year later on the patient with mitochondrial myopathy due to complex III deficiency who responded to the vitamin K3 and vitamin C treatment showed that the clinical metabolic improvement was maintained by this therapy. An increased dose of vitamin K3 to 80 mg daily improved the bioenergetic state of the patient’s muscles at rest; postexercise recovery was less responsive to the increased dose. The higher dose of vitamin K3 did not produce any side effects (Argov et al., 1986). A study conducted in mitochondria of mouse aortic smooth muscle cells and insulin-stimulated adipocytes reported that Mitochondrial GLUT10 facilitates dehydroascorbic acid import and protects cells against oxidative stress. They also found that when GLUT10 expression in mitochondria is inhibited, protection from oxidative stress is compromised. Therefore, it was concluded that the loss of function of GLUT10 mutations may lead to arterial abnormalities. These results also reinforce the importance of vitamin C in ROS production in degenerative diseases (Lee et al., 2010). Multiple studies have established that vitamin C supplementation improves endothelial function. The effect of vitamin C supplementation seems to depend on health status, with greater effects in those at higher cardiovascular disease risk (Ashor et al., 2014). Vitamin C supplementation of between 300
1000 mg daily, is associated with significant reductions of blood pressure in patients with primary hypertension (Guan et al., 2020). Systemic inflammation and increased oxidative stress in congestive heart failure (CHF) are factors associated with enhanced endothelial cell (EC) apoptosis and the development of endothelial dysfunction. To investigate the effects of antioxidative vitamin C therapy on EC apoptosis, a clinical study was conducted. In a group of 34 patients with CHF, administration of vitamin C was shown to suppress apoptosis of ECs in vivo. The study results show that vitamin C interferes with apoptosis signaling in ECs stimulated by TNF-a, Ang II activation, and serum of patients with CHF. The antiapoptotic activity of vitamin C was associated with the prevention of mitochondrial cytochrome c release and suppression of subsequent caspase activation in activated ECs. Vitamin C administration to CHF patients significantly reduced plasma levels of circulating apoptotic

9.8 Vitamin C and exercise (physiology/inflammation/recuperation)

microparticles to baseline levels, whereas the placebo had no effect (P , .005). Also, vitamin C administration suppressed the proapoptotic activity on EC of the serum of CHF patients (P , .001) (Ro¨ssig et al., 2001).

9.7 Safety of vitamin C Vitamin C has low toxicity and does not cause serious adverse effects at high intakes (Institute of Medicine, 2000). The most common adverse effects of oral vitamin C intake are diarrhea, nausea, abdominal cramps, and other gastrointestinal disturbances because of the osmotic effect of unabsorbed vitamin C in the gastrointestinal tract (Jacob & Sotoudeh, 2002). High vitamin C intake also has the potential to increase urinary oxalate and uric acid excretion, which could contribute to the formation of kidney stones, especially in individuals with renal disorders. However, studies evaluating the effects on urinary oxalate excretion of 30 mg to 10 g/day of vitamin C have had conflicting results, so it is not clear whether vitamin C plays a relevant role in kidney stone development. Furthermore, in a group of 16 healthy individuals, intravenous doses ranging from 0.2 to 1.5 g/kg body weight were given and less than 0.5% of ascorbic acid was recovered as urinary oxalic acid in people with normal renal function (Robitaille et al., 2009).

9.8 Vitamin C and exercise (physiology/inflammation/ recuperation) Athletes and people that exercise often take vitamin C supplements with the idea to reduce free radicals because intense muscular contractile activity can result in oxidative stress, as indicated by altered muscle and blood glutathione concentrations and increases in ROS-related protein, DNA, and lipid peroxidation. There is, however, considerable debate regarding the beneficial health effects of vitamin C supplementation in exercise. A study reported that vitamin C supplementation decreases training efficiency because it prevents some cellular adaptations to exercise. These factors are peroxisome proliferator-activated receptor coactivator 1, nuclear respiratory factor 1, and mitochondrial transcription factor A. It was also reported that vitamin C also prevented the exercise-induced expression of cytochrome c (a mitochondrial protein released into the cytosol and used as a mitochondrial activity marker) and of the antioxidant enzymes, superoxide dismutase and glutathione peroxidase (Gomez-Cabrera et al., 2008). These results should not be surprising since vitamin C in small and moderate doses works as an antioxidant while in high doses might work as a pro-oxidant. This study was carried out in rats. Although the redox biology between rats and humans is somewhat similar, there are differences that

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may affect the extrapolation of the results. Rats produce their own vitamin C, which makes it difficult to interpret the supplemental doses of these reported results. Human studies showing no effects on these parameters were conducted with oral dosages of vitamin C ranging between 500 mg/day and 1000 mg/day that were expected to predominantly produce an antioxidant effect (Gonzalez et al., 2010; Theodorou et al., 2011; Yfanti et al., 2010; Zeviar et al., 2014). Another important methodological issue of the pertinent studies is the utilization of untrained or recreationally trained individuals that may differ from highly trained elite athletes. The wide divergence among studies addressing the effect of vitamin C supplementation on exercise adaptations could be due to several variables involved, such as the uniqueness of each study in terms of type of exercise (aerobic or anaerobic), species (rat or humans), age (young or old), tissue (blood or muscle), oxidant biomarker, and training endpoints examined. Other factors that could explain some of the diversity of results include nutrition, subject characteristics, exercise characteristics, and experimental error arising from the complexity of the techniques employed in redox status analysis. Finally, the lack of consensus may be partially explained by the biological variability of redox biochemistry and the subject’s biochemical individuality. Hormesis is a dose-response physiological phenomenon characterized by lowdose stimulation and high-dose inhibition, leading to the biphasic, or triphasic response curve, which is the opposite of a high linear dose. In physiology, the basic parameters that should be considered are deficiency, homeostasis, and toxicity. Physiologically, homeostasis is the hermetic zone (Hayes, 2007). Pharmacologically it is the therapeutic window the key for effectiveness or toxicity. The conceptual framework provided by hormesis can potentially reconcile differences that emerge among relevant studies regarding the effect of antioxidant supplementation on exercise adaptations. In general, metabolic resilience, physiological balance, and quantum biology may further complicate any interpretation of these studies. The ROS produced seem to work as signaling molecules. ROS can stimulate intracellular signaling leading to mitochondrial biogenesis under vitamin C supplementation. The insufficient antioxidant protection may arise from the difficulty in raising intramuscular vitamin C concentration to a sufficiently high level for suppressing overall oxidative stress. Four weeks of supplementation with vitamin C prevented adverse changes of the indices of oxidative stress and inflammation after intense eccentric exercise by increasing the serum total antioxidant capacity at baseline. Thus, by taking precautions, athletes can use supplementation of vitamin C in order to prevent loss of antioxidant capacity and oxidative stress of intense unconventional exercise-induced inflammatory effects (Ahmadi, 2016; Righi et al., 2020). One of the most recognized features of vitamin C is its antioxidant action. Vitamin C protects cells, especially muscle cells from harmful effects of highly reactive free radicals produced when the rate of oxygen consumption is

9.8 Vitamin C and exercise (physiology/inflammation/recuperation)

substantially increased during strenuous exercise. Exercise that forcibly contracts the muscle may result in damage to the muscle structure and postexercise soreness. This muscle soreness peaks one to three days after the exercise bout and is often referred to as delayed on-set muscle soreness (DOMS). The pain associated with this condition is thought to be a consequence of micro traumatization of connective tissue and microfibers. It has been reported that oral vitamin C (in doses of about 200 mg) may significantly reduce DOMS (Torre et al., 2021). It has been suggested with some preliminary evidence, that vitamin C may benefit muscle function and reduce plasma levels of malondialdehyde, a secondary product of lipid peroxidation, in addition to diminishing muscle soreness (Gabrial et al., 2018). Vitamin C also appears to offer protection against ultrastructural damage following reperfusion injury (Hao et al., 2016). Infusion of vitamin C before elective percutaneous coronary intervention (PCI), reduced oxidative stress in both studies (Basili et al., 2010; Wang et al., 2014) and even periprocedural myocardial injury in the larger study (n 5 532). In a systematic review of eight randomized controlled trials vitamin C administration reduced cardiac injury as measured by troponin and CK-MB increases, along with increased antioxidant reserves, diminished ROS, and reduced inflammatory markers. Improvement of the left ventricular ejection fraction. All these clinical trials reported no significant adverse effects from vitamin C. Given these results, it has been suggested that vitamin C should be considered as an adjunct therapy for PCI for the prevention of reperfusion injury (Khan et al., 2020). Following cardiac surgery, oxidative stress promotes myocardial abnormalities and arrhythmias. High-dose IV vitamin C administered before and after surgery decreased oxidative stress and myocardial injury (Dingchao et al., 1994). In a recent metaanalysis, the use of vitamin C significantly decreased the incidence of paroxysmal atrial fibrillation (95% CI: 0.36
0.62; P , .00001) (Hu et al., 2017). Reduction in inflammation may partly explain the decreased soreness present in the vitamin C supplement group. Reduction in plasma concentration of proinflammatory cytokines such as 1L-6, TNF-alpha, and 1L-8, have also been reported following high dose oral supplementation of ascorbate in doses between 250 and 1500 mg (Fischer et al., 2004; Gholizadeh et al., 2021; Nieman et al., 2004). High doses of vitamin C may also reduce cortisol (Brody et al., 2002). Athletes supplemented orally with 500
1,500 mg of vitamin C in the week before and on the day of the race, significantly attenuated the postexercise serum cortisol responses (Peters, Anderson, & Nieman, Fickl, et al., 2001; Peters, Anderson, & Theron, 2001). High doses of vitamin C may inhibit the synthesis of cortisol, possibly through a pro-oxidant effect. However, some animal studies also suggest that ascorbate in the adrenals might have a role in the conversion of cholesterol into pregnenolone which is a precursor of cortisol (Bjo¨rkhem et al., 1978). As we have pointed out in other publications (Gonzalez et al., 2005; Gonzalez et al., 2012), ascorbic acid may act as a pro-oxidant under certain conditions. Interestingly, declines in plasma ascorbic acid correspond to a rapid and dramatic increase in plasma cortisol and ACTH concentrations (Redmann et al., 1995). It is conceivable,

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that both cortisol and ascorbic acid have a regulatory influence on gluconeogenesis, an important energy mechanism. Moreover, it has been reported (Cheraskin et al., 1976) that subjects consuming less than 100 mg of ascorbic acid per day showed a higher fatigability mark than those ingesting more than 400 mg of ascorbate per day when performing the same tasks. It has also been documented that in cancer patients, high doses of ascorbic acid produce substantial benefits ranging from increased energy to the reduction of pain and tumor burden (Carr & McCall, 2017; Carr et al., 2014; O’Leary et al., 2020; Raymond et al., 2016; Vineetha et al., 2021; Zasowska-Nowak et al., 2021). A study with terminal cancer patients that evaluated health-related quality of life after high dose vitamin C, demonstrated significant reductions in fatigue, nausea, vomiting, pain, and appetite loss after administration of vitamin C (P , .005) (Yeom et al., 2007). Cancer patients receiving intravenous vitamin C reported improvements in fatigue, pain, and mood while receiving ascorbic acid (Bazzan et al., 2018). Another study with a prospective, randomized design in 350 patients with breast cancer who received 25 grams IV vitamin C per week, showed significant improvement in the severity of nausea, fatigue, tumor pain, loss of appetite, and fatigue (Mansoor et al., 2021).

9.9 Vitamin C as an ergogenic factor (performance) Vitamin C may target the mitochondria and increase electron flux, thus increasing the production of ATP and therefore normalizing the apoptosis function. A greater amount of vitamin C available to the mitochondria may optimize the production of ATP as well as cell-to-cell communication and cell differentiation (Gonzalez et al., 2010; Zeviar et al., 2014). Using this concept of mitochondrial enhancement and optimization of the RedOx reactions and signaling with cofactors and phytochemicals (Metabolic Correction and Physiological Modulation therapy) may become a key element of the medicine of the 21st century (Gonzalez et al., 2018). Ascorbic acid is distributed in varying concentrations throughout the body and is involved in a variety of metabolic reactions related to exercise such as the synthesis and activation of neuropeptides, the formation and repair of collagen, and the synthesis of carnitine. Once these facts are taken into account, it is conceivable that exercise may enhance the utilization, metabolism, and excretion of ascorbic acid, thus increasing the body’s requirements for vitamin C. The current recommended daily allowance for vitamin C is between 75 and 90 mg daily (Institute of Medicine, 2000). These doses are too low to detect any possible ergogenic activity of vitamin C. As physiologic need is increased by any significant physiologic stress, these doses will be allocated preferentially for other physiologic needs and may not be available for optimization of mitochondrial function or other ergogenic purposes. This was proposed by Bruce Ames in his Triage Theory (Ames, 2006) which suggests that modest micronutrient deficiencies and insufficiencies are key in the development of chronic diseases, these deficiencies

9.9 Vitamin C as an ergogenic factor (performance)

and or insufficiencies are common even in affluent nations like USA (Reider et al., 2020). We used high dose intravenous ascorbic acid as an ergogenic agent in the Basketball Pre-Olympic tournament of the Americas of 2003 with the Puerto Rico National basketball team. This intervention seemed to promote a rapid recovery and increased energy in the players. After this therapy, the Puerto Rico team performance improved, and it seemed that intervention helped them classify for the 2004 Olympic games in Athens, in which the Puerto Rican national basketball team gave the USA dream team their first defeat in an Olympic competition. The question that follows is: what is the proposed mechanism by which ascorbic acid (in large doses) produces these ergogenic effects? Albert Szent-Gyo¨rgyi, discoverer of vitamin C and Nobel prize winner, believed that the real physiological significance of vitamin C should be ultimately looked for in the electron transport system (Gonzalez et al., 2012). Chemically, ascorbic acid exhibits redox characteristics as a reducing agent. Physiologically, ascorbic acid provides electrons for enzymes and other electron acceptors. Szent-Gyo¨rgyi explained that dead tissue had a full complement of electrons, while living tissue maintained a deficit of electrons. Vitamin C assures a continuing electron exchange among body tissues (as it does in plant tissue for the photosynthesis process), cell mitochondria, and molecules. All body functions are directly controlled and regulated by this physiological flow of electrons. Furthermore, this flow of electricity/photons through the body also establishes and maintains the subtle magnetic fields in the body that appear to be involved in maintaining the healthy state (Gonzalez et al., 2019). Vitamin C may be the most important stimulus to this flow of electrons. A greater amount of vitamin C in the body enhances the flow of electrons, optimizing the ability of the cells to maintain aerobic energy production and metabolic intermediaries that facilitate cell-to-cell communications. The disease exists when this flow is impaired, and death occurs when this flow stops. In support of this theory, it has been documented that osteoblast cells treated with ascorbic acid had a fourfold increase in respiration and a threefold increase in ATP production that provides the high-energy electrons necessary for aerobic metabolism (Komarova et al., 2000). This redox activity of ascorbate at the level of the plasma membrane may be important not only in mitochondria energy production, but also in the healthy regulation of cell metabolism. This healthy regulation of cell growth implies both muscle growth and bone density and strength as well as maintaining a cellular environment that supports the differentiated state (Choi et al., 2019; Kim et al., 2018; Peters et al., 2020), and may be protective of the malignant transformation and entropic disease state (Gonza´lez et al., 2002; Levine et al., 2011). The most commonly used dietary supplement ingredients for mitochondrial disorders include antioxidants, such as vitamin C, vitamin E, and alpha-lipoic acid electron donors and acceptors, such as CoQ10 and riboflavin; compounds that can be used as alternative energy sources, such as creatine and compounds that can conjugate or bind mitochondrial toxins, such as carnitine (Arrigoni-Martelli & Caso, 2001; Barcelos et al., 2020; Kidd, 2005; Valero, 2014; Wesselink et al., 2019).

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In health, the balance between ROS and the antioxidant defenses lies slightly in favor of the ROS, so that they are able to fulfill their biological roles and maintain the necessary electron flow of life. Taken together, the effect antioxidant supplementation has on skeletal muscle adaptation to exercise training is still equivocal. On one side, supplementation might produce possible interference with hormesis where stressors induce ROS that act as intracellular signaling molecules to promote adaptations that equip the cell to better tolerate future stress. On the other side, supplementation may prevent excessive exercise-induced stress sufficient to chronically elevate ROS to levels that impair function and cause damage. This does not preclude the potential for acute antioxidant supplementation to enhance the performance of certain types of exercise and sports. The level of ROS required to impair muscle performance is likely to be lower than that which would hamper muscle adaption. In conclusion, biochemical individuality, form, quantity of macro- and micronutrients, and nutrient timing are important variables to consider when evaluating possible ergogenic substances.

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CHAPTER

Roles of dietary fiber and gut microbial metabolites short-chain fatty acids in regulating mitochondrial function in central nervous system

10

Huajun Pan and Zhigang Liu College of Food Science and Engineering, Northwest A&F University, Yangling, China

10.1 Introduction It has been reported that the gut-brain axis plays a pivotal role in mediating brain health, especially in the pathogenic process of dementia, anxiety, depression, and autism (Foster et al., 2017). This chapter will focus on the beneficial effects of shortchain fatty acids (SCFAs), gut microbial metabolites fermented from dietary fiber, on the mitochondrial function in the central nervous system (CNS). Dietary fiber is traditionally defined as the portions of plant foods that are resistant to digestion by human digestive enzymes, including several nonstarch polysaccharide substances such as cellulose, hemicellulose, β-glucans, mucilages, gums, and the nonpolysaccharide lignin (Anderson et al., 2009; Burton-Freeman, 2000). Gut microbiota, which has evolved a symbiotic relationship with the host (Myhrstad et al., 2020), provides fermentation of the nondigestible dietary fibers, therefore supporting the growth of specialist microbes that produce SCFAs and gases. The SCFAs, including acetate, propionate, and butyrate, are the major microbial metabolites produced by microbiota in the colon which also play essential roles in the function and metabolism in the CNS. In this chapter, we will review the cognitive, appetitive, and psychological functions of SCFAs in CNS and delve into the roles of SCFAs in the mechanism of mitochondrial metabolism, serving as an energy metabolite to produce ATP, a G proteincoupled receptor (GPCR) activator, and a histone deacetylase (HDAC) inhibitor.

10.2 Gut microbiota and short-chain fatty acids Dietary fibers are naturally occurring compounds with diverse compositions and are present in all plant-based food, including cereals, tubers, and agro-industrial Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00024-2 © 2023 Elsevier Inc. All rights reserved.

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byproducts (Jha & Berrocoso, 2015). According to the definition of the British Nutrition Foundation in 2018, dietary fiber refers to a group of substances in plant foods that cannot be completely broken down by human digestive enzymes. Gut microbiota produce thousands of complementary enzymes with diverse specificities, while humans only produce around 17 gastrointestinal enzymes to digest food glycans, which enables them to ferment dietary polysaccharides into hostabsorbable SCFAs (El Kaoutari et al., 2013). For example, acetate, the most abundant form in SCFA, is produced from acetyl-CoA during glycolysis and can also be transformed into butyrate by the enzyme butyryl-CoA, the acetyl-CoA transferase (Duncan et al., 2004). SCFAs are carboxylic acids with aliphatic tails of 1
6 carbons, among which acetate (C2), propionate (C3), and butyrate (C4) are the main metabolites produced by aerobic fermentation of dietary fibers in the intestine (Parada Venegas et al., 2019). Besides these, other SCFAs, such as isobutyric (C4), valeric (C5), and isovaleric (C5), are present in lower amounts. The beneficial bioactivity of SCFAs focuses on energy metabolism, anti-inflammation, and immune system regulation (Wang et al., 2017).

10.3 Short-chain fatty acids regulate peripheral organizational activities The most direct effect of SCFAs is their role as energy fuel. It has been estimated that when taken up, a large part of the SCFAs is used as a source of energy, and this could provide nearly 10% of human daily caloric requirements (Bergman, 1990). Glucose synthesis from propionate accounts for 69% of total glucose production, and the synthesis of palmitate and cholesterol in the liver from fecal acetate and butyrate as substrates when synthesis from propionate is absent (den Besten et al., 2013). Moreover, research suggested that any hepatic changes associated with SCFA administration were large because of the metabolism of propionate by the liver, which is a gluconeogenic substrate and inhibits the utilization of acetate for lipid and cholesterol synthesis (den Besten et al., 2013). Potential upregulation of this pathway is likely to be responsible for any observed changes in hepatic function. SCFAs produced by human gut microbiota are playing important roles as energy resources not only of gastrointestinal cells like intestinal epithelial cells but also of the whole body. For instance, butyrate is particularly important to colon health because it is the primary energy source for colonocytes (Wong et al., 2006). Also, the liver can use acetate for energy. SCFAs are vital metabolites in the mechanism of energy metabolism. In addition to the roles in energy consumption, SCFAs also modulate different processes in the gastrointestinal tract function. Research has shown that certain construction of SCFAs acetate and butyrate enhanced the viability of human islets

10.3 Short-chain fatty acids regulate peripheral organizational activities

and beta cells and prevented metabolic stressor streptozotocin-induced cell apoptosis, viability reduction, mitochondrial dysfunction, and the overproduction of reactive oxygen species (ROS) and nitric oxide (Hu et al., 2020). These rescue effects of SCFAs were accompanied by preventing the reduction of the mitochondrial fusion genes MFN, MFN2, and OPA1. In peripheral tissues, SCFAs act on leukocytes and endothelial cells through at least two mechanisms. One is the activation of GPCRs (GPR41 and GPR43), the other one is the inhibition of a HDAC (Vinolo et al., 2011a). SCFAs regulate leukocyte functions, including the production of several cytokines, including TNF-α, IL-2, IL-6, and IL-10, eicosanoids, and chemokines such as MCP-1 and CINC-2. According to extracorporeal results, SCFAs could induce directional migration of neutrophils, which is dependent on the activation of GPR43. This receptor could be activated by SCFAs, just as in the case of GPR41 and GPR109A. Evidence has shown that SCFAs elicited GPR43-dependent activation of PKB and MAPKs in neutrophils (Vinolo et al., 2011b). SCFAs also reduced the surface expression of chemoattractant receptors such as C5aR and CXCR2 (Vinolo et al., 2011a). Experimental studies of SCFAs in kidney cells have been conducted. Results showed that when glomerular mesangial cells were induced by high glucose and lipopolysaccharide (LPS), the pharmacological concentrations of SCFAs accompanied with GPR43 agonist diminish renal inflammation by decreasing MCP1and IL-1b (Li et al., 2017). SCFAs inhibited ROS generation induced by high glucose and LPS in GMCs and HK-2 human kidney epithelial cells after hypoxia (Andrade-Oliveira et al., 2015). Notably, they have been recognized as potential mediators involved in the effects of gut microbiota on intestinal immune function, which finally impact the CNS through the microbiota-SCFA-brain axis. It is widely known that peripheral insults that cause a systemic inflammatory response could affect ongoing inflammation in the CNS mainly by microglial activation, production of inflammatory molecules, as well as recruitment of peripheral immune cells into the brain. Afterward, these insults would shape a cerebral inflammatory milieu which could affect neuronal function seriously. Chronic functional bowel syndrome could enhance gut-brain axis dysfunction, neuroinflammation, cognitive impairment, and vulnerability to dementia, for instance (Daulatzai, 2014). SCFAs activated AMP-activated protein kinase and ameliorated ethanol-induced intestinal barrier dysfunction in certain cell lines (Elamin et al., 2013). The translocation of bacterial products could increase the production of cytokines and impact the blood-brain barrier during gut pathologies with increased permeability of the gut barrier, leading to more harmful effects (Dinan & Cryan, 2017). Studies have also shown that SCFA intake could cause phase changes in the peripheral clocks with stimulation timing dependency and fiber-containing diets on circadian clock phase adjustment in mouse peripheral tissues, including liver, kidney, and the submandibular gland (Tahara et al., 2018). Evidence is presented that mitochondrion is implicated in the programmed cell death process induced by acetate. The acetate-induced apoptosis is found to be independent of oxidative phosphorylation, for it was not inhibited by oligomycin treatment (Ludovico et al., 2002).

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10.4 Effects of short-chain fatty acids on modulating the central nervous system function Apart from the regulation effect of SCFAs on peripheral organizations, they could exert influence on the CNS directly. Brain disorders have been linked to imbalances in the gut microbial composition, but whether these changes in the microbiota are induced by CNS signaling or whether brain dysfunction is driven by alternations in the gut microbiota, remains to be more specifically elucidated. SCFAs can cross the blood-brain barrier (BBB), according to the cell culture model, possibly owing to the abundant expression of MCTs on endothelial cells (Kekuda et al., 2013; Mitchell et al., 2011). Using PET imaging, it was shown that about 3% of intravenously infused C11-acetate was immediately taken up in the rat brain (Frost et al., 2014). This was the direct evidence of their ability to cross the BBB. The modulation of gut physiology by the CNS, such as motility, secretion, blood flow, nociception, and immune function during neurological stressors, have been well documented. SCFAs impact the cognitive, appetitive, and psychological function of the CNS. SCFAs cross the BBB by monocarboxylate transporters located on endothelial cells and influence BBB integrity by upregulating the expression of tight junction proteins (Silva et al., 2020). For example, acetate could suppress appetite and activate hypothalamic neuronal patterning (Frost et al., 2014). It has been shown that several bacterial strains can modify the levels of neurotransmitter precursors in the gut lumen and independently modulate the synthesis of numerous neurotransmitters, including r-aminobutyric acid (GABA), serotonin (5-HT), dopamine (DA), and noradrenaline (NA) (Calvani et al., 2018). These neurotransmitters could influence the activation of microglia, resident immune cells in the brain that are essential for modulating neurogenesis, then make microglia respond to local signals within the brain and receive input from the periphery, including the gastrointestinal tract (Abdel-Haq et al., 2019). SCFAs might influence microbiota-gut-brain interactions by signaling to the host via free fatty acid receptors (FFARs) FFAR2 and FFAR3, as well as GPR109A and olfactory receptor 51E2. Once activated, they will result in further signaling cascades, including phospholipase C, mitogen-activated protein kinase (MAPKs), phospholipase A2 (PLA2), and nuclear factor-kB (NF-κB) pathways (Dalile et al., 2019). Above all, SCFAs might be directly or indirectly involved in communication along the microbiota-gut-brain axis because of their neuroactive properties and their effects on other gut-brain signaling pathways, including the immune and endocrine systems (Stilling et al., 2016). SCFA could be used as interventional substances to target microbiota-gut-brain interactions in humans (Dalile et al., 2019). It has been well documented that propionate is closely associated with mitochondrial dysfunction, including both beneficial and toxic effects, depending on

Effects of SCFAs

the concentration, exposure duration, and microenvironment redox state. The expression of the mitochondrial biogenesis-related proteins PGC-1α, TFAM, SIRT3, and COX4 was significantly increased after propionate treatment. Transcriptome analysis revealed that mRNA expression in the notch signalingrelated genes ASCL1 and LFNG changed after propionate treatment, and positively correlated protein expression changes were also observed (Kim et al., 2019). Butyrate is reported to be a substrate for energy production, a regulator of energy metabolism, and an HDAC inhibitor. According to research results, dietary administration of butyrate has been shown to increase energy expenditure and favor insulin sensitivity in mice models through induction of PGC1α and AMPactivated protein kinase, and consequently, mitochondrial fatty acid oxidation increases (Chriett et al., 2017).

10.4.1 Short-chain fatty acids influence cognitive and psychological function on mitochondria in the brain Several reports suggested connections between the intestinal microbiota and gutbrain axis disturbances since gastrointestinal symptoms like bloating, constipation, and diarrhea have been positively correlated with the severity of neurological disorders like autism and Parkinson’s disease (PD) (Mulak & Bonaz, 2015). Comparative studies were also conducted on whether the levels of SCFAs in fecal specimens from autistic and PD patients are impaired in individuals without deficits in neurological function (Sampson et al., 2016). The intestinal microbiota influences CNS functions and host behaviors that likely involve immune hormonal, and neural pathways. Alterations in the intestinal microbiome are suggested to be a risk factor for neurological disorders like Autism Spectrum Disorders (ASD), PD, anxiety, and depression. Modulation of the microbiome (e.g., psychobiotics) represents an emerging therapeutic modality that may be addressed to improve the quality of life of those affected by neurodegenerative and neurodevelopmental disorders. SCFAs might exert positive effects on psychological functioning via interactions with GPCR or HDAC and influence the brain via direct humoral effects, indirect hormonal and immune pathways, and neural routes. Results of one of our recent studies showed that treatment with the microbiotaderived SCFAs could alleviate the behavioral deficits in the offspring of obese mice dams (animal model: C57BL6 mouse) and indicated that the microbiotametabolites-brain axis might underlie maternal obesity-induced cognitive and social behavior dysfunctions (Liu et al., 2021a). High dietary fiber intake, therefore, could be a promising intervention for obesity-induced cognitive function deficits. Studies showed that pre- or perinatal infection, hospitalization, or early antibiotic exposure, which may alter gut microbiota, therefore, influencing the production of SCFAs, had been suggested as potential risk factors for ASD. Propionate, for example, was confirmed to produce reversible behavioral, electrographic,

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neuroinflammatory, metabolic, and epigenetic changes closely resembling those found in ASD in rodent models (MacFabe, 2015). Major effects of SCFAs may be through the alteration of mitochondrial function via the citric acid cycle and carnitine metabolism or the epigenetic modulation of ASD-associated genes. Research has also shown that butyrate-enhanced mitochondrial function during oxidative stress in cell lines from ASD diagnosed boys (Rose et al., 2018). Apart from ASD, SCFAs also benefit patients with depression a lot. Research has been carried out to confirm that brain inflammatory processes could be affected through the modulation of gut permeability and blood LPS levels. Aberrant mitochondria functionality coupled to adverse cellular homeostasis could be a key mediator for the effect of the intestinal microbiota on the progression of depression (Chen & Vitetta, 2020). Animal studies provide direct evidence of the effects of SCFAs on neuropsychiatric disorders and psychological functioning, whereas human studies are rare due to methodological limitations. SCFAs should be quantified in the systemic circulation, and the effects on psychological functioning and psychopathology are an outcome of interest (Dalile et al., 2019).

10.4.2 Short-chain fatty acids influence appetitive function on mitochondria in the brain A significant body of evidence suggested that SCFAs had a beneficial role in appetite. Research done to investigate the effect of intravenous and colonic infusion of acetate in vivo, showed that most of the acetate was absorbed by the heart and liver, and a small amount crossed the BBB and was taken up by the brain when a significant reduction in energy intake and weight gain was observed (Cani et al., 2005). Therefore, it suggests that acetate-induced hypothalamic neuronal activation in the arcuate nucleus following intraperitoneal administration. It is counterintuitive idea that SCFAs have a beneficial effect on energy homeostasis but could also increase energy harvest. Metabolizable energy gained from SCFAs via the colonic fermentation process of nondigestible carbohydrates may outweigh the beneficial effect associated with their consumption. A study carried out by Isken et al. (2010) demonstrated that a long-term (45 weeks) intervention of soluble guar fiber could increase body weight and markers of insulin resistance in mice, which could be partly explained by the enhanced production of SCFAs. As discussed above, diets consisted mainly of vegetable matter would have a large fermentable component (FCs), and their consumption would have stimulated gut hormone release. As for humans, evidence suggested that the fermentation process significantly contributed to digestible energy when nondigestible carbohydrates are consumed of over 20 grams (Behall & Howe, 1995). Research showed that consuming an evening meal consisting of FCs significantly increased circulating peptide YY concentrations and decreased ghrelin concentrations at breakfast time (Nilsson et al., 2013). Also, a study concerning long-term (2
12 weeks)

References

supplementation with oligofructose showed that it increased feelings of satiety and reduces feelings of hunger, as well as reducing energy intake and the total area under the curve for ghrelin (Cani et al., 2006). Notably, changes take 9
12 months to develop, which suggests that time is required for gut microbiota to adapt to the extra fermentable content of the diet. To summarize, SCFAs, as the major gut microbial metabolites of the dietary fiber generated in the colon, play essential roles in regulating gut barrier function, liver metabolism, kidney function, and immune balance in the peripheral systems. Moreover, SCFAs also modulate the CNS function, including improving cognitive and social dysfunctions and suppressing appetite. Our recent research indicated that supplements of mannan oligosaccharide, as well as its metabolites, SCFAs, significantly alleviated the cognitive deficits and balanced the HPA axis (Liu et al., 2021b). However, more data are needed on whether these beneficial effects of SCFAs are associated with the mediating roles of SCFAs on mitochondrial function in the neurons or in the glial cells.

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Dalile, B., et al. (2019). The role of short-chain fatty acids in microbiota-gut-brain communication. Nature Reviews Gastroenterology and Hepatology, 16(8), 461
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644. den Besten, G., et al. (2013). Gut-derived short-chain fatty acids are vividly assimilated into host carbohydrates and lipids. American Journal of Physiology-Gastrointestinal and Liver Physiology. Dinan, T. G., & Cryan, J. F. (2017). The microbiome-gut-brain axis in health and disease. Gastroenterology Clinics of North America, 46(1), 77
89. Duncan, S. H., et al. (2004). Contribution of acetate to butyrate formation by human faecal bacteria. The British Journal of Nutrition, 91(6), 915
923. El Kaoutari, A., et al. (2013). The abundance and variety of carbohydrate-active enzymes in the human gut microbiota. Nature Reviews. Microbiology, 11(7), 497
504. Elamin, E. E., et al. (2013). Short-chain fatty acids activate AMP-activated protein kinase and ameliorate ethanol-induced intestinal barrier dysfunction in Caco-2 cell monolayers. The Journal of Nutrition, 143(12), 1872
1881. Foster, J. A., Rinaman, L., & Cryan, J. F. (2017). Stress & the gut-brain axis: Regulation by the microbiome. Neurobiology of Stress, 7, 124
136. Frost, G., et al. (2014). The short-chain fatty acid acetate reduces appetite via a central homeostatic mechanism. Nature Communications, 5, 3611. Hu, S., et al. (2020). Acetate and butyrate improve beta-cell metabolism and mitochondrial respiration under oxidative stress. International Journal of Molecular Sciences, 21(4). Isken, F., et al. (2010). Effects of long-term soluble vs. insoluble dietary fiber intake on high-fat diet-induced obesity in C57BL/6J mice. The Journal of Nutritional Biochemistry, 21(4), 278
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667. Kim, S. A., et al. (2019). Propionic acid induces mitochondrial dysfunction and affects gene expression for mitochondria biogenesis and neuronal differentiation in SH-SY5Y cell line. Neurotoxicology, 75, 116
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derived short-chain fatty acids and kidney diseases. Drug Design, Development and Therapy, 11, 3531. Liu, Q., et al. (2021b). Mannan oligosaccharide attenuates cognitive and behavioral disorders in the 5xFAD Alzheimer’s disease mouse model via regulating the gut microbiotabrain axis. Brain, Behavior, and Immunity, 95, 330
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1480, e12. Silva, Y. P., Bernardi, A., & Frozza, R. L. (2020). The role of short-chain fatty acids from gut microbiota in gut-brain communication. Frontiers in Endocrinology (Lausanne), 11, 25. Stilling, R. M., et al. (2016). The neuropharmacology of butyrate: The bread and butter of the microbiota-gut-brain axis? Neurochemistry International, 99, 110
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876. Vinolo, M. A., et al. (2011b). SCFAs induce mouse neutrophil chemotaxis through the GPR43 receptor. PLoS One, 6(6)e21205. Wang, Y., et al. (2017). Berberine-induced bioactive metabolites of the gut microbiota improve energy metabolism. Metabolism: Clinical and Experimental, 70, 72
84. Wong, J. M., et al. (2006). Colonic health: Fermentation and short chain fatty acids. Journal of Clinical Gastroenterology, 40(3), 235
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Dietary bioactive compounds and mitochondrial function

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11

Mitochondria-targeted antioxidants: coenzyme Q10, mito-Q and beyond

Guillermo Lo´pez-Lluch Andalusian Centre for Developmental Biology (CABD-CSIC), CIBERER, Pablo de Olavide University, Seville, Spain

11.1 Introduction Mitochondrial dysfunction is associated with most of the chronic metabolic diseases and with the progression of the systemic deterioration associated with aging (Hernandez-Camacho et al., 2018; Lopez-Lluch, 2021). Dysfunctional mitochondria not only miss the capacity to produce energy and to control many metabolic activities (Lopez-Lluch et al., 2018), but also produce high levels of reactive oxygen species (ROS) that generate oxidative damage in cells and organs (Miquel, 1998). Accumulation of damaged mitochondria contributes to a process in which dysfunctional mitochondria increase mtROS production that oxidizes molecules, mainly in the mitochondria, which increases the damage in mitochondria creating even more ROS release. This vicious cycle can be blocked by increasing mitochondrial turnover through inducing mito/autophagy and mitochondrial biogenesis and/or by the treatment with mitochondrial targeted-antioxidants able to reduce oxidative damage. The free radical theory of aging associates the unbalance in the production and elimination of ROS with the degenerative processes that accompany aging. This can be applied to the loss of functionality found in many chronic metabolic diseases (Lopez-Lluch et al., 2018). In fact, healthy aging, in which the decay of functionality of organs is slower, has been associated with the control of ROS production and the maintenance of a balanced antioxidant capacity (Maurya et al., 2016). Molecular oxygen is the final acceptor of electrons in the mitochondrial electron transport chain (mETC) where it is finally reduced to metabolic water by complex IV. During their transit through the mETC, some electrons can escape from protein complexes and react with molecular oxygen, producing superoxide anion. Under balanced metabolic conditions, low ROS levels are necessary to modulate the induction of antioxidant defenses in a hormetic response (Ba´rcena et al., 2018). In this defensive response, many antioxidant enzymes are involved. In cells, the main antioxidant activities are carried out by superoxide dismutase Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00013-8 © 2023 Elsevier Inc. All rights reserved.

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(SOD), which converts superoxide to hydrogen peroxide and catalase or glutathione peroxidase (GPx), which eliminate hydrogen peroxide producing water (Remacle et al., 1992). Other enzymes, such as thioredoxins and glutaredoxins, play a regulatory role by modulating protein activities through cysteine-redox dependent mechanisms (Arne´r & Holmgren, 2000; Ogata et al., 2021). In the lipidic milieu of cell membranes, the main antioxidant activity depends on lipidic molecules such as α-tocopherol or coenzyme Q (CoQ) (Barroso et al., 1997a,b; Takayanagi et al., 1980). CoQ is not only involved in the transport of electrons in the mETC and in many other metabolic activities in mitochondria (Lopez-Lluch, 2021). The reduced form of CoQ, ubiquinol, is crucial for protecting all cell membranes against oxidative damage (Lopez-Lluch et al., 2010; Takayanagi et al., 1980). In this chapter, the importance of the CoQ-dependent antioxidant activity in mitochondria and the use of mitochondria targeted CoQ-derived compounds in the maintenance of mitochondrial activity and the treatment of diseases is reviewed.

11.2 Importance of coenzyme Q in mitochondria CoQ is a vital factor for mitochondria in which its main function is to transport electrons through the mitochondrial respiratory chain from complexes I and II to complex III (Lopez-Lluch et al., 2010). But CoQ not only transfers electrons between mtETC complexes, it is also an essential factor to stabilize and control the assembling of these complexes in more efficient structures known as supercomplexes (Scialo et al., 2016). Further, CoQ acts also as electron acceptor of other dehydrogenases involved in many other different metabolic processes in mitochondria (Lopez-Lluch, 2021). In any case, the reduced form of CoQ, ubiquinol, is reoxidized back to ubiquinone by complex III in the mETC, contributing to the generation of the proton motile force needed to synthesize ATP. The main form of CoQ in humans is CoQ10, but in other organisms the isoprenoid tail can be shorter depending on the number of isoprene units in the tail. In mammals, the relationship between CoQ9 and CoQ10 has been associated with longevity and the generation of superoxide in mitochondria, linking oxidative stress with lifespan. Although in invertebrates, the degree of superoxide levels are not clearly associated with longevity, in vertebrates, a higher CoQ10/CoQ9 ratio is associated with a lower rate of superoxide generation and greater longevity (Lass et al., 1997). On the other hand, mitochondrial dysfunction and deficiency of CoQ10 have been associated with aging and many chronic diseases indicating an important role of CoQ10 in the control of ROS production and the onset and the severity of these diseases (Navas et al., 2021). It is widely accepted that aging is accompanied by the accumulation of dysfunctional mitochondria in cells and tissues. The accumulation of deficient

11.3 CoQ10 prevents oxidative damage

mitochondria depends on an increase in oxidative stress and a reduction of mito/ autophagy and mitochondrial turnover. As a marker of the decline of mitochondrial function during mammalian aging, mutated mtDNA genes accumulate (Greaves et al., 2012), impairing the activity of mtDNA-codified proteins (Larsson, 2010) that further aggravates the malfunction of mitochondria in a vicious cycle. In this vicious cycle, secondary CoQ deficiency associated with aging can appears in response to the OXPHOS dysfunction (Kuhl et al., 2017). In fact, downregulation of genes involved in CoQ synthesis (COQ genes), accompanied by the decrease in levels of CoQ9, have been found in conditional knock-out mouse strains showing impaired mtDNA gene expression (Kuhl et al., 2017). All these mutants show cardiomyopathy and a very short lifespan in comparison with wild type (wt) animals. In these animals, many components of the CoQsynthome, COQ3, COQ5, COQ6, COQ7, COQ8A, COQ9 and COQ10A, show a clear decrease in mitochondria, whereas the decline in mitochondrial activity progresses indicating that deficient mitochondria can lose the capacity to synthesize adequate amounts of CoQ, aggravating mitochondrial dysfunction. Therefore, mitochondrial dysfunction can generate a reduction in CoQ levels but, also, CoQsynthome deficiency can also accelerate mitochondrial dysfunction. To block this vicious cycle, upregulation of mitochondrial biogenesis, induction of mitochondrial proteases and chaperones, and metabolic changes can be induced as compensatory adaptive mechanisms to maintain the mitochondrial homeostasis of cells (Hansson et al., 2004). Further, supplementation with CoQ10 in diseases associated with mitochondrial dysfunction and CoQ depletion can be considered as protective therapy against accelerated mitochondrial dysfunction.

11.3 CoQ10 prevents oxidative damage CoQ10 shows strong hydrophobicity due to the long tail of 10 isoprene units (a total of 50 carbons). Due to this, it is incorporated from diet and nutritional supplements or treatments as a lipid and, for this reason, the highest incorporation is reached in lipid-based formulations where CoQ10 is dissolved in a fat, mainly phospholipidic, matrix (Lopez-Lluch et al., 2019). Once incorporated in to blood plasma, CoQ10 is mainly found in low-density lipoproteins (LDLs) and probably for this reason, it is easily incorporated into cells and organs expressing LDLreceptors. In cultured cells, exogenous CoQ10 is incorporated by cells in a serumdependent mechanism through its binding to lipoproteins, although shorter forms such as CoQ6 did not use this mechanism (unpublished results). Once in cells, CoQ10, CoQ9, and CoQ6 reach mitochondria through the endomembrane system (Fernandez-Ayala et al., 2005a). This mechanism explains why these compounds incorporate primarily to the lyso-endosomal system and reach mitochondria at a similar level. However, the influence of the CoQ molecule on the mtETC activity depends on the length of the tail and, the shorter the length of

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the isoprene tail, the more disturbances of the mtETC activity, and the higher oxidative stress (Fernandez-Ayala et al., 2005b). For this reason, shorter forms of CoQ cannot be considered suitable molecules to restore mtETC activities. Apart from its effect in restoring mtETC activity in deficient cases (for a more in-depth review, see (Hernandez-Camacho et al., 2018; Navas et al., 2021; Salviati et al., 2017; Yubero et al., 2016)), CoQ10 protects cell membranes against oxidative-damage-dependent apoptosis and mainly ferroptosis (Stockwell et al., 2020). Under stressful conditions, CoQ10 in cell membranes, prevents the activation of apoptotic signaling such as with ceramides and reduces cell death (Barroso et al., 1997b). In this role, CoQ10 acts in combination with known antioxidants such as α-tocopherol or ascorbic acid (Barroso et al., 1997a). The antioxidant activity of CoQ also prevents lipid peroxidation in mitochondrial membranes acting as the key antioxidant factor as demonstrated by the inhibition of lipid peroxidation in mitochondrial membranes depleted of α-tocopherol (Mellors & Tappel, 1966). The mtETC-dependent lipid peroxidation is also controlled by the concentration of ubiquinol, adding an essential function of CoQ to mitochondrial protection (Takayanagi et al., 1980). Further, massive production of superoxide both intracellularly by mitochondria or intracellular redox activities, or extracellularly by NADPH oxidases, can produce cell damage that is avoided by the tandem CoQ/α-tocopherol in cell membranes (Stoyanovsky et al., 1995). Taken into consideration the role of oxidative stress and the accumulation of oxidative damage in aging (Mehdi et al., 2021), prevention of oxidative damage of membranes (Bello et al., 2005) and DNA (Quiles et al., 2005) explains why supplementation with CoQ10 increases longevity and avoids senescence (Marcheggiani et al., 2019).

11.4 Structure of coenzyme Q and mitochondrial-targeted coenzyme Q-related compounds CoQ10 is a strong hydrophobic molecule due to its long tail of ten isoprene units. Mitochondrial-targeted related compounds show shorter side-chains with a tail of no more than ten carbons (Fig. 11.1). An important difference with the natural molecule, is that the hydrophobicity of these CoQ10-related compounds is very low in comparison to the natural molecule. The different hydrophobicity of these compounds affects their activity in mitochondria and their relationship with CoQdependent activities like mtETC. In general, CoQ-associated compounds have an identical head to any CoQ but they differ in the nature of the hydrophobic tail (Fig. 11.1). Idebenone (IDE) (2,3dimethoxy-5-methyl-6-(10-hydroxy)decyl-1,4-benzoquinone) is a short-chain synthetic CoQ analog that crosses cell membranes easily with a ten-carbon decyl tail, with a hydroxyl group at the tenth carbon. It is considered a potent free-radical scavenger (Mordente et al., 1998). Similarly, decylubiquinone (2,3-dimethoxy-5-

11.4 Structure of coenzyme Q and mitochondrial-targeted coenzyme

FIGURE 11.1 Structure of CoQ10-dependent molecules used in human therapies or in preclinical studies.

methyl-6-decyl-1,4-benzoquinone) shows the same structure but lacks the hydroxyl group at the tenth carbon. MitoQ, (10-(6’-ubiquinyl)decyl triphenyl phosphonium), was described by the group of Dr. M. Murphy in 2001 as a mitochondrial-targeted antioxidant that selectively blocks mitochondrial oxidative damage (Kelso et al., 2001). The compound is a derivative of CoQ that enters selectively into mitochondria through the binding of a lypophilic triphenylphosphonium cation to the ubiquinone head through a ten-carbon aliphatic chain (Kelso et al., 2001). SKQ1, (10-(6-plastoquinonyl) decyltriphenylphosphonium bromide) is a cationic plastoquinone derivative that uses the same cation as MitoQ to enter mitochondria. SKQ1 is used as an antioxidant at extremely low concentrations (Khailova et al., 2015). It seems that the reduced form, SKQ1H2, is better as an antioxidant than MitoQ (Antonenko et al., 2008a,b). EPI-743 or Vatiquinone (2-((R,6E,10E) 2 3-hydroxy-3,7,11,15-tetramethylcyclohexadeca-6,10,14-trien-1-yl) 2 3,5,6-trimethylcyclohexa-2,5-diene-1,4-dione) is an α-tocopherol derivative. It has been designed as a potent antioxidant showing the same capacity of redox cycling as CoQ10 and IDE (Enns et al., 2012). Other new antioxidants compounds targeted to mitochondria are: BGP-15 (N-[2-hydroxy-3-(1-piperidinyl)propoxy] 2 3-pyridinecarboximidamide dihydrochloride), which accumulates in mitochondria and reduces ROS levels (Sumegi et al., 2017), and XJB-5
131, (4-hydroxy-2,2,6,6-tetramethyl piperidine1-oxyl nitroxide), a bi-functional antioxidant that comprises a radical scavenger bound to a mitochondrial-targeting peptide based on the modification of the Leu-DPhe-Pro-Val-Orn segment of the antibiotic, gramicidin S (Zhiyin Xun et al., 2012).

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These compounds are not based on the structure of CoQ10 and are only used as oxidative radical scavengers.

11.5 Idebenone reduces reactive oxygen species levels and bypasses complex I-deficiency Water-soluble homologs of CoQ and vitamin E can be reduced by antioxidant enzymes such as a plasma-membrane redox system maintaining an antioxidant redox cycle that blocks oxidative damage (Kagan et al., 1998). In its function, the length of the tail of these molecules affects their capacity to reduce oxidative damage. For these compounds, the shorter the length of the tail, the lower the insertion in cell membranes (Kagan et al., 1990). However, as it is more hydrophilic, there is a higher antioxidant activity in liposomes, although natural α-tocopherol was the most stable compound in the membrane and did not produce disturbances nor membrane damage (Kagan et al., 1986, 1990). These experiments demonstrated that more hydrosoluble antioxidants are important to avoid initiation phases of oxidative damage. However, when initiated, the lipid-peroxidation cascade is blocked with higher efficiency by more stable liposoluble compounds (Kagan et al., 1990). In this activity, NQO1 and NQO2 can use short-chain quinones as substrates and then maintain a redox cycle of these quinones improving their activity as antioxidants and superoxide scavengers (Haefeli et al., 2011). For this reason, IDE is a strong antioxidant, acting outside the mitochondrial membrane, probably at the outer face of the inner mitochondrial membrane, where it directly scavenges superoxide (Geromel et al., 2002). In fact, IDE is located at the lipid/water interface of membranes (Gomez-Murcia et al., 2016). It seems clear that the main mechanism of action of IDE is to avoid oxidative stress and then reduce oxidative damage in mitochondria (Genova et al., 2003). As early as 1984, it was proposed that IDE protected cell membrane against lipid peroxidation (Suno & Nagaoka, 1984). Further, the NQO1-dependent redox cycle of IDE can explain its capacity to maintain an antimycin A-sensitive transfer of electrons from cytosolic NAD(P)H to mETC. This would explain its effect in the recovery of mitochondrial activity, specifically in mETC complex I-deficient diseases (Haefeli et al., 2011). In fact, it have been shown that the capacity of IDE to rescue mitochondrial function, depends on the activity of NQO1 (Erb et al., 2012), and this effect is also associated with the capacity of the reduction of lipid peroxidation under defective complex I activity. Further, an activating effect of IDE on glycerol phosphate oxidation in brown adipose tissue was found, whereas NADH oxidoreductase activity was inhibited (Rauchova et al., 2008). Interestingly, IDE and CoQ1 were able to reduce glycerol phosphate and complex II-dependent ROS production, evidence of their role as ROS scavengers (Rauchova et al., 2006).

11.6 MitoQ a strong antioxidant that protects against apoptosis

On the other hand, exposure of cells in culture to IDE (around 25 μM) can also induce cell death (Tai et al., 2011) in dopaminergic neurons. This effect has also been associated with the activity of NQO1. It has been proposed that optimizing NQO1 expression in cells may be a key to improve IDE therapeutic potential. NQO1 is found mainly in glia in the brain and these cells can probably be considered the target of the treatment and neuroprotective effects (Jaber & Polster, 2015). The therapeutic use of IDE must be carefully taken into consideration in the context of a functional complex I. With functional complex I, IDE can reduce with one electron by flavin in complex I, generating the semiquinone form that can increase ROS levels. In this case, IDE increases ROS but also inhibits the reduction of physiological CoQ10. On the other hand, in the case of a deficiency of CoQ10, IDE can preserve NADH oxidation, generating NAD1, and by this mechanism maintain mitochondrial activity (King et al., 2009). Interestingly, mitochondrial-located NQO1-dependent reduction of 2-methoxy-1,4 naphtoquinone, another CoQ10 analog, also oxidizes NAD1, maintaining substrate-level phosphorylation and bypassing complex I inhibition (Ravasz et al., 2018). The effect of IDE in brain mitochondrial activity also depends on the activity of NQO1. The levels of NQO1 are high in astrocytes and low in neurons. For this reason, IDE is able to support oxygen consumption in complex I, inhibiting astrocytes but not in cortical neurons (Jaber et al., 2020).

11.6 MitoQ a strong antioxidant that protects against apoptosis and induces mitophagy The number of publications about this compound has increased during the last three years, indicating a rise in its importance in many different processes in which oxidative damage is one of the key factors. MitoQ moves into mitochondria where it is quickly reduced to the quinol form (Ross et al., 2008). It reduces ROS generation and mitochondrial thiol oxidation, preserving (by these mechanisms) mitochondrial function after GSH depletion (Lu et al., 2008). As the mechanism of action, MitoQ is able to reduce ROS production from reverse electron transport, explaining why malonate inhibits its protective effect. However, it increases the production of superoxide derived from the forward transport, probably by disturbing supercomplexes’ activities (O’Malley et al., 2006). Its different activities can be associated with its location at the inner surface of the inner mitochondrial membrane. For this reason, MitoQ can interact with complex II, but poorly with complex III and ETF:quinone oxidoreductase (James et al., 2007). The capacity of MitoQ as an antioxidant is strong and it is considered an important antioxidant against ferroptosis. In cell models of neuronal HT22 and MEFs cells, ferroptosis induced by GPX4-inhibition, generates mitochondrial fragmentation, decreases mitochondrial redox potential and reduces mitochondrial

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respiration. However, the presence of MitoQ preserves mitochondrial integrity and function and prevents ferroptosis (Jelinek et al., 2018). In an old-mice model of nitrosamine stress, the treatment with 500 μM mitoQ in water during five weeks was able to supress synaptosomal lipid peroxidation and nitrosamine levels, and also reduce caspase 3 and caspase 9 activities (Maiti et al., 2018). MitoQ also reversed ONOO(2)-dependent mitochondrial complexes I and IV inhibition, increased ATP production and reduced mitochondrial potential loss, indicating a high protective effect against mitochondrial damage in both in vitro and in vivo models (Maiti et al., 2018). In beef heart mitochondria, oxidized mitoQ can be reduced by mtETC and blocked by the complex II inhibitor malonate (Kelso et al., 2001). Complex III inhibitor, myxothiazol, inhibits their reoxidation, indicating the interaction of this compound with components of the mtETC. However, in KO yeast for CoQ synthesis, the addition of mitoQ did not restore respiration whereas, CoQ2 did, indicating that the compound is unable to restore mtETC activity in the absence of CoQ. Most likely, the surface location of mitoQ at the matrix face of the mitochondrial inner membrane blocks the capacity of the compound to maintain normal mtETC activity in the absence of CoQ. For all these effects, MitoQ is considered a tool to determine the effect ROS have on apoptosis (Dhanasekaran et al., 2004; Saretzki et al., 2003). For example, this compound was used to unveil the critical role of mtROS in heart cell death (Kalivendi et al., 2005), or endothelial cells (Dhanasekaran et al., 2004). In endothelial cells, a decrease in mtROS levels by MitoQ also blocked the activation of ERK2 and the proliferating response to hypoxia (Schafer et al., 2003), indicating the role of mtROS in the control of endothelial response to hypoxia.

11.7 Pharmacokinetics of mitochondrial-targeted antioxidant One of the key aspects of these compounds is their pharmacokinetics. The hydrophobicity of CoQ10 and its large molecular weight, reduces the capacity of nutritional CoQ10 to reach blood plasma (Bhagavan & Chopra, 2007). Recently, it has been demonstrated that nutritional CoQ is taken by intestinal epithelium through Niemann-Pick Like 1 protein, a transporter involved in the intestinal absorption of fat-soluble components (Nashimoto et al., 2020). For this reason, fatsolubilized formulations are preferred to powder-based presentations (Bhagavan & Chopra, 2007; Lopez-Lluch et al., 2019). From them, chilomicrons are taken by liver and exogenous CoQ10 assembled to lipoproteins in hepatocytes to be released into blood plasma. On the other hand, IDE is quickly metabolized by CYP enzymes and disappears in plasma within a few hours (Becker et al., 2010). Quickly, the metabolites are sulfated and glucuronidized even at the intestine, and shorter forms QS10,

11.7 Pharmacokinetics of mitochondrial-targeted antioxidant

QS8, QS6 and QS4 can be found in plasma (Fig. 11.2) (Bodmer et al., 2009). Contrary to CoQ10, animal experiments have demonstrated that IDE can cross the blood-brain barrier and distribute to cells and mitochondria (Torii et al., 1985). Interestingly, QS10 has been proposed to be more active than IDE in the treatment of complex I-defects and in CoQ10 deficiencies (Giorgio et al., 2018). In any case, IDE incorporation into different types of delivery systems (liposomes, cyclodextrins, microemulsions, self-micro-emulsifying drug delivery systems, lipid-based nanoparticles, polymeric nanoparticles) tries to increase its bioavailability (Montenegro et al., 2018). Further, treatment of patients, especially Friedreich’s ataxia patients (FRDA), have demonstrated it’s safety and tolerance without showing secondary effects (Di Prospero et al., 2007b). Up to 60 mg/kg/ day in 11 different clinical studies demonstrated improvement in cardiac hypertrophy and some neurological symptoms in FRDA patients (Meier & Buyse, 2009).

FIGURE 11.2 Products of the metabolization of Idebenone.

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As in the case of IDE, animal studies have demonstrated that the bioavailability of MitoQ can be limited by internal epithelial cell metabolism and assimilation (Li et al., 2007a). Similarly to IDE, the bioavailability of mitoQ through intestine epithelial cells depends on lipophilicity, affinity of the transporter, protein binding (albumin) and affinity for phase 2 metabolizing enzymes (Li et al., 2008). After oral ingestion, MitoQ can be found quickly in rat plasma in different forms: hydroxylated, desmethyl and the glucuronide and sulfate conjugates of the quinol form indicating a rapid metabolization (Li et al., 2007b). This suggests that mitoQ undergoes enterohepatic recycling that ends in the glucuronide form of the compound as the main metabolite. On the other hand, MitoQ shows a sustained and low bioavailability after oral treatment, however it quickly disappears after intravenous injection, probably indicating a fast incorporation into the tissues (Li et al., 2007b). MitoQ is also a well-tolerated compound. Chronic supplementation with high doses of mitoQ in C57BL/6 mice during 28 weeks did not produced negative effects indicating the compound was safe (Rodriguez-Cuenca et al., 2010). In vivo experiments with C57BL/6J female mice demonstrated that the mesylate β-cyclodextrin form of MitoQ is able to increase β-oxidation of fatty acids and ATP generation in gastrocnemius muscle at the same time that it reduces oxidative stress and maintains NRF2, catalase, and MnSOD levels (Ju et al., 2017). These effects indicate its safety and also its capacity to reach tissues reflective of the treatment with CoQ10. In humans, its use even in healthy older adults has also demonstrated its high-tolerance and safety (Rossman et al., 2018).

11.8 Therapeutic use of idebenone IDE is already used as a therapeutic compound in the treatment of some diseases, especially in complex-I deficient diseases such as FRDA and Leber Hereditary Optic Neuropathy (LHON).

11.8.1 Therapeutic use of idebenone in Friedreich ataxia The main disease in which IDE has shown high effectiveness is FRDA. FRDA is a degenerative disorder caused by reduction of frataxin levels that results in mitochondrial dysfunction and oxidative damage (Seznec et al., 2004). Frataxin decrease causes a respiratory deficiency due to mitochondrial iron accumulation and oxidative stress. The increase in ROS levels ends in the reduction of complex I activity and in mitochondrial dysfunction (Calabrese et al., 2005). Treatment with IDE has demonstrated its efficacy and its capacity to delay the progression of the disease in many aspects. However, the effect of IDE on this disease is controversial, as some studies showed a limited dose response in the improvement of the international cooperative ataxic rating scale (ICARS)

11.8 Therapeutic use of idebenone

(Di Prospero et al., 2007a), whereas other studies did not report significant differences even after five years of treatment (Pineda et al., 2008). Further, the effect of IDE may depend on the organ affected. Whereas, no effect was found in muscle mitochondria in FRDA patients (Schols et al., 2001), 12 months of treatment in children improved cerebellar ataxia (Artuch et al., 2002). In cardiac function, long trials with IDE resulted in positive effects in ventricular hypertrophy in these patients (Mariotti et al., 2003). Although even in these cases, other studies have found no effect of 5 mg/kg/day of IDE in ventricular hypertrophy (Lagedrost et al., 2011), or an effect on neurological symptoms (Lynch et al., 2010; Rinaldi et al., 2009). Perhaps these studies represent a dose-response dependent effect being intermediate (15 mg/kg/day) and high (40 mg/kg/day), safe, and more beneficial for cardiac hypertrophy and neurological symptoms (Schulz et al., 2009). Most likely, the combination of IDE with iron chelators such as deferiprone are the best strategy to improve the protection against cardiovascular and neurological disorders in FRDA patients (Velasco-Sanchez et al., 2011). Moreover, a combination of IDE with darbepoetin-α and riboflavin also improves symptoms in FRDA patients (Arpa et al., 2013, 2014).

11.8.2 Idebenone treatment of leber hereditary optic neuropathy and other neuropathic diseases IDE is also used in the treatment of LHON (Tonagel et al., 2021) and other related neuropathy diseases. LHON is a mitochondrially-inherited disease usually caused by different pathogenic mitochondrial DNA mutations affecting mtETC complex I proteins such as ND4, ND1 or ND6 (Yu-Wai-Man et al., 2016). The effect of IDE on this disease is independent of the type of mutation that causes the disease (Jorstad et al., 2018). IDE has been successfully used in the treatment of LHON if it is used early in the progression of the disease (Carelli et al., 2011; Klopstock et al., 2011). Its effect seems to be dependent on the maintenance of mitochondrial activity when complex I is not functional. Incubation of LHON-patients’ fibroblasts during 24 h with IDE, improved complex I activity although respiration seems to be impaired, indicating a conflicting effect probably because the phenolic head of IDE can consume oxygen (Angebault et al., 2011). The reduced form of IDE, idebenol, can feed electrons to complex III in cibrids from patients showing the LHON MTND1 mutations, improving the complex I deficiency (Giorgio et al., 2012). In vitro studies on fibroblasts from LHON patients showed a positive effect on IDE, reducing ROS levels and increasing ATP production. These effects were less clear with CoQ1, decylubiquinone (decylQ), or CoQ10 (Yu-Wai-Man et al., 2017). Further, the effect of IDE seems to depend on the presence of NQO1, since in mouse retina NQO1-deficient cells, the treatment generated cell death (Varricchio et al., 2020).

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In clinical trials, a 11778/ND4 LOHN patient improved visual capacity with nine months of treatment with 900 mg IDE/day (Sabet-Peyman et al., 2012). Other studies have demonstrated that IDE is a valuable agent to treat visual impairment both in adolescents (900 mg during 24 weeks) and adults (LysengWilliamson, 2016). A recent study has demonstrated that IDE treatment promotes the recovering of vision and the maintenance of good residual vision if the treatment is initiated early and maintained during, at least, 24 months (Catarino et al., 2020). Further, the combination of IDE with vitamin B2 and vitamin C accelerates the recovery of vision in these patients (Mashima et al., 2000). Importantly, IDE preserves or re-establishes retinal ganglion cell function during acute phases of the disease, protecting cells against apoptosis (Klopstock et al., 2013). IDE has also been used in the treatment of other optic diseases such as dominant optic atrophy or age-related macular degeneration. IDE exerted protective effect in patients suffering from OPA1-mutation dependent dominant optic atrophy (Barboni et al., 2013). This effect was associated with the reduction of oxidative stress due to OPA1 deficiency (Yarosh et al., 2008). On the other hand, age-related macular degeneration is the leading cause of blindness due to the degeneration of the retinal pigment epithelium. This disease has also been associated with oxidative stress. In a culture model using ARDE-19 cells, IDE increased the survival and reduced cell death and senescence by stabilizing the BAX/Bcl-2 ratio and probably reducing oxidative stress (Arend et al., 2015).

11.8.3 Therapeutic use of idebenone in other oxidative-damage related diseases IDE has also been used in the treatment of other diseases in which oxidative stress in involved. For example, in Leigh syndrome (LS), IDE did not produce an effect on respiratory failure but did reduce against oxidative damage (Haginoya et al., 2009). In cardiovascular diseases (CVD), IDE can prevent oxLDL-induced endothelial dysfunction and also inhibit mitochondrial dysfunction induced by oxLDL (Lin et al., 2015). Further, due to its capacity to prevent complex I deficiency in ischemia/reperfusion, IDE can maintain mitochondrial capacity during this process (Perry et al., 2019). In the case of mitochondrial cardiopathy, a case report showed that IDE produced a clear improvement in the clinical status of the patient (Lerman-Sagie et al., 2001). IDE also improved cardiovascular activity in Duchenne muscular dystrophy (DMD) patients after 12 months of treatment affecting the left ventricular (LV) wall, the region most affected by this disease (Buyse et al., 2011). Further, IDE reduced the loss of respiratory function (Buyse et al., 2015; Mayer et al., 2017) probably through preserving inspiratory muscle function (Buyse et al., 2017). In fact, in the DMD long-term IDE study (DELOS), IDE treatment protected respiratory function associated with a reduced risk of bronchopulmonary complications and a reduced need for systemic antibiotics (McDonald et al., 2016).

11.9 Therapeutic activity of MitoQ

Another interesting effect of IDE has been associated with the treatment of Type 2 diabetes. It has been considered as a cytoprotective agent by interacting with Shc protein and reducing its competition with IRS1, improving by this mechanism, insulin signaling (Dassano et al., 2019; Tomilov et al., 2018). IDE obstructs p52Shc interaction with the insulin receptor, permitting the binding of IRS1 to the receptor and thus, increasing insulin sensitivity. In a recent study in mice, two IDE analogs showed more capacity than IDE to increase sensitivity (Hui et al., 2020). Currently, the exact mechanism by which IDE blocks this interaction is under study. In recent years, IDE has gained importance in the treatment of degenerative diseases. Treatment with IDE increases lifespan in a mice model of neurodegenerative diseases associated with motor dysfunction; Parkinson’s and Huntington’s Diseases (PD and HD) (Gerhardt et al., 2011). Neuroinflammation is currently considered an important factor in the progression of these diseases (Clark & Kodadek, 2016) and IDE has demonstrated capacity to alleviate it by decreasing ROS levels (Yan et al., 2018). In fact, it has been proposed that IDE can act as an active agent by inhibiting NLRP3 via SIRT3/SOD2/mtROS pathway although the direct capacity of IDE cannot be discarded (Jiang et al., 2020). Recent studies in a mouse model have also demonstrated the capacity of IDE to reduce the release of pro-inflammatory cytokines in spontaneous chronic murine colitis (Shastri et al., 2020a) and in acute murine colitis by inducing antioxidative antiinflammatory pathways (Shastri et al., 2020b). The capacity of IDE to reduce ROS levels may decrease NLRP3 activation and therefore, the release of IL-1β and TNFα in septic rats (Akpinar et al., 2021). Further, mitochondrial dysfunction and oxidative stress has also been associated with autoimmune diseases such as lupus erythematosus. In a murine model of lupus, treatment with IDE reduced mortality and attenuated glomerular inflammation and fibrosis, reducing markers of inflammation such as IL-17A and IL-18. It seems clear that the main effect of IDE as therapeutic compounds is through reducing oxidative stress and ROS-dependent cell damage and inflammation.

11.9 Therapeutic activity of MitoQ MitoQ has been used as a putative therapeutic compound for many diseases in which oxidative stress and mitochondrial dysfunction are involved. Many studies have been carried out in rodents in which oxidants are introduced to determine the putative protective effect of MitoQ in the development of the disease.

11.9.1 MitoQ use in inflammation and immune response One of the most promising therapeutical uses of MitoQ is in the prevention of inflammation and the control of the side effects of pathogen infections. MitoQ

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has been proposed to fight against inflammation associated with sepsis, indicating that oxidative stress from mitochondria could be a key factor in this inflammatory response (Lowes et al., 2008). MitoQ decreased oxidative stress and protected mitochondria in an endothelial cell model and in a rat model of sepsis. Treatment with mitoQ reduced the release of pro-inflammatory cytokines and increased the release of the anti-inflammatory IL-10 (Lowes et al., 2008). Further, the release of endotoxins in bacterial infections can affect cardiac mitochondrial activity, which affects cardiac contractility. In adult rats, endotoxin-induced reduction of state 3 respiration rates and control ratio in cardiomiocytes was prevented by MitoQ administration, including protection against caspase 3 and 9-dependent apoptosis (Supinski et al., 2009). In LPS-induced intestinal injury for 30 min, MitoQ was able to reduce diamine oxidase activity, D-lactate release, and histological damage through inhibition of oxidative stress and the inflammatory response. Interestingly, MitoQ induced antioxidant activity through activating the NRF2 response as indicated by NOQ1 and HO-1 levels and increasing SOD and GSH activity, accompanied by a reduction in the markers of inflammation such as IL-1β, IL-6, TNFα, and NO levels (Zhang et al., 2020). It seems clear that the common denominator of the protective effect of MitoQ against inflammation is the reduction of oxidative stress and the protection against mitochondrial dysfunction. For example, in a dextran sulfate sodium-induced mouse model of colitis, MitoQ significantly reduced oxidative damage and reduced mitochondrial injury and the subsequent activation of the NLRP3inflammasome and the release of inflammatory cytokines IL-1β and IL-18 (Dashdorj et al., 2013). In inflammation associated with hemorrhagic shock, liver damage is accompanied by the increase of superoxide release. Treatment with MitoQ was able to reduce TNFα and IL-6 release and modulate oxidative damage and morbidity. In this case, TPP itself, also reduced hepatic necrosis and the effect on TNFα and IL-6 was also found with TPP alone, probably indicating an indirect role (Powell et al., 2015). On the other hand, in diabetes, mitoQ but not TPP, reduced superoxide production and increased GPx1 presence in polymorphonuclear leukocytes. MitoQ, but not TPP blocked the rolling velocity of leukocytes from diabetic patients on the endothelium and reduced the rolling flux and adhesion, restoring the levels found in control. Further, in these cells, MitoQ also reduced TNFα levels and NF-κB p65 activity (Escribano-Lopez et al., 2016). On the other hand, high doses of MitoQ can produce the contrary effect. In fact, MitoQ and other antioxidants such as NAC, show different effects on the inflammatory response, depending on the dose. At low doses, these compounds inhibit NF-κB through activating IKKα, resulting in the inhibition of TFNα release and ICAM-1 induction. However, high levels of NAC or MitoQ can cause inhibition of IKKα by glutathionylation and then, produce the opposite effect increasing NF-κB (Mukherjee et al., 2007). In the case of viral infections, MitoQ has been used mainly to protect against side effects of infection or inflammation. For example, MitoQ treatment was used in a chronic hepatitis C (HCV) clinical trial to prevent oxidative stress and

11.9 Therapeutic activity of MitoQ

mitochondrial damage. MitoQ (40
80 mg/day) in HCV patients decreased ALT and AST levels indicating a reduction in liver damage (Gane et al., 2010). Venezuelan equine encephalitis virus TC-83 used as an in vitro model of virus-dependent cell damage, produces pro-inflammatory cytokine release. This cytokine release has been associated with mitochondrial changes resulting in the induction of mitophagy in microglial cells. In this case, treatment with MitoQ mesylate reduced the levels of cytokine released and reduced neuroinflammation (Keck et al., 2018b) affecting mainly microglia (Keck et al., 2018a). A similar effect was found in the case of respiratory syncytial virus (RSV) infection. RSV induces mitochondrial redistribution in infected cells and changes to complex Idependent mitochondrial bioenergetics. In Complex I knock out (KO) cells, respiration is low whereas RSV proliferation is higher in comparison with wt cells. This effect correlated with the increase in ROS levels found in KO cells. By blocking ROS levels MitoQ mesylate reduced the proliferative capacity of RSV (Hu et al., 2019), indicating a putative antiviral capacity of mitoQ through controlling mtROS levels. Its activity of decreasing mitochondrial dysfunction can be important in the maintenance of immune activity in autoimmune diseases and aging. For example, in Lupus erythematosus, mitochondrial dysfunction has been related with this disease through an increase of mtROS in T-cells and neutrophils. This increase in mtROS is involved in the oligomerization of Mitochondrial Antiviral-Signaling Protein (MAVS) that end the increase in interferon (IFN)-I (mainly IFN-α and β) release (Buskiewicz et al., 2016). An increase in mtROS in neutrophils induces neutrophil extracellular traps (NETs) that also increase in Lupus and affects kidney function (Fortner et al., 2020). In the lupus-prone MRL-lpr mice 200 μM, MitoQ was used from weaning to 11 weeks. As suspected, MitoQ reduced ROS in neutrophils and also the NET formation. This decrease reduced MAVS oligomerization and subsequently, the release of IFN-I (Fortner et al., 2020). This opens up the possibility to use MitoQ and other mitochondria-targeted antioxidants in the reduction of symptoms of this disease. In the case of aging, MitoQ has been considered an anti-senescence compound for immune cells (Marthandan et al., 2011). Interestingly, treatment with MitoQ has been proposed in the treatment of COVID-19 disease to reduce cytokine storms and to restore the function of T cells, especially in aged individuals (Ouyang & Gong, 2020).

11.9.2 MitoQ as a treatment in neurodegenerative diseases Human studies and animal models for Alzheimer’s, Huntington’s and Parkinson’s diseases have been widely used to determine if MitoQ can be used to delay the progression of the diseases. The capacity of the compound to pass through the blood-brain barrier and accumulate in microglia and neuron mitochondria warrants the idea about its use to prevent oxidative damage and restore mitochondrial function in these diseases (Oliver & Reddy, 2019).

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In most cases, the mechanism of action of MitoQ is to prevent mitochondrial dysfunction and mtROS increase due to different inducers of the disease. For example, in C. elegans model of Alzheimer’s disease (AD), MitoQ extended lifespan, delayed β-amyloid induced paralysis, ameliorated depletion of cardiolipin (CL), and protected Complex IV and I in the electron transport chain (etc) but did not protect against oxidative stress or moderate ROS levels (Ng et al., 2014). However, in mice, MitoQ attenuated β-amyloid-induced neurotoxicity and apoptosis by preventing mitochondrial damage (McManus et al., 2011). In this model, two to seven month-old 3xTg-AD mice treated with MitoQ mesylate showed an improvement of AD symptoms from developing. Further, in AD-developed female mice, MitoQ also improved memory retention and a decrease in brain oxidative stress, synapse loss, astrogliosis, and microglial cell proliferation (neuroinflammation) and increased lifespan. These results indicate a role of ROS and inflammation in AD progression (Young & Franklin, 2019). In a cell model of HD with striatal neurons stably expressing mutant hungtingtin (Htt), (STHDhQ111/Q111), the treatment with MitoQ produced a downregulation of fission proteins, Drp1 and Fis1, whereas it increased MFN1, MFN2 and OPA1, indicating a change in mitochondrial dynamics to fusion (Yin et al., 2016). Further, in a mouse model of autoimmune encephalomyelitis, preventive treatment with MitoQ reduced neuronal cell loss in the spinal cord, likely through the inhibition of oxidative stress and axonal and glia inflammation (Mao et al., 2013). In addition, in an amyotrophic lateral sclerosis mouse model, SOD1 (G93A), MitoQ prolonged lifespan of the animals by again, reducing ROS levels (Miquel et al., 2014). All these models indicate that MitoQ is a promising agent in the treatment of neurodegenerative diseases mainly associated with aging or with mitochondrial dysfunction. However, in humans, the studies are scarce and results are not clear. For example, in a study performed in 128 newly diagnosed PD patients, MitoQ did not produce any effect on the progression of the disease as determined through the Unified Parkinson’s Disease rating Scale (Snow et al., 2010), although this disease has been associated with dysfunction of mitochondria by complex I deficiency (Flønes et al., 2018). MitoQ probably cannot restore the mitochondrial dysfunction associated with this disease. On the other hand, MitoQ has been also considered in the treatment of neurodegeneration caused by external agents. In the case of organophosphate pesticides such as dichlorvos, MitoQ protected against cell damaging by suppressing DNA fragmentation, cytochrome c release, and caspase 3 activation. MitoQ also reduced protein, lipid, and DNA oxidative damage, and changes in mitochondrial structure (Wani et al., 2011). Other studies used a mice model of Pb accumulation in brain, in which MitoQ efficiently alleviated peroxynitrite-mediated mitochondrial complexes II, III and IV inhibition, increasing the levels of ATP, restoring mitochondrial redox potential, and reducing caspase 3 and 8 activation and apoptosis (Maiti et al., 2018).

11.9 Therapeutic activity of MitoQ

Interestingly, in recent years, the use of MitoQ has been considered to prevent oxidative damage in neurological damage induced by traumatic brain injury (TBI). In a mouse model of TBI, MitoQ protected against injury by increasing antioxidant capacity of cells via inducing SOD and GPx and decreasing lipoperoxidative damage. As in other cases, MitoQ induced NRF2 activation, translocation to the nucleus, and induction of the expression of HO-1 and NQO1 enzymes (Zhou et al., 2018). In rats suffering subarachnoid hemorrhage (SAH), another model of TBI, MitoQ reduced oxidative stress 1 h after SAH and protected neurons from death in the short- and long-term. In this model, MitoQ reduced Keap1, Romo1, Bax, and Caspase 3 activation, whereas it increased NRF2, Bcl-X, and PINK1/PARKIN, PHB2, and LC3II indicating the inhibition of apoptosis and the induction of mitochondrial mitophagy and turnover (Zhang et al., 2019a). Further, MitoQ attenuated blood-brain barrier disruption avoiding further damage of the central nervous system (Zhang et al., 2019b). Inflammation is also a main factor in TBI damage. After intracerebral hemorrhage, M1 microglia promotes inflammatory injury whereas M2 microglia inhibits neuroinflammation. In this situation, MitoQ attenuated neurological deficits and reduced inflammation, edema and hematoma volume. Further, it also reduced M1 markers at the same time that it increased M2 markers in an effect attributed to the inhibition of mtROS that blocks the activation of inflammasome as has also been found in FeCl2-treated microglia (W Chen et al., 2020a). In this last model, MitoQ also blunted the loss of oligodendrocytes and their precursor cells reducing demyelination and axon swelling, processes that are associated with white matter injury (W Chen et al., 2020b). All these effects have permitted to take into consideration the use of this compound and other antioxidants in the mitigation of the damage produced by oxidative stress in TBI (Ismail et al., 2020). Treatment of peripheral neurological diseases with MitoQ has also been considered. In progressive optic neuropathy (PoAG), one of the leading causes of irreversible blindness, TGFβ2 and the subsequent remodeling of extracellular matrix are markers of early pathology. As in other cases, the increase of ROS and oxidative damage accompany the pathology. In a cell culture model of primary or transformed human trabecular meshwork, cells treated with TGFβ2, activation of Smad2/3 signaling, and collagen mRNA were markers of fibrosis. The presence of MitoQ (10 nM) attenuated the effect of TGFβ2 on ROS decreasing Smad2/3 signaling and reducing collagen synthesis. These effects open up the opportunity to use MitoQ in the treatment of this disease (Rao et al., 2019). Another interesting effect in which MitoQ has shown therapeutic capacity is the improvement of pain sensitivity and glial activation after chemotherapy with Vincristine. Vincristine produced oxidative stress in spinal cord tissues in mice. Oxidative damage was inhibited by MitoQ and also the release of proinflammatory cytokine. Vincristine increased fission through activating Drp-1 by phosphorylation and also by increasing Fis-1 levels, an effect that was abrogated by MitoQ (XJ Chen et al., 2020). Similarly, in peripheral neuropathy associated

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with diabetes induced by high fat diet, MitoQ 0.93 g/kg diet during 12 weeks in obese and type 2 diabetic rats, produced positive effects. MitoQ improved motor and/or sensory nerve conduction velocity, density of cornea and intraepidermal nerve fiber and thermal nociception. These results also indicate that mitochondrial dysfunction and oxidative damage is important in this effect of diabetes and that MitoQ can used to prevent these consequence (Fink et al., 2020).

11.9.3 Rare diseases Many rare diseases are caused by mutations causing dysfunctional enzymes involved in important metabolic processes. In most of these processes, mitochondrial dysfunction, and oxidative damage are key in the onset and evolution of the disease. In some of these diseases, MitoQ, through restoring mitochondrial fusion or reducing oxidative damage, could be a putative therapeutic compound. For example, in spinocerebellar ataxia type 1 (SCA1), the unstable polyglutamine expression produces premature degeneration of Purkinje cells ending in dysfunction of motor coordination and death around 10
15 years. There are currently no therapies for this disease. In search of putative therapies, in a model of SCA1 disease (54Q/2Q) in mice, MitoQ was able to restore mitochondrial function and prevent SCA1-associated pathology acting at both presymptomatic and symptomatic stages, preventing oxidative damage of DNA and Purkinje cell loss (Stucki et al., 2016). Further, in the rare disease X-linked adrenoleukodystrophy (X-ALD), caused by a defective peroxisomal ABCD1 transporter, MitoQ prevented axonal degeneration and locomotor disability (Guha et al., 2021). In the Charcot-MarieTooth disease, the IGDAP-1-null mouse model was used to demonstrate that MitoQ prevented weight gain and ameliorated the motor coordination deficiency halting the decay in mitochondrial function probably by decreasing ROS levels (Nuevo-Tapioles et al., 2021). Moreover, Slc4a11 knockout mice, a model of the congenital hereditary endothelial dystrophy, also show high rates of ROS that produce endothelial cell damage. In these animals, MitoQ reversed aberrant lysosomal function reducing edema and endothelial cell loss (Shyam et al., 2021). It seems clear that the antioxidant protection produced by MitoQ improves the evolution of these diseases in which oxidative damage and probably inflammation are key. For example, in Kawasaki disease (KD), Ogg2/2 mice, is the leading cause of acquired heart disease among children. Its pathophysiology depends on the NLRP3/IL-1β pathway. Another mice model of KD vasculitis uses Lactobacillus casei cell wall extract (LCWE) as an inducer of the disease. LCWE-injected mice showed impaired autophagy/mitophagy and increased ROS and DNA damage in cardiovascular lesions. Blockage of autophagy increased inflammation, indicating the importance of mitochondrial turnover in the pathology. Treatment of these animals with MitoQ reduced vascular tissue inflammation, ROS production, and systemic 8-OHdG indicating, again, the importance of the reduction of oxidative damage and inflammation on the progression of the disease (Marek-Iannucci et al., 2021).

11.9 Therapeutic activity of MitoQ

11.9.4 Ischemia/reperfusion and organ transplantation In organ transplantation, ischemia/reperfusion is one of the main factors that increase oxidative stress and damage. In kidney transplantation, the addition of MitoQ to the kidney after cold storage and rewarming in the University of Wisconsin (UW) preservation solution offered protection against cold storagedependent injury. MitoQ decreased superoxide production, prevented mitochondrial dysfunction, significantly improved cell viability, and preserved renal morphology (Mitchell et al., 2011). MitoQ administration 15 min before kidney ischemia in mice protected the kidney from damage and dysfunction indicating the importance of ROS in this process (Dare et al., 2015a). MitoQ reduced the severity of renal damage in rats under renal ischemic/reperfusion injury (Liu et al., 2018). The same effect was found in the case of cardiac transplants (Dare et al., 2015b). In the case of the heart, mitoQ reduced ROS-dependent damage, innate immune response, and cardiac injury (Dare et al., 2015b). In intestinal epithelial cells, MitoQ protected against I/R-induced damage helping in the stabilization of the intestinal barrier. MitoQ reduced oxidative stress, DNA damage, and inflammatory response. As in other cases, this effect was again dependent of NRF2 activity that induced HO-1, NQO1, and γ-GCLC (Hu et al., 2018).

11.9.5 Liver fibrosis Liver fibrosis is induced by the treatment with CCl4 exposure in rodents. In this model, MitoQ blunted fibrosis probably through reducing the activation of Hepatic Stellate Cells (HSC), TGFβ release, and activation of the Smad 2/3 pathway. Further, MitoQ also decreases necrosis, apoptosis, and inflammation probably by decreasing ROS (Rehman et al., 2016). CCl4 also produces cirrhosis in rats. Treatment of rats with MitoQ (5 mg/kg/day) or its tail decylTPP for two weeks deactivated HSC, and decreased their proliferation but did not produce effects against apoptosis. However, this effect was considered sufficient for MitoQ treatment for cirrhosis (Vilaseca et al., 2017).

11.9.6 Metabolic syndrome and related diseases Metabolic syndrome (Ms) is defined as a high-prevalence disorder caused by the presence of three or more of the following clinical criteria: abdominal region associated obesity, high blood pressure, hypertriglyceridemia, low HDL cholesterol, and/or increased fasting glucose (Alberti, 2009; Shaw et al., 2005). Ms has been associated with many aging-related chronic diseases such as CVD (Mottillo et al., 2010), type 2 diabetes mellitus (Kahn & Flier, 2000), and cancer (Cowey & Hardy, 2006). In these processes, mitochondrial dysfunction, high ROS levels and oxidative damage play a crucial role in the impairment of cell functions (LopezLluch et al., 2018). For this reason, MitoQ has been extensively used in the

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treatment of Ms and related diseases mainly in order to restore mitochondrial activity, decrease oxidative damage, and reduce ROS-related inflammation. Many of the studies on the effect of MitoQ in Ms have been performed in mice models. In high fat-fed ApoE2/2 mice and ATM1/2/ApoE2/2 mice as a model, treatment with MitoQ prevented the accumulation of fat and the increase of cholesterol and glucose in plasma. Further, oxidative damage of DNA was also reduced, indicating that the reduction of ROS levels at mitochondria is important in the evolution of the disease (Mercer et al., 2012). In male Sprangue-Dawley rats, MitoQ attenuated body weight gain and glucose intolerance in a model of Ms. This effect revealed a key role of mitochondrial stress in the evolution of this disease (Feillet-Coudray et al., 2014). MitoQdependent reduction of body weight and fat accumulation was associated with the control or the hypothalamic axis, by reducing the intake of food in these animals. However, improvement of liver capacity by virtue of its antioxidant properties was considered the main factor (Fink et al., 2014). In relation to this, in obesogenic diet-fed rats, MitoQ produced major effects in liver mitochondrial phospholipids and mitochondrial function. MitoQ increased CL synthase gene expression and CL content and modified the content of phospholipids in mitochondria in comparison with HF-diet. CL content positively correlated to fluidity in mitochondrial membrane, redox potential and respiration and also the activity of ATP synthase and negatively correlated to mtROS production indicating a clear protective role of mitochondrial function (Fouret et al., 2015). Further, MitoQ also prevented liver damage after mechanical induction of liver cirrhosis by bile duct ligation. It is clear that oxidative stress causes inflammation that is involved in the initiation and evolution of the pathological characteristics of Ms. In SpragueDawley, MitoQ (10 mg/kg/day) administered from the third to the 28th day of this procedure was able to prevent inflammation, hepatocyte necrosis, fibrosis, decreased levels of TNFα and other inflammatory markers such as TGFβ, IL6 and IL-1β or markers of oxidative damage (Turkseven et al., 2020). However, whereas in all these works MitoQ, normally at 250
500 μM concentration in water, attenuated weight gain by decreasing food intake and mitigated oxidative stress at the onset of feeding with high fat (HF). In already obese mice, MitoQ was not tolerated and animals lost weight very quickly and the benefit of MitoQ treatment was limited (Fink et al., 2017). This indicates that the use of MitoQ in already obese animals must be carefully studied although many studies indicate that MitoQ is a good therapeutic candidate to reduce liver fat accumulation and oxidative damage (Fink et al., 2021). Diabetes is one of the main diseases associated with Ms and produces many side effects in organs. Pancreatic β-cell failure is a consequence of glucose intolerance in type 2 diabetes. In a cell model of pancreatic β-cells (RINmIF and HITT15 β-cells), MitoQ prevented oxidative damage and changes in protein mtETC levels, lipid accumulation, apoptosis, and the activation of NF-κB. These results indicated that mtROS and oxidative damage are key in the dysfunction of these

11.9 Therapeutic activity of MitoQ

cells and point to MitoQ as a putative treatment in the prevention of pancreatic β-cell dysfunction in type 2 diabetes (Lim et al., 2011). Kidney dysfunction is also an important pathological consequence of diabetes. In db/db mice, high glucose was responsible for mitochondrial abnormalities such as defective mitophagy, mtROS levels and mitochondrial fragmentation, and reduced PINK1/Parkin expression. In these animals, MitoQ partially reversed these effects including defective mitophagy. Further, in HK-2 cells treated with high glucose, MitoQ reduced tubular injury through increasing mitophagy and mitochondrial control via Nrf2/PINK1 axis (Xiao et al., 2017). In the Akita mouse model, MitoQ also improved tubular and glomerular function without significantly affecting renal filtration. However, MitoQ also prevented interstitial fibrosis. These studies indicate that mitochondrial-targeted antioxidants can prevent mitochondrial-associated damage in diabetic nephropathy especially avoiding mtROS-dependent inflammatory processes and fibrosis (Chacko et al., 2010). Diabetes also produces damage in endothelial cells causing many different effects but mainly cardiovascular-related diseases. In a diabetes model of endothelial dysfunction, MitoQ treatment increased NRF2 activity and HO-1 levels in high glucose-induced cell dysfunction and also improved mitochondrial membrane potential decreasing mtROS production and thus, protecting cells against damage and apoptosis (Yang et al., 2021). The antioxidant role of MitoQ is central in its effect in the prevention of Msrelated cardiovascular dysfunction. In fact, in a rat model of Ms, mitochondrial oxidative stress and dysfunction are considered key factors in the disruption of coronary collateral growth. In this model, treatment with MitoQ, rescued collateral growth by reducing ROS levels (Pung et al., 2012).

11.9.7 Therapeutic potential of MitoQ in the treatment of cardiovascular diseases Prevention of endothelial damage caused by Ms or only by deterioration during aging can reduce the possibility to suffer CVD disease. Many experiments in cells, animal models, and also in humans have recently demonstrated the therapeutic use of MitoQ in the prevention of this disease. It is known that MitoQ attenuates age-related arterial endothelial dysfunction, in another example of the role of ROS in age-related dysfunction (Gioscia-Ryan et al., 2014). MitoQ also restored mitochondrial health in aged carotid endothelial cells from old mice (Gioscia-Ryan et al., 2014). Further, stiffening of blood vessels is associated with age-related damage. In a mice model of aortic stiffening, only four weeks of treatment with MitoQ (250 μM in water) was able to partially revert this process by increasing elastin region without affecting collagen or oxidative damage (GiosciaRyan et al., 2018). In studies carried out in humans, MitoQ improves vascular endothelial function by decreasing oxidative stress. In twenty 60
79 years old healthy

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participants, six weeks of treatment with MitoQ (20 mg/day) showed good tolerance and improved brachial artery flow-mediated dilation, a marker of vascular dysfunction. The effect of MitoQ was associated with a decrease in ROSdependent endothelial dysfunction since MitoQ reduced the levels of oxidized LDL in plasma (Rossman et al., 2018). Further, in a cell model with HUVEC’s cells treated with smoke extract, MitoQ restored endothelial barrier integrity by preventing cadherin disassembly and actin skeleton remodeling and reducing inflammatory response by downregulating NF-κB and NLRP3 activities (Chen et al., 2019). Heart failure is also a therapeutic target for MitoQ treatment. Eight weeks-old C57BL/6 mice under ascending aortic constriction suffered damage in mitochondrial structure, an impaired mitochondrial network, and reduced MFN2 levels in cardiomyocytes. Treatment with MitoQ (1.34 mg/day 1 day before damage) protected against mitochondrial dysfunction and maintained MFN2 levels. MitoQ also alleviated dysregulation of a MFN2-associated lncRNA (Kim et al., 2020). In a rat model of heart failure induced by pressure overload by aortic constriction, low doses of MitoQ (10 μM in water) restored the levels of mitochondrial potential, mitochondrial respiration, and reduced ROS levels (Ribeiro Junior et al., 2018). MitoQ also prevented fibrosis caused by heart failure. In C57BL/6 mice under LV pressure overload, MitoQ blunted TGFβ1 and NOX4 upregulation, two factors involved in fibrosis. MitoQ also prevented NRF2 downregulation and TGFβ1 release from fibroblasts, indicating that its mechanism of action was to block the interplay between TGFβ1 and mitochondria-associated redox signaling (Goh et al., 2019). In general, all these studies indicate that MitoQ can be an important therapeutic agent in the treatment of CVD and to reduce damage after heart failure.

11.9.8 Other uses of MitoQ Another putative therapeutic use of MitoQ is focused on the prevention of oxidative damage due to contamination or smoking. MitoQ attenuated cigarette smokeinduced effects in the airway epithelium, inflammation, and oxidative stress. It also reduced the release of pro-inflammatory cytokines such as TNFα and IL-6 (Yang et al., 2020). Contamination-dependent damage can also be prevented by MitoQ. Particulate matter with aerodynamic diameter below 2.5 μM produces aortic fibrosis. MitoQ reduced mitochondrial dysfunction associated with ROS in this model (Ning et al., 2021). Further, MitoQ also reduced cigarette smokerelated senescence decreasing mtROS and increasing SIRT1 activity (Zhang et al., 2021). Protection of offspring against oxidative damage due to smoking during pregnancy has also been demonstrated. In pregnant rats, MitoQ was able to reduce oxidative damage in offspring and the levels of IL-1β (Sukjamnong et al., 2017). This effect reduced the impact of maternal smoking on renal development and mitochondrial density in male mice offspring (Sukjamnong et al., 2018). Further,

11.10 Other mitochondria-targeted compounds

cigarette smoke exposure in Balb/c mice six weeks prior to mating and along gestation and lactation, produce metabolic disorders in offspring (Li et al., 2019). Smoke exposure generated glucose intolerance, hepatic steatosis, and oxidative stress in mitochondria. MitoQ treatment (500 μM) in water reduced hepatic oxidative stress by reducing ROS and increasing GPx levels. MitoQ also promoted mitochondrial biogenesis and turnover. Again, MitoQ reduced levels of TNFα, and fibrosis (Li et al., 2019). Interestingly, MitoQ has been proposed as an interesting therapeutic compound in the prevention of damage for radiation in space. Mouse brain is damaged by 56Fe iron ions resembling space radiation. Production of ROS overpassed antioxidant defenses and induced mitochondrial dysfunction. MitoQ (5 mg/kg/day intraperitoneal) was able to reduce radiation-induced oxidative stress and oxidative damage. To this effect, MitoQ increased MFN2 and OPA1 levels whereas decreased DRP1 levels, indicating the induction of mitochondrial fusion. MitoQ also decreased oxidative damage and cytochrome c release, indicating protection against apoptosis (Gan et al., 2018). These results permit the consideration of MitoQ as a putative preventive agent against space radiation-dependent cell damage.

11.10 Other mitochondria-targeted compounds IDE and MitoQ are the most CoQ10-related mitochondrial targeted antioxidant compounds used in clinical trials and in models for human disease therapies. In the search for more efficient and mitochondrial-targeted antioxidants, other compounds have been used in different studies. Notwithstanding its similarity with IDE, decylQ is less used in clinical studies. However, it has also demonstrated capacity to avoid mitochondrial dysfunction due to oxidative damage in cell cultures. In diethylmaleate treated HL-60 cells that ends in GSH depletion, decylQ blocked mitochondrial-dependent apoptosis by reducing oxidative stress produced in mitochondria (Armstrong et al., 2003). In animal studies, decylQ showed a possible therapeutic capacity in the treatment of metabolic disease (Murad et al., 2007), similarly to the effect of IDE, in the treatment of LHON (Sala et al., 2008), in the maintenance of mitochondrial activity in neurological diseases, (Telford et al., 2010) or even in cancer therapy (Cao et al., 2020). The mitochondria-targeted plastoquinone SKQ1 has been associated with the inhibition of mitochondrial superoxide production at specific sites of complexes I and III rather than through scavenging superoxide and lipid peroxyl radicals directly. Its direct interaction with SOD has been discarded, since the radical scavenging properties of SKQ1 are very low in comparison with SOD (Jezek et al., 2017). However, its putative use in therapy has been considered for liver protection against oxidative damage (Zaparina et al., 2021), protection against

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chemotherapy-dependent cell damage (Sacks et al., 2021), and the treatment of different eye diseases, among others (Baiula & Spampinato, 2021; Baturina et al., 2021; Qu et al., 2021; Telegina et al., 2020). EPI-743, also known as Vatiquinone, was designed as a potent antioxidant showing the capacity of redox cycling of CoQ10 and IDE (Enns et al., 2012). It is considered an anti-aging compound that uses NQO1 to maintain a redox cycle to increase antioxidant protection (Shrader et al., 2011). In fact, EPI-743 has been proposed as a redox compound able to modulate the activity of NQO1 (Enns & Cowan, 2017). Further, as a mechanism of action, this compound acts as an antioxidant preventing ferroptosis-associated with mitochondrial-dependent degenerative diseases (Kahn-Kirby et al., 2019). In humans, studies in children with LS, another complex I-deficiency dependent disease, EPI-743 prevented the deterioration of speech, chorea and cognitive abilities (Gagnon, 2010). In fact, in these patients, FDA approved its use. Treatment with this compound improved neurologic and neuromuscular symptoms (Kouga et al., 2018; Martinelli et al., 2012) and glutathione redox unbalance (Pastore et al., 2013). EPI-743 has shown similar effects to IDE in LHON, reversing the loss of ganglions and improving visual loss (Sadun et al., 2012). However, in FRDA patients, EPI-743 counteracts the accumulation of toxic compounds whereas IDE also improved mitochondrial protection, probably indicating different targets (Petrillo et al., 2019). In any case, the treatment with this compound has also shown positive effects in the progression of FRDA (Zesiewicz et al., 2018). Further, Brown-Vialetto-Van Laere syndrome and Fazio-Londe diseases have been recently renamed as “riboflavin transporter deficiency (RTD).” RTD comprises autosomal recessive diseases caused by mutations of SLC52A2 and SLC52A3 genes that encode riboflavin transporters. In RTP, EPI-743 restored redox status and improved neurite length and calcium-intracellular flux in motor neurons (Marioli et al., 2020), indicating the importance of oxidative stress in these diseases. Other targeted antioxidants do not have a structural relationship with CoQ10, although they show promising effects in the treatment of the diseases treated with CoQ10-related antioxidants. SS31 is a tetrapeptide used as a promising treatment for neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Hungtinton’s diseases. It scavenged free radicals including H2O2 and inhibited lipid peroxidation. It also induced changes in mitochondrial dynamics and turnover (Yin et al., 2016). Further, it also ameliorated sepsis-induced heart injury by reducing oxidative damage and derived inflammation (Liu et al., 2019). XJB-5
131 was synthesized as an electron scavenger and SOD mimic by binding the SOD mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) to hemigramicidin S (Macias et al., 2007). This compound suppresses oxidative damage and motor decline in a mouse model of Huntington’s disease (Z. Xun et al., 2012), improving physiology and behavior in an age- and sexdependent mechanism (Polyzos et al., 2018). Further, it has also been used in the

References

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11.11 Conclusions Many diseases are associated with an excess of production of mtROS due to the accumulation of defective mitochondria. Defective mitochondria results in the accumulation of damaged mitochondria that causes a pressure in autophagy/mitophagy and proteostasis, hallmarks of the development of a many chronic diseases and aging. Mitochondrial CoQ10-related antioxidants protect mitochondria and cells against oxidative damage reducing mitochondrial dysfunction and breaking down the vicious cycle in which defective mitochondria produces more mtROS that produce and augment oxidative damage. In many mitochondrial and chronic diseases, selective inhibition of mitochondrial oxidative damage is an obvious therapeutic strategy. The common denominator of these compounds is based on the reduction of ROS levels but also in the induction of the mitochondrial turnover and dynamics, maintaining more efficient mitochondria in cells. This effect can explain many of the effects of these compounds on metabolism, antioxidant response and inflammation.

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9. Available from https://doi.org/10.1016/j.expneurol.2019.020.009. Zhang, Y., Huang, W., Zheng, Z., Wang, W., Yuan, Y., Hong, Q., Lin, J., Li, X., & Meng, Y. (2021). Cigarette smoke-inactivated sirt1 promotes autophagy-dependent senescence of alveolar epithelial type 2 cells to induce pulmonary fibrosis. Free Radical Biology & Medicine, 166, 116
127. Available from https://doi.org/10.1016/j. freeradbiomed.2021.020.013. Zhao, Z., Wu, J., Xu, H., Zhou, C., Han, B., Zhu, H., Hu, Z., Ma, Z., Ming, Z., Yao, Y., Zeng, R., & Xu, G. (2020). Xjb-5-131 inhibited ferroptosis in tubular epithelial cells after ischemia-reperfusion injury. Cell Death and Disease, 11, 629. Available from https://doi.org/10.1038/s41419-020-02871-6. Zhou, J., Wang, H., Shen, R., Fang, J., Yang, Y., Dai, W., Zhu, Y., & Zhou, M. (2018). Mitochondrial-targeted antioxidant mitoq provides neuroprotection and reduces neuronal apoptosis in experimental traumatic brain injury possibly via the nrf2-are pathway. American Journal of Translational Research, 10, 1887
1899.

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Flavonoids, mitochondrial enzymes and heart protection

12

Sneha Sivadas, Nandakumar Selvasudha, Pooja Prasad and Hannah R. Vasanthi Natural Products Research Laboratory, Department of Biotechnology, Pondicherry University, Pudhucherry, India

12.1 Introduction Mitochondria are membrane-bound organelles found in nearly all eukaryotes that provide most of the biochemical energy required to drive the cell’s metabolic activities. Therefore, it is well known as the “energy plants” or “the cell’s powerhouse” that supply more than 90% of the energy needed for cellular respiration. The mitochondria are thought to have descended from symbiotic ancestors, indicating a bacterial evolutionary basis, and carry a residual genomic DNA (mtDNA), which encodes 13 proteins required for mitochondrial respiration. The rest of the mitochondrial proteins are encoded by nuclear genes (Young & Francis, 2017). Mitochondria are crucial for proper cell metabolism and production of ATP molecules and drive various cell signaling pathways and the synthesis of multiple biomolecules such as amino acids, fatty acids, glucose, and heme (Olowookere, 2021). Apart from its role in cellular metabolism, it plays a crucial role in calcium homeostasis, cell signaling, apoptosis, and redox pathways, thus making mitochondria an essential organelle in the life and death of a cell. Defects in any of the mitochondrial pathways resulting in mitochondrial dysfunction leads to the development and evolution of many human diseases, notably cancer, cardiovascular disorders, neurodegenerative diseases, diabetes, traumatic brain damage, and inflammation (Mertens et al., 2021). Mitochondrial participation in these disorders has been related to the organelle’s vital part in the cascade of events that end in cell death via several programmed and nonprogrammed cell death processes. In the mechanism of transferring electrons to make ATP, the mitochondria are the principal source of reactive oxygen species (ROS) generation, which can lead to apoptosis and function as a signaling molecule. The production of ROS can pose a threat to mitochondrial DNA (mtDNA) and proteins, therefore, the mitochondria must have quality control mechanisms to protect any cell or organ from being damaged (Hernansanz-Agustı´n & Enrı´quez, 2021). Several studies have attested the potential of different flavonoids that effectively protect the cells from various stressors that cause mitochondria-mediated Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00002-3 © 2023 Elsevier Inc. All rights reserved.

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cell damage. Clinical evidence suggests that specific molecules can reduce the severity of diseases related to oxidative stress and mitochondrial dysfunction (Maaliki et al., 2019). Since flavonoids have several biological effects that contribute to a healthy lifestyle, their antioxidant properties enhance their nutritional value to play a preventive role in treating various chronic diseases, especially cardiovascular diseases (CVDs) (Rees et al., 2018). The generation of ROS leading to apoptosis is a crucial element in cardiovascular disease. The flavonoids act via the mitochondrial target site and maintain calcium homeostasis to protect the heart during mitochondrial dysfunction. Furthermore, flavonoids neutralize ROS by inhibiting mitochondrial enzymes and chelating the generated toxic components in the cells (Kicinska & Jarmuszkiewicz, 2020). Numerous experimental studies have proven flavonoids’ antioxidant characteristics against cardioprotection. This chapter summarizes and discusses the fundamentals of flavonoids and mitochondrial enzymes and the various roles in regulating cardiovascular ailments and protecting the beating heart.

12.2 Mitochondria and mitochondrial enzymes in cellular functions The mitochondria constitute vital cellular programs that disseminate and trigger intracellular events that result in overall cellular and metabolic responses in the body. Energy metabolism, the primary function in a cell, is mediated through a complex interconnected metabolic network through cytosolic glycolysis, the triboxycyclic acid (TCA) cycle, and mitochondrial oxidative phosphorylation (OXPHOS) using amino acids, lipids, and carbohydrate derivatives as multiple energy sources (Morio et al., 2021). Thus, mitochondria are respiration sites that provide chemical energy via OXPHOS. It consumes almost 85%
90% oxygen in cells to allow OXPHOS, the principal metabolic pathway for the generation of ATP. The various cellular events taking place in the functional mitochondria are shown in Fig. 12.1. Studies have found that retrograde signaling in mitochondria undergoes regulation of several gene expressions, facilitating diverse cellular functions (Strobbe et al., 2021). The retrograde response is activated during the aging process and through other pathophysiological conditions such as cardiovascular disease as a cause of increasing mitochondrial dysfunction. It is a result of a progressive decline in the mitochondrial membrane potential. Although mitochondria regulate a wide array of cellular functions, a recent study found the role of TCA cycle intermediates in biosynthetic signaling for DNA methylation, chromatin modifications, hypoxic response, and immunity (Martı´nez-Reyes & Chandel, 2020). The cytochrome C (Cyt c)-induced cell death, regulation of mitochondrial fission and fusion by activation of AMP-activated protein kinase (AMPK), and the activation of transcription factors by the formation of ROS, activation of immune responses

12.3 Mitochondria as an essential organelle for cardiovascular health

TOM

APOPTOSIS (Myricetin, Quercetin, Kaempferol)

CPT1

Cyt C Bak/Bax

Pyruvate

CPT2

NLRP3 INFLAMASOME ACTIVATION (Apigenin, Catechin, Ferulic Acid) PROTEIN SYNTHESIS Protein import & processing

TIM

mtDN A

UCP FADH2 NADH

ROS

RIBOSOME

PINK1/Parkin

FeS

CypD

ENERGY METABOLISM

ROS

H2O Solutes I

H+

Succinyl CoA H+

Na+

ISC

(Resveratrol, Quercetin, Puerarin)

H+

ATP ANT

H+

V

Na+ Ca2+ Exchange

mPTP VDA VDAC C

BIOSYNTHESIS OF FeS CLUSTERS & COFACTORS REGULATE Ca2+ EFFLUX

Acetyl CoA TCA

β-[O]

Matrix

MITOPHAGY

FA CoA

Glycolysis

ATP Ca2 +

Mitochondrial Function (Naringenin, Baicalein)

HEME SYNTHESIS (Quercetin) FREE RADICAL SCAVENGING REDOX Signalling (Hesperitin, Naringin, Luteolin)

FIGURE 12.1 Mechanism of action of flavanoids for cardioprotection. ANT, Adenine nucleotide translocase; β-[O], Beta oxidation; Ca21, Calcium ions; CytC, Cytochrome C; CPT1&2, Carnitine palmitoyltransferase 1&2; CypD, Cyclophilin D; Endo G, Endonuclease G; FA CoA, Fatty-acyl-CoA; ISC, Iron-Sulfur (Fe-S) clusters; mPTP, mitochondrial permeability transition pore; mtDNA, mitochondrial DNA; NLRP3, NLR family pyrin domain containing 3; OXPHOS, Oxidative phosphorylation; ROS, reactive oxygen species; TCA, tricarboxylic acid cycle; TIM, translocase of the inner membrane; TOM, translocase of the outer membrane; UCP, uncoupling protein; VDAC, voltagedependent anion channel.

by the release of mtDNA, are the four eminent mechanisms by which mitochondria communicate with the rest of the cell. Studies have also found another mechanism to control cell fate and function by the TCA cycle metabolites released from the mitochondria. Mitochondrial enzymes also regulate amino acid synthesis and steroid synthesis such as pregnenolone, and defective mitochondrial enzymes are observed in neurodegenerative diseases and hepatic diseases (Murali Mahadevan et al., 2021).

12.3 Mitochondria as an essential organelle for cardiovascular health Myocardial function depends primarily on the production of oxidative energy. It requires more energy than skeletal cells and consumes 20-fold more oxygen by 60
70 beats per min heartbeats in healthy cells. Mitochondria are abundant in cardiac cells due to the greater energy needs, and they produce around 6 kg of ATP every day through the OXPHOS process (Das et al., 2017). The beating heart maintains a constant flow of chemical energy to execute its mechanical pumping of blood towards different body organs. It also requires the regulation of

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intracellular and trans-sarcolemmal ionic fluxes and concentration gradients in cells. Mitochondrial fatty acid oxidation is the most common substrate used in the normal healthy human heart, accounting for 60%
80% of cardiac ATP synthesis, with glucose, lactate, and ketone bodies contributing less. Apart from mitochondrial energy, cardiac cells also provide energy in the form of phosphocreatine, generated by mitochondrial creatine kinase, utilizing ATP (Poznyak et al., 2020). Mitochondrial dynamics, including fission, fusion, and autophagy, are tightly controlled mechanisms required for energy production and the organelle’s structural integrity. Mitochondrial ROS generation and energy imbalance are critical targets causing damaging effects, leading to mitochondrial dysfunction, which disrupts cellular lipids, proteins, enzymes, and DNA. Aberrations in the mitochondrial structure and its activities leads to increased cardiovascular anomalies such as dilated cardiomyopathy and hypertrophic cardiomyopathy (HCM), atherosclerosis, ischemiareperfusion injury, hypertension, heart failure, and other disorders such as diabetes. Apoptosis, which promotes cardiovascular damage, is also associated with mitochondrial dysfunction. Fig. 12.2 illustrates how numerous risk factors affect the cardiomyocyte mitochondria, resulting in cellular and genetic alterations contributing to various cardiovascular disorders.

12.4 Role of mitochondrial enzymes in cardiomyocytes 12.4.1 Mitochondrial enzymes for scavenging reactive oxygen species Mitochondria have emerged as an antioxidant system for scavenging ROS generated under physiologic and pathologic conditions in normal cell metabolism. Fuel molecule oxidation produces reduced electron carriers (NADH and FADH2), transporting electrons to the respiratory system. The transmission of electrons to the molecular oxygen, the terminal receiver, is associated with proton pumping across the inner mitochondrial membrane, resulting in a proton motive force that causes the generation of ATP (Ushio-Fukai et al., 2021). The discharge of electrons due to incomplete reduction of molecular oxygen in the electron transport chain (ETC) leads to ROS formation. Several sites such as the peroxisomes, cytosol, plasma, phagosomes, endoplasmic reticulum (ER) membranes, and mitochondria generate ROS. However, the mitochondria produce larger amounts of ROS, leading to intracellular oxidative stress in living organisms (Zorov et al., 2014). However, low levels of ROS generated in mitochondria are critical in signaling pathways and are required for proper mitochondrial activity and cell homeostasis. ROS, mainly hydrogen peroxide (H2O2), plays a key role in regulating cellular functions via redox signaling at physiological levels. Still, an excess generation of ROS can cause oxidative distress and damage of macromolecules in the cell. NADPH-dependent glutathione (GSH) and thioredoxin (Trx)-dependent

12.4 Role of mitochondrial enzymes in cardiomyocytes

Age & Sex

Metabolic disorders

Environmental stress

Lifestyle modifications Functional Heart

Oxidative Stress Energy Imbalance ER Stress

Mitochondrial dysfunction in cardiomyocyte

Atherosclerosis

Myocardial Infarction

Ischemic Cardiomyopathy Reperfusion Injury

Heart Failure

FIGURE 12.2 Role of mitochondria in the functional heart.

peroxidase systems are two mechanisms for mitochondrial ROS scavenging. Furthermore, catalase, an enzyme found in high concentrations in the peroxisomes, certainly contributes to ROS catabolism in the mitochondria of the liver and, to a lesser extent in the heart mitochondria. While ROS are catabolized by the peroxidase systems, glutathione reductase (GR) and thioredoxin-reductase (TrxR) oxidize GSH and Trx, respectively, before reducing them to the functional form, at the cost of NADPH oxidation (Napolitano et al., 2021). Despite the efficiency of the OXPHOS process, 1% to 3% of electrons may leak from the etc. during normal respiration in lesser amounts, specifically from complexes I and III, which rapidly decrease O2 and produce superoxide (O22), converting to H2O2 by superoxide dismutase and thus generating ROS. Thus, Complexes I and III in the mitochondria are considered to be the primary contributors to ROS formation. However, complex II and Cyt c oxidase may also be implicated in some circumstances. ROS formed at complex I is located near mtDNA and redox-sensitive

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proteins in the respiratory chain resulting in damaging oxidation reactions. In contrast, complex III participates primarily in signal transduction mechanisms (BenShachar, 2017). Other mitochondrial proteins implicated in ROS generation involve glycerophosphate dehydrogenase, ketoglutarate, and pyruvate dehydrogenase. Apart from the mitochondria, H2O2 is created by plasma membrane NADPH oxidases, xanthine oxidase, amino acid oxidase, and metabolic activities of lipid peroxidation and several cytosolic enzymes. However, ROS formed inside the mitochondria accounts for about 90% of total ROS generated in the cell (Hernansanz-Agustı´n & Enrı´quez, 2021). ROS, particularly H2O2, involves regulating critical cellular functions via redox signaling at physiological levels, but its excess concentration leads to oxidative stress and macromolecular damage. Cells also produce toxic nitric oxide synthase (NOS) from nitric oxide (NO) in addition to ROS. NO is produced in biological tissues by NOSs, which convert arginine to citrulline via a five oxidative electron process, resulting in NO production (Hossain et al., 2015). It plays a crucial role in oxidative signaling involving various physiological activities such as blood pressure regulation, neurotransmission, cell defense mechanisms, smooth muscle relaxation, and immunological regulation responsible for cardioprotection. NO rapidly disperses through the cytoplasm and plasma membranes owing to its solubility in aqueous and lipid environments (Venditti et al., 2013). In the ETC, electron movement is inhibited by ETC inhibitors such as rotenone, antimycin A, and myxothiazol at specific ETC sites. An upsurge in mitochondrial ROS production has been used to discover site-responsive errors in the transport of electrons (Brand, 2016).

12.4.2 Mitochondrial enzymes for apoptosis in cardiomyocytes Mitochondria are a prime target for oxidative damage and play a crucial role in activating apoptosis in mammalian cells. DNA damage, oxidative stress, and ischemia in mitochondria are the key factors of programmed cell death and necrosis (Li et al., 2021). The two main apoptotic pathways involve extrinsic and intrinsic pathways. The intrinsic cell signals include oxidative stress by ROS, NO, GSH, Cyt c, calcium iron, and ER stress. In contrast, extrinsic cell materials involve cytokines, analgesics, hormones, pathogen mediators, and native activity molecules. The intrinsic pathway, also known as the mitochondrial pathway, involves pro-and antiapoptotic proteins and signals to initiate a cell death program. The intrinsic mitochondrial mechanisms are mediated by the B cell lymphoma 2 (BCL-2) protein family (Hongmei, 2012). The extrinsic pathway, also termed as the death-receptor pathway, is triggered by ligating transmembrane death receptors such as Fas, TNF, TRAIL, and Dr3
6 receptors with their ligands from outside the cell. The activation of caspases, belonging to the cysteine protease group may fragment various cellular targets and destroy cell components and is crucial in apoptosis regulation.

12.4 Role of mitochondrial enzymes in cardiomyocytes

Cyt c is an integral part of the pro-apoptosis signal molecular factor translocated in the inner mitochondrial membrane, which activates initiator caspase 9. A heme group linked to Cyt c forms holocytochrome c, a redox intermediary for electron transport between complex III and IV (Banerjee et al., 2021). UV or X-ray radiation can trigger mitochondrial depolarization and membrane permeabilization, resulting in an increase in ROS, Cyt c release, and caspase-9 and caspase-3 activation. Apoptosis-inducing factor, which is translocated in the inner mitochondrial membrane, and endonuclease G, which is found in the inner mitochondrial membrane and the matrix, are two more apoptogenic proteins released from mitochondria during apoptosis. These mitochondrial proteins are not devoted killers, instead, they conduct various essential mitochondrial tasks required for optimal cell growth. In healthy cells, to prevent unwanted activation of the apoptotic machinery, it is preferable to remove interactive mitochondrial proteins from their target proteins (Flanagan et al., 2016).

12.4.3 Mitochondrial enzymes in autophagy Mitophagy is a mechanism that allows damaged mitochondria to be eliminated from the cell selectively via autophagy, a process by which defective and inoperative proteins are removed by self digestion. The hydrolytic destruction of mitochondrial macromolecules like lipids, proteins, and DNA after an autophagosome, a double-membrane structure, has sequestered them. The autophagosome is formed and regulated by various autophagy-related proteins (ATG), and specific mitochondrial proteins are required to recognize and mark mitochondria for mitophagy. This degradation pathway entails the phagophore recognizing, tethering, and engulfing the mitochondrion components to form the autophagosome, which is then subsequently fused with the lysosome for digestion (Kumar & Reichert, 2021; Qiu et al., 2021). The first defined mechanism of mitophagy was discovered in yeast. The process involves the autophagic pathway and specific mitophagy proteins such as Atg32 present outside the mitochondrial membrane, which act as a central receptor for mitophagy. It binds through its WXXL domain to ATg8 located on the phagophore’s surface. The scaffold protein Atg11 contributes to the recruitment of mitochondria breakdown by interacting with Atg32 and the mitochondrial fission protein Dnm1. This leads to phagophore expansion and autophagosome formation around the mitochondria and then combines with the vacuole, degrading the mitochondrion (Idelchik et al., 2017). Similarly, NIX-dependent mitophagy is another mechanism during reticulocyte maturation regulated by mitochondrial protein BNIP3L/NIX. NIX belongs to the BH3 family and is attached to the outer mitochondrial membrane. It binds to the MAP1LC3 phagophore membrane protein and facilitates autophagosomes leading to lysosomal degradation. Studies found that mitophagy mediated by BNIP3L/NIX is needed to differentiate proinflammatory macrophages and retinal ganglion cells. NIX is expressed in a wide range of cell types and organs, and its role is also implicated in the pathogenesis of cancer and hepatic and cardiac

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muscle cells. Hypoxia regulates BNIP3 and NIX expression in tumor cells, and dysregulation of BNIP3 or NIX expression is linked to tumor growth. In heart muscle, hypoxia and Gq-dependent signaling regulate BNIP3 and NIX, respectively, and their expression is linked to impaired myocardial function (EstebanMartı´nez & Boya, 2018; Melser et al., 2015). The involvement of Parkinson’s disease in a PINK1/PARKIN-dependent mitophagy pathway is a widely accepted mechanism. PTEN-induced putative kinase 1 (PINK1) is localized in the outer membrane of the mitochondria and phosphorylates PARKIN, a member of the E3 ubiquitin ligase family at the Ser65 position, and recruits it to the mitochondria for ubiquitylation. PARKIN mediates the interaction of two polyubiquitin chains connected by Lys 63 and Lys 27. It also ubiquitinates several mitochondrial proteins such as VDAC, TOM20, and mitofusins (MFN1/2) at the mitochondrion. The process also relies on autophagy-related proteins such as Atg5, Beclin 1, and Atg12 and recruits autophagic adaptor p62/ SQSTM1 and LC3 for engulfing by autophagosome and clearance of mitochondrion through mitophagy (Yao et al., 2019). Mitochondria use the fission method in the form of mitochondrial-derived vesicles to shed damaged proteins and mtDNA when it exposes mild chronic mitochondrial stress, and acute stress depends on PINK1/PARKIN mediated mitophagy. Hormones also play an essential role in either promoting or preventing mitochondrial degradation. Thyroid hormones, for example, suppress mitochondrial breakdown in the heart but fasting and glucagon enhances mitochondrial degradation in hepatocytes via mitophagy and nonselective autophagy. AMPK signaling plays a crucial role in coordinating mitophagy. The AMPK and mTORC1 signaling pathways are two important energy-sensing protein molecules which interconnect mitochondrial regulation to the rest of the body and control mitochondrial biogenesis and degradation (Geisler et al., 2010; Yao et al., 2019).

12.5 Structure and function of dietary flavonoids Flavonoids are naturally occurring compounds with various phenolic structures found in fruits, vegetables, cereals, grains, cocoa, bark, roots, stems, flowers, and certain beverages such as tea, wine, and other dairy products. They contain wellknown bioactive antioxidants with several potential health benefits making them an essential component with various applications (Shahidi et al., 2019). Flavonoids are categorized into distinct groups based on diversity in the basic structure and functional group of the heterocyclic C ring and its oxidation and unsaturation state such as flavanones, flavones, flavonols, flavans (flavan-3-ols), isoflavones, and anthocyanins. These secondary metabolites are synthesized and localized in the vacuoles (Chen et al., 2018; Di Lorenzo et al., 2021). With reports on nearly 10,000 plant flavonoids, they stand in the third position in relation to their bioactivity. The majority of foods include flavonoids in the form of

12.6 Pharmacokinetic profile (ADME) of flavonoids

polymers/glycosides. The hydroxyl functional groups present on all three rings are likely places for carbohydrates to connect. Flavonoid glycosides are formed due to the binding of sugar molecules to flavonoids, whereas, aglycones are formed due to the absence of sugar molecules. Apart from flavan-3-ols, other forms of flavonoids exist as glycosides. The difference in the biological actions and interactions with mitochondrial enzymes is attributed to the structural intricacy of flavonoids, that is, due to acetyl and malonyl group linkages to the glucose molecules and hydroxyl group positions. For instance, the micromolar range of 7-hydroxy groups containing flavonoids is observed to bond firmly with xanthine oxidase (Sosa et al., 2017). Hydroxyl groups present in the exterior of the quercetin ring structure (which has a hydroxyl group at the 7th position) intermingle with Arg880 /Glu802 of the enzyme through the H-bond. The hydrophobic binding of quercetin and the conjugated three-ring structures provide close fitting and are beneficial for treating inflammatory disease states like atherosclerosis. Studies have found that most classes of flavonoids have heart-protective effects because of their ability to prevent lipid peroxidation (Mozaffarian & Wu, 2018). The sources of different categories of flavonoids and their structure are depicted in Table 12.1 to understand the structure-activity relationship of the different types of flavonoids. Flavonoid-rich plant foods are extensively reported as potential bioactive compounds with significant biological potential, partaking in various vital signaling pathways related to chronic diseases. There is already a broad range of flavonoid supplements available in the market. The various herbal supplements containing flavonoids are reported for their beneficial effects in managing metabolic disorders, including metabolic syndrome, CVDs, and diabetes mellitus (Khan et al., 2021). Further, several flavanols are known for their antihypertensive and hypolipidemic effects, thereby enhancing endothelial protection (Ro´z˙ a´nska & RegulskaIlow, 2018). Moreover, the vital role of different classes of flavonoids is related to their antioxidant and free radical scavenging activity, which alleviates oxidative stress, the primary pathological event in most chronic diseases such as cancer, diabetes, arthritis, stroke, and cardiovascular disorders.

12.6 Pharmacokinetic profile (ADME) of flavonoids After ingesting the dietary flavonoids, the absorption depends on its physicochemical properties to pass food to the circulatory system from the gut lumen. The absorption depends on the structure of flavonoids as they exist in two forms, aglycone or glycoside. The aglycone possesses higher bioavailability, and therefore, the small intestine quickly absorbs it than the glycosides by more significant membrane interactions. In most cases, flavonoids are administered orally and exist as glycosides (Cassidy & Minihane, 2017; Williamson et al., 2018). The hydrophilic glucosides undergo deglycosylation and enter through Na 1 -dependent glucose

311

Table 12.1 Different sources of flavonoids and its bioactivity. Class

Flavonoids

Flavonols

Kaempferol Quercetin

Rutin

Resveratrol

Flavones

Apigenin

Diosmetin

Hesperidin

Biochanin A

References

Antioxidant vasoprotective cytoprotective

Ganeshpurkar and Saluja (2017), David et al. (2016)

Parsley, oregano, celery, green peppers

Anticancer, antibacterial, blood pressure reduction

Ruviaro et al. (2020), Yan et al. (2017)

Citrus fruits, oranges mushrooms

Anticancer, antiinflammatory, antiobesity

Arinç et al. (2015), Barreca et al. (2020)

Soya bean alfalfa sprouts and other legumes

Neuroprotective, anticancer, antibacterial antiinflammation, antiaging

Manayi (2021), Aboushanab et al. (2021)

Hesperitin

Isoflavones

Diadzein

Bioproperties

Green leafy vegetables, wild leeks, berries, apples, red wine, onions

Tangeritin

Flavanones

Naringenin

Dietary sources

Genistein

Flavanols

Catechin

Theaflavin

Peonidin

Anticancer, antiinflammatory, antidiabetic, hepatoprotective

O’Neill et al. (2021), Fan et al. (2017)

Grapes, Tomatoes, Tart cherries, Berries

Antioxidant Antiadipodgenic

Liu et al. (2021), Zeb (2021)

Epicatechin

Anthocyanin

Malvidin

Apples, pears, apricot, green tea, cocoa, chocolates

Cyanidin

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CHAPTER 12 Flavonoids, mitochondrial enzymes and heart protection

cotransporter (SGLT1) in the small intestine. In plants, except the catechin subclasses, all flavonoids are conjugated to sugars as β-glycosides, thus, no free aglycone flavonoid is present in urine or plasma. The conjugation of flavonoids in the liver is through sulfation, glucuronidation, methylation or metabolism of the phenolic compounds. In the epithelial cells, cytosolic β-glucosidase hydrolysis occurs by active SGLT1 (Jain et al., 2019). Alternatively, luminal hydrolysis in the brushborder of the small intestine epithelial cells by the enzyme lactase phlorizin hydrolase (LPH) removes the added sugar and forms an aglycone. LPH significantly exhibits greater specificity for Flavonoid-O-glycosides than C-glycosides, which are more resistant to hydrolysis. O-glycosides include galactosides, glucosides, xylosides, rhamnosides, and arabinosides. The enhanced lipophilicity and vicinity to the biological membrane lead the released aglycone to enter the epithelial cells through passive diffusion (Wang et al., 2018). Phase 2 metabolism constantly proceeds in the small intestine epithelial cells, followed by deglycosylation. The flavonoids are released with the bile and those not absorbed by the small intestine undergo degradation by the intestinal microflora in the colon leaving the flavonoid ring structure (Wang et al., 2018). Thus, the corresponding aglycone flavonoids released are absorbed in the large intestine, or they are degraded further. Acidic conditions in the stomach degrade the oligomeric flavonoids to monomers and dimers. Their sugar moiety primarily determines the flavonoid glycosides’ bioavailability which has to be reduced by dimerization. Isoflavones have the highest bioavailability of all flavonoid subclasses. Onions absorb four times more quercetin than an apple or a cup of tea. Thus, bioavailability differs for certain groups of flavonoids. Quercetin metabolites are found in plasma in concentrations ranging from 0.7 to 7.6 M (Rodrı´guez-Garcı´a et al., 2019).

12.7 Structure activity relationship of flavonoids for cardioprotective activity Quantitative structural activity interactions are effective predictors of chemical action in biological systems. Therefore, natural product derivatives with a wide range of biological activity could be synthesized for possible health impacts. Several flavonoids have been proven for their potential therapeutic role in the management of CVDs, which include endothelial dysfunction, coronary artery disease, cardiac fibrosis, myocardial infarction, ischemic reperfusion injury, etc. Kru¨pple like factor-2 expression is a class of transcription factor that regulates cell proliferation and differentiation. Numerous research studies reported the specific variation of Kru¨pple like factor-2 expression by flavonoids (Martı´nezFerna´ndez et al., 2015). A study of apigenin vs. naringenin revealed that the presence of C2 5 C3 double bond provides a dual structure concerning ET-1/Enos expression and produced gene-dependent effects. Hydroxylation/glycosylation at the 3rd position of a flavonoid decline ET-1/Enos by two fold as in rutin/

12.7 Structure activity relationship of flavonoids

quercetin vs. luteolin and 1.35-fold gene expression was observed with 4carbonyl moiety containing flavonoid (quercetin vs. catechin/ epicatechin/). The varied functional groups are one of the reasons for altered electronic delocalization properties of different subclasses of flavonoids and their reaction with the respective receptors. 4-carbonyl group (ring C), C2 5 C3 double bond, hydroxylation substitution, and protein binding mechanisms were proven by research with paraoxonase 1 (rePON1) containing 12 flavonoids(Atrahimovich et al., 2013). PON1 is an HDL-associated lactonase with antiatherogenic and antioxidant activities. PON1 protects against LDL oxidation induced by macrophages while also increasing HDL adherence to macrophages, which boosts HDL’s potential to facilitate cholesterol efflux. PON1 interactions are more significant for flavonols and flavones due to the C2 5 C3 double bond in ring C, the coplanar structure of 3-hydroxyl moiety, and a 4-carbonyl oxygen atom and molecular planar structure and all together subsiding electron delocalization effect between rings A and B. Therefore, various subclasses of flavonoids can be studied in-depth for their typical cardioprotective activity by elucidating the individual structure-activity relationship. Flavones: It has a 2-phenylchromogen-4-one backbone with a double bond between C2 and C3. Apigenin and luteolin represent this class and are found in celery, parsley, chamomile tea, fenugreek, garlic, onion, pepper, and citrus fruits (Clark et al., 2015). Studies have found that its capacity to surge vasodilation and accumulation of cAMP diminishes blood pressure by hampering cAMP-specific phosphodiesterase (Su et al., 2015). Vascular relaxation through NO is flooded due to the activation of endothelial NOS. Flavonols: It includes isorhamnetin, kaempferol, myricetin, and quercetin found in broccoli, onions, tea, blueberries, and apples that help control systolic blood pressure (Kumar & Pandey, 2013). The hydroxyl group at the third position on flavanols is deficient in flavones. Hence, these are considered flavone-derived substances. Quercetin and kaempferol have antihypertensive effects through modulating the renin-angiotensin-aldosterone system and improving endothelial function. Quercetin lowers blood pressure in diabetic patients and reduces oxidative tension in the heart and kidneys by causing protein kinase C-dependent vasodilation at the renal level (Oyagbemi et al., 2018). Likewise, gallocatechin, epicatechin, and catechin, proanthocyanidin, and procyanidins like thearubigins and theaflavins (oligomers), are the members of Flavan-3-ols found in cocoa, apricots, chocolates, red wine, red grapes, and tea. Studies reported that it could reduce mean arterial pressure, insulin resistance, and LDL-C and improve HDL-C levels (Clark et al., 2015). Catechins are aglycones that have positive effects on vascular function and thus have a cardioprotective effect (Ried et al., 2012). Epigallocatechin-3-gallate is a potent antioxidant that exhibits antiinflammatory and antiatherogenic properties. In a study, epicatechin declined systolic and diastolic blood pressure and was found to diminish myocardial rigidity in rats with HCM (Ellinger et al., 2012). Flavanones and flavones isolated from citrus fruits such as eriodictyol, hesperidin, and naringenin alleviate ischemic stroke and produce antioxidant effects by

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blocking free radicals (Khan et al., 2018; Mahmoud et al., 2019). The fundamental structure of flavanones is a 2-phenylchromogen with a saturated C2 5 C3 double bond. They are highly reactive and can be altered by methylation, hydroxylation (Koirala et al., 2016), and glycosylation. Naringenin lowers blood pressure, reduces NO levels, and defends against endothelial dysfunction (Ikemura et al., 2012). A flavanone derivative of hesperidin and its metabolites, produced in the gut after hesperidin digestion, has an antihypertensive effect. Their antiinflammatory action reduces the formation of atheroma plaques, moderates calcium ions levels, increases NO, and leads to smooth muscle vascular relaxation (Yamamoto et al., 2013). Anthocyanidins The cardioprotective potential of anthocyanins is generally related to its antioxidant properties. The brightly colored elements such as red wine, cranberries, red cabbage, and cherries containing cyanidin, malvidin, delphinidin, peonidin, pelargonidin, and petunidin compounds reduce the risk of myocardial infarction (Clark et al., 2015). They are polyhydroxy or polymethoxy derivatives of 2-phenylbenzo phyryllium. The cardiovascular functions of this class of flavones include endothelium-dependent vasodilation and the reduction of acute myocardial infarction (McKay et al., 2010). Recently, it has been reported that some anthocyanins may protect the heart against ischemia/reperfusioninduced injury by activating signal transduction pathways and supporting the functions of the mitochondria acting solely as antioxidants. This is possibly due to the decrease in cytosolic Cyt c preventing apoptosis and sustaining electron transfer between NADH dehydrogenase and Cyt c, supporting OXPHOS in ischemia-damaged mitochondria (Liobikas et al., 2016). Isoflavones: genistein, daidzein, and glycitein are isoflavones found in dairy products, soybean, egg, and meat to treat metabolic disorders. It resembles mammalian estrogens and behaves as an agonist for estrogen receptors. The difference is between isoflavones and flavones in the presence of a phenyl group at the C3 and C2 positions, respectively. Daidzein produces a cardioprotective action by augmenting NO synthesis and prostaglandins while reducing oxidative stress (Lu et al., 2014), whereas genistein possesses antihypertensive effects. Further genistein, daidzein, and biochanin, isoflavones present in soybeans, peanuts, and horse gram have been linked to their lipid-lowering property, promoting cardiac protection (Malarvizhi et al., 2021).

12.8 Biological action of flavonoids in cardioprotection Flavonoids exhibit embracing biological properties in the cardioprotection which is used to prevent and invade most of the pathological conditions of the heart. The important biological effects exerted by the various types of flavonoids have been elaborated in this section and the mechanism of action of a few newly reported flavonoids are given in the Table 12.2. Antiplatelet, antioxidant,

12.8 Biological action of flavonoids in cardioprotection

Table 12.2 Dietary flavonoids for mitochondrial health. Flavonoids

Sources

Wogonin

Scutellaria baicalensis Georgi

Vicenin-2

Scolymoside

Hesperetin

Kurarinone Orientin Vitexin

Morin

Increase Nrf2, decrease Ho-1 and Nqo-1signalling pathway, reduce oxidative stress and Inflammation Cyclopia subternata Antiaggregatory action in a Vogel thrombin-induced platelet aggregation Artichoke (Cynara Anticoagulation action scolymus L. leaves and leaf extracts) Citrus fruits Boost nitric oxide synthase adhesion and reduce nitrous oxide levels Sophora flavescens Inhibits iNOS, ROS, suppress roots cytokines, CCL2, TNFα, IL1β Papaver orientale Reduce ROS, inhibit cyt c, PI3K/ Akt signaling pathway Passion flower, Reduce inflammatory cytokines Bamboo leaves and and MAPK pathway. Pearl millet Waxy barley, White Enhance BCl2, Apoptosis, enhance mulberry, Guava mitochondrial enzymes

Phloretin

Apple tree leaves and the Manchurian apricot Formononetin Leguminous plants and fabaceae Delphinidin

Pelargonidin Myrecytin

Mode of action

Berries, cereals, grapes, blackcurrants Strawberries, radishes Moringa oleifera leaves

Resveratrol

Grapes, peanuts

Naringenin

Citrus fruits

Activates AMPK/Sirt3 pathway, reduces mitochondrial ROS

References Shi et al. (2021), Bei et al. (2020), Bojic´ et al. (2019) Bojic´ et al. (2019)

Bojic´ et al. (2019)

Yamamoto et al. (2013), Testai (2015) Han et al. (2010) Lu et al. (2011) Dong et al. (2013)

Benito-Román et al. (2015), Cheng et al. (2016) Han et al. (2020)

Downregulates IGF-1/IGF-1 R pathway and induce mitochondrial apoptosis Downregulates STAT1

Huang et al. (2014)

Upregulates Bax and Bid and downregulates Bcl-2 and Bcl-xL Activates PGC-1α and SIRT1 pathways, enhances mitochondrial activity Redox signaling, mitochondrial membrane potential, AMPK -Kir6.2/K-ATP signal pathway Activation of calcium-activated potassium channel (mitoBK) in IR injury, Modulates AMPK-SIRT3 signaling pathway.

Karthi et al. (2016)

Scarabelli et al. (2009)

Jung et al. (2017)

Das et al. (2014)

Moghaddam et al. (2020)

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antiinflammatory, antihypertensive, antiatherogenic, hypoxia, necrotic and apoptotic activity, and mitophagy are some of the other key biocharacteristics of flavonoids, which are briefly mentioned below.

12.8.1 Antiplatelet activity Antiplatelet agents aid in the prevention of heart attacks and strokes. The C-ring, responsible for various flavonoid subclasses, is essential for the antiaggregatory activity, wherein the hydroxyl groups at position six make the flavonoid dynamic in action. In the case of nonmethylated flavonoids, 2 C2 5 C3 augments antiaggregatory activity. Methylation of rings A and B reduces antiplatelet activity. Catechins and isoflavones have an antiplatelet activity that ranges from high to low (Boji´c et al., 2011). It reduces platelet aggregation by participating in arachidonic acid metabolism. Thromboxane A2, the central molecule produced by arachidonic acid metabolism, has receptors on the surface that cause platelet aggregation. Flavonoids have been shown to antagonize thromboxane A2 receptors in studies, implying that flavonoids indirectly reduce thromboxane A2 levels by inhibiting COX 1 enzyme. Genistein and daidzein, nonglucuronidated flavonoids, interfere with platelet aggregation due to their interaction with specific receptors on the thromboxane A2 surface (Mun˜oz et al., 2009). Upsurge in intracellular calcium levels and inositol pathways initiate oxidative stress that leads to collagen-induced platelet aggregation. Few flavonoids, including quercetin, catechin, and kaempferol, have been demonstrated to lower oxidative stress by inhibiting NADPH-oxidase (Rolnik et al., 2020). Flavonoids such as catechin and quercetin upregulate NO and downregulate the IIB/IIIa glycoprotein complex expression, thereby inhibiting platelet aggregation in some situations. Flavonoid glycosides, in addition to flavonoid aglycones, were found to have an antiaggregatory action. Scolymoside (luteolin-7-O-rutinoside) demonstrated an anticoagulation action elicited by 5 mol/L thrombin concentration, while its aglycone luteolin demonstrated a similar effect in an ADP-induced platelet aggregation assay (MINaAC 5 7.6 mol/L). Wogonin, a flavone derived from Scutellaria baicalensis Georgi, showed antiaggregatory activity in platelet aggregation tests produced by thrombin in its glycosidic form, wogonin-7-O-glucuronide, at 5 mol/L. Vicenin-2, a C-glycoside found in Cyclopia subternata Vogel, showed antiaggregatory action in a thrombin-induced platelet aggregation experiment at a dose of 5 μmol/L (Boji´c et al., 2019).

12.8.2 Antioxidant activity Stable radical formation in flavonoids reduces the active nitrogen/oxygen moiety because of its electron contribution to hydroxyl, peroxynitrite, and peroxyl radicals. Specific hydroxyl groups in distinct tautomeric forms or the presence of a conjugated ring system that is resonance stabilized may be responsible for the antioxidant characteristics of flavonoids. A lower saccharide link, characteristic

12.8 Biological action of flavonoids in cardioprotection

hydroxylation arrangement (catechol moiety in ring B), conjugation of C2 5 C3 double bond with C4-carbonyl moiety, methoxyl groups confers higher antioxidant properties. Planarity and electron modifications affect the dissociation constant of the phenolic hydroxyl group, allowing the whole fragment to connect to relevant molecular targets (e.g., enzymes) more efficiently. Hydrophobicity is another component that influences bio-membrane absorption (Wang et al., 2018). Das and coworkers determined the order of functional groups in the flavonoids based on their antioxidant capacity. that is, “20 ,4 0 -diOH,40 -OH 30 ,40 -diOH .2, 3double bond in conjugation with 4-carbonyl substitution, 3,5-diOH in conjugation with 4-carbonyl substitution, 3-OH in conjugation with 4-carbonyl substitution, 5OH in conjugation with 4-carbonyl substitution, and 3,5-diOH in conjugation with 4-carbonyl substitution” (Das et al., 2014). ROS production was prevented due to the bonding between enzymes and flavonoids, which regulate the formation of free radicals. Flavonoids also protect lipids against peroxidation when subjected to oxidative stress. Effects on lipid oxidation is induced at the hydrophobic part of the membrane due to the interaction between nonpolar molecules and flavonoids. It prevents oxidants from entering the hydrophobic area, hence shielding the membrane structure. This shielding effect was demonstrated mainly with epicatechin, and epicatechin greatly affected free radical elimination (Hamid et al., 2020). Inflammatory cytokines and bacterial endotoxins promote NOS expression, resulting in NO production and oxidative damage. Polyphenols action on metabolic enzymes of arachidonic acid synthesis viz. cyclooxygenase, lipoxygenase, NO synthetase reduce the inflammatory impact by lowering the synthesis of NO, prostaglandins, and leukotrienes. Flavonoids reduce xanthine oxidase ROS. Flavonoids’ carbohydrate fragments play a significant part in their antioxidant effect. Compared to the glycosides, aglycones are efficient, but the reduction in the antioxidant activity of flavonoids is reported with the increasing aglycone quantity attached to the glycosides (Ruparelia et al., 2017).

12.8.3 Anti-inflammatory activity An inflammatory response is a general complex mechanism that often occurs in any disease condition, primarily involving the microvessels, immune cells, and molecular mediators. In particular, CVDs predominantly have an association with inflammation. The flavonoids suitably provide vasodilation through the improvement of blood flow at the site of injury. Flavonoids are primarily known for vasodilation except for the class of flavonols as they lack the keto group. Accordingly, during CVDs, the specific properties of flavonoids could play their role through their anti-inflammatory action. For example, quercetin could aid in this mechanism by suppressing various inflammatory enzymes such as cyclooxygenase, lipoxygenase, and proinflammatory mediators. In addition to this, quercetin also reduces the inflammation process by suppressing endothelial leukocyte adhesion and other signaling pathways. Most importantly, the hydroxylated

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flavonoids are effectively inhibiting the synthesis of leukotrienes, reducing the histamine release. Aglycone is one such flavonoid that could impede the process of neutrophil degranulation, followed by decreasing the concentration of arachidonic acid (Mirsafaei et al., 2020). Other studies have highlighted the properties of flavonoids in antiinflammatory action through proper inhibition of prostaglandin synthesis, NOS, and phosphodiesterase. The beneficial effects of anthocyanidins have been elucidated for dropping the rate of acute myocardial infarction. These soluble pigments present in the fruits and vegetables aid its action through endothelium-dependent vasodilation (McKay et al., 2010). Some other examples of bioactive molecules from natural food sources like resveratrol from grapes, lutein from broccoli, methylxanthine, and theobromine from cocoa have been widely studied for their antiinflammatory properties in preventing CVDs (Cium˘arnean et al., 2020). Hesperidin and diosmin have been shown in vivo to inhibit prostaglandin production. Flavonoids in sophora and others such as, morelloflavone, bilobetine, amentoflavone function through inhibiting arachidonic acid release. Quercetin inhibits the cyclooxygenase and lipoxygenase enzymes by interfering in the leukotrienes, prostaglandins, and thromboxanes production. By reducing specific inflammatory mediators, NO, eicosanoids, or prostaglandin production, Resveratrol induces anti-inflammatory action (Cium˘arnean et al., 2020). The tannins, luteolin, and apigenin found in Cymbopogon citratus (DC) Stapf. act as anti-inflammatory, antioxidant, and vasorelaxation agents (Simo˜es et al., 2020) and are proven to exhibit cardioprotection in isoproterenol-induced cardiotoxicity (Gayathri et al., 2011). Flavonoids such as hesperidin, rutin, naringenin, quercetin from Anchusa italica Retz. and Heliotropium taltalense Phil. also possess the same potential (Wang et al., 2020). The cardioprotective effect of the flavonoid rich fraction of D. bulbifera Linn in isoproterenol induced myocardiac infarction and was modulated by the energy producing mitochondrial enzyme and endogenous antioxidant enzymes (Jayachandran et al., 2009).

12.8.4 Antihypertensive activity Flavones exhibits antihypertensive effects by activating the cAMP/protein kinase A cascade, subsequently activating NO synthase, resulting in increased endothelial NO concentration. This process, which is controlled by potassium and calcium channels, causes vasodilation (Barrientos et al., 2020; Puzserova & Bernatova, 2016). This is possible by activating the ion channels present in the mitochondrial membrane called mitoK, mitoKATP. Here, the flavonoid compounds like baicalein, puerarin, and naringenin exhibit cardioprotection by activating the Ca12 channels and then modulating Ca21 uptake. The opening of calcium/potassium channels likely results in the vasorelaxant effects. Naringenin, a flavanone, is produced due to hyperpolarization of membrane and vascular smooth muscle relaxation. Potassium channels, which are triggered by calcium, have an impact on them (Grijalva-Guiza et al., 2021). Hesperetin, a flavanone,

12.8 Biological action of flavonoids in cardioprotection

induces vasodilation via hesperetin-7
O-betaglucuronide, its metabolite, boosting NOS adhesion and decreasing nitrous oxide levels. As a result, NO levels rise in the blood promoting cardioprotection (Testai, 2015; Yamamoto et al., 2013). Flavonols like quercetin and kaempferol produce antihypertensive effects through influencing the renin-angiotensin-aldosterone system, smooth muscle contraction, and endothelial dysfunction (through endothelin-1), as well as NO-synthase (Larson et al., 2012). Flavan-3-ols (epicatechin) produce antihypertensive action by surging NOsynthase activity and reducing superoxide production in the left ventricle and aorta (Fusi et al., 2017). Additionally, epicatechin works by diminishing the level of arginase-2, which is accountable for NO reduction (Ried et al., 2012). Daidzein, an isoflavone, produces vasodilation via the same processes as other flavonoids but differs from them in that it stimulates the formation of prostaglandins (Lu et al., 2014). Tyrosine kinase Pyk2 inhibition by Genistein, an antihypertensive phyto molecule, aids calcium ion channel activation and its signaling pathways. It also helps to decrease pulmonary hypertension by reducing smooth muscle hypertrophy in the pulmonary arteries (Mita et al., 2013).

12.8.5 Antiatherogenic activity A flavonoid-rich diet can reduce the risk of ischemic stroke by lowering blood pressure, decreasing lipid oxidation, and increasing endothelial function, and drinking red wine after a meal lowers lipid hydroperoxides, a highly atherogenic component (Bouyahya et al., 2020). Animal model research discovered that the resveratrol treatment might minimize the volume of injured tissue following ischemia, presumably due to decreased lipid oxidation. Polyphenols in grapes may reduce the number of ischemia-affected neurons (Fraga et al., 2019). A study revealed flavonoids inhibit lipid buildup in the iliac artery in rabbits with hypercholesterolemia (Fernandes et al., 2017). It reduces cell distress caused by cardiac or cerebral ischemia by raising endothelial NO concentrations. An experimental investigation found that patients with diabetes who ate procyanidin (an antioxidant present in citrus fruits) had lower levels of oxidized LDL-C (Fraga et al., 2019). Polyphenols in grape derivatives also can reduce plasma lipid levels, including postprandial lipemia. It is inferred that the structural difference contributes to many pharmacological actions and influences interactions of flavonoids with active sites through synergistic or antagonistic effects. It is challenging to prove flavonoids’ cardioprotective properties based on structural characteristics; therefore, more compounds must be evaluated before definitive conclusions concerning SAR in flavonoids can be drawn (Arriagada et al., 2020; Zuo et al., 2020).

12.8.6 Hypoxia, necrotic and apoptotic activity Mitochondrial fission is induced by membrane depolarization through anion channels in the presence of ROS, and the resulting mitochondrial fragmentation

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provokes Cyt c release, which elicits cellular death/apoptosis. In common, the cells, especially mitochondria, neutralize the effects produced by ROS through complex enzymatic reactions. Ser/Thr moiety is phosphorylated by Src kinase (complex I enzymes) to reduce ROS production by etc. complexes (Ge et al., 2015). Amplified antioxidant enzymes expression in the deteriorating hearts is achieved by inhibiting histone deacetylase and reducing guanine by Boxybutyrate dehydrogenase (Uchihashi et al., 2017). Hypoxia is the oxygenabsent state which critically leads to a variety of cardiac pathologies. It typically results from ROS formation due to mitochondrial dysfunction and would cause unstable angina, myocardial infarction, and other CVDs. Some proteins such as hypoxia-inducible factors and chemical hypoxia mimetic agents (e.g., Cobalt chloride) play essential mechanisms in regulating the state of hypoxia (Shi et al., 2019; Uchihashi et al., 2017). Flavonoids have different metabolisms, pharmacokinetics, and bioavailabilities and are found to exhibit their role in hypoxia/reoxygenation cardiac injury. Recently, genistein has been studied for its excellent activity in protecting cardiomyocytes. It is a soybean-derived isoflavone of the Leguminosae family and can fight against the chemical hypoxia-induced injury through apoptosis. It is possible through the inhibition of the mitochondrial apoptotic pathway. In addition to this, it also has diverse biological functions, including regulation of lipid homeostasis and insulin resistance (Shi et al., 2019). Improved cardioprotection has been achieved by using the nano-encapsulated quercetin during hypoxia-reoxygenation through the preservation of mitochondrial function. This system could effectively overcome the problem of delivering the antioxidants as the metabolization rate hinders them. Comparing the encapsulated quercetin containing the poly (lacticco-glycolic) acid and free one, the former shows the ability to preserve the function of the mitochondria and the synthesis of ATP through suppression of oxidative stress (Lozano et al., 2019). The cardioprotective role of Borreria Hispida in a postischemic insult was mediated by the flavonoids; a polyphenol in the extract as evidenced by downregulating of HO-1 (Vasanthi et al., 2009). It is interesting to note that flavanoids such as rutin and quercetin, are abundant in Borreria Hispida and are responsible for the cardioprotective action in hypoxia-induced cardiomyocytes (Sundaram & Vasanthi, 2019). During mitochondrial apoptosis, Cyt-c and other pro-apoptotic elements are freed from the mitochondria into the cytoplasm. Further, it has been exquisitely demonstrated that significant Cyt-c inhibition from mitochondria after both hypoxia/reoxygenation and ischemia/reperfusion leads to cardiomyocytic apoptosis and mitochondrial dysfunction. Mitochondrial bioenergetics is the essential component behind the survival and function of cardiomyocytes. Vitexin treatment to the cardiomyocytes accordingly enhances the mitochondrial activity in a broader aspect. Additionally, after the hypoxia/reoxygenation, Drp1, a critical regulator in the mitochondrial fission process, is drastically increased. In contrast, the mitofusin-2 (fusion-promoting factor) level decreased after hypoxic

12.9 Concluding remarks

/reoxygenation or ischemic/reperfusion. In this case, vitexin has promising application as it reduces the expression of both Drp1 and mitofusin-2 (Xue et al., 2020).

12.8.7 Mitophagy The function of mitochondria especially in programmed cell death depends on mitochondrial fusion and fission reactions. Also, it destroys dysfunctional organelles through the process called mitophagy (autophagy), controlled by intracellular calcium and the sympathetic nervous system (Tilokani et al., 2018). Among fusion and fission reactions, fission has a very close relationship with cellular dysfunction/degradation. Ubiquitin ligase protein parkin is a key protein in mitophagy that translocate into the membrane, initiates ubiquitination, enters into lysozymes, and engulfs damaged organelles. Inter-mitochondrial communication between adjacent mitochondria is found in heart muscles which have “kissing junctions” that allow ions/protein exchanges (Siasos et al., 2018). Modifications in mitophagy led to the pathogenesis of several cardiomyopathies, and many studies prove it. It was demonstrated that the Becn1 1 / 2 genotype reduced in contrast to the overexpression of BECN1 and the pathological cardiac remodeling in a mouse model of pressure overload. It proposes that mitophagy/autophagy shows a noteworthy part in the progression of cardiac disease (Bravo-San Pedro et al., 2017). Frataxin, a mitochondrial protein, regulates the association of sulfur/iron mass-containing protein. Fascinatingly, stimulation of this frataxin expression triggers mitophagy. Filomeni and coworkers noted that supplementation of kaempferol modified impaired mitophagy (Filomeni et al., 2012). Puerarin can mitigate mitophagy defects through PINK1/Parkin pathway and alleviate palmitate-induced mitochondrial dysfunction (Chen et al., 2018). Another important flavonoid, quercetin, can improve mitochondrial dysfunction by inducing Parkin-dependent mitophagy (Liu et al., 2018; Yu et al., 2016). Naringin, by hindering the Parkin translocation into mitochondria exert shielding action in cerebral I/R injury and thus helps in ONOO
mediated excessive mitophagy (Feng et al., 2018). Likewise, a grape-derived antioxidant (Resveratrol) regulated mitochondrial fusion and fission, leading to mitophagy, via the Sirt1-Sirt3-Foxo3PINK1-PARKIN signaling network (Das et al., 2014).

12.9 Concluding remarks The mitochondria play a pivotal role in maintaining a healthy heart due to several cellular processes. Natural defense mechanisms preserve the cardiomyocytes from any stressful event such as hypoxia, oxidative stress, or ischemia. If healing is not possible, cellular survival relies upon the clearance of damaged mitochondria

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(mitophagy) or by pharmacological agents that antagonize cellular events like necrosis and apoptosis primarily or exclusively in the mitochondria. Flavonoids, categorized as polyphenolic secondary metabolites, are the third most abundant bioactive compounds involved in mitochondrial repair mechanisms. Almost 10,000 flavonoids, as evidenced by numerous research studies, have been explored for their practical use on cardiac health, and are outlined in this chapter. It achieves its function through counteracting inflammation, oxidative stress, and curbing intracellular signaling pathways of mitochondrial enzymes. Currently, flavonoids have focused on long-term studies both in preclinical and clinical conditions, and many studies advocated for the adjunctive treatment for the management of chronic diseases, especially cardiovascular disorders. In the case of the advancement of illnesses that distress huge populations, diet-based medicine appears to be exceptional. The use of bioactive substances like flavonoids could subsidize the economic burden and diminish the adverse effects of conventional treatment. Although flavonoid research has developed progressively, the few obstacles in terms of their poor bioavailability hinder its commercialization. Present-day research goals to address these issues by developing novel drug delivery systems may account for heart protection and thereby a healthy life.

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CHAPTER

Tea polyphenols stimulate mt bioenergetics in cardiometabolic diseases

13

Ravichandran Srividhya Department of Biotechnology, IIT Madras, Chennai, Tamil Nadu, India

13.1 An introduction to cardiometabolic diseases Cardiometabolic diseases are a category of noncommunicable diseases related to changes in lifestyle. Noncommunicable diseases have been called a global emergency in the rising arena of academic and political community (Nugent & Fottrell, 2019). Cardiovascular diseases (CVDs) are considered to be one of the leading causes of death worldwide, with .80% CVD-related deaths in low- and middle-income countries. Metabolic syndrome refers to a cluster of pathological conditions that provoke a major risk for CVD and other metabolically-associated syndromes like Type II Diabetes Mellitus (Kassi et al., 2011) and has also emerged as a health problem in modern society, associated with enormous social, personal, and economic burdens in both the developing and developed countries of the world. Literature cites that genetic defects are implicated in many cardiometabolic diseases (Kataoka et al., 2013). Metabolic syndrome has associated risk factors for cardiovascular risk by 50%
60% higher than people who do not possess any. Cardiovascular risks associated with metabolic syndrome includes the involvement of factors like genetic disposition, obesity, insulin resistance and inflammation (Qiao et al., 2007; Hasan et al., 2022). Cardiometabolic diseases are linked to several risk factors, in particular: obesity, hypertension, diet, tobacco, air pollution, and physical inactivity (Benziger et al., 2016). Cardiometabolic diseases have comorbidities and are multifactorial in nature with diverse factors including changes in living environments, diets, lifestyles, genetic, and epigenetic factors (Malik et al., 2013). Globally, the incidence of CVD-related deaths increased by 14.5% between 2006 and 2016 (Yan et al., 2019). Cardiometabolic diseases are constituted by multifactorial disorders with a wide spectrum of different factors including changes in living environments, diets, lifestyles, genetic, and epigenetic factors (Malik et al., 2013). With the escalation of obesity, diabetes and hypertension, there has been a parallel increase in the incidence and prevalence of cardiometabolic complications (Piche´ et al., 2020; Hasan et al., 2022). According to (Hasan et al., 2022), changes in metabolic status is a reliable parameter on par with rising BMI. People who are Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00012-6 © 2023 Elsevier Inc. All rights reserved.

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healthy but obese had lower risks for diabetes, coronary heart disease, stroke, and mortality than unhealthy subjects, but increased diabetes risks on obese rather than healthy lean people. Cardiometabolic risk factors confer much higher risk than obesity per se. Cardiovascular system is a specialized system which encompasses the major function of transporting substances like amino-acids, electrolytes, oxygen, carbon dioxide, hormones and other metabolic products to and from the cells through the bloodstream. With effective neurohormonal regulation, it has a main function of maintaining homeostasis and also to provide adequate nourishment for fighting various types of diseases (Tu et al., 2019). Any malfunction in the cardiovascular system is reflected by the metabolic status of the organism. They are characterized as multifactorial diseases wherein a number of factors like genetic, diet, lifestyle and environment are involved. Cardiometabolic diseases refer to a group of cardiovascular disorders often observed with metabolic syndrome. Several dysfunctions like hyperglycemia, dyslipidemia, hypertension, hyperinsulinemia, and the oxidative stress and proinflammatory cytokines associated with them are capable of driving endothelial dysfunction which is a hallmark of cardiometabolic diseases. The molecular mechanisms of CVD includes accumulation of reactive oxygen species (ROS), imbalance of vasoconstriction/vasodilation, chronic inflammation, and premature senescence, which are closely related to sirtuin-mediated regulation (Conti et al., 2017). Mitochondrial function and its relationship/changes to the dietary interventions have been widely studied owing to the role of mitochondria as the powerhouse of the cell and its involvement in the catabolism and anabolism of macromolecules and their energy metabolism in the body. In fact, mitochondria are critical to the maintenance of metabolic flexibility, efficient switches in metabolism, depending on the environmental demand (starve/feed cycles) (Smith et al., 2018). Several molecular cruxes that exist in mitochondria vouch for many metabolic pathways and refluxes paving way for its indispensable role in all the metabolic pathways of a cell. The metabolic disturbances in the body could have an impact on the molecular interplay that convene a severe effect on the physiological status of the cell. The role of mitochondria in the maintenance of the molecular stability of cell is a known fact. The ways by which mitochondria control the metabolic events in a cell are solely governed by genetic, physiological, and molecular factors. How could diet play a major role in metabolically driven functions? Can diet rich in functional foods and phytonutrients like green tea polyphenols be considered an alleviator of metabolic stress in the body? This review could highlight the importance of tea polyphenols in stimulating mitochondrial bioenergetics in cardiometabolic diseases.

13.2 Structure and bioenergetics of mitochondria A mitochondrion is a subcellular organelle which has 2
10 copies of mtDNA (Lan et al., 2008). Unlike many other organelles, mtDNA is replicated inside the

13.2 Structure and bioenergetics of mitochondria

organelle without the involvement of the nucleus. The human mtDNA consists of a 16.5 kb, double-stranded, circular DNA molecule (Anderson et al., 1981) which encodes 13 polypeptide genes that code for essential components of the respiratory chain or the electron transport chain. MtDNA also encodes the 12S and 16S ribosomal RNA (rRNA) genes and the 22 transfer RNA (tRNA) genes required for mitochondrial protein synthesis (Reddy and Beal, 2005). Some mitochondrial proteins which are coded by the nuclear DNA (nDNA) include several metabolic enzymes, DNA and RNA polymerases, ribosomal proteins, and mtDNA regulatory factors, such as mitochondrial transcription factor A. Nuclear mitochondrial proteins are synthesized in the cytoplasm and are subsequently transported into mitochondria. The special feature of Mitochondrial DNA (mtDNA) is that it is maternally inherited, and substantially more susceptible to mutations than nDNA owing to its lesser complexity in chromatin organization and repair capacity compared to the nDNA. Mitochondria represent the crucial bioenergetic and biosynthetic factories of the cell which are indispensable for normal function and physiology (Zong et al., 2016). The special feature of mitochondria is that it has its own DNA. The human mitochondrial genome is a double-stranded circular structure that is about 16.6 kb pairs in length. It contains 37 genes that code 2 rRNAs, 22 tRNAs, and 13 mitochondrial proteins of the respiratory chain (Carew et al., 2003). One of main features that differentiate the mitochondrial genome from the nuclear genome is the intrinsic susceptibility to damage. mtDNA is substantially more susceptible to mutations than nDNA as it is less protected due to its complex chromatin organization as discussed above, limited repair capacity, and also ROS generated by the electron transport chain being in its proximity (Wallace, 1999). There are multiple copies of mtDNA in each cell and mutations can affect either all of them (this is termed as homoplasmy) or a proportion (termed as heteroplasmy) which have a role in many mitochondrial diseases. However, it is important to point out that mitochondrial proteins involved in oxidative phosphorylation (OXPHOS) and ATP production are vastly encoded by nDNA. Several metabolic pathways like the TCA cycle, OXPHOS, fatty acid oxidation, urea cycle, amino acid metabolism, etc., occur in the mitochondria. Mitochondrial intermediates are often found as substrates in various anabolic pathways like gluconeogenesis, amino acid synthesis, nucleotide biosynthesis, fatty acid synthesis, cholesterol biosysnthesis, heme biosynthesis, etc., A pictorial representation of metabolic pathways occuring in mitochondria is shown in Fig. 13.1. Therefore, mitochondria are the source and site of ROS, hence the ROS generated by the electron transport chain imposes damage to mtDNA (Wallace, 1999). Mitochondrial number and morphology are controlled by an equilibrium of mitochondrial fusion and fission (Meyer et al., 2017) that is vital for metabolism, energy production, Ca21 signaling, ROS production, apoptosis, and senescence (Serasinghe and Chipuk, 2017). Fusion allows the exchange of mitochondrial components including mtDNA between different mitochondria. MtDNA due to their proximity to the respiratory chain, and a lack of protective histones have

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FIGURE 13.1 Metabolic pathways in mitochondria. (A) depicts the role of mitochondria in the oxidation of glucose, fatty acids and amino acids. (B) illustrates the anabolic pathways of glucose, cholesterol, amino acids, nucleotide and heme in mitochondria.

13.3 Mitochondria and its role in metabolism

FIGURE 13.2 Role of mitochondria in electron transport chain and oxidative phosphorylation. From Garcı´a-Garcı´a, F. J., Monistrol-Mula, A., Cardellach, F., & Garrabou, G. (2020). Nutrition, bioenergetics, and metabolic syndrome. Nutrients, 12(9), 1
39. https://doi.org/10.3390/nu12092785 Mitochondria and respiration.

a very high mutation rate that is about ten times faster compared to the nDNA. Mitochondrial membranes are composed of important proteins that are involved in the respiratory chain. Any alterations in the mitochondrial membrane constituents may lead to a decline in the bioenergetic function of mitochondria and this may be a contributing factor in a variety of pathological conditions including heart ischemia/reperfusion (Tengattini et al., 2008). The role of mitochondria and its electron transport chain proteins and their role in OXPHOS and respiration is shown in Fig. 13.2.

13.3 Mitochondria and its role in metabolism Mitochondria is regarded as the powerhouse of the cell and its widespread importance in the nutrient metabolism of the cell is demonstrated elaborately (Garcı´aGarcı´a et al., 2020). They are semiautonomous organelles with a unique genetic system that provide the chemical energy required for biosynthesis, respiration, secretion, and mechanical movement in organisms; they are also important organelles that generate intracellular free radicals and regulate apoptosis (Spinelli & Haigis, 2018). Mitochondria are dynamic in nature whose main function is the

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production of ATP through OXPHOS (Picard et al., 2011). Mitochondrial respiration involves the channelization of high-energy molecular intermediates through a series of enzymatic reactions, transferring chemical energy from food substrates and oxygen into a transmembrane electrochemical potential (Nicholls & Fergusson, 2013). They also regulate various cellular functions such as apoptosis, calcium homeostasis, and production of ROS, among others. A misleading functionality in any of the above could lead to an imbalance between oxidative stress and antioxidant defense which leads to macromolecular changes in postmitotic cells like cardiomyocytes, neurons, and skeletal muscles. Organisms with excessive mitochondrial dysfunction are incapable of sustaining life and adapting to dietary and physiological perturbations (Picard et al., 2018). The mitochondria is a well-known subcellular organelle which communicates with other organelles like the endoplasmic reticulum and the molecular crosstalk of mitochondria with other pathways has major impacts on cellular metabolism. For instance, ER, and mitochondria are tightly linked through contact points on their membranes, formed between the voltage-dependent anion channel, glucoseregulated protein 75 and inositol 1,4,5-triphosphate receptor. This crosstalk has been identified as an important regulator of mitochondrial dynamics, lipid and calcium homeostasis, autophagy, apoptosis (Kornmann, 2013) and disturbances in it, characterized by either an increase or a reduction of ER-mitochondria interactions, and this been reported in several neurodegenerative and metabolic diseases (Filadi et al., 2017). Mitochondria also have roles in cell death pathways like apoptosis and necroptosis (Thornton & Hagberg, 2015). What defines the respiratory state of the cell? ATP generation and its demand and/or availability of substrates for ATP generation defines the respiratory state in normal cells. However, there are several factors that influence the respiratory activity of a cell. Some of them are ROS, cell signaling, intracellular Ca2 1 , and the redox state of the electron transport chain. A list of bioenergetic parameters defines the bioenergetic profile of healthy mitochondria, they are: basal respiration, proton leak, coupling efficiency, respiratory control ratio, reserve respiratory capacity and nonmitochondrial respiration (Roy Chowdhury et al., 2012). Thus, most of the parameters are related to the electron transport chain of the mitochondria. A healthy mitochondrion always dictates the well-being of a cell’s physiological state. When there is a compromised antioxidant state, redox imbalance or a state of huge oxidative stress, the mitochondrion lose its metabolic capacity and becomes fragile. Several processes in the mitochondria help to keep the number, integrity, morphology, and physiological strength of the mitochondria. The socalled “mitochondrial metabolic stress” is a ruined condition of the mitochondria or the cell per se. Under such conditions, the main affected process is the respiratory chain and OXPHOS which synthesizes ATP for the cells. Thus, because the main function of mitochondria is the respiratory chain, any anomalies in the respiratory chain might spare the production of ATP, increase the production of ROS and, also lowers the antioxidative capacity, thereby leading to apoptosis (Van Houten et al., 2006). The bioenergetic status of a cell highly

13.4 Mitochondria and metabolic stress

depends on the morphological and physiological status of the mitochondria (Benard & Rossignol, 2008) and also contributes to many complex functional aspects of mitochondria. The functional and morphological behavior of mitochondrial networks largely influences the bioenergetic status of cells, tissues, and organs (Benard & Rossignol, 2008) and confers properties typical of complex systems, such as robustness, redundancy of function, and plasticity (Aon & Cortassa, 2012). These properties provide the system with the adaptive flexibility needed to adjust to changing stresses and metabolic demands.

13.4 Mitochondria and metabolic stress A metabolic syndrome is a combined dysfunction of several physiological disorders in the body. Several metabolic risk factors including abdominal obesity, insulin resistance, hypertension, and atherogenic dyslipidemia contribute to metabolic syndrome. Various genetic and environmental components (Hebebrand & Hinney, 2009) and changes of lifestyle (Hamilton et al., 2007) contribute to it as well. However, the fundamental mechanisms underlying metabolic syndrome remain largely unclear. Nutrient overloading which is seen in obesity conditions can lead to endoplasmic reticulum stress in the mitochondria. Several evidences show that mitochondrial stress decreases the mitochondrial capacity by decreasing its membrane potential and also electron transport-coupled ATP synthesis. Along with this, conditions like elevation of cytosolic Ca2 1 levels is also reported which could lead to the upregulation of genes involved in Ca2 1 transport and storage, as well as activation of the Ca2 1 -responsive factor calcineurin (Kelly & Scarpulla, 2004). In the biogenesis of mitochondria, calcineurin plays an important role in the expression of peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α (Schaeffer et al., 2004). There exists a strong interlinking mechanism between the mitochondria and ER which is fundamental for the maintenance of cellular homeostasis and survival (Giorgi et al., 2009). Transfer of lipids between the two organelles is a major site of metabolic crosstalk in the cell (Beller et al., 2010). Furthermore, there is dynamic exchange of Ca2 1 ions between these two organelles, which regulates processes such as ER chaperone-assisted folding of newly synthesized proteins, regulation of mitochondria-localized dehydrogenases involved in the ATP-producing Krebs cycle, and the activation of Ca2 1 -dependent enzymes that execute cell death programs (Franzini-Armstrong, 2007). ER stress during prolonged conditions, may induce a slow but sustained increase of free Ca2 1 in the mitochondrial matrix, which may induce the pro-apoptotic mitochondrial membrane permeabilization (Deniaud et al., 2008). There exists a homeostasis in the mitochondria which is represented by the steady-state balance between mitochondrial biogenesis and degradation. Some of the aspects that maintain homeostasis are mitochondrial division (fission) and

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fusion (Meyer et al., 2017), mitochondrial crest remodeling (Lesnefsky et al., 2016), mitochondrial biosynthesis (Pfanner et al., 2019), mitochondrial autophagy (Morales et al., 2020), and mitochondrial oxidative stress (Forrester et al., 2018). Any disturbance to mitochondria could lead to its dysfunction and henceforth to the malfunction of bioenergetics and related molecular pathways in the system. A regular adaptive regimen is indispensable in lieu of maintaining the regular functions of mitochondria during oxidative or metabolic stress conditions. One such adaptation is Mitochondrial biogenesis which is helpful in counteracting mitochondrial dysfunction/toxicity. Mitochondrial biogenesis is a very intricate process, and several proteins are involved in the process. The changes in protein expression and disruption of the mitochondrial stress response signaling by environmental factors, such as nutrition, are likely to induce mitochondrial function changes associated with metabolic dysregulation. Though the mechanisms involved in cardiovascular senescence is not thoroughly understood, the role of mitochondria in cardiometabolic diseases is identified well. Dutta and his coworkers claim that dysfunctional mitochondria are not only less bioenergetically efficient, but also generate increased amounts of ROS, interfere with cellular quality control (QC) mechanisms, and are more prone to triggering apoptosis. In this context, the removal and replacement of damaged/dysfunctional mitochondria are crucial for the maintenance of cellular homeostasis (Ding and Yin, 2012).

13.5 Mitochondrial fission and fusion The structural composition as well as the functional integrity of mitochondria is governed by the two important processes via mitochondrial fission and fusion. Table 13.1 highlights the important proteins involved in these processes. These two machineries are tightly controlled. Mitochondrial fission eliminates the defective mitochondria from the healthy ones by a process called as mitophagy. Drp1 and Fis1 are the important proteins involved in fission process in mammalian system. Drp1 is also involved in apoptosis wherein the role involves Bax oligomerization and cytochrome c release. Mitochodrial fusion is also a tightly regulated process which involves fusion of both outer membrane and inner membrane. The mitofusins, Mfn1 and Mfn2, exist as transmembrane dynamin-related GTPases (Santel & Fuller, 2001) wherein, the outer membrane fusion requires the mitochondrial outer membrane proteins like mitofusins namely Mfn1 and Mfn2, whereas the inner mitochondrial fusion mainly involves inner mitochondriallocalized protein, Opa1. These fission and fusion processes are not only crucial for the morphology and function of mitochondrial networks which determine the mitochondrial morphology, size and shape but also for transmitting redoxsensitive signals, redistributing metabolites and proteins, maintaining mtDNA integrity, performing metabolic processes, and regulating QC and cell death pathways (Green et al., 2011; Calvani et al., 2013).

13.6 Polyphenols as functional food

Table 13.1 Proteins involved in mitochondrial biogenesis and their functions. Protein

Location

Function

Mitofusin 1 (Mfn1)

Outer mitochondrial membrane Outer mitochondrial membrane

Fusion (Malka et al., 2005)

Mitofusin 2 (Mfn2)

Optic atrophy-1 (Opa1) Dynamin related protein (Drp1) Fission protein 1 (Fis1)

Inner mitochondrial membrane Cytosol Outer mitochondrial membrane

Fusion (Zorzano et al., 2010) Structural & functional connection with ER stress? (de Brito & Scorrano, 2009) Regulation of respiration (Liesa et al., 2009) Fusion (Malka et al., 2005) Cristae remodeling (Frezza et al., 2006) Protects from apoptosis Regulation of respiration (Liesa et al., 2009) Fission (Yoon et al., 2003; Santel & Fuller, 2001) Regulation of apoptosis (Frank et al., 2001) Fission (Yoon et al., 2003; Santel & Fuller, 2001)

Mitochondrial fission and fusion processes maintain the morphology and integrity of the mitochondria as discussed earlier. The expression, availability, and activity of the proteins that constitute fission and fusion machineries are tightly controlled. Mitochondrial fission dictates the QC of the mitochondria wherein the defective organelles are discarded or trashed by a process called mitophagy. Mitochondrial fission is a single step process in the mammalian system. On the other hand, fusion is a two-step process which involves the fusion of both outer and inner membranes. Both fission and fusion are cyclic events (Twig et al., 2008). According to Cagalinec et al. (2013), the rate of a mitochondrial fission depends on the length of mitochondria, whereas the rate of mitochondrial fusion depends upon mitochondria motility. The two processes are highly interlinked. For instance, mitofusins involved in fusion pathway, activates the Diacylglycerol-Protein kinase C pathway to phosphorylate further downstream effectors like Drp1 which mediates fission. Fission activation is again followed by fusion. Thus, the two pathways coherently determine the healthy state of the mitochondria. For easy understanding, the readers are requested to go through the captivating figures, which explain the mitochondrial fission and fusion process (Fig. 13.3). Table 13.1 explains the proteins that are involved in mitochondrial biogenesis and their physiological functions in the cellular milieu.

13.6 Polyphenols as functional food Foodstuff can be regarded as functional if it is satisfactorily demonstrated to affect beneficially one or more target functions in the body, beyond adequate

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Mitochondrial Fission

A

Drp1

B BSE

GTPase Domain

B- Insert

BSE Stalk

N

Adaptor Protein

BSE Stalk

C

Mitochondrial Fusion

A

HR1

B

Mfn1/2

GTPase Domain N

TM

TM

HR2 Mfn1/2

C

Opa1 Opa1

MIS N

TM

Coiled Coil Domain GTPase Domain GED S1 S2

C

FIGURE 13.3 Mitochondrial fission and fusion pathways. (A) illustrates the fission processes that occurs in the mitochondria with the role of proteins like Drp1. (B) denotes the fusion processes that occur in the mitochondria highlighting the role of factors like Mfn1/2 and Opa1. From Babbar, M., & Saeed Sheikh, M. (2013). Metabolic stress and disorders related to alterations in mitochondrial fission or fusion. Molecular and Cellular Pharmacology, 5(3), 109
133. https://doi.org/ 10.4255/mcpharmacol.13.11 Co-ordinated control of fission and fusion pathways of mitochondria.

nutritional effects in a way which is relevant to either the state of well-being and health or the reduction of the risk of a disease (Wu & Wei, 2002). In other words, functional foods are those which are taken along with diet, which provide basic nutrition. Secondary metabolites have known beneficial effects on human health and one such category of functional food is the flavonoid. They form a large proportion of secondary metabolites which display a broad array of biological and pharmacological properties (Wink, 2015). Several polyphenols

13.6 Polyphenols as functional food

FIGURE 13.4 Chemical structure of green tea polyphenols. From Journal of Agricultural and Food Chemistry (2019), 67(4), 1029
1043. Structure of catechins.

are grouped under the category of functional food. Polyphenols are a group of chemical substances widely distributed in the plant kingdom and are a part of human diets (Cheynier, 2005; Scalbert et al., 2005). They are characterized by the presence of more than one phenol group per molecule. The main polyphenol dietary sources are fruits, vegetables, and beverages (fruit juice, wine, tea, coffee, and chocolate). The total intake of polyphenols is approximately 1 gram per day, depending on lifestyle and dietary preferences (Scalbert and Williamson, 2000). Polyphenols can be divided into four subgroups: flavonoids, anthocyanins, proanthocyanidins, and xanthones. The flavonoids constitute a large family of compounds including flavanols (e.g., catechins and epicatechins found in cocoa and dark chocolate), flavones (e.g., apigenin and luteolin found in parsley, celery and broccoli), flavonols (e.g., quercetin and kaempferol found in citrus fruits), flavanones (e.g., naringenin found in orange and grapefruits), anthocyanidins, proanthocyanidins and isoflavones (e.g., genistein found in soy beans) (Fig. 13.1) (Mukhtar & Ahmad, 2000). A schematic diagram for the classification of secondary metabolites and general structure of flavanoids is given in Figs. 13.5 and 13.6.

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FIGURE 13.5 General structure and classification of flavonoids. From Lewandowska, H., Kalinowska, M., Lewandowski, W., Tomasz, M., Ste˛pkowski, K., & Brzo´ska (n.d.). The role of natural polyphenols in cell signaling and cytoprotection against cancer development. Skeletal framework of flavanoids and their classification into broader categories.

13.7 Tea and its health benefits Tea is the highly consumed beverage after water (Cabrera et al., 2006). About three billion kilograms of tea is produced and consumed yearly (Khan & Mukhtar, 2007). Tea, brewed from the plant Camellia sinensis is consumed in different parts of the world as green, black or oolong tea. Of the tea produced worldwide, 78% is black tea, which is usually consumed in the Western countries, 20% is green tea, which is commonly consumed in Asian countries, and 2% is oolong tea which is produced (by partial fermentation) mainly in southern China. Brewed tea contains many compounds, especially polyphenols, and several studies show that polyphenolic compounds present in tea reduce the risk of a variety of diseases (Yang & Wang, 1993; Mukhtar & Ahmad, 2000).

13.7 Tea and its health benefits

FIGURE 13.6 Broad classification of secondary metabolites. From Lewandowska, H., Kalinowska, M., Lewandowski, W., Tomasz, M., Ste˛pkowski, K., & Brzo´ska (n.d.). The role of natural polyphenols in cell signaling and cytoprotection against cancer development. Classification of flavanoids from parent compounds.

The amount of tea consumed, and its bioavailability dictates its potential health benefits, which are due to the presence of catechins. Following oral administration of tea catechins to rats, the four principal catechins were identified in the portal vein, indicating that tea catechins are absorbed intestinally (Okushio et al., 1996). The functional catechins which are widely recognized as the main functional components in tea include catechin, epicatechin, epigallocatechin (EGC), epicatechin-3-gallate (ECG), epigallocatechin-3-gallate (EGCG) and gallocatechin gallate (GCG) (Cyboran et al., 2015). Green tea also contains gallic acid and other phenolic acids such as chlorogenic acid and caffeic acid, and flavonols such

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as kaempferol, myricetin, and quercetin (data as per USDA). The structure of green tea polyphenols is given in Fig. 13.4. Green tea polyphenols have proven to be beneficial in counteracting conditions of oxidative stress predominantly seen in cardiovascular diseases and cancer (McKay & Blumberg, 2002). Metabolically active elements of tea is attributed to their epicatechin derivatives which have been proven to possess potent functions in alleviating cardiometabolic stress in humans (Ruijters et al., 2013). Green tea polypenols have noted antioxidant properties. Tea polyphenols are commonly known as catechins, and are flavonoid compounds with a basic structure of α-phenyl-benzopyran, which is about 18% to 36% of the dry weight of tea leaves (Khan & Mukhtar, 2007). The biosynthetic pathways of catechin formation from plant flavonoids includes certain steps. Structurally, catechins and epicatechins have slight variations, while catechin and epicatechin differ at the stereochemistry around the 2 and 3 positions of the central heterocyclic ring: catechin is 2,3-trans whereas epicatechin is 2,3cis. The flavonoid precursors of both catechin and epicatechin are of the 2,3-trans configuration. Catechin is formed by direct reduction of 2,3-trans-leucoanthocyanidin by leucoanthocyanidin reductase whereas epicatechin is formed via the reduction of an achiral anthocyanidin by the enzyme anthocyanidin reductase, allowing for the introduction of 2,3-cis stereochemistry (Blumberg et al., 2014). Their structural configuration allows them to be effective free radical and singlet oxygen scavengers. Several reports have quoted the beneficial effects of green tea polyphenols in the prevention/treatment of cardiovascular, hepatic, renal, neural, pulmonary and intestinal diseases, cancer, diabetes, arthritis, shock, and decreases in ischemia/reperfusion injury and drug/chemical toxicity in various organs and tissues and many of these effects are presumably due to their antioxidant and antiinflammatory properties (Hu¨gel & Jackson, 2012; Zhong et al., 2002; Darvesh & Bishayee, 2013; Frank et al., 2001; Vinson, 2000; Lin et al., 1998; Hara, 1994; Zhong et al., 2003). Heart diseases form a major category of cardiometabolic disorders wherein an excessive preadipocyte differentiation and lipid accumulation leads to fat metabolic disorders, which in turn leads to a range of associated chronic diseases, such as atherosclerosis. Catechins from green tea have been proven indispensable in promoting the lipid metabolism thereby decreasing fat deposition and accumulation leading to blocks.

13.8 Cytoprotective actions of green tea polyphenols Tea is characterized by the presence of polyphenols (especially catechins), phenolic acids, amino acids, proteins, and fats. The catechins most commonly found in tea include several catechins and these compounds constitute up to 30% of the dry leaf weight of tea. Potent antioxidant action, modulation of cell signaling, stabilization of membranes, improvement of endothelial function, reduction of the blood pressure, and protection of mitochondria, the main organelles responsible

13.8 Cytoprotective actions of green tea polyphenols

for cellular energy supply, are being proposed as possible mechanisms of the beneficial effects of (2)-epicatechin (Fraga & Oteiza, 2011) apart from their antioxidant, lipoprotective and antiinflammatory activities (Musial et al., 2020). The antiinflammatory activities of catechins are found to be related to the decrease in the activity of IKK-β protein, which is involved in the phosphorylation of IκB-α. This has an ameliorating effect on the NF-κB signaling pathway. The major tea catechin EGCG has demonstrated its antiinflammatory activities in the MAPK pathway by inhibiting the phosphorylation of p38 kinase. Such roles of catechins in decreasing the activities of stress-related kinases have major effects on transcription factors like AP-1 (Rahman et al., 2006). Tea polyphenols have been shown to be effective in many signal transduction pathways involved in the prevention of cardiovascular diseases (Stangl et al., 2007). Food-derived bioactives and food-based antioxidants and neutraceuticals have gained importance owing to their physiological and clinical relevance in functional properties. As natural antioxidants and chemopreventive agents, dietary polyphenols are found in most of the components in human diets including fruits, vegetables, grains, tea, essential oils, and their derived foods and beverages. Phenolic compounds render protection against oxidative stress related diseases like metabolic diseases, cardiovascular diseases, and neurodegenerative diseases as evidenced by epidemiological, clinical and nutritional studies (Scalbert et al., 2005). Polyphenols are compounds having one or more aromatic (benzene) rings with hydroxyl groups. The antioxidant and antiinflammatory activities as well as other biological functions of polyphenols have been largely attributed to the particular chemical structures. The aromatic feature and highly conjugated system with multiple hydroxyl groups make these compounds good electron or hydrogen atom donors, neutralizing free radicals and other ROS (Zhang and Tsao, 2016). Earlier studies involving polyphenols report that some polyphenols exert protective effects in many diseases, including cardiovascular diseases (CVD) and metabolic syndrome, both triggered by oxidative stress (Chiva-Blanch & Badimon, 2017). In purview of CVD protection, tea polyphenols are mainly focused on the effects of EGCG, including the prevention of LDL oxidation, reduction of platelet aggregation, lipid regulation, and inhibition of proliferation and migration of smooth muscle cells (Cabrera et al., 2006). Cardiovascular diseases apply an intricate process of excessive preadipocyte differentiation and lipid accumulation leading to plaque/atherosclerosis (Elagizi et al., 2018). Jiang et al. (2019) has stated that (1)-catechin can effectively inhibit the differentiation of 3T3-L1 preadipocytes and promote lipid decomposition in the mature adipocytes by the regulation of C/EBPs/PPARγ/ SREBP1C and cAMP/PKA signaling pathways. The molecular pathways which stimulated the hydrolysis of glycerol in adipose tissue include lipid mobilization, hormone-senstitive lipase phosphorylation, thus promoting lipid decomposition (Lampidonis et al., 2011). Fig 13.7 outlines the role of catechins in lipolysis.

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FIGURE 13.7 Role of catechins in lipolysis. From Yang, J., Shijie, D., Feng, L., Chen, Z., Dongxiao, S.-W., Yilun, C., & Dapeng, L. (2019). Effects of (1)-catechin on the differentiation and lipid metabolism of 3T3-L1 adipocytes. Journal of Functional Foods, 62, 103558. https://doi.org/10.1016/j.jff.2019.103558. The action of catechins in promoting lipolysis by stimulating protein kinase A thereby increasing hormone-sensitive lipase to stimulate lipid degradation by stimulating its mobilization and metabolism.

13.9 Effects of nutraceuticals on cardiometabolic disorders The transformation of nutritional science has brought into the limelight, key compounds and pathways for how diet influences health and well-being. Among these, some of the most exciting advances are occurring in the areas of flavonoids, which are bioactive phytochemicals found in a range of plant foods, and dairy foods, including milk, yogurt, and cheese. Nutraceuticals have implications in oxidative stress and inflammation. Evidence has been documented for their role in intervening with such diseases associated with metabolic stress. Many compounds with a polyphenolic structure such as flavonoids, isoflavones, phenolic acids, and lignan contribute to increased plasma antioxidant capacity, decreased oxidative stress markers, and reduced total and LDL cholesterol. They modulate genes associated with metabolism, stress defense, detoxification, and transporter proteins. The antiinflammatory and antioxidant nature of these compounds play a pivotal role in their cytoprotective actions in metabolic stress. The mechanism of action of these compounds involve their polyphenol (catechol) structure, redox status, and their interaction with other biomolecules. As bioactive phytochemicals, they play an important therapeutic role in attenuating oxidative damage induced by a metabolic syndrome associated with atherogenic dyslipidemia, and a proinflammatory, pro-thrombotic state, at a subcellular level. In previous studies, administration of flavonoids exerted vasorelaxation effects and lowered blood pressure (Clark et al., 2015). Nitric oxide (NO) metabolism could be a key pathway by which flavonoids regulate vascular health (Bondonno et al., 2015). By increasing the expression of endothelial NO, flavonoids mitigate the bioavailability of NO through the AMPK pathway (Croft, 2016). Several

13.10 Molecular mechanisms of flavonoids in cardiometabolic diseases

NO-independent mechanisms like inhibition of vascular calcium ion channels have also been studied in vitro (Fusi et al., 2017). Tea polyphenols have been reported to exert lipid-lowering effects. Markers like LDL cholesterol and total cholesterol have been lowered by tea polyphenols (Zheng et al., 2011). Green tea polyphenols have lipid-lowering effect in the body. Proven evidence in this context state that the antioxidant activity of polyphenols is responsible for a reduction of lipid peroxidation, determining a qualitative reduction of oxy-LDL (oxidized LDL). Gren tea polyphenols are effective nutraceuticals which have proven effects in activating several enzymes in lipogenesis, and interfering with cholesterol biosynthesis by inhibiting HMG-CoA reductase. This leads to micellar solubilization and absorption of endogenous cholesterol (Way et al., 2009). The lipid lowering effect of polyphenols especially green tea catechin, is attributed to the galloyl moiety of tea catechins (Ikeda, 2008).

13.10 Molecular mechanisms of flavonoids in cardiometabolic diseases The cytoprotective effects of flavonoids have been widely studied in various diseases especially in metabolic stress, related to the heart. The catechin rings of tea polyphenols fall under the subcategory of flavan-3-ols under the broad category of flavonoids. Molecular mechanisms of cytoprotection of flavanols include antioxidant protection, antiinflammation (Kyung-Joo et al., 2003), metal chelation (Hasan et al., 2022), and by regulation of lipid metabolism (Kim et al., 2012). Several molecular mechanisms of cytoprotection have been studied for flavonoids, especially polyphenols with respect to their roles in cardiometabolic diseases. Polyphenols have a structural advantage wherein the presence of the chemical structure of polyphenols is characterized by the presence of several hydroxyl groups on different sites of a carbon atom, which would have chances to interact with reactive oxidizing species, thus counteracting oxidative stress. The 2,3-double bond and the unsaturated 4-oxo group in the C-ring facilitates electron delocalization of o-dihydroxyl catechol within the B-ring. Pathways involving NF-κB is portrayed as an important pathway in their mechanism of action. This transcription factor has a primary function of regulating the expression of antiapoptotic genes and also to activate certain proinflammatory cytokine and chemokines (Iliopoulos & Hirsch, 2013). Under normal conditions, NF-κB binds to the inhibitory protein inhibitor of NF-κB (I-κB) in the cytoplasm. During oxidative stress and conditions like compromised antioxidant defense mechanism, I-κB rapidly phosphorylates and releases NF-κB to transfer it into the nucleus and the resultant oxidative stress responses lead to the development of inflammation and organ damage (Potoyan et al., 2015; Nanji et al., 2003). Excessive oxygen free radicals cause activation of NF-κB and regulate the expression of

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inflammation and immune-related genes, exacerbating apoptosis of vascular smooth muscle cells and causing inflammatory responses (Mitjans et al., 2009). The antioxidant system is also activated and there is a profound reduction in the formation of ROS (Liu et al., 2014). The antioxidant mechanisms of catechins particularly due to flavan-3ols is due to their capacity of mitigating with nuclear factor kappa-B (NF-κB), a transcription factor that activates inflammatory cytokines in tissue injury or ischemia (Bharrhan, 2011). Otherwise, the oxidative damage to cardiac tissue and vascular endothelium would lead to a serious damage. The role played by EGC has been attributed to its mitigating efficacy at the level of proliferation of vascular smooth muscle cells, induced by interleukin-1-beta (IL-1β, a potent proinflammatory cytokine), thereby preventing atherosclerosis. Studies have also shown that in endothelial cells, flavanone metabolites have been shown to affect the expression of a number of genes related to atherogenesis and especially those involved in cell adhesion, cytoskeleton organization, inflammation, and chemotaxis (Chanet et al., 2013). Clinical evidence for the mechanism of action of catechins demonstrate the cardioprotective capacity of catechins like EGCG. For instance, in a study on pulmonary fibrosis, EGCG protected the cardiac capacity by reducing collagen deposition and wet-dry lung weight ratio in Wistar rats along with the restoration of activities of antioxidant enzymes like GST, NADPH, and quinone oxidoreductase 1 (NQO1) (Sriram et al., 2009). The protective effect of EGCG is attributed to the Nrf2 signaling pathway. Nrf2 interacts with Keap1 to reduce oxidative stress and improves the antioxidant functions by acting on target enzymes like Heme oxygenase 1 (HO1), NADPH, NQO1, and glutathione S-transferase (GST) (Wang et al., 2015) thereby playing a master role in controlling the antioxidant capacity of the cells (Jaramillo & Zhang, 2013).

13.11 Molecular mechanisms of action of tea polyphenols Cell signaling pathways are regulated by tea polyphenols. For instance, EGCG regulates apoptosis induced by oxidative stress via the protein kinase B (Akt) and c-Jun N-terminal kinase signaling pathways. ECG upregulates mitogen-activated protein kinase and antioxidant response element gene expression, thereby enhancing the ability of the cell’s antioxidant defense system (Nie et al., 2002). The redox status of mitochondria is a major concern in determining the overall antioxidant status of the cell since mitochondria are the power resources of the cell. Three members of the Sirtuin family (SIRT3, SIRT4, and SIRT5) are located in the mitochondrion (Tang et al., 2017). These sirutins play a major role in the redox status of the mitochondria, which in turn regulates the cardiometabolic status of the organism. Studies show that the mitochondrial sirtuin family is involved in insulin resistance with diabetes mellitus (Koentges et al., 2015; Zeng et al., 2015); The chapter focusses on the role of polyphenols in counteracting the

13.11 Molecular mechanisms of action of tea polyphenols

oxidants thereby augmenting the sirutins in maintaining the redox balance of the mitochondria and in turn, the cell. Hypertension, a common risk factor for diseases, such as cardiovascular, cerebrovascular, and kidney disease, has become a major risk factor for premature death and disability worldwide (Forouzanfar et al., 2017). The roles of SIRT3 in hypertension are well documented. Reduced SIRT3 levels promote glycolysis via two mechanisms. First, the peptidylprolyl isomerase D (cyclophilin D) is in a highly acetylated state in the absence of SIRT3, which activates hexokinase II (HK2), a key glycolytic enzyme in the mitochondrial outer membrane. HK2, phosphorylates glucose to produce glucose-6-phosphate (G6P), the first step in most glucose metabolism pathways (Wei & Dai, 2013). Second, loss of Sirt3 increases ROS production, which stabilizes hypoxia-inducible factor- (HIF-) 1α, a transcription factor that regulates glycolytic gene expression (Paulin et al., 2014). One of the important catechin polyphenols present in green tea, (2)-epicatechin and its derivatives, have been proven efficient in the prevention of cardiovascular diseases in humans (Ruijters et al., 2013). Plausible beneficial effects of epicatechin has been matched to its potent antioxidant action, modulation of cell signaling, stabilization of membranes, improvement of endothelial function, reduction of the blood pressure, and protection of mitochondria and other organelles responsible for cellular energy supply (Fraga & Oteiza, 2011). Since mitochondria are the major sites of cellular energy supply, modulation of their functional activity may be very important for preserving cell viability under normal conditions and during metabolic stress. Impaired mitochondrial function represents an early manifestation of endothelial dysfunction and has been reported to be a contributor of cardiovascular diseases (Moreno-Ulloa et al., 2013). A polyphenol-rich diet would specifically improve the cardiac function by protecting the endothelial cells from oxidative stress and also improve their function. A decrease in blood pressure (Galleano et al., 2013), and augmentation of nitric oxide in endothelial cells (Brossette et al., 2011) have been stated as the contributing factors in the intervention of polyphenol for improving the physiological function in cardiometabolic diseases. Cardiovascular function is improved by EGCG via the eNOS pathway. EGCG elevates cGMP levels through a specific receptor 67LR that stimulates the Akt/eNOS pathway. This pathway leads to vasodilation which contributes to improvement of cardiovascular function. Fig. 13.8 summarizes the role of EGCG in improving vasodilation via eNOS pathway. Kim et al. have shown that EGCG is a key player in improving the metabolic and vascular functions. They have quoted that EGCG activates a kinase called AMPK which regulates key enzymes involved in energy metabolism and endothelial functions. Figs. 13.5 and 13.6 illustrate the classification of secondary metabolites and general structure of flavanoids. AMPK is a key molecule that regulates enzymes involved with energy metabolism and endothelial functions. Fig. 13.9 represents the actions of EGCG in energy metabolism and endothelial functions via AMPK pathway,

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FIGURE 13.8 Effect of EGCG on vasodilation in cardiovascular diseases. From Kim, H.S., Quon, M.J., Kim, J.A. (2014). New insights into the mechanisms of polyphenols beyond antioxidant properties; lessons from the green tea polyphenol, epigallocatechin 3-gallate. Redox Biology 10;2:187
95.

The mitochondrial electron transport chain is the main source of ROS, and SIRT3 enhances the ability of the mitochondria to cope with ROS in multiple ways. The key superoxide scavenger, Mn superoxide dismutase (SOD2), can reduce superoxide production and protect against oxidative stress. SIRT3 directly regulates the activity of SOD2 by deacetylation (Dikalova et al., 2017; Xie et al., 2017; van de Ven et al., 2017). SIRT3 deacetylates and activates the TCA cycle enzyme IDH2 and helps replenish the mitochondrial pool of NADPH (Someya et al., 2010). NADPH is a key reducing factor that affects glutathione reductase, a part of the antioxidant defense system against cellular oxidative stress (Someya et al., 2010). In addition, SIRT3 promotes effective electron transport via deacetylation of etc complex components, which indirectly reduces ROS production (van de Ven et al., 2017). Molecular intervention of EGCG activates certain metabolic pathways in metabolic diseases like CVD. The production of ROS and/or the formation of EGCG quinone by EGCG oxidation could activate the nuclear factor erythroid 2 p45-related factor 2 (Nrf2)-dependent cytoprotective enzymes (Yang et al., 2018), and this would be beneficial in the protection against oxidative stress. There are proven evidences which claim the mitoprotective activity of

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FIGURE 13.9 Role of EGCG in energy metabolism and endothelial functions via the AMPK pathway. From Kim, H.S., Quon, M.J., Kim, J.A. (2014). New insights into the mechanisms of polyphenols beyond antioxidant properties; lessons from the green tea polyphenol, epigallocatechin 3-gallate. Redox Biology 10;2:187
95.

EGCG, a green tea catechin by exhibiting its antioxidant activity (Meng et al., 2008). Thus, the augmenting effect of green tea polyphenols confer cytoprotection against metabolic-stress induced damage to the heart, thereby protecting it from deterioration or damage. The multifunctional effects of green tea polyphenols have proven beneficial in decreasing ROS production, maintaining redox balance and activating certain metabolic pathways which are helpful to the cardiac cells to counteract the demolishing effects of free radicals thereby correcting them and offering them a shield to work against the metabolic insults riggered in the body.

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14

A review of quercetin delivery through nanovectors: cellular and mitochondrial effects on noncommunicable diseases

Omar Lozano1,2,3, Diego Solis-Castan˜ol1, Sara Cantu´-Casas1, Paolo I. Mendoza Muraira1 and Gerardo Garcı´a-Rivas1,2,3,4 1

Tecnologico de Monterrey, School of Medicine and Health Sciences, Ca´tedra de Cardiologı´a y Medicina Vascular, Monterrey, Mexico 2 Tecnologico de Monterrey, Hospital Zambrano Hellion, Biomedical Research Center, San Pedro Garza Garcı´a, Mexico 3 Tecnologico de Monterrey, The Institute for Obesity Research, Monterrey, Mexico 4 Tecnologico de Monterrey, Functional Medicine Center, Hospital Zambrano Hellion, TecSalud, San Pedro Garza Garcı´a, Mexico

14.1 Introduction Noncommunicable diseases are the leading causes of mortality worldwide (WHO, 2020). For example, in Mexico cardiovascular diseases (CVD) and malignant neoplastic disease are the leading causes of mortality (Aceves et al., 2020). For the treatment of these diseases, new molecules as coadjuvants have been explored, which could be of easy access and with remarkable effects, such as in the case of nutraceutical products, including flavonoids such as quercetin (QCT). QCT is a flavonoid found in multiple fruits and vegetables, and possesses properties, which include anti-inflammatory, antioxidant, and antineoplastic effects (Davis, Murphy, Carmichael, 2009, 2009). For example, QCT in cancer therapy has become the main research focus for using it as a pharmacologic agent (Saavedra-Leos et al., 2021), while it also has been explored to reduce the cardiac damage produced in events related to cardiac and cerebral ischemia/reperfusion (Ghosh et al., 2017; Lozano et al., 2019). However, one of its main limitations lies in its low water solubility, (2.15 mg/L) (Srinivas et al., 2010), and high metabolization rate, for example 12 h after oral administration of QCT in rats, , 1% was found in blood with an absorption of 0.3%
0.5% of unaltered QCT (Formica & Regelson, 1995). Such restrictions are reflected in low bioavailability, which has led to issues translating benefits Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00006-0 © 2023 Elsevier Inc. All rights reserved.

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observed in vitro into clinical studies with natural compounds (Watkins et al., 2015). Therefore, increasing the bioavailability of QCT is a desired quality, and it can be achieved through the use of nanoparticles (NPs) for its encapsulation (G. E.-S. Batiha et al., 2020). Such encapsulation may not only yield improved in vivo bioavailability, but also elicits controlled and sustained release depending on the used nanomaterial (Watkins et al., 2015). On the other hand, noncommunicable diseases are linked to low-grade inflammation, oxidative stress, and metabolic impairment. Interestingly, many of these factors are associated with mitochondrial damage, suggesting that mitochondria play an essential role in the pathophysiology of CVD and cancer (Diaz-Vegas et al., 2020). This chapter is centered around the studies of nanoencapsulated QCT (nanoQCT) and its applications in the mitochondria. In this regard, a search on Pubmed was performed, including the keywords “Quercetin,” “mitochondria” and “nano .” Original research articles of nanostructures delivering QCT with effects on the mitochondria were analyzed. The search resulted in 12 articles, 7 of which were published in the past five years, and most of the literature was based on noncommunicable diseases. The delivery of nanoQCT to the cell and mitochondria has been studied in scenarios such as, cytotoxicity enhancement for cancer therapy, protection against cardiovascular ischemia/reperfusion, gastric ulcers, and preservation of sperm quality. This review begins with a presentation of QCT and its cellular and mitochondrial effects, followed by the literature review on nanoQCT and its applications under the aforementioned biomedical contexts and their effects on mitochondria, then finalized by an analysis of the nanomaterials used to encapsulate it and its use rationale.

14.2 Quercetin metabolism, biodistribution and pharmacokinetics The polyphenolic flavonoid QCT (C15H10O7) is an abundant antioxidant found on fruits and vegetables. It is most abundant in red onions (32 mg/100 g), capers (234 mg/100 g), and dill (55 mg/100 g). It is absorbed by the gastrointestinal tract at the small intestine via enzymatic deglycosation. Next, QCT is transformed at various tissues, for example via catechol-O-methyltransferase, it produces 30 -Omethyl-QCT on the upper small intestine. After being absorbed by the small intestine, QCT enters the circulation and follows several enzymatic reactions, such as O-methylation, and glucuronidation, and/or sulfation in the liver. 40 -Omethyl-QCT beta-oxidation results in benzoic acid, the final metabolite before urine excretion (Amanzadeh et al., 2019). After sulfation or glucuronidation in liver, the second phase of QCT metabolism is its metabolites which can be excreted in the bile, then either reabsorbed in the small intestine or continue through the gastric tube for fecal excretion (Murota & Terao, 2003). Afterwards, the metabolites can be de-glucoronated or de-sulfated in peripheral tissues for

14.3 Mechanism of protection of quercetin

posterior methylation. In regard to QCT distribution, it has been found that glucorinated QCT can cross the blood brain barrier and be posteriorly methylated by some cells such as macrophages. The most frequent QCT metabolites found in plasma are: sulfated, sulfo-methylated, glucorinated, and glucorono-methylated QCT (Day et al., 2001). However, one of its main limitations lies in its low water solubility, 2.15 mg/L (Srinivas et al., 2010), and high metabolization rate; 12 h after oral administration of QCT in rats , 1% was found in blood with an absorption of 0.3%
0.5% of unaltered QCT (Formica & Regelson, 1995). Such restrictions are reflected in low bioavailability, which has led to issues translating benefits observed in vitro into clinical studies with natural compounds (Watkins et al., 2015). Two important factors that contribute to QCT’s low bioavailability are related to its first pass metabolism, that happens in the liver and the small intestine. In a study after oral administration of QCT, only 6.7% was extracted as unchanged QCT by the small intestine, and in regard to liver metabolism, QCT was injected intravenously into portal circulation and afterwards only 52.6% unchanged QCT was obtained, demonstrating how much first pass metabolism by the liver and gut reduces QCT bioavailability (Chen et al., 2005). It has also been reported that the pharmacokinetic properties of QCT at 200 mg had Cmax and Tmax of 2.3 6 1.5 μg/mL and 0.7 6 0.3 h, respectively (G. E. Batiha et al., 2020).

14.3 Mechanism of protection of quercetin in noncommunicable diseases 14.3.1 Quercetin as an antioxidant compound The antioxidant activity of QCT comes from its structure; it has a 3-OH and 5OH group in its “A” ring, a catechol moiety in its B-ring, and an oxo function carbonyl group in its C-ring, see inset of Fig. 14.1. Such antioxidant activity arises first from its capacity to scavenge free radicals and to bind to transition metal ions. For example, QCT has been used to reduce the ischemia-reperfusion injury, where by binding to free radicals, it impedes nitric oxide binding to free radicals, thus reducing the formation of peroxynitrite, which damages the cell membrane (Baghel et al., 2012). Additionally, as a reactive oxygen/nitrate species (R/NOS) scavenger effect, it has been found that high doses (50
100 μM) of QCT increase concentrations of superoxide dismutase (SOD) and catalase, while low doses (0.1
1 μM) of QCT decrease SOD, but increase levels of glutathione peroxidase (Alı´a et al., 2006). It has been reported that QCT can inhibit glutathione-S-transferase P1
1 at micromolar levels, where the coadministration of ascorbic acid prevents such inhibition (van Zanden et al., 2003). In addition, the administration of QCT has beneficial effects on the mitochondria, such as the preservation of respiratory chain complexes, ATP levels, and reduced lipid peroxidation and swelling in the 3-nitropropionic acid model of Huntington’s disease

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FIGURE 14.1 Protective effects of quercetin (QCT) on the cell. QCT exerts its effects through activation of SIRT-1 and SIRT-3 mechanisms, as well as direct reactive oxygen species (ROS) scavenging. For example, QCT increases SIRT-3 mediated deacetylation of SOD, decreasing mitochondrial superoxide formation, while activation of SIRT-1 decreases proinflammatory mediators. Inset: chemical structure of QCT.

(Sandhir & Mehrotra, 2013). Also, QCT has been found to reduce proinflammatory cytokines such as TNF-α and IL-6, as well as pro-apoptotic proteins such as caspase 3 (Min et al., 2007). Despite QCT having a high antioxidant activity, some structure-related limitations come from its low solubility in water due to its hydrophobic phenol rings. Low solubility of QCT and fast metabolization rate has made it necessary for researchers to find innovative solutions for its transport and delivery, such as using NPs to increase its stability and solubility until their uptake (Lozano et al., 2019). QCT is implicated in several processes involving sirtuins, especially sirtuin1 (SIRT1). Sirtuins are enzymes found in most eukaryotic and prokaryotic cells, and in humans there are 7 isoforms that play a role in the metabolic status of the cell. Most sirtuins have strong deacetylase activity, except for SIRT5
6

14.3 Mechanism of protection of quercetin

(Trevin˜o-Saldan˜a & Garcı´a-Rivas, 2017). SIRT1 and SIRT3 have been implicated in several cell processes such as apoptosis and cell survival, regulation of reactive oxygen species (ROS), as well as hypertrophic and fibrotic processes. Meanwhile, SIRT1 deficiency leads to the activation of pro-inflammatory and procarcinogenic processes such as the activation of NF-κβ and TNF-α (Zhu et al., 2011). A study on oocytes from humans and aged mice found that QCT improved ROS scavenging by 33% through SIRT3 via the acetylation of the SOD2 K68 residue, reducing apoptosis, with a 25% reduction of Caspase 3, and improving autophagy with a 10% increase of LC-3 (Cao et al., 2020). SIRT1 plays an important role in mitochondrial biogenesis by deacetylation and increases the activity of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), which acts as an important regulator of mitochondrial biogenesis. This can used as therapeutic ground for mitochondrial diseases, cancer, or even neurodegenerative disorders. For example, the relation between QCT and an increase in SIRT1 and PGC-1α was studied in mice treated for seven days with QCT doses, 12.5 and 25 mg/kg. Results from biopsies of soleus muscle and brain showed a twofold higher mRNA expression of SIRT1 and PGC-1α activity in the QCT-treated group compared to the placebo (control) group (Davis, Murphy, Carmichael & Davis, 2009). Fig. 14.1 presents a summary of the protective effects of QCT in the cell. For neurodegenerative diseases, the beneficial role of QCT, in addition to the direct antioxidant effect, activation of SIRT1, and inducing autophagy, has been associated with the pathway activation of Nrf2-ARE and the antioxidant and antiinflammatory enzyme paraoxonase 2 (PON2), as well as QCT acting as a phytoestrogen (Costa et al., 2016).

14.3.1.1 Effects of nanoquercetin in cardiovascular ischemiareperfusion injury The antioxidant effect of nanoQCT has shown promising results for preventing cerebral and cardiac ischemia-reperfusion injury. A study conducted in young and aged rats was designed to evaluate the effectiveness of QCT as a method to protect the brain from ischemia-reperfusion injury (Ghosh et al., 2017). In the study, 42 nm QCT-loaded poly(lactic-co-glycolic) acid (PLGA) NPs functionalized with triphenylphosphonium were used. Results were similar in terms of changes for both rat groups. The results present a significant difference in edema reduction after ischemia-reperfusion injury in the groups treated with the NPs, compared to no difference in the groups treated with free QCT. After cerebral ischemiareperfusion, the groups treated with NPs showed, on brain mitochondria, a twofold increase in SOD and a 30% increase in catalase compared to the untreated group, reaching similar levels as the control. The groups treated with free QCT did not show significant changes with respect to the untreated group. These effects were correlated to the preservation of ΔΨm, having a twofold increase in the NP treated groups with respect to the untreated groups. Regarding cardiac hypoxia-reoxygenation, the effectiveness of QCT delivery has been assessed with 90 nm nanoQCT NPs, PLGA being the material of the

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NPs (Lozano et al., 2019). It was shown that, when H9c2 cells were challenged with antimycin A, it resulted in cells with 24 h of nanoQCT administration with lower mitochondrial O22 (4.65-fold) vs. untreated group (5.69-fold) and H2O2 rate production (1.15-fold) when compared to the untreated group (1.73-fold). Similarly, under hypoxia-reoxygenation, improved cell viability was found in the group treated 24 h before with nanoQCT (77%) vs. untreated group (65%), as well as a reduction in thiol groups (B70%) vs. untreated group (40%). Free QCT did not reach mitochondrial or cellular protection levels compared to its nanoencapsulated counterpart. The electron transport chain capacity showed that treatment with nanoQCT preserved it, while only a preservation of 35% and 40% were found for the untreated and free QCT groups, respectively. Also, it was shown that the ΔΨm was preserved 80% in the nanoQCT group, while it was only preserved 35% and 50% for the untreated and free QCT groups, respectively.

14.3.1.2 Effects of nanoquercetin in prevention of gastric ulcers The effects of QCT as an antioxidant are clear when it comes to the reduction of ROS in certain doses. A study was conducted to assess the protection effect of 15 nm PLGA-QCT NPs with regard to the pro-inflammatory capabilities of ethanol, which increases the concentration of ROS via the upregulation of myeloperoxidase (Chakraborty et al., 2012). Male Sprague Dawley rats were treated orally with encapsulated or free QCT, prior to orally administering ethanol to induce gastric ulcers. Analyzing the gastric tract, compared to the control, rats treated with nanoQCT vs. free QCT (2.5 mg/kg) resulted in a 5% and 13% increased in lipid peroxidation and ROS, respectively for the former, and 61% and 2860% increase, respectively for the latter. Similarly, glutathione levels were reduced 8% and 58% for nanoQCT and free QCT, respectively, compared to the control. The mitochondria of the gastric tissue were protected in nanoQCT administration, the only treatment that preserved complex I and II activities of NADH and succinate dehydrogenase, respectively, as well as preserving the ΔΨm. MMP-9, a sign of gastric ulcer damage, was increased 2.60-fold in untreated rats, and treatments with nanoQCT and free QCT resulted in a 1.20- and 2.50- fold increase. Similarly, it was shown that the group treated with nanoQCT reduced the levels of extramitochondrial cytochrome-c by 60% compared to the untreated group.

14.3.1.3 Effect of nanoquercetin on sperm quality and fertility The effects of QCT alone, or loaded in nanoliposomes (NLs) or in a nanostructured lipid carrier (NLC) were tested on sperm rooster cryopreservation and fertility performance, showing advantages related to the preservation of mitochondrial function using nanoQCT (Najafi et al., 2020), where all reported comparisons of QCT, free of nanoencapsulated, were done at 15 μM. All of the sperm motility parameters were higher in the QCT-loaded NLCs group compared to the control group (67% increase vs 52%). Viability was highest in QCT-loaded NLC group (72.92%) compared to the control group (58.46%) and QCT-loaded NLs group

14.3 Mechanism of protection of quercetin

(64.27%). Interestingly there was not a significant statistical difference in viability for all groups (71.99% in free QCT). Membrane functionality was greater in the NLC-loaded QCT group (65%) compared to the control (49.31%) and free QCT groups (53.68%). The mitochondrial activity of the sperm was higher (66.04%) for the QCT-loaded NLC group compared to the control (44.65%), free QCT (62.38%), and NLs (51.92%) groups . Compared to the control, free QCT, QCT-loaded NLs , and QCT-loaded NLC groups showed an increase in total antioxidant capacity (1.57-fold, 1.29-fold and 1.61-fold respectively). The QCTloaded NLC group had the highest living spermatozoa (63.50%) compared to control group (41.31%) and had the lowest rate of death (16.57%) compared to the control group (32.12%).

14.3.2 Quercetin as an anticancer agent Besides the antioxidant properties of QCT, its antineoplastic activity has been extensively documented. The classic mechanism associated is the inhibition of glycolysis, which leads to proapoptotic activity in tumor cells (Lang & Racker, 1974). However, recently another mechanism driving antineoplastic activity has been described involving protective autophagy. QCT antagonizes proliferation by cellular arrest in the G1 phase, which is achieved through downregulation of B1 cyclin and CDK1 (Reyes-Farias & Carrasco-Pozo, 2019). QCT has been shown to downregulate β-catenin, as well as stabilize HIF-1α and induce caspase 3 activity, all of this contributing to the loss of cell viability associated with QCT administration (Reyes-Farias & Carrasco-Pozo, 2019). Autophagy is promoted by QCT through the production of autophagic vacuoles, formation of acidic vesicular organelles, and activation of autophagy genes. LC3, an autophagy marker that aggregates in autophagosomes, is also increased by QCT, as found in the administration of the flavonoid gastric cancer cells, in addition to other autophagosome components such as the ubiquitin-like conjugation system ATG12-ATG5 (Wang et al., 2011). The AKT, m-TOR, PI3K pathway is greatly affected by QCT. It acts by impeding the activation of mTOR, which inhibits the formation of the mTOR complex 1 followed by a downregulation of several proteins such as p70, S6K, and 4E-BP1, which under normal conditions decrease autophagy through the inhibition of the vesicular double membrane formation (Wang et al., 2011). This results in greater autophagy activity. In addition, HIF-1α, increased in several conditions such as hypoxia, plays an important role in the promotion of autophagy by QCT. HIF-1α has been shown to increase under QCT administration in MKN28 cells, which acts by inhibiting the mTOR pathway and increasing levels of BNIP3/BNIP3L, resulting in an impediment of the Beclin 1 interaction with Bcl-2/Bcl-xL, which under physiological conditions inhibits apoptosis and autophagy progression (Wang et al., 2011). Fig. 14.2 summarizes the evidence of pathways involved in QCT-promoted autophagy in cancer cells. Cellular apoptosis in cancer cells is activated under QCT administration; this effect is mainly accomplished by the arrest of the PI3K/Akt/IKK/NF-κβ pathway

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FIGURE 14.2 Pathways of QCT effects on cancer cells: cell cycle arrest, promotion of autophagy via increasing HIF-1α and induction of BNIP3/BNIP3L, and increase in apoptosis by reduction in PI3K/Akt and increase of β-catenin pathways.

(Chirumbolo, 2013). NF-κβ is known to promote antiapoptotic gene transcription which is one of the main contributors to cancer cells. Activation of NF-κβ comes through phosphorylation by IKβ kinase (IKK). IKK is formed by the subunits IKKα, IKKβ, and IKKγ; where IKKβ is the main activator of NF-κβ. QCT downregulates IKK, decreasing the activity of NF-κβ, thus contributing to an apoptotic and inflammatory effect. PI3K/Akt also serve as important activators of the IKβ subunit, and the action of QCT in the PI3K/Akt pathway further decreases NF-κβ through the upstream downregulation of PI3K, which impedes activation of Akt and further reduces IKβ phosphorylation. The apoptosis promoted by QCT through the inhibition of the PI3K/Akt pathway, decreases HDM2 activity, which acts as a negative modulator of p53 through E3 ubiquitin ligase proteolysis. Contributing to this effect is the increase of PTEN by QCT (Chirumbolo, 2013). PTEN is a well-known oncosuppressor with phosphatase activity, which acts on the PI3K pathway by degradation of phosphatidylinositol triphosphate, causing an inhibition of protein kinase B (PKB/ Akt), resulting in reduced cell proliferation and survival signals (Hlobilkova´ et al., 2003).

14.3 Mechanism of protection of quercetin

QCT has also been reported to reduce chemoresistance by acting as a coadjuvant with antineoplastic drugs, such as Temozolomide (TMZ) (Thangasamy et al., 2010). Using DB-1 melanoma cells, TMZ treatment did not induce apoptosis, linking the localization in the nucleus of ΔNp73. However, co-administration of TMZ with QCT promoted apoptosis, with ΔNp73 redistributed to the cytoplasm and nucleus, and associated with increased transcriptional activity of p53.

14.3.2.1 Effects of nanoquercetin against tumor cells The pathways of nanoQCT delivery to cancer cells have been elucidated. In a study on C6 glioma cells, the administration of nanoQCT using PEG2000-DSPEcoated NLs, promoted with respect to control cells, increased time- and dose-dependent cell death, up to 12-fold, cytosolic ROS (3-fold), reduced phosphorylated p53 (up to 70%), increased necrotic cell death rate (4-fold), downregulated phosphorylated STAT3 (8%) and JAK2 (30%), while increasing cytochrome c (10%) and caspase 3 (15%), and reducing Bcl-2 (50%) and Bax (30%) (Wang, Wang, Chen, et al., 2013). The inhibitor of JAK2/STAT3, AG490, partially reversed the necrosis-induced cell death by 40% compared to the cells treated with the QCT NLs. This led to the conclusion that QCT NLs-induced cell death was mediated by JAK2/STAT3 and mitochondrial pathways. Additionally, doseand time-dependent effects were observed for decreasing C6 glioma cell viability when administered by QCT-NLs, including up to 50% reduction in ΔΨm, abolishing ATP levels and doubling LDH release compared to controls (Wang et al., 2012). Studies with QCT encapsulated in other materials have been found to reach the mitochondria. In a study the delivery of QCT was done through a LyP-1 functionalized regenerated silk fibroin (RSF)-based nanoparticle of 203.2 nm (Zhang et al., 2020). These NPs have a triple bioresponsive drug release, increasing its release at lower pH, or higher ROS and GHS conditions. The NPs internalized by 4 T1 cells were found within the mitochondria. Anticancer activity was observed by a . 80% decrease in viability of 4 T1 cells, compared to a 30% decrease in primary peritoneal macrophages, after 24 h of administration. The main cell death pathway was apoptosis (50.8%) through mitochondrial damage, as assessed by measuring ΔΨm. Migration of 4 T1 cells was reduced (4.50-fold) in the Lyp1QU-NP treated group compared to the control group, finding reduced PKM2 (sevenfold), MMP2 (1.40-fold) and MMP9 (twofold), and an increase in autophagy (13-fold) as assessed by LC3II1 puncta per cell. Using a BALB/c mice model of breast cancer, those administered with the nanoQCT NPs had 83% less tumor volume at day 21 than the control group. There was a 13-fold decrease in tumor growth rate compared with the control, and a 7.50-fold reduction in tumor weight compared to the control at day 21. Furthermore, a 21-fold decrease in metastatic nodes on the lung were found in the mice injected with NPs. LyP-1-Cy7NPs showed an increased biodistribution of the tumor (fourfold on day 3) and reduced systemic presence, compared to Cy7-NPs.

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A different approach to reducing tumor sizes is mediated by the inhibitory effects of topotecan (TPT) and QCT on triple negative breast cancer (TNBC, MDA-MB-231) and multidrug resistant type breast cancer cells (MCF-7) (Murugan et al., 2016). Using 70 nm mesoporous silica NPs to coencapsulate TPT and QCT, functionalized with the cRGD homing peptide, it was found that QCT and TPT drug release were inversely proportional to pH, having 80% and 75% released at 50 h at pH 5.0, respectively, compared to 10% released at 50 h at pH 7.4 for both compounds. After 48 h of NP administration to MDA-MB-231 and MCF-7 cells, with 2 μg/mL of drug, cell viability was reduced to 10% and 40% respectively, vs. 70% in both cells by administration of free TPT 1 QCT in both cells, compared to the control. The ΔΨm on these cells was reduced due to membrane depolarization and increased ROS. In vivo antitumor activity of the NPs showed that for induced tumors in female athymic nude mice, the tumor volume had an eightfold decrease in size, compared to the control group, or a fourfold decrease compared to either QCT or TPT treatment group after 24 days of intravenous administration of 5 mg/kg per treatment. Another study was conducted where QCT aimed to treat cancer cells in rats by using 100 nm mitochondria-targeted self-assembled NPs based on amphiphilic triphenylphosphine QCT conjugates functionalized with a pH responsive bond to PEG (TQ-PEG) (Xing et al., 2017). It was shown that the apoptosis rate of the TQ-PEG NPs was about 6% higher compared to the control. H22 tumor-bearing mice treated with TQ-PEG NPs showed the highest tumor suppression, 2.93-fold decrease in weight compared to the control, or a 2.66-fold decrease compared to free QCT. Major organs were neither damaged nor inflamed after treatment with TQ-PEG NPs. A summary of the reported results of nanoQCT and its effects on the mitochondria is presented in Table 14.1.

14.4 Nanomaterials for quercetin encapsulation The encapsulation of QCT has been done using a wide variety of materials, most of which are of polymeric origin. In this section, each material for QCT encapsulation is reviewed, along with the advantages it presents for this endeavor. The most used material for the encapsulation of QCT is PLGA, see Fig. 14.3. PLGA is most commonly used as a polymeric matrix due to its biocompatibility and biodegradability, with degradation products in the form of the metabolites lactic and glycolic acid, which are incorporated into the cell mitochondria Krebs cycle (Lu¨ et al., 2009). Also this polymer has suitable physical properties for controlled drug release, as well as its flexibility for surface functionalization, such as with moieties for active targeting (Danhier et al., 2012). This type of polymer has been used to deliver QCT to a wide range of targets: H9c2 (cardiac myoblast)

Table 14.1 Summary of studies delivering QCT by nanoencapsulation to the cell and mitochondria. Focus

Nanomaterial Biological Key results model

Cancer PEG2000cytotoxicity DPSE QUENLs LyP-1-QU TPT-MSNNH2-PAA-CSQT TQ-PEG

Gastric PLGA ulcer prevention Ischemia- PLGA reperfusion PLGA-TPP

Sperm motility

NLs and NCL

C6 glioma cells, 6-week-old female BALB/c mice, Female athymic nude mice of 5
6 week old H22 tumorbearing mice Male Sprague dawley rats Neonatal rat ventricular myoblast H9c2 Young and old male Wistar rats Adult ROSS 308 roosters at 30 weeks of age

Mitochondrial mechanism

References

Antitumor activity via the JAK2/ STAT3 signaling pathway by 40% compared to the control. Tumor size reduction at 83% and a 13-fold decrease in tumor growth in treated mice compared to control. Tumor size had a threefold decrease Apoptosis rate was 9% higher in treated models

High levels of mitochondrial ROS promoted p53 expression, inhibited expression of Bcl-2, upregulated Bax protein expression, and promoted C6 glioma cell apoptosis or necrosis via the mitochondrial pathway. Loss of ΔΨm Loss of ΔΨm Increased mitochondrial ROS

Wang, Wang, Luo, et al. (2013); Wang, Wang, Chen, et al. (2013); Wang et al. (2012); Zhang et al. (2020); Murugan et al. (2016); Xing et al. (2017)

90% prevention of gastric ulcer development compared to control

Increase of ΔΨm and reduction of mitochondrial swelling

Chakraborty et al. (2012)

Improved cell viability by 70% in hypoxia-reoxygenation Rats under cerebral ischemiareperfusion treated with N1QC showed a 10% edema reduction versus control and a 2-fold increase in mitochondrial membrane potential versus control Motility in sperm was higher with QCT treatment (67% increase vs 52%)

Reduction of mitochondrial ROS, preservation of ΔΨm, electron transport chain, and ATP production Reduction of mitochondrial ROS, preservation of ΔΨm, reduction of apoptotic signals

Lozano et al. (2019); Ghosh et al. (2017)

Increase of mitochondrial activity and membrane functionality

Najafi et al. (2020)

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FIGURE 14.3 Nanomaterials used for encapsulation and delivery of QCT.

cells (Lozano et al., 2019), gastric tissues (Chakraborty et al., 2012), and brain tissues (Ghosh et al., 2017). Another commonly used material is polyethylene glycol (PEG), see Fig. 14.3. This material is mainly used for surface modification of many NPs, such as magnetite NPs, because of its protective effect by forming a steric layer, thus granting NPs a high solubility in water, it is mostly biocompatible, nonantigenic, and a protein-resistant polymer (Tai et al., 2016; Torchilin & Trubetskoy, 1995), thus granting stealth properties to the immune system when NPs traverse the bloodstream (Blanco et al., 2015). This polymer has been used for surface modification for the delivery of QCT with different purposes: for drug delivery of anticancer drugs, inducing programmed cell death on C6 glioma cells through

14.4 Nanomaterials for quercetin encapsulation

PEG2000-DPSE NPs (Wang, Wang, Luo, et al., 2013), and as a strategy to provide protection from the immune system and particle stability to amphiphilic triphenylphosphine
QCT NPs (Xing et al., 2017). Another surface functionalization material is the triphenylphosphonium cation (TPP 1 ), see Fig. 14.3. This is a compound that is known for crossing into the mitochondrial matrix space due to its high lipophilic properties, effectively delivering cargo to the mitochondria (Marrache & Dhar, 2012; Marrache et al., 2014; Smith et al., 2003). It is remarkable in the accumulation of QCT NPs with TPP 1 in vitro (Ghosh et al., 2017), and has also been used to deliver it via amphiphilic triphenylphosphine QCT conjugates modified by PEG (Xing et al., 2017). Chitosan (CS), see Fig. 14.3, is a cationic biopolymer, derived from chitin, which can be dissolved in aqueous solvents at low pH; it is biocompatible and bioactive (Zhou et al., 2014). Due to its biocompatibility, it allows encapsulation and chain grafting of drugs and active ingredients. The slow biodegradability of chitosan NPs has shown that it can release drugs in a controlled and sustained way (Rajitha et al., 2016; Sivashankari & Prabaharan, 2017). This polymer has been used mainly for drug delivery of a variety of compounds such as ibuprofen (Popat et al., 2012) for inflammation, as well as a drug carrier and gatekeeper with poly(acrylic acid), loading TPT and encapsulating QCT (Murugan et al., 2016) for eliminating breast cancer cells. Poly(acrylic acid) (PAA) (see Fig. 14.3), is an anionic polyelectrolyte, which is soluble in aqueous media at natural pH (Nayak & Bhattacharyay, 2021). It has been used in addition to chitosan as an anionic inner-cationic outer layer, conjugated with QCT (Murugan et al., 2016). A drug carrier that has gained prominence, are the mesoporous silica NPs (MSNs), see Fig. 14.3. These NPs are composed of SiO2 and tend to have pore widths between 2 nm and 50 nm. This material offers several advantages such as high specific surface area, large pore volume, uniform pore size distribution, good biocompatibility, as well as tunable pore size (Kao & Mou, 2013; Kneuer et al., 2000; Shafiee et al., 2021), and an easily controllable morphology (Rouquerol et al., 1994; Wang & Huang, 2014; Xu et al., 2019; Zhao et al., 2020). MSNs have been used to deliver QCT to MDA-MB-231 and MCF-7 cells (Murugan et al., 2016). NLs are also commonly used for the nanoencapsulation of QCT. A material used for the preparation of liposomes can be Dimyristoyl phosphatidylcholine (DMPC), see Fig. 14.3. DMPC is a synthetic phospholipid analog of natural lecithin (Zinchenko et al., 2011), often used for bilayer studies (Dutagaci et al., 2014; Gurtovenko et al., 2004). It has been applied for the nanoencapsulation of dihydroquercetin modified with beta-cyclodextrin (Zinchenko et al., 2011). NLC have been prepared by combining QCT with a liquid lipid, Miglyol, a solid lipid, Precirol, and added the aqueous surfactant Poloxamer 407 (Najafi et al., 2020). Miglyol see Fig. 14.3, was used because the addition of triglycerides to the lipid matrix enables emulsification, thus preventing the formation of aggregates (Mahant et al., 2018). Precirol, see Fig. 14.3, is a solid lipid found to have a

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moisturizing effect, commonly used to prepare NLCs (Fang et al., 2008; Huang et al., 2008). The surfactant Poloxamer 407, see Fig. 14.3, is a nontoxic copolymer which can self-assemble into micelles in aqueous solutions at concentrations greater than the critical micelle concentration and at temperatures greater than the lower critical solution temperature (Yu et al., 2018). QCT has also been loaded into RSF-based NPs and they functionalized their surface with LYP-1 (X. Zhang et al., 2020). RSF, see Fig. 14.3, is a natural macromolecule derived from silk. This macromolecule has good biocompatibility, is nontoxic, nonpolluting, biodegradable, and nonirritating (Asakura et al., 2017; Yang et al., 2020), which has been used for drug delivery (Li et al., 2017; Zhang et al., 2020). Lyp-1, see Fig. 14.3, is a cryptic CendR peptide that specifically binds to p32 receptors, and its primary receptor is the protein p32/gC1q-R/ HABP1; overexpressed in tumor cells, this kind of peptide recognizes lymphatics and tumor cells in certain tumors, such as, breast carcinoma cells (Zhang et al., 2020), brain metastatic tumor (Zhang et al., 2018), lymphatic metastatic tumor (Yan et al., 2012), and pancreatic cancer cells (Jiang et al., 2017).

14.5 Conclusions The use of nanoQCT has been used in diverse areas, including cancer, cardiac, cerebral, gastric, and in the preservation of rooster sperm. Experimentation has included in vitro and in vivo scenarios, where the delivery of QCT in a nanostructure has shown improved results to the protection of the target cells and their mitochondria. Such protection may be associated to localized, controlled and sustained release of it, mitochondrial targeting, and protection of QCT by the nanostructure. This body of knowledge, even if limited in quantity, should provide a proof of principle that there are grounds to explore, under a preclinical setting, studies for the treatment of noncommunicable diseases with an efficient NP transporting QCT, which may lay the groundwork for the development of the next generation of nutraceutical-based treatments.

Acknowledgments This work was supported by CONACYT Grants 256577, 258197, Fronteras de la Ciencia (0682), and Ciencia Ba´sica (A1-S-23901 and A1-S-43883); and Tecnologico de Monterrey grant ILUT002
20IT22001.

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6074. Available from https://doi.org/10.2147/IJN.S92162. WHO. (2020). The top 10 causes of death. Retrieved from. Available from https://www. who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death. Xing, L., Lyu, J. Y., Yang, Y., Cui, P. F., Gu, L. Q., Qiao, J. B., & Jiang, H. L. (2017). pH-Responsive de-PEGylated nanoparticles based on triphenylphosphine-quercetin self-assemblies for mitochondria-targeted cancer therapy. ChemCommun (Cambridge), 53(62), 8790
8793. Available from https://doi.org/10.1039/c7cc04058j. Xu, C., Lei, C., & Yu, C. (2019). Mesoporous silica nanoparticles for protein protection and delivery. 7(290). doi:10.3389/fchem.2019.00290. Yan, Z., Wang, F., Wen, Z., Zhan, C., Feng, L., Liu, Y., & Lu, W. (2012). LyP-1conjugated PEGylated liposomes: A carrier system for targeted therapy of lymphatic metastatic tumor. Journal of Controlled Release: Official Journal of the Controlled Release Society, 157(1), 118
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Yu, Y., Feng, R., Yu, S., Li, J., Wang, Y., Song, Y., & Li, S. (2018). Nanostructured lipid carrier-based pH and temperature dual-responsive hydrogel composed of carboxymethyl chitosan and poloxamer for drug delivery. International Journal of Biological Macromolecules, 114, 462
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422. Available from https://doi.org/10.1134/S0006350911030298.

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Creatine monohydrate for mitochondrial nutrition

15

Maher A. Kamel1, Yousra Y. Moussa2 and Mennatallah A. Gowayed3 1

Department of Biochemistry, Medical Research Institute, Alexandria University, Alexandria, Egypt 2 Faculty of Science, Department of Biochemistry, Alexandria University, Alexandria, Egypt 3 Faculty of Pharmacy, Department of Pharmacology and Therapeutics, Pharos University in Alexandria, Alexandria, Egypt

15.1 Creatine monohydrate In the European Union, creatine monohydrate is defined as “food” according to the regulation of the European Parliament and the council as it is a “substance or product intended to be, or reasonably expected to be ingested by humans.” In the United States, creatine monohydrate and its salts are classified as “dietary supplements” under the 1994 “Dietary Supplements Health and Education Act” (http:// www.fda.gov/opacom/laws/dshea.html) (Pischel & Gastner, 2007). Creatine (Cr) is considered a mitochondrial nutrient because it acts as an energy-boosting compound by increasing creatine/phosphocreatine stores and Adenosine triphosphate (ATP) production. The system functions as a spatial energy buffer between the cytosol and mitochondria, and variation in this ratio is related to mitochondrial dysfunction (Lapointe, 2014; Tarnopolsky, 2008).

15.1.1 Structure Creatine is an amino acid with the chemical name methylguanidine acetic acid and with the empirical formula C4H9N3O, molecular weight 131.133, and CAS number 57-00-1 (Fig. 15.1). Cr is found ubiquitously in mammalian cells where it is converted into phosphocreatine (PCr). It is de novo synthesized intracellularly by the liver, kidney, and pancreas from the amino acids: arginine, glycine, and methionine, or is supplied exogenously via the diet. It is shuttled into cells via a sodium-dependent creatine transporter (CRT) and is actively transported into the mitochondria (Lapointe, 2014; Wyss & Kaddurah-Daouk, 2000).

Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00004-7 © 2023 Elsevier Inc. All rights reserved.

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FIGURE 15.1 Chemical structure of creatine.

FIGURE 15.2 Creatine de novo synthesis. AGAT, L-Arginine:glycine amidinotransferase; GAA, guanidinoacetate; GAMT, guanidinoacetate methyltransferase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine.

15.1.2 De novo synthesis of creatine Creatine de novo synthesis in mammalian cells requires three amino acids, methionine, glycine, and arginine, and two enzymes, L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1), and guanidinoacetate methyltransferase (GAMT, EC 2.1.1.2) (Fig. 15.2). The pathway consists of just two enzymecatalyzed reactions. In the first reaction, AGAT catalyzes the transfer of the amidino group of arginine to the amino group of glycine to form ornithine and guanidinoacetic acid (GAA). Then GAMT catalyzes the methylation of GAA on the original glycine nitrogen using S-adenosylmethionine as the methyl donor to yield Cr and S-adenosylhomocysteine (Da Silva et al., 2009). The kidney,

15.1 Creatine monohydrate

pancreas, liver, and some regions in the brain contain AGAT (Kreider & Stout, 2021). However, the activity of AGAT is predominant in the kidneys, whereas GAMT is highly active in the liver which suggests that GAA is synthesized primarily in the kidney and then transported to the liver where it is methylated to form Cr. Synthetized Cr is distributed via circulation to the organs of usage (mainly the muscles and brain) (Wyss & Kaddurah-Daouk, 2000). In humans, the total Cr pool is approximately 120 g in the average-sized adult male (70 kg). The daily turnover rate of Cr to creatinine (Crn) has been estimated to be about 1.6% of the total Cr pool. The daily requirement for Cr supplied through the diet or from the endogenous synthesis in a 70 kg adult man is 2 g/day. The endogenous Cr synthesis provides about 50% of the daily need of Cr (Brosnan & Brosnan, 2016). The remaining daily need of Cr is obtained from diet (red meat and fish) or dietary supplements (Kreider & Stout, 2021).

15.1.3 Supplementation form The primary dietary sources of Cr are red meat and fish which contain about 3 to 5 g of Cr/kg uncooked meat or fish. However, the heat cooking may degrade some of the Cr found in food. The commercially available Cr was firstly extracted from animal flesh, which was an expensive process. Nowadays, commercial Cr is produced through chemical synthesis, which uses many biomolecules as the principal starting materials (Kamath et al., 2005). One of these methods is based on guanylation (also, called amidination) of the amino acid sarcosine or its salts (sarcosinates). The guanylation agents are cyanamide, O-methylisourea, or S-methylisothiourea salts (Fig. 15.3). Cyanamide is used mainly for commercial manufacturing of Cr in Western countries, for example, Europe and the United States, whereas Omethylisourea or S-methylisothiourea salts are preferably used in the Eastern world, for example, Japan and China (Pischel & Gastner, 2007). The dose for the rapid increase of muscle Cr stores is 5 g of Cr monohydrate four times daily for 5
7 days (i.e., 0.3 g/kg/day). Supplementation with 2
3 g/day of Cr for 30 days effectively increases muscle Cr stores (Harris et al., 1992; Hultman et al., 1996). Cr is required at higher doses (e.g., 15
20 g/day for 2
4 weeks) to diffuse through the blood-brain barrier (BBB) to significantly increase Cr content in the brain in healthy individuals (Dolan et al., 2019). Patients with a deficiency in endogenous Cr synthesis (deficiency in AGAT and/or GAMT enzymes) may need to consume about 20
30 g of Cr/day to increase and maintain elevations in the brain Cr content (Schulze, 2003).

15.1.4 Tissue distribution of creatine The tissues store Cr in both free and phosphorylated forms (PCr). The total Cr content or pool in the body is the amounts of both free Cr and PCr. It was assumed that the body Cr content in adult men ranges between 109
169 g while

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FIGURE 15.3 Chemical synthesis of creatine.

in adult women it ranges between 70
112 g (Janssen et al., 2000). Most of the body’s Cr (95%) is stored in skeletal muscle where the predominant form is PCr (60%
70%) while the remaining 30%
40% is free Cr. The remaining 5% of total body Cr is found in the heart muscles, smooth muscles, brain, and testes. The synthesized or supplemented Cr reaches target tissues through the bloodstream, and the transport into the cells is mediated through a solute carrier protein called sodium/chloride dependent CRT (also known as SLC6A8) (Balestrino et al., 2009).

15.1.5 Catabolism Inside the cells, the creatine kinase (CK) catalyzes the reversible phosphorylation of Cr to produce PCr. In the tissues that require large and intermittent amounts of energy, several CK isozymes are ubiquitously expressed in different cellular compartments (e.g., sarcomere, cytosol, mitochondria) connecting places of ATP synthesis with sites of ATP consumption. This is known as the “Phosphocreatine-shuttle system” (PCr-Shuttle) (Wallimann et al., 1992). Cr is spontaneously degraded to creatinine in a monomolecular and nonenzymatic reaction that depends on temperature and pH (Uzzan et al., 2009). Creatinine might diffuse out of the cells to be excreted by the kidneys into the urine with a mean excretion rate of 23.6 mg/kg/day (about 1.7% of the total Cr pool per day) (Fig. 15.4) (Wyss & Kaddurah-Daouk, 2000).

15.2 Creatine in cellular and mitochondrial bioenergetics

FIGURE 15.4 Creatine catabolism.

15.2 Creatine in cellular and mitochondrial bioenergetics Cellular bioenergetics are balanced and tightly regulated for efficient energy use. ATP is the primary source of chemical energy, and its hydrolysis (by ATPases) is highly exergonic. Maintenance of cell homeostasis depends on mechanisms that adjust the ATP generation processes according to the demand for energy which may increase the rate of ATP hydrolysis within seconds by several orders of magnitude, but the intracellular ATP levels remain constant. This stability paradox (Hochachka, 1999; Hochachka, 2003) is explained by many shreds of evidence throughout the last five decades by the action of the so-called PCr-Shuttle or Cr-PCr circuit. This system is a fast and efficient energy-supporting and backup system that connects the sites of energy consumption with those of energy production via phosphoryl transfer networks (Dzeja & Terzic, 2003; Dzeja et al., 1996; Saks et al., 2006; Wallimann et al., 1992). The efficient cellular bioenergetics requires efficient ATP production (in mitochondria) and sending to the sites of its use (cytoplasmic organelles like myofibrils) through proper hydrolysis with ATPases. The continuity of these processes requires efficient coupling between the two compartments (mitochondria and cytoplasm) and immediate removal of the products of ATP hydrolysis (Adenosine diphosphate, ADP; Inorganic phosphate, Pi; and Hydrogen ion, H1) to avoid the

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inhibition of ATPases (Guimaraes-Ferreira, 2014). It was found that the mitochondrial and cytoplasmic compartments are efficiently coupled by a wellorganized energy transfer system constituted by Cr and different isoforms of CK in the so-called PCr-shuttle system proposed by Bessman in 1972 (Bessman, 1972). The functional evidence shows that energy is preferentially emitted by the mitochondrion as PCr and preferentially utilized by the myofibril in the same form. The evidence also shows that Cr, not ADP, is emitted by the myofibril and received by the mitochondrion (Bessman, 1986).

15.2.1 Creatine kinase isoenzymes CK isozymes are the core of the PCr-shuttle system. There are four major CK isozymes located with distinct subcellular locations: two cytosolic isoforms: muscle CK isoform (CK-M) and brain CK isoform (CK-B) and two mitochondrial isoforms mitochondrial Cr kinase (mtCK). As illustrated in Fig. 15.5, the cytosolic CKs exist as homo-dimers (CK-MM and CK-BB) under physiological conditions however, hetero-dimers (CK-MB) have also been identified (Eppenberger et al., 1967; Teixeira & Borges, 2012). The brain-type (CK-BB) is distributed mainly in the brain, heart, smooth muscle, nervous system, and other tissues, whereas the muscle-type CK (CK-MM) is the major isoform in the differentiated skeletal muscle tissue (Teixeira & Borges, 2012; Wallimann & Hemmer, 1994). The heterodimer isoform (CK-MB), appeared during fetal and neonatal development of skeletal muscle but also persisted in the heart during adult life (Dawson & Foss, 1965; Eppenberger et al., 1964; Teixeira & Borges, 2012). The two mitochondrial isoenzymes are the sarcomere-specific mitochondrial Cr kinase (smtCK) which is expressed only in skeletal and heart muscle and the ubiquitous mitochondrial Cr kinase (umtCK) which is expressed in many tissues with the highest levels in the brain, gut, and kidney (Payne & Strauss, 1994;

FIGURE 15.5 Creatine kinase isoenzymes classification and distribution.

15.3 Creatine/mitochondrial creatine kinase system

Teixeira & Borges, 2012). Both mtCK isoforms usually exist as octamers, however they can be dissociated into dimers (Bong, 2008; Teixeira & Borges, 2012). The mtCK is functionally associated with oxidative phosphorylation and is localized between inner and outer mitochondrial membranes in colocalization with the adenine nucleotide translocase (also known as SLC25A4), and with the voltage-dependent anion channel (Schlattner et al., 2018). This compartmentation of CKs involves the direct or indirect association of CK with ATP-providing (mitochondria and glycolysis) and ATP-consuming processes (ATPases and ATP-dependent cell functions), forming distinct compartments that facilitate a direct exchange of ADP and ATP between associated CK and its substrates (PCr and Cr) and the respective association partners of CK, without mixing with the bulk cytosol.

15.2.2 The phosphocreatine “shuttle” system in cell energy homeostasis The PCr-shuttle system (or Cr-PCr circuit) acts as an energy buffer to maintain equilibrium ATP potential and is fundamental in promoting cellular bioenergetics through energy transfer in the form of PCr (synthesized from ATP by the action of mitochondrial CK at the site of production) and the rapid resynthesis of ATP from ADP at the site of consumption (by means of cytosolic CK action). Mg ADP2 1 PCr22 1 H1 3MgATP22 1 Cr

In the PCr-shuttle system (Fig. 15.6), high-energy phosphate is transferred from the ATP formed through oxidative phosphorylation in the mitochondria (production site) to Cr, via the action of the mtCK, thus generating PCr and ADP. PCr diffuses into the cytoplasm, where under the action of cytosolic isoforms of CK, it generates ATP and Cr. ATP is then used by the ATPases (site of use), while Cr returns to the interior of the mitochondria by crossing the mitochondrial membrane, besides being present at higher levels in the intracellular medium. The aim of using Cr as a mitochondrial nutrient is to act as an energyboosting compound by increasing Cr/PCr stores and consequently preventing ATP depletion. The Cr/PCr system functions as a spatial energy buffer between the cytosol and mitochondria, and a variation in this ratio is related to mitochondrial dysfunction (Lapointe, 2014; Tarnopolsky, 2008).

15.3 Creatine/mitochondrial creatine kinase system in health and disease 15.3.1 In cardiac and skeletal muscles of athletes Sports organizations consider Cr as a natural food substance and hence it is never considered a banned substance for athletes (Thomas et al., 2016).

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FIGURE 15.6 The PCr-shuttle system. ANT, Adenine nucleotide translocase; CK-B, creatine kinase in the brain; CK-M, creatine kinase in muscle; Cr, creatine; CRT, creatine transporter; etc, electron transport chain; mt-CK, mitochondrial creatine kinase; PCr, phosphocreatine; VDAC, voltage-dependent anion channel.

15.3.1.1 Effects of creatine monohydrate on the skeletal muscle mitochondria Cr supplementation has shown to be the most suitable for sports with more anaerobic characteristics like sprinting, weight lifting, ice hockey, and football (Volek & Kraemer, 1996). Enhancing muscle bioenergetics is considered the primary mechanism by which Cr is able to improve the anaerobic persistence in the short term. The ability of Cr to enhance muscle growth upon long-term administration together with resistance training is again owing to the Cr ability to promote muscle bioenergetics, but also to its hypertrophic mechanism. As a result of the increased mechanical load, the muscle in turn produces new proteins as compensation. Those muscle proteins cause muscle hypertrophy and hence increase the ability of the muscle to generate more power and force in the long-term (Prevost et al., 1997; Volek & Kraemer, 1996). To perform all this muscle action during exercise, a series of reactions evolve to generate ATP and energy. Once Cr enters the bloodstream, it is transported to the muscle fibers through sodium/chloride transporters as shown in Fig. 15.7. The intracellular store of total muscle Cr (Cr 1 PCr) increases. Kreider et al. (Kreider et al., 2003) showed that supplementing Cr monohydrate (20 g/day) for 5 days will cause an increase in the content of free-Cr by 30%, PCr by 17%, and total-Cr

15.3 Creatine/mitochondrial creatine kinase system

FIGURE 15.7 Potential bioenergetic mechanisms of creatine inside a skeletal muscle. CK, Creatine kinase; Cr, creatine; CRT, creatine transporter; mtDNA, mitochondrial DNA; NFR1, nuclear respiratory factor 1; PCr, phosphocreatine; PGC-1α, peroxisome proliferatoractivated receptor-gamma coactivator-1 alpha; TFAM, mitochondrial transcription factor A.

by 20%. PCr stores synthesized by the PCr shuttle system in the mitochondria are responsible for replenishing the muscle ATP reservoir during the first 5 s in a sprint run, where a PCr donates its phosphate to ADP in the presence of Cr kinase enzyme. To provide more fuel to continue the run, enzymatic activation of adenylate kinase takes place to produce ATP from ADP. Administration of Cr in this phase will then provide more PCr, which can make more temporary ATP sources available and increase muscle strength and power before becoming exhausted (Stout et al., 2009). The mitochondrial pool of Cr serves as a reservoir in case the plasma Cr level drops, keeping the cytosolic level of Cr elevated during high-contractile muscle activity (Nash et al., 1994). Recently, a study by our research group examined the effect of Cr supplementation (0.5 g/kg/day for 5 weeks) with and without moderate-intensity exercise (1 h swimming with a metal ring) on the genes controlling mitochondrial biogenesis in rat cardiac and soleus muscles (Gowayed et al., 2020). The Cr supplementation efficiently boosts both mitochondrial biogenesis and functions and hence provides more ATP for muscle consumption, thus improving its muscle performance. The increased expression of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α) in the

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improvements in the soleus muscle of exercised rats receiving Cr was associated with increased serum pyruvate, but not lactate level, providing another energy source for the skeletal muscle. As clarified by Riesberg et al. (2016), when PCr level decrease in the muscle during the re-phosphorylation process of ADP, phosphofructokinase is produced to speed glycolysis and produce pyruvate. Our data suggested that in the presence of Cr, pyruvate could serve directly as a substrate for the mitochondrial Tricarboxylic Acid Cycle (TCA cycle), without being converted to lactate, to catabolize glucose and produce ATP (Fig. 15.7). Other bioenergetic mechanisms of Cr include its antioxidant and antiinflammatory potential because it has the ability to protect the mitochondria of myoblasts against oxidative stress that protects from DNA damage and prevents cell death (Deminice & Jordao, 2012; Riesberg et al., 2016). The ability of Cr to decrease skeletal muscle necrosis and enhance mitochondrial respiration has been observed in several contexts (Passaquin et al., 2002; Pearlman & Fielding, 2006). This antioxidant effect of Cr is believed to be the cause of its anticatabolic activity. As stated by Parise et al. (Parise et al., 2001) Cr administration decreased leucine oxidation in young adults, a marker of muscle protein catabolism, and also the urinary 3-methylhistidine in aging adults, as a marker of whole-body protein catabolism (Candow et al., 2008), Fig. 15.8.

FIGURE 15.8 Bioenergetic mechanisms of creatine in the muscle to promote muscle hypertrophy and physical performance. MRFs, Myogenic regulatory factors; mtDNA, mitochondrial DNA; mTOR, mammalian target of rapamycin; NFR1, nuclear respiratory factor 1; PCr, phosphocreatine; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator-1 alpha; ROS, reactive oxygen species; TFAM, mitochondrial transcription factor A.

15.3 Creatine/mitochondrial creatine kinase system

15.3.1.2 Effects of creatine monohydrate on the cardiac muscle mitochondria It has long been believed that cardiomyocytes cannot endogenously synthesize Cr (Lygate, Bohl, Ten Hove, et al., 2012; Zervou et al., 2016) and proteins required for Cr synthesis have to be actively taken up from the blood by Cr transporters. However, AGAT, the first enzyme in the process of Cr synthesis, was detected in the heart and its level is comparable to any other tissue synthesizing Cr under normal conditions (Cullen et al., 2006). Further studies have confirmed that AGAT is being converted into Cr in isolated heart muscle (Fisher & Wilhelmi, 1937; Nekhorocheff, 1955). As a key element for energy storage and transport inside the heart, the downregulation of CK activity together with decreased Cr levels in the myocardium are excellent prognostic indicators of heart failure. Tokarska-Schlattner et al. (2012) have shown the strong membrane-stabilizing influence of Cr/PCr against oxidative and mechanical stress. Moreover, Cr causes overexpression of mtCK to reduce the oxidative stress and keep the intact mitochondria (Lygate, Bohl, Ten Hove, et al., 2012), and it also reduces cardiovascular risk factors like lipid and homocysteine peroxidation (Deminice et al., 2008).

15.3.2 In muscle disorders 15.3.2.1 Mitochondrial myopathy Mitochondrial myopathy is a metabolic disease of the skeletal muscle, where the mitochondrial DNA is mutated (Tarnopolsky & Raha, 2005). As compensation, the mitochondria undergo proliferation (Tarnopolsky, 2006), as well as upregulation of mtCK to overcome the stress associated with mitochondrial dysfunction (Schlattner et al., 2006). However, the newly formed mtCK seems to be defective, as individuals suffering from mitochondrial myopathy show low exercise tolerance and a decrease in peripheral O2 extraction (Tarnopolsky, 2006). Studies have shown that Cr supplementation could ameliorate the defects presented in the muscle biopsy, increase grip strength, decrease dorsiflexor muscle fatigue (Tarnopolsky et al., 2004) and improve cyclic endurance (Borchert et al., 1999) in patients with mitochondrial myopathy. Rodriguez et al. (2007) used a combination of Cr, coenzyme Q10, and alpha-lipoic acid in patients with mitochondrial myopathy and noted lower lactate levels and a decrease in oxidative stress markers. Alpha-lipoic acid was found to enhance the Cr uptake into the muscle (Burke et al., 2003), favoring the use of Cr monohydrate in combination therapy. Also, it was suggested that Cr supplementation could enhance the mitochondria energy shuttle (Tarnopolsky, 2011), and mitochondrial biogenesis (Gowayed et al., 2020).

15.3.2.2 Ischemia/infarction Cr supplementation has gained attention due to its ability to maintain the myocardial bioenergetics during ischemia (Balestrino et al., 2016). In an ischemic heart,

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all organs do not receive enough oxygen and nutrients and hence lose their ability to synthesize ATP. Preclinical data have shown protection against ischemia in hearts presupplemented with moderate doses of Cr (Lygate, Bohl, Ten Hove, et al., 2012; Webster et al., 2012). Recently, Park et al. (Park et al., 2021) have shown overexpression of mtCK during an ischemic animal model. This increased expression protects the heart from hypoxia and reactive oxygen species (ROS) production by activating mitochondrial function and enhancing cell viability. The mitochondrial ATP level increases to preserve the membrane potential of the mitochondria. This activates the PGC-1α pathways to enhance the mitochondrial biogenesis, as reported earlier by Gowayed et al. (2020).

15.3.2.3 Sarcoma and chemotherapy Several anticancer medications are known to generate oxidative stress in multiple biological tissues to induce apoptosis (Conklin, 2000). One of the main sites of ROS generation is the electron transport chain of the cardiac mitochondria. Such disruption is the cause of acute and chronic cardiotoxicity during chemotherapy. This selective cardiotoxicity caused by the chemotherapeutic agents is owed to the structure of the cardiac mitochondria possessing both cytosolic and inner matrix NADPH dehydrogenase. Hydrophilic chemotherapeutics like doxorubicin can penetrate the outer mitochondrial membrane and enter the cytosol, where it is reduced by NADPH dehydrogenase to the more lipophilic deoxyaglycone, which is able to enter the inner mitochondrial membrane and disrupt the mitochondrial bioenergetics (Conklin, 2004). This explains the susceptibility of the heart to chemotherapeutic agents more than any other tissue and how they are retained in cardiomyocytes for longer periods. Moreover, the cardio-specific phospholipid, cardiolipin, is present in the inner mitochondrial membrane and shows high affinity for the chemotherapeutics (Goormaghtigh et al., 1990). As the creatine/CK system is closely related to tumor metabolism due to its regulation of ATP mitochondrial production, the use of creatine as an anticancer supplement has gained attention over several years (Miller et al., 1993; Onda et al., 2006). Both, sarcomeric mtCK and CK-M have shown a decreased amount and expression as malignancy progresses (Onda et al., 2006; Patra et al., 2008). Studies reported that Cr in the cardiac muscle could protect cardiac mitochondria from the side effects of anticancer medication, like methylglyoxal (Roy et al., 2003). The anticancer effect of methylglyoxal with ascorbic acid has shown a prominent increase upon creatine administration in an in vivo model of cancer reaching an 80% recovery rate; an effect that was associated with increased expression and content of both, mtCK and CK-M (Patra et al., 2012).

15.3.3 In pregnancy and gestation The fact that the fetus depends on the placental transmission of Cr and there is a continuous change in Cr synthesis as well as excretion as pregnancy progresses (Dickinson et al., 2014; Ellery, Walker, et al., 2016), increases the need towards

15.3 Creatine/mitochondrial creatine kinase system

using Cr supplementation during pregnancy. In several animal studies, Cr supplementation improved fetal survival and neonatal organ function (Ellery et al., 2017; Ellery, Larosa, et al., 2016; Larosa, Ellery, Snow, et al., 2016). Thomure et al. (Thomure et al., 1996), has reported increased expression of CK-BB and ubiquitous mitochondrial CK (umtCK) in the placenta across gestational trimesters. This explains the increased metabolism of the placenta in the third trimester of pregnancy (Thomure et al., 1996). Ellery et al. (2015) studied the Cr homeostasis in pregnant women towards mid to late gestation and has shown a complete alteration in plasma Cr levels, renal excretion, and increased mRNA expression of essential components in the gastrocnemius muscle, highlighting the role of Cr for fetus development. Interestingly, newborns have been shown to possess ten times more CK levels in the serum than adults, which declines after four days of birth and reaches normal levels by ten weeks of age. Such effects have been attributed to be stress-induced during delivery (Gilboa & Swanson, 1976; Rudolph & Gross, 1966). Giving dietary Cr to mothers before giving birth has resulted in survival of female and male fetuses from birth asphyxia by 12% and 19%, respectively (Ellery, Larosa, et al., 2016). Larosa et al. (Larosa, Ellery, Parkington, et al., 2016) used a model of birth asphyxia to investigate the effect of Cr supplementation on the diaphragm muscle. While Cr is still not considered an essential supplement during pregnancy, one should look at the effect of low maternal Cr amount on fetal development. Research in this area is still lacking.

15.3.4 Creatine and central nervous system mitochondria 15.3.4.1 Creatine: the devoted energy provider for neuronal mitochondria The PCr-shuttle is predominantly involved in cellular energy buffering and energy transport in neurons, which have a fluctuating and continual demand of energy (Bessman & Carpenter, 1985; Meyer et al., 1984). CKs are expressed in the adult and developing human brain and spinal cord, which provides unwavering evidence that they are implicated in a series of events in the central nervous system (CNS). The CK isoenzymes are present in different areas of the human brain including the hippocampus, cerebellum, cerebral cortex, and choroid plexus, additional compelling evidence of the implication of CKs in reserving brain function and the integrity of neurons (Kaldis et al., 1996; Lin et al., 1994). Functional impairment of the mitochondrial PCr-shuttle leads to detrimental effects on energy metabolism, which is phenotypic for many neurodegenerative and age-related diseases (Wallimann et al., 1992). Mice with a gene knockout of cytosolic CK-B showed diminished cognitive function and exhibited lower memory formation demonstrated by reduced open-field habituation, slower spatial learning acquisition, sensory and motor dysfunction (Jost et al., 2002). Moreover, Cr-deficiency causes mental retardation, delay in speech and brain

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atrophy (Curt et al., 2015). Studies showed loss of CK-BB activity in the brain of patients suffering from Alzheimer’s disease (AD), resulting in disruptions in the energy supply in glia cells, neurons, and synaptic transmitters (Aksenov et al., 2000; David et al., 1998). Other studies demonstrated that Alzheimer’s patients have reduced levels of brain PCr in early stages of the disease and decreased oxidative metabolism in later stages compared to healthy individuals, reinforcing the notion that the AD is related to energetic stress of the brain and implicating Cr and PCr further in the onset and progression of AD (Chen et al., 2021; Pettegrew et al., 1994). In addition to the vital role of Cr in balancing the brain energy production, storage, and utilization, Cr modulates an array of cellular functions in the CNS (Fig. 15.9). Cr preserves membrane potential as it mediates the flow of ions across the membrane. Therefore, Cr synchronizes a series of cellular pathways such as cytoprotection in addition to axonal and dendritic elongation. Cr is

FIGURE 15.9 Functions of creatine in CNS. AGAT, L-Arginine:glycine amidinotransferase; Arg, arginine; CK, creatine kinase; Cr, creatine; CRT, creatine transport; GAA, guanidinoacetate; GABAA-R, GABAA receptor; GAMT, guanidinoacetate methyltransferase; Gly, glycine; SAH, S-adenosyl homocysteine; SAM, S-adenosyl methionine.

15.3 Creatine/mitochondrial creatine kinase system

suggested to be a neurotransmitter as it can be synthesized in neurons, released in an action potential-dependent manner, and is a partial agonist of the postsynaptic GABAA-Receptor and possibly other receptors. CRT is also presynaptically expressed, possibly for recapture and recycling of Cr. Furthermore, Cr is one of the main CNS osmolytes, and it preserves the balance of osmotic pressure within neurons. Hypo-osmotic conditions, lead to Cr efflux while hyperosmotic conditions promote the inflow of Cr into cells (Hanna-El-Daher & Braissant, 2016). Over the past few decades, neurological approaches have revealed that exogenous Cr supplementation reduces neuronal cell loss in experimental models of acute and chronic neurological diseases. Accordingly, clinical trials have documented promising therapeutic effects of Cr supplementation (Balestrino et al., 2002). Cr supplements promote differentiation of neuronal precursor cells that might be of importance for improving neuronal cell regeneration strategies and can protect against traumatic brain injury (Dolan et al., 2019) through its role in mitochondrial bioenergetics. Such findings evoked a scientific curiosity to exploit the role of Cr and CK in the CNS and their implication in pathological conditions.

15.3.4.2 Creatine, mitochondrial bioenergetics, and neurodegenerative disorders As we mentioned, adequate mitochondrial functions are crucial for the integrity of neurons, particularly under stress conditions, when neurons are highly activated and require an excess supply of energy. Disruptions in mitochondrial functions impair the cellular ATP supply which subsequently alters neuronal excitability and leads to Ca21 overload and cell death (Rossi et al., 2019). The damage in mitochondria, alterations in mitochondrial Ca21 handling, and its consequent neuronal dysfunction are cooperatively responsible for neuronal degeneration. Defects in mitochondrial bioenergetic pathways are the main feature of neurodegenerative diseases (NDs), such as AD, Parkinson’s disease (PD), and amyloid lateral sclerosis (ALS) (Rossi & Pizzo, 2021). In the last decade, the “mitochondrial cascade hypothesis” has been established as the most reliable theory in the onset and progression of AD. Various mitochondrial defects have been observed in AD cases. These defects cover a whole spectrum of alterations in mitochondrial morphology, dynamics, and movement, mitochondrial oxidative stress, impaired mitochondrial metabolism, and mitophagy. One of the many of these dysfunctional processes could lead to synaptic dysfunction and neuronal death. The outcome of such impairments is catastrophic not solely on single neurons but on the structure of the brain as a whole (Swerdlow et al., 2014). In other words, mitochondrial deficits are a predominant feature in AD (Flannery & Trushina, 2019). This mitochondrial dysfunction leads to impairments in the electron transport chain (etc) complexes, another feature of the AD mitochondrial profile (Parker et al., 1994). One of the compromised complexes is complex I, which transfers electrons from nicotinamide adenine dinucleotide 1 hydrogen to coenzyme Q within the etc (Giachin et al., 2016). The therapeutic benefits of Cr supplementation

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in AD may be attributed to its ability to increase complex I levels and enhance oxidative respiration in brain mitochondria (Snow et al., 2020). Since NDs lead to loss of physical activity as they progress, then the peaking interest in using Cr supplements as a countermeasure that delays the progression of NDs and helps control the symptoms is utterly justified, as Cr supplements can improve muscle strength, mass, and endurance (Kreider et al., 2017). Moreover, Cr particularly targets distinct neurometabolic pathways and pathological characteristics induced by NDs rendering it a perfect candidate for therapeutic intervention. Cr exhibits antiapoptotic, antiexcitotoxic, and direct antioxidative properties (Lewitt, 2004). Previous studies have reported that homocysteine accumulation may eventually lead to peripheral nerve damage and that Cr exhibits neuroprotective and antioxidant properties by reducing homocysteine levels (Bereket-Yu¨cel, 2015; Lawler et al., 2002). Therefore, studies have been exploiting Cr and Cr monohydrate supplements in managing and treating several NDs namely PD (Allen, 2012; Hass et al., 2007). However, research is still lacking information on how such Cr supplementation could affect mitochondrial dysfunction and bioenergetics in NDs, which could be very promising in better understanding the NDs’ pathophysiology.

15.3.4.3 Creatine, neuronal mitochondrial dysfunction, and amyotrophic lateral sclerosis ALS is an ND that results in the progressive loss of motor neurons that control voluntary muscle activity. It is the most common type of motor neuron disease and is always fatal (Zucchi et al., 2020). Individuals diagnosed with ALS suffer from muscle weakness, atrophy, and difficulty with speech, among many other debilitating symptoms. With no cure available, most ALS medications are intended to minimize pain and fatigue (Gittings & Sattler, 2020). Studies investigating in vivo and in vitro models and patient tissues linked neuronal mitochondrial dysfunction to the onset and progression of ALS (Gautam et al., 2019; Jiang et al., 2015; Manfredi & Xu, 2005). Postmortem tissues from ALS patients showed increased mitochondrial density and impaired mitochondrial morphology (Delic et al., 2018). Optimal mitochondrial functions are required for the proper functions of motor neurons to provide them with a consistent energy supply. The damage to neuronal mitochondria impairs that crucial energy production mechanism by compromising ATP production. The extent of this damage is mirrored in a devastating chain of events: calcium-mediated excitotoxicity of neurons, an increase in oxidative stress due to irregular production of ROS, and activation of the intrinsic apoptotic pathway with the inevitable outcome of motor neurons degeneration ultimately leading to ALS (Manfredi & Xu, 2005; Wang et al., 2013). Several authors have provided a rationale for the use of Cr supplementation as a viable treatment option for individuals diagnosed with ALS (Tarnopolsky & Beal, 2001; Wyss & Schulze, 2002). Plausible explanations for its ability to enhance the quality of life in patients with ALS include protection against neuronal loss in the substantia nigra and motor cortex (Beal, 2011), as well as decreased oxidative stress

15.3 Creatine/mitochondrial creatine kinase system

(Klopstock et al., 2011), and mitochondrial dysfunction (Hervias et al., 2006) commonly observed with this condition. Despite this initial enthusiasm and encouraging animal work (Klivenyi et al., 1999), clinical trials in humans have reported disappointing results. Only three published trials utilizing a double-blind, randomized design have evaluated the effects of Cr supplementation in ALS patients, and all three showed no benefits beyond the placebo (Jan Groeneveld et al., 2003; Rosenfeld et al., 2008; Shefner et al., 2004). All three studies utilized doses of 5
10 g/day, measured muscle strength, and reported that Cr was well tolerated with no major side effects. One major drawback of those studies was that patients were in the late stages of the disease. Therefore, the speculation that the use of Cr as a therapeutic intervention in the early stages of the disease might yield better outcomes is still a highly promising notion, taking into consideration its expected counteractive effect on the mitochondrial neuronal dysfunction.

15.3.4.4 Creatine, neuronal mitochondrial dysfunction, and multiple sclerosis Multiple sclerosis (Ms) is a chronic inflammatory demyelinating disease of the CNS. Symptoms of Ms involve the autonomic, visual, motor, and/or sensory system. The underlying cause of Ms is unknown, yet a dysfunction of the immune system accompanied by the failure of the myelin-producing cells and a breakdown of the BBB have been implicated in its etiology, with many other genetic and environmental factors involved (Dobson & Giovannoni, 2019). Among other features, impaired metabolism of high-energy phosphate-generating Cr has been implicated in the pathogenesis of Ms. Particularly, a lack of Cr in white-matter astrocytes in Ms may lead to glutamate-mediated oligodendrocyte damage and impaired myelination (Cambron et al., 2012). These fundamental findings in addition to the fact that Cr plays an important role in cellular energy buffering and energy transport in the CNS have encouraged scientists to use brain Cr as a surrogate biomarker of Ms progression, and even to administer oral Cr as a therapeutic strategy against the disease (Andres et al., 2008). A study suggested that Cr metabolism in Ms could be impaired due to decreased CK-B levels measured in brain slices from patients who had died with secondary progressive Ms (Steen et al., 2010). Disruption in energy metabolism affecting neurons and/or glial cells was illustrated by a reduction in the apparent diffusion coefficient of Cr-PCr as evaluated by diffusion-weighted Mr spectroscopy trial (Bodini et al., 2018). Cr metabolism in Ms appears compromised in other tissues besides the brain. Patients with Ms demonstrated considerably lower serum CK-B levels than in healthy individuals (Vassilopoulos & Jockers-Wretou, 1987), with a diminished enzyme activity indicating impaired synthesis of ATP in Ms brain or other tissues. Along with serum, reduced mean CK-B levels were also found in the CSF of patients with Ms (Matias-Guiu et al., 1986; Pfeiffer et al., 1983), suggesting inadequate tissue bioenergetics and/or demyelination. An interesting trial provided

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evidence of slowed PCr re-synthesis in the skeletal muscle following exercise in Ms patients (Kent-Braun et al., 1994). Cr administration in Ms was initially evaluated on a very limited number of patients, where eight individuals with relapsing-remitting Ms received 20 g/day of Cr for five days while the other eight Ms patients received a placebo. A needle biopsy was performed on the vastus lateralis muscle before and after the treatment. The authors reported that Cr ingestion had no significant effect on intramuscular ATP, on Cr stores (including total Cr, PCr, and free Cr) and work performance although the mean PCr concentrations increased more in the Cr group than in the placebo group (Lambert et al., 2003). The lack of a substantial effect in this trial could be attributed to the small sample size, short duration of the intervention, and perhaps the restricted capacity of dysfunctional muscle for Cr transport (Tarnopolsky et al., 2001). Another study investigated the ability of Cr supplementation to enhance mitochondrial-mediated oligodendrocyte survival in the demyelinating state caused by Ms. Chronic oligodendrocyte loss is a hallmark feature of the neuronal demyelinating induced by Ms and it leads to axonal dysfunction and neurodegeneration subsequently leading to the formation of lesions. In this study, they found that creatine-treated mice exhibited increased oligodendrocyte density suggesting that creatine may promote the survival of oligodendrocytes within the lesion. They also observed that creatine-treated mice had significantly fewer oligodendrocytes compared with PBS-treated mice which further implicates Cr supplementation in the inhibition of caspase-dependent oligodendrocyte apoptosis. The results of this study demonstrated that creatine-mediated oligodendrocyte survival did enhance CNS remyelination, a finding that may revolutionize therapeutic approaches in demyelination disorders such as Ms (Chamberlain et al., 2017). Further research is needed to expand our understanding of the implication of Cr misbalance in Ms, its impact on the neuronal mitochondrial dysfunction and to investigate unexplored compounds that may target relevant Cr pathways involved in Ms.

15.3.4.5 Creatine treatment and mitochondria: could it be the hope for patients with Parkinson’s disease? Evidence strongly suggests that we should predominantly target enhanced mitochondrial biogenesis to develop innovative therapeutic strategies to treat PD. A recent study investigated mitochondrial dysfunction in parkin-deficient human dopaminergic neurons. The PRKN gene is responsible for making the protein parkin which is responsible for preserving the integrity of mitochondrial biogenesis. Parkin is believed to trigger mitophagy or the destruction of damaged mitochondria. The study demonstrated mitochondrial dysfunction caused by defects in the mitochondrial biogenesis, associated with impaired mitophagy provoked by the lack of parkin (Kumar et al., 2020). The study also proved that the defects in mitochondrial biogenesis are caused by the upregulation of the parkin interacting substrate and the subsequent downregulation of PGC-1α, a notion that is reinforced by other studies (Ge et al., 2020; Kumar et al., 2020).

15.3 Creatine/mitochondrial creatine kinase system

The therapeutic effects of Cr in treating and managing the symptoms of PD or slowing the progression of the disease remain partially obscure. Researchers have been relentless about finding conclusive data that can end the debate of whether Cr can offer a solution to PD or not. Previous studies have documented conflicting results regarding the effects of Cr on the symptoms and the progression of PD (Bender et al., 2006; Investigators, 2008; Kieburtz et al., 2015; Li et al., 2015), and it remains undetermined whether Cr treatment can improve clinical outcomes when compared to placebo. Different studies used different strategies of Cr treatment. In one study, Cr was administered at a loading dose of 20 g/d for six days, followed by 2 g/d for six months, and finally 4 g/d for two years (Bender et al., 2006). Another study combined Cr (10 g/d) with coenzyme Q10 (300 mg/d) (Li et al., 2015). Patients in other studies received 10 g/d of Cr (Investigators, 2006; Investigators, 2008; Kieburtz et al., 2015). Disease progression was primarily measured using the Unified Parkinson’s Disease Rating Scale (UPDRS) (Ivey et al., 2012). The total mental Activities of Daily Living and Motor UPDRS scores were also assessed. The data in those studies were conflicting, yet undeniably tempting. Further work should be performed, and more data should be collected to reach conclusive evidence. These conflicting data are still offering some hope and scientists should not overlook unexplored possibilities. Recent metanalyses have been trying to analyze and collect data from all previous studies to reach an inclusive understanding of the effects of Cr supplements on PD and to conclude whether Cr supplements can ameliorate the symptoms of PD and can delay the disease progression. The conflict of data in studies of PD and Cr does not help in resolving this debate (Avgerinos et al., 2018; Mo et al., 2017). More studies and analyses followed similar approaches to find the answer to an intriguing and equally frustrating question: can Cr supplements offer some hope for PD patients? The answer to this question remains ambiguous, yet scientists strongly believe that Cr treatment may offer an innovative approach in treating PD. Thus, their efforts are relentless as they continue the journey that began several decades ago (Bender et al., 2006; Li et al., 2015; Xiao et al., 2014; Xu et al., 2019). Current studies are thoroughly investigating Cr supplements and their effects on mitochondrial biogenesis as potential therapeutic approaches in the treatment of NDs. Many factors should be taken into consideration during current and future studies such as the stage and rate of progression of the disease and the age of the onset of the disease in addition to factors like the patients’ genders, medical history, ethnicities, and demographics.

15.3.5 Creatine and adipocyte-specific functions of the mitochondria It has been established that mitochondria in many tissues, including skeletal muscle, heart muscle, and neurons are crucial regulators of energy supply that ensure

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the integrity of these cells. Similarly, the mitochondria of adipose tissues function as a provider of cellular energy, but they also perform functions that are specific and tailored to the requirements of adipocytes. Studies have illustrated some of these adipocyte-specific functions as adipogenesis, lipid metabolism, and thermogenesis (Boudina & Graham, 2014). Furthermore, recent studies have revealed that in addition to the role of mitochondria in adipocytes to regulate the wholebody energy homeostasis, mitochondria mediate insulin sensitivity and glucose metabolism (Keuper et al., 2014; Vernochet et al., 2014). These adipocytespecific roles of mitochondria made recent studies explore potential molecular regulation to enhance mitochondrial function in adipose tissues and exploit that notion in developing new therapeutic strategies in treating metabolic diseases and the obesity (Lee et al., 2019). The adipocytes express CK isoenzymes, and they contain the CK substrates PCr and Cr. In adipocytes, the PCr-shuttle is responsible for maintaining balanced thermogenic homeostasis. Brown adipocytes (BAT) have Cr kinase activity of the same magnitude as its activity in cardiac muscles or neurons (Berlet et al., 1976). However, the precise Cr kinase isozyme responsible for most of this activity is currently unknown. The two mitochondrial Cr kinases, smtCK, and umtCK are enriched in human BAT at the mRNA and protein level, and the genes encoding these kinases are regulated in coordination by the uncoupling protein 1 (UCP1) (Jash et al., 2019; Mu¨ller et al., 2016). There is increased interest in exploring the mechanisms by which white adipose tissue (WAT) can be recruited to resemble the phenotypical and functional characteristics of the more metabolically active BAT in a process known as “browning”, which leads to the development of beige adipocytes with morphological and functional resemblance to brown fat. Like brown fat, beige adipocytes are assumed to express UCP1 and are thermogenic due to the UCP1-mediated H 1 leak across the inner mitochondrial membrane (Bertholet et al., 2017; Sanchis-Soler et al., 2020). Non-shivering thermogenesis was previously thought to be UCP1-dependent, however, recent evidence demonstrates the contribution of UCP1-independent mechanisms in thermogenesis. Namely, futile Cr cycling has been identified as a contributor to WAT thermogenesis (Bertholet et al., 2017).

15.3.5.1 Creatine metabolism in adipose tissue In adipocytes, Cr is generated by both intracellular synthesis and sequestration from the circulation (Kazak et al., 2017), taken up by a CRT (Fitch et al., 1968). Futile Cr-cycling is identified as a major molecular mechanism of Cr-dependent thermogenesis in adipose tissue. A futile cycle is a metabolic pathway that is not generating useful work but dissipates free energy as heat. This alternative thermogenic mechanism appears to utilize Cr-dependent futile cycling of ATP, depending on the ATP synthase activity (Kazak & Spiegelman, 2020). This cycle drives hydrolysis of a molar excess of ATP with respect to Cr, resulting in a surplus of oxygen consumption under ADP-limiting conditions. A Cr-dependent increase in

15.3 Creatine/mitochondrial creatine kinase system

ADP-limited respiration was observed in inguinal beige fat mitochondria following one week of cold exposure (Kazak et al., 2015).

15.3.5.2 Creatine and obesity Impaired mitochondrial biogenesis is a fundamental attribute of the pathophysiology of several metabolic disorders such as obesity, insulin resistance, and type 2 diabetes (Wada & Nakatsuka, 2016). Mitophagy is induced to counteract impaired metabolic cellular state, oxidative stress, inflammation, and insulin resistance. This process preserves the integrity of mitochondrial function, biogenesis, and morphology by degrading damaged mitochondria and limiting mitochondrial dysfunction. Predictably, studies reported a higher accumulation of dysfunctional mitochondria in obese subjects compared to control ones. As we established, mitochondrial biogenesis and mitochondrial mitophagy are two opposing cellular pathways, that cooperate to ensure optimum mitochondrial content. Thus, these findings suggest that mitophagy might be negatively controlled by excessive fat accumulation induced in obese status (Chattopadhyay et al., 2015; Kraunsøe et al., 2010). Obesity occurs when energy intake surpasses energy consumption. Excess energy is mainly stored as triglycerides in WAT. The expansion of WAT takes place through increases in the size and/or a number of adipocytes. Several factors may affect energy consumption and therefore affect metabolic rates in the adipose tissue (Longo et al., 2019). Decreased levels of Cr in thermogenic adipocytes as a result of inhibited Cr biosynthesis and/or Cr transport reduces thermogenesis and causes obesity. In humans, levels of Cr in adipocytes correlate with lower body mass index and increased insulin sensitivity. Data indicate that adipocyte Cr abundance depends on the removal of Cr from the circulation. Such findings indicate that enhancing Cr uptake into adipocytes may offer an effective approach to counteract obesity and obesity-related metabolic disorders (Kazak et al., 2017; Kazak et al., 2019). Cr supplementation increased energy expenditure during high-fat feeding in mice and the BAT Cr abundance was significantly higher in animals receiving Cr supplementation. These data demonstrate that adipocyte Cr transport is critical for supporting diet-induced thermogenesis in vivo. This is caused by several elements, which are the interaction between adipocyte Cr with certain components of a high-fat diet, an increase of Cr uptake into BAT on a high-fat diet, or a combination of the two elements (Kazak et al., 2019). Furthermore, studies are exploring the various effects of Cr monohydrate supplementation in treating obesity and obesity-related diseases. Specific effects of Cr supplementation in correlation with other factors like sex, age, exercise, diet, and the distinct routes of action of Cr supplements are investigated in anatomically different types of adipose tissue (Ferretti et al., 2018; Forbes et al., 2019). A study by Ryan et al. demonstrated that Cr monohydrate supplementation increased WAT mitochondrial markers and induced WAT browning in male and female SpragueDawley rats in a sex-specific manner (Ryan et al., 2021). Their findings provided

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further insight into the different responses of Cr supplementation depending on the sex. Their results highlighted the efficacy of Cr supplementation in increasing mitochondrial proteins and strongly proposed Cr as a preventive or therapeutic approach to treat obesity and obesity-related metabolic diseases (Ryan et al., 2021). The benefits of Cr supplementation could be ameliorated when combined with a healthy diet and resistance training (Forbes et al., 2019), while a high caloric diet can drastically diminish those beneficial effects (Ferretti et al., 2018). Scientists are relentlessly exploiting Cr supplements and their effects on fat mass as they can help end the crisis of global obesity, especially when combined with other alterations in lifestyle habits like exercise and a balanced diet.

15.4 A promising future Our knowledge is always challenged, but our determination has never wavered. We are recruiting what we know so far about mitochondria and their role in chronic disorders and NDs, and we are exploiting every possibility to develop various therapeutic approaches to treat and manage these devastating diseases. The future of Cr and Cr monohydrate to enhance mitochondrial bioenergetics seems utterly promising despite the obstacles of the present.

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CHAPTER

Arginine and neuroprotection: a focus on stroke

16 Yasutoshi Koga1,2

1

Department of Pediatrics and Child Health, Graduate School of Medicine, Kurume University, Kurume, Fukuoka, Japan 2 Cognitive and Molecular Research Institute of Brain Diseases, School of Medicine, Kurume University, Kurume, Fukuoka, Japan

16.1 Introduction Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) (OMIM 540000), characterized by an early onset of stroke-like episodes (SLEs) before age 40, was first described by Pavlakis and colleagues in 1984 (Pavlakis et al., 1984). At least 40 distinct mitochondrial DNA mutations have been associated with MELAS and about 80% of MELAS patients have an A3243G mutation in the mitochondrial tRNALeu(UUR) gene (OMIM 59005) (Goto et al., 1990; Iizuka & Sakai, 2005). There are three hypotheses of the pathophysiology of SLEs that have been proposed: (1) mitochondrial angiopathy, (2) mitochondrial cytopathy, and (3) nonischemic neurovascular cellular mechanism. Mitochondrial angiopathy with degenerative changes in small arteries and arterioles, which has been reported in many MELAS patients (Koga et al., 1988; Ohama et al., 1987; Takahashi et al., 2005), is suggested by the observation of strong succinate dehydrogenase (SDH) activity in the wall of blood vessels (SDH-reactive blood vessels, SSVs) (Hasegawa et al., 1991; Sakuta & Nonaka, 1989). Mitochondrial cytopathy and nonischemic neurovascular cellular mechanisms both suggest that SLEs are caused by mitochondrial dysfunction in brain cells, and together can be referred to as mitochondrial cytopathy theory (King et al., 1992). Based on a hypothesis that SLEs in MELAS are caused by segmental impairment of vasodilatation in intracerebral arteries, we use L-arginine in MELAS patients during the acute phase to cure the symptoms or to decrease the frequency and/or the severity of the SLEs (Koga et al., 2002; Koga et al., 2005; Koga et al., 2006). This review aims to give an updated overview on the actual knowledge about the pathogenic mechanism of mitochondrial angiopathy related to SLEs in the clinical and pathophysiological levels, and proposed the tips for the pitfalls in the treatment of L-arginine on MELAS.

Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00026-6 © 2023 Elsevier Inc. All rights reserved.

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16.2 Mitochondrial angiopathy in MELAS MELAS is the most common subtype of mitochondrial disorders. MELAS patients showed ragged-red fibers by modified Gomori-Trichrome staining in muscle (Fig. 16.1), which are also called ragged-blue fibers by SDH staining (Sakuta & Nonaka, 1989). Small artery in the muscle section showed cytochrome c oxidase (COX) positive and SDH hyperreactive staining designated as a strongly SSVs (Hasegawa et al., 1991), because they are rich in mitochondria. By electron microscopy analysis, such SSVs area showed enlarged abnormal mitochondria with occasional complicated cristae with aggregation in the smooth muscle cells as well as endothelial cells of the small artery (Koga et al., 1988; Sakuta & Nonaka, 1989). In the autopsy cases, there was a striking increase in the

FIGURE 16.1 Muscle pathology in MELAS. MELAS showed ragged-red fibers (RRFs) by modified Gomori-trichrome (mGT) staining in muscle, which are also called ragged-blue fibers (RBFs) by succinate dehydrogenase (SDH) staining. Small artery in the muscle section showed cytochrome c oxidase COX positive and designated as a strongly SDH-reactive vessels (SSVs). By electron microscopy analysis, SSVs area showed enlarged abnormal mitochondria with occasional complicated cristae with aggregation in the smooth muscle cells as well as endothelial cells of small artery. COX, cytochrome c oxidase; H&E, hematoxylin and eosin; mGT: modified Gomoritrichrome; NADH, nicitinamide andenine dehydrogenase; RBFs, ragged-blue fibers; RRFs, ragged-red fibers; SDH, succinate dehydrogenase.

16.3 Endothelial dysfunction in MELAS

number of abnormally shaped mitochondria in the smooth muscle and endothelial cells in the brain artery, which were most prominent in the pial arteries and intracerebral arterioles and small arteries (Kishi et al., 1988; Ohama et al., 1987). SSVs area sometimes showed pathological narrowing or occlusion which may be related with abnormal microcirculation in the intracerebral artery. Although infarct-like lesions histopathologically and SLEs clinically may not be caused simply by the occlusion or obliteration of small vessels, this mitochondrial angiopathy, which can be severe in pial arterioles and small arteries, seems to explain the distribution of multiple areas of necrosis (Tanahashi et al., 2000). All findings, described here, suggest that mitochondrial angiopathy is a unique and common abnormality in all MELAS brains examined. The reason why RRFs and SSVs change have strongly induced in MELAS has not been elucidated.

16.3 Endothelial dysfunction in MELAS Nitric oxide NO, the smallest signaling molecule, is produced by three isoforms of NO synthase (NOS; EC1.14.13.39), type 1 or neuronal, type 2 or inducible, and type 3 or endothelial (eNOS). All of those utilize L-arginine and molecular oxygen as substrates and require the cofactors including NADPH, FAD, FMN, and BH4. It keeps blood vessels dilated, controls blood pressure, manages the microcirculation in the cerebral blood flow, and has numerous other vasoprotective and antiatherosclerotic effects (Fo¨rstermann & Sessa, 2012). NO regulating the physiological vascular tone, is mainly generated by eNOS from circulating L-arginine and molecular oxygen (Fig. 16.2). Since MELAS has defective respiratory chain enzyme activities, the accumulation of superoxides, free-radicals, or a high NADH/NAD ratio inhibits the NOS reaction to cause the decreased production of NO at endothelial cells or smooth muscle cells in the artery. We measured the plasma concentrations of amino acids in patients in the acute stroke phase or interictal phase of MELAS and in controls (Koga et al., 2005). Mean plasma concentrations of L-arginine and citrulline were significantly lower in both acute and interictal phases of MELAS than that in controls. Concentrations of L-arginine at the acute phase were also significantly lower than those at the interictal phase, while those of citrulline did not show a significant phase-related change. A significant decrease of L-arginine at the acute phase is not correlated with the concentration of urea cycle intermediates including ornithine and citrulline, indicating that the decrease of L-arginine is not related to urea cycle activities but an unknown cause. In addition, asymmetrical dimethyl-arginine (ADMA), a risk factor of ischemic heart disorders, was relatively increased in MELAS patients (Koga et al., 2005), which may lead to a negative effect on the endothelial NOS activity. Physiologically, we have demonstrated that MELAS patients have a decreased vasodilation capacity in small arteries examined by flow-mediated vasodilatation methods (Koga et al., 2006). Why L-arginine decreased at the acute phase of SLEs in MELAS should be clarified in the future.

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Cytokine Steroids Endothelial Cell

L-arginine NADPH/NADP

-

L-arginine

inactivation

NOS NO

Respiratory chain deficiency

Superoxide

L-citrulline

Cytokine Steroids Smooth muscle layer L-arginine

ADMA

inactivation

NOS

+

NADPH/NADP L-arginine

sGC NO +

Physiological vasodilation

GTP cyclic GMP

FIGURE 16.2 Nitric oxide regulated the vascular relaxation. Nitric oxide (NO), a free radical that regulates the physiological vascular tone, is mainly generated by eNOS from circulating L-arginine and molecular oxygen. Since MELAS patients have defective respiratory chain enzyme activities, the accumulation of superoxides or free-radicals, or a high NADH/NAD ratio, inhibits the NO synthetase reaction to cause a decreased production of NO at the endothelial cells or smooth muscle cells in the artery. MELAS showed NO-dependent endothelial dysfunction.

16.4 Neuroimaging of stroke-like episodes in MELAS Unlike thrombotic or embolic stroke usually seen in adult patients, the SLEs in MELAS are characteristic because they affect young people and are often triggered by febrile illnesses, migraine-like headaches, seizure, psychological stress, cold exposure, and dehydration (Coman et al., 2008; Koga et al., 2012). They have the following features; (1) distribution of the brain lesions incongruent to a vascular territory, (2) preferential involvement of the cerebral cortex, (3) predilection to the posterior brain, (4) reversible vasogenic edema, and (5) slowly progressive spread of the brain lesions (Iizuka et al., 2003b). Many neuroimaging studies have been reported at different time series from the onset of SLEs in MELAS through the use of in vivo imaging tools. MRI scans of acute stroke-like events show an increased signal on T2-weighted or on fluid attenuation inversion recovery. Acute changes in these regions may fluctuate, migrate, or even disappear during the time course. The acute phase of stroke-like lesions in MELAS appear as a high signal on DWI with normal or increased ADC values, suggesting

16.5 Clinical study of L-arginine in MELAS

vasogenic edema which support the mitochondrial angiopathy theory (Ikawa et al., 2009; Sproule & Kaufmann, 2008; Yoneda et al., 1999). On the contrary, many case reports found a decrease in ADC, which suggests mitochondrial cytopathy theory is correct (Oppenheim et al., 2000). Recently, there is another report where increased and decreased ADC portions are mixed in stroke-like lesions, suggesting that there might be different levels of mitochondrial energetic transport impairment, correlated with cellular dysfunction. Specifically, this would be a mild energy failure resulting in moderate cellular dysfunction, responsible for vasogenic edema (high ADCs), and a severe energy failure resulting in irreversible cellular failure with cytotoxic edema (low ADCs) (Wang et al., 2003). Cerebral angiograms in MELAS patients taken within several days after the onset of SLEs have confirmed the absence of large vessel pathology by demonstrating normal results (Gerriets et al., 2004; Stoquart-Elsankari et al., 2008). Tsujikawa describes the longitudinal change of rCBF in the stroke-like lesion after symptom onset by Magnetic Resonance Spectroscopy (Mrs) and CASL perfusion imaging (Tsujikawa et al., 2010) showing the bi-phasic change of hyperperfusion up to two weeks after the onset of SLEs and hypoperfusion from two to at least up to five weeks after the onset of SLEs. Ikawa et al. developed a novel double imaging method using positron emission tomography with 62Cu-ATSM and 18FDG to visualize the regional oxidative stress (Ikawa et al., 2007; Ikawa et al., 2009). Most of the reported studies about perfusion status in MELAS analyzed by single photon emission tomography (SPECT) have generally revealed that there is focal vasodilatation and hyperperfusion, and such luxury perfusion lasted for a couple of months (Kaufmann et al., 2004; Mo¨ller et al., 2005). In the chronic stage several months or years later, the decreased tracer accumulation was reported in the same region because of the irreversible and atrophic change in the brain. However, only a few SPECT studies which were taken at the hyperacute stage 24 h or less after the onset of SLEs, showed hypoperfusion (Gropen et al., 1994; Iizuka et al., 2003a; Koga et al., 2005; Koga et al., 2006). In addition, we found that the hypoperfusion and the hyperperfusion areas are both demonstrated in the MELAS patients not only at a hyperacute phase but at an interictal phase, demonstrating that MELAS has an inappropriate cerebral circulation (Nishioka et al., 2008).

16.5 Clinical study of L-arginine in MELAS After L-arginine supplementation by either intravenous injection or oral administration, the impaired endothelial dependent vasodilation capacity normalized to the age-matched control (Koga et al., 2006). Although plasma levels of Larginine are 100 times higher than km of eNOS 3.0 μM (Cardounel et al., 2007), L-arginine can influence NO production and partially reverses the impairment of endothelium dependent vasodilation in response to acetylcholine in diverse patient groups (Hishikawa et al., 1991; Kernohan et al., 2005; Lucotti et al., 2009; Ruel

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et al., 2008; Tsao et al., 1994). This unexpected response to L-arginine in spite of a large intracelluler excess of L-arginine has been termed “the arginine paradox” (Bode-Bo¨ger et al., 2007). Several researchers demonstrated that the extracellular L-arginine, not intracellular L-arginine, is the major determinant of NO production in endothelial cells. It is likely that once transported inside the cell, Larginine can no longer gain access to the membrane-bound eNOS (Karbach et al., 2011; Shin et al., 2011). We speculated that the application of L-arginine antagonizes ADMA, which is relatively higher than a normal control, the endogenous NOS inhibitor to normalize the calculating the L-arginine/ADMA ratio. Administration of L-arginine to patients with MELAS during acute SLEs led to improved microcirculation and reduced tissue injury from ischemia and improved clinical and Mrs findings. Kubota et al. reported that L-arginine infusion protects from the accumulation of lactate by Mrs analysis in SLEs in MELAS (Kubota et al., 2004). L-arginine administration during the acute phase of MELAS might be a potential therapy to reduce brain damage due to mitochondrial energy failure. Shigemi et al. reported that intravenous administration of L-arginine during the acute phase of the SLEs reduced symptoms immediately, and oral supplementation of L-arginine successfully prevented further SLEs (Shigemi et al., 2011).

16.6 Superacute intervention by L-arginine We evaluated the therapeutic effects of an L-arginine infusion at the superacute phase of SLEs on clinical and the serial neuroimaging analysis in two MELAS patients (Kitamura et al., 2016). All the clinical symptoms disappeared within 30 min after Larginine infusion without using any anticonvulsants. The MRI obtained at 18 h after the onset showed high intensity signal in T2WI, and DWI and low intensity signal in ADC in the right and left hemisphere of the cerebellum and right occipital region showing vasogenic edema. However, those abnormal findings completely normalized on MRIs at 7 days and one month later. We speculate that L-arginine, if it is used at the superacute phase, may protect neurons in the cortex from permanent cell death, by improving either regional microcirculation of cerebral blood flow or energetic status, permeability of BBB, or interaction by astrocyte-neuron coupling which controls the cerebral circulation.

16.7 Therapeutic regimen of L-arginine for MELAS In the Japanese cohort study in 2009, we have identified 233 patients with MELAS in across Japan, and among those, 96 patients completed a 5-year nationwide, multicenter, prospective cohort study (Yatsuga et al., 2012). This epidemiologic study on MELAS patient groups revealed the natural course of patients with MELAS (time from diagnosis to death: 7.3 6 5.0 years; mortality rate: 20.8%).

16.7 Therapeutic regimen of L-arginine for MELAS

FIGURE 16.3 Plasma L-arginine levels at SLE in the clinical trial. During a 2-year period of clinical trials of oral supplementation of L-arginine, red circle showed plasma levels when patients had SLEs. The highest plasma level of L-arginine at SLEs in patients showed 167 μmol/L in this trial, suggesting that when the trough level of enrolled patients is controlled over 167 μmol/L, MELAS patients may have not any SLEs in this study. Modified from original Figure 3 in Journal of Neurology.

To evaluate the efficacy of L-arginine infusion on the acute phase of SLEs, and L-arginine oral administration on the interictal phase in order to decrease the severity and frequency of SLEs in MELAS, we have done the two years clinical trials between May 9, 2009 to June 30, 2011 (Koga et al., 2018) using 25 MELAS patients in this cohort study. We have found that oral L-arginine extended the interictal phase (P 5 .0625) and decreased the incidence and severity of ictuses. Intravenous L-arginine improved the rates of four major symptoms: headache, nausea/vomiting, impaired consciousness, and visual disturbance. The maximal plasma arginine concentration was 167 μmol/L (Fig. 16.3), when an ictus developed. Neither death nor bedriddenness occurred during the 2-year clinical trials, and the latter did not develop during the 7-year followup despite the progressively neurodegenerative and eventually life-threatening nature of MELAS. Although both trials have failed to achieve the primary outcomes,

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1.0

9 years follow-up study

0.8

IV trial group

0.6

PO trial group

0.4

Natural history Adult MELAS All MELAS

0.2

Survival probability

Juvenile MELAS 0.0

424

0

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2

Number at risk PO trial group 13 13 13 IV trial group 10 10 10

3

4

5

6

7

8

9

Elapsed time (years) 12 11 9 9

11 9

11 8

11 8

11 8

11 8

FIGURE 16.4 Comparison of survival curve in clinical trial with L-arginine and natural course without L-arginine in patients with MELAS. Black lines: A Kaplan
Meier survival curve is shown in the cohort study and adopted (Yatsuga et al., 2012). The dashed line indicates the juvenile form and the solid line indicates the adult form. The results of the log-rank analysis were significant. The juvenile form was associated with a higher risk of mortality than the adult form (hazard ratio, 3.29; 95% CI, 1.32
8.20). Those survival curves are recalculated with normalized severity score of the patients in the clinical trial study. Red lines: A Kaplan
Meier survival curve is shown in the clinical trial and 7-year followup study (Koga et al., 2018; Ikawa et al., 2020). The dashed line indicates the intravenous study and the solid line indicates the oral study. The surviving curve in clinical trial is significantly improved with those seen in the natural history. Modified from original Figure 2 in Journal of Neurology.

no patient died or became bedridden at the completion of the 2-year clinical trial. Subsequently, patients were followed up for seven years. The nine year follow-up study of our MELAS patients (two years in clinical trial and following seven years follow up) showed significant improvement in their surviving curve compared to a group that standardized age, gender, and severity in a cohort study by Kaplan-Meyer analysis (Fig. 16.4). No treatment-related adverse events occurred, and the formulations of L-arginine were well tolerated. Since plasma level of L-arginine varied from patient to patient, even we administered 0.5 g/kg/dose of L-arginine/HCl/TID, it is important to monitor the plasma trough level to prevent the additional SLEs in MELAS. One-hundred and 67 μmol/L of plasma L-arginine level is the threshold to prevent the SLEs completely in our study.

16.8 Contraindication in the treatment of MELAS

FIGURE 16.5 Pathogenic mechanisms of mitochondrial angiopathy in MELAS. Functional occlusion and pathological occlusion may contribute the SLEs seen in MELAS (Koga et al., 2012; Koga et al., 2018).

Our data provides physicians with the following insights of paramount clinical relevance: (1) plasma arginine concentration: 168 μmol/L may prevent the ictuses through the optimal normalization of endothelial dysfunction; (2) the bedriddenness rate was 0% at the completion of both the 2-year clinical trials and the 7-year followup, in marked contrast to 5.2% in the 5-year cohort study (Yatsuga et al., 2012), and natural history study (Kaufmann et al., 2011); (3) the mortality rates improved; (4) sudden death occurred in two patients with juvenile-onset MELAS, implying the pathogenic feature of MELAS; (5) MELAS was well controlled in the 2-year clinical trials as indicated by minor changes in the JMDRS scores; and (6) the regimen was not sufficient to stem disease progression during the 7-year follow-up as evidenced by increases in the JMDRS scores. Our results afford clinical evidence on effective pharmacotherapy for patients with MELAS, an unmet desire of pediatricians and neurologists who treat patients with MELAS (Koenig et al., 2016) (Fig. 16.5).

16.8 Contraindication in the treatment of MELAS According to the pharmacological effects of L-arginine as well as the experience of the therapeutic reaction to the drugs in MELAS, several therapeutic conditions

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seem to be a contraindication in this disorder. In our cohort study (Yatsuga et al., 2012), migraine headache is the most common presenting symptom in patients with MELAS, and it is often very difficult to distinguish its association with MELAS from the classical migraine headache. Triptans compound, a silver bullet for the classical migraine headache, is presumed to suppress headache by constricting the dilated vessels of the dura and brain, inhibiting the release of neuropeptides, or inhibiting the propagation of pain signals by C fibers from the dura and vessel walls (Cutrer et al., 1995). To investigate the mechanism of headache in patients with MELAS based on the neuronal hyperexcitability hypothesis, Iizuka injected 3 mg of sumatriptan subcutaneously for intractable headache in two MELAS patients. One patient developed the transient chest compression following injection without abnormal change on EKG, and the other MELAS showed epileptic seizure three days after the injection, and on the same day, she developed the continuous spread of the SLEs in the surrounding brain (Iizuka et al., 2003b). According to the angiopathy associated with MELAS, I think triptans compound is a contraindication for MELAS. In addition, MELAS, treated with steroid pulse therapy at the acute phase of SLEs, becomes dementia much faster than those without steroid therapy. Since MELAS has an endothelial dysfunction, they have a high risk of dementia by the vasculopathy. They showed hypoperfusion at posterior cingulated cortex which is similar to Alzheimer’s disease. Among various steroid therapies, pulse therapy such as hydrocortisone 30 mg/kg/shoot for three days, and steroid suppository for MELAS patients complicated with hemorrhoids should be avoided. We have a MELAS patient with hemorrhoid and had SLEs soon after he used the steroid suppository.

16.9 Concluding remarks The possible pathogenic mechanism of SLEs in MELAS may not be as simple as described above. Mitochondrial angiopathy also has been demonstrated in brain and muscle pathology and vascular physiology. Although the results of neuroimaging studies are controversial and are difficult to evaluate, there are several specific findings which may lead to the pathophysiology of SLEs in MELAS. We have to elucidate what initiates SLEs in MELAS in the future by developing the animal model. Currently, L-arginine therapy, to cure the symptoms of SLEs at acute phase, and to prevent or decrease the severity of SLEs at interictal phase of MELAS, is the most promising therapy for this incurable disorder. Global clinical trials of L-arginine on MELAS using randomized double-blind placebo control protocols may be done in near future.

16.12 Summary points

16.10 Applications to other neurological conditions In this study, we found reduced L-arginine status in MELAS patients. Reduced Larginine levels in blood was correlated with deficiency of endothelial dependent vasodilation and related to SLEs in MELAS. Migraine-type headache, which is always associated with patients who have an A3243G mutation in the mitochondrial tRNALeu(UUR) gene, is also effective for L-arginine supplementation. Reduced L-arginine status is also seen in other subtypes of mitochondrial disorders including Kearns-Sayre syndrome, Leigh syndrome, and deficient status of respiratory chain enzymes. Triptans (serotonin receptor agonists) which are usually used for classical migraine headaches, is the contraindication for migrainetype headaches associated with A3243G mutation carriers (Iizuka et al., 2003b). Steroid pulse therapy for acute phase of MELAS should also be avoided, since it accelerates brain atrophy and promotes early dementia onset.

16.11 Key facts of arginine and neuroprotection: a focus on stroke 16.11.1 Key fact of neuroprotection in MELAS MELAS has a segmental arterial occlusion pathologically. MELAS has an endothelial dependent vasodilation abnormality, physiologically. MELAS has a decreased status of plasma L-arginine which harmonizes the endothelial dysfunction. MELAS has a hypoperfusion status in superearly stage of SLEs. L-arginine infusion at superacute stage of SLEs improved symptoms quickly and normalized neurological abnormality associated with SLEs. L-arginine supplementation decreased severity and frequency of ictus in SLEs. Maintaining plasma arginine concentration at least 168 mmol/L may prevent the ictuses. L-arginine supplementation improves mortality rates through therapeutic regimens.

16.12 Summary points • • •

MELAS has an endothelial dependent vasodilation abnormality, pathologically and physiologically. L-arginine supplementation normalized the endothelial function. Maintaining plasma arginine concentration of at least 168 mmol/L may prevent the ictuses.

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2874. Koga, Y., Povalko, N., Nishioka, J., Katayama, K., Yatsuga, S., & Matsuishi, T. (2012). Molecular pathology of MELAS and L-arginine effects. Biochimica et Biophysica Acta, 1820, 608
614. Kubota, M., Sakakihara, Y., Mori, M., Yamagata, T., & Momoi-Yoshida, M. (2004). Beneficial effect of L-arginine for stroke-like episode in MELAS. Brain & Development, 26, 481
483. Lucotti, P., Monti, L., Setola, E., La Canna, G., Castiglioni, A., Rossodivita, A., Pala, M. G., Formica, F., Paolini, G., Catapano, A. L., Bosi, E., Alfieri, O., & Piatti, P. (2009). Oral L-arginine supplementation improves endothelial function and ameliorates insulin sensitivity and inflammation in cardiopathic nondiabetic patients after an aortocoronary bypass. Metabolism: Clinical and Experimental, 58, 1270
1276. Mo¨ller, H. E., Kurlemann, G., Putzler, M., Wiedermann, D., Hilbich, T., & Fiedler, B. (2005). Magnetic resonance spectroscopy in patients with MELAS. Journal of the Neurological Sciences, 229
230, 131
139. Nishioka, J., Akita, Y., Yatsuga, S., Katayama, K., Matsuishi, T., Ishibashi, M., & Koga, Y. (2008). Inappropriate intracranial hemodynamics in the natural course of MELAS. Brain & Development, 30, 100
105. Ohama, E., Ohara, S., Ikuta, F., Tanaka, K., Nishizawa, M., & Miyatake, T. (1987). Mitochondrial angiopathy in cerebral blood vessels of mitochondrial encephalomyopathy. Acta Neuropathologica, 74, 226
233. Oppenheim, C., Galanaud, D., Samson, Y., Sahel, M., Dormont, D., Wechsler, B., & Marsault, C. (2000). Can diffusion weighted magnetic resonance imaging help differentiate stroke from stroke-like events in MELAS? Journal of Neurology, Neurosurgery, and Psychiatry, 69, 248
250. Pavlakis, S. G., Phillips, P. C., DiMauro, S., De Vivo, D. C., & Rowland, L. P. (1984). Mitochondrial myopathy, encephalopathy, lactic acidosis, and SLEs: A distinctive clinical syndrome. Annals of Neurology, 16, 481
488. Ruel, M., Beanlands, R. S., Lortie, M., Chan, V., Camack, N., deKemp, R. A., Suuronen, E. J., Rubens, F. D., DaSilva, J. N., Sellke, F. W., Stewart, D. J., & Mesana, T. G. (2008). Concomitant treatment with oral L-arginine improves the efficacy of surgical angiogenesis in patients with severe diffuse coronary artery disease: The Endothelial Modulation in Angiogenic Therapy randomized controlled trial. The Journal of Thoracic and Cardiovascular Surgery, 135, 762
770. Sakuta, R., & Nonaka, I. (1989). Vascular involvement in mitochondrial myopathy. Annals of Neurology, 25, 594
601. Shigemi, R., Fukuda, M., Suzuki, Y., Morimoto, T., & Ishii, E. (2011). L-arginine is effective in SLEs of MELAS associated with the G13513A mutation. Brain & Development, 33, 518
520. Shin, S., Mohan, S., & Fung, H. L. (2011). Intracellular L-arginine concentration does not determine NO production in endothelial cells: Implications on the "L-arginine paradox". Biochemical and Biophysical Research Communications, 414, 660
663. Sproule, D. M., & Kaufmann, P. (2008). Mitochondrial encephalopathy, lactic acidosis, and SLEs: Basic concepts, clinical phenotype, and therapeutic management of MELAS syndrome. Annals of the New York Academy of Sciences, 1142, 133
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Stoquart-Elsankari, S., Lehmann, P., Perin, B., Gondry-Jouet, C., & Godefroy, O. (2008). MRI and diffusion-weighted imaging followup of a stroke-like event in a patient with MELAS. Journal of Neurology, 255, 1593
1595. Takahashi, N., Shimada, T., Murakami, Y., Katoh, H., Oyake, N., Ishibashi, Y., Nishino, I., Nonaka, I., & Goto, Y. (2005). Vascular involvement in a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and SLEs. The American Journal of the Medical Sciences, 329, 265
266. Tanahashi, C., Nakayama, A., Yoshida, M., Ito, M., Mori, N., & Hashizume, Y. (2000). MELAS with the mitochondrial DNA 3243 point mutation: A neuropathological study. Acta Neuropathologica, 99, 31
38. Tsao, P. S., McEvoy, L. M., Drexler, H., Butcher, E. C., & Cooke, J. P. (1994). Enhanced endothelial adhesiveness in hypercholesterolemia is attenuated by L-arginine. Circulation, 89, 2176
2182. Tsujikawa, T., Yoneda, M., Shimizu, Y., Uematsu, H., Toyooka, M., Ikawa, M., Kudo, T., Okazawa, H., Kuriyama, M., & Kimura, H. (2010). Pathophysiologic evaluation of MELAS strokes by serially quantified MRS and CASL perfusion images. Brain & Development, 32, 143
149. Wang, X. Y., Noguchi, K., Takashima, S., Hayashi, N., Ogawa, S., & Seto, H. (2003). Serial diffusion-weighted imaging in a patient with MELAS and presumed cytotoxic oedema. Neuroradiology, 45, 640
643. Yatsuga, S., Povalko, N., Nishioka, J., Katayama, K., Kakimoto, N., Matsuishi, T., Kakuma, T., & Koga, Y. (2012). MELAS Study Group in Japan., MELAS: A nationwide prospective cohort study of 96 patients in Japan. Biochimica et Biophysica Acta, 1820, 619
624. Yoneda, M., Maeda, M., Kimura, H., Fujii, A., Katayama, K., & Kuriyama, M. (1999). Vasogenic edema on MELAS: A serial study with diffusion-weighted MR imaging. Neurology, 53, 2182
2184.

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17

Nutraceuticals for targeting NAD 1 to restore mitochondrial function

Antje Garten1 and Gareth G. Lavery2 1

Hospital for Children and Adolescents, Center for Pediatric Research Leipzig, Leipzig, Germany 2 Department of Biosciences, Nottingham Trent University, Nottingham, United Kingdom

17.1 Nicotinamide adenine dinucleotide as redox cofactor and signaling molecule in mitochondria Vitamin B3 comprises a group of precursors for nicotinamide adenine dinucleotide (NAD), an essential coenzyme and signaling molecule for maintaining energy metabolism. Nicotinamide (NAM) and nicotinic acid (NA) have long been known for their preventive and therapeutic effects on pellagra, discovered by Conrad Elvehjem in 1937 (Elvehjem, 1940; Elvehjem et al., 2002), a disease caused by a diet deficient in vitamin B3. NAD, as well as its precursors, NA and NAM, are readily available in a balanced diet including vegetables, fruits, whole grains, meat, fish, and dairy products. During the last 30 years, the effects of additional NAD precursors and metabolites on augmenting cellular and mitochondrial NAD levels have been characterized. These include nicotinamide riboside (NR) (Bieganowski & Brenner, 2004) and nicotinamide mononucleotide (NMN) (Revollo et al., 2004), which can also be found in food (Trammell et al., 2016; Mills et al., 2016), and nicotinic acid riboside (NAR) which is generated through the action of 50 nucleotidases dephosphorylating nicotinic acid mononucleotide (Kulikova et al., 2015). Recently, reduced forms of NAD precursors, NR hydride and NMN hydride, have been identified as potent augmenters of cellular NAD concentrations (Zapata-Pe´rez et al., 2021; Yang et al., 2019, 2020; Sa´nchezGarcı´a et al., 2019). NAD can also be synthesized from the amino acid tryptophan by the tissue-specific de novo or kynurenine pathway (Bender, 1983). Mitochondria are the organelles with highest NAD concentrations (approx. 400 μM, 40%
70% of the total cellular NAD pool) (Alano et al., 2007; Di Lisa et al., 2001), while the ratio of NAD 1 /NADH is low compared to other subcellular compartments. NAD 1 /NADH functions as a coenzyme that catalyzes the transfer of hydride ions in metabolic reactions (Warburg & Christian, 1936) and therefore, is essential for mitochondrial and cellular energy metabolism. A multitude of in vitro and in vivo studies using either specific inhibitors for, or knocking Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00019-9 © 2023 Elsevier Inc. All rights reserved.

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out NAD biosynthesis enzymes, have shown the detrimental effects of deficient NAD 1 /NADH concentrations on mitochondrial function (Kristian et al., 2011; Jokinen et al., 2017; Wiley et al., 2016). NAD 1 is also a substrate for enzymes, including sirtuins (Sirts1
7), NAD glycohydrolases/cyclic ADP-ribose synthases (CD38, CD157), as well as mono-ADP-ribosyltransferases and poly(ADP-ribose) polymerases (PARPs) (P et al., 1963; S et al., 2000; DJ et al., 1992; HC & R, 1991). Some of these NAD 1 dependent enzymes are regulators of mitochondrial function and consequently influence cellular energy metabolism and stress responses. Being both a cofactor for metabolic reactions and a substrate for enzymes involved in the regulation of gene expression and the generation of second messengers, NAD links energy metabolism to cellular signaling. In this chapter, we want to give an overview on mitochondrial NAD metabolism, and ways to influence mitochondrial function by supplementation with NAD precursors and their potential impact on human disease.

17.2 Cellular and mitochondrial nicotinamide adenine dinucleotide metabolism In mammalian cells, NAD 1 is generated in a tissue-specific manner, either de novo via the kynurenine pathway from tryptophan, via the Preiss-Handler pathway from NA, via the salvage pathway from NAM, or via the nicotinamide ribose kinase pathway from NR (Bieganowski & Brenner, 2004; Ratajczak et al., 2016). NAD that is taken up from the diet can enter cells either intact (Pittelli et al., 2011; Billington et al., 2008), or after hydrolysis by NAD degrading enzymes. CD73 cleaves NAD to NMN and adenosine monophosphate and subsequently dephosphorylates the molecules to NR and adenosine (Grozio et al., 2013), CD38 and its paralog CD157 convert NAD to cyclic ADP ribose and NAM (Malavasi et al., 2008). CD157 also hydrolyzes NR and NAR to NAM and NA, respectively (Preugschat et al., 2014). Plasma membrane transporters exist for tryptophan (SM & D, 2011; Kanai et al., 1998) and NA (Bahn et al., 2008; Gopal et al., 2007). Recently, a plasma membrane NMN carrier was identified (Grozio et al., 2019), the biological relevance of which has however been questioned (Schmidt & Brenner, 2019). NMN was shown to be dephosphorylated to NR prior to cellular uptake (Ratajczak et al., 2016). Uptake of NR and NAR into cells is mediated by equilibrative nucleoside transporters (Kropotov et al., 2021). The NAD biosynthetic pathways converge at the reaction of NMN and ATP to NAD, which is catalyzed by NMN adenylyltransferases (NMNAT) 1
3 (Berger et al., 2005). These isoenzymes have different subcellular localizations, with NMNAT1 found in the nucleus, NMNAT-2 in the Golgi apparatus and the cytosol and NMNAT3 in mitochondria. The compartmentalized setup of NAD biosynthesis points to a differential regulation of subcellular NAD pools and NAD-dependent signaling (Cohen, 2020). In preadipocytes, for example,

17.2 Cellular and mitochondrial nicotinamide adenine

extracellular signals for induction of adipogenesis increases cytosolic NAD 1 biosynthesis by upregulation of NMNAT-2, which leads to lower availability of NMN for nuclear NAD 1 biosynthesis. Consequently, the nuclear NAD 1 consumer PARP-1 is less active and cannot repress the adipogenic transcription factor CCAAT/enhancer binding protein β by ADP-ribosylation. The missing repressive effect then leads to adipocyte differentiation (Ryu et al., 2018). All NAD 1 consuming enzymes generate NAM as one of their products. NAM can be salvaged via the action of nicotinamide phosphoribosyltransferase (NAMPT) and converted to NMN by transfer of a phosphoribosyl group from phosphoribosylpyrophosphate. NAM can also be methylated by nicotinamide-N-methyltransferase to 1-methylnicotinamide (MNA). This reaction predominantly takes place when NAM concentrations are higher than 5 nM, which correspond to the KM value of NAMPT for NAM (Burgos & Schramm, 2008). MNA has been implicated in the etiology of Parkinson’s disease and coronary artery disease by inducing mitochondrial dysfunction through inhibiting mitochondrial complex I (G et al., 2018; T et al., 2002). Another study, however, showed no negative effect of MNA on complex I activity, but rather an extension of C. elegans lifespan through increased reactive oxygen species production (K et al., 2013). Further experimental clarification is clearly required to resolve these contrasting findings on the effects of MNA. NAD 1 , NADH, and NAD precursors cannot freely diffuse across mitochondrial membranes, but nuclear/cytosolic and mitochondrial compartments are connected via the malate/aspartate and the glycerol-3-phosphate shuttles. It is still debated which components of NAD biosynthesis pathways are actually present and active in mitochondria. NAD was shown to be synthesized directly in mitochondria (Berger et al., 2005; Yang et al., 2007), although blocking of NAMPT activity by the specific inhibitor FK866 was shown to leave the mitochondrial NAD pool unaffected (Pittelli et al., 2010). Evidence from Nmnat3 knockout mice shows that Nmnat3 is dispensable for the maintenance of mitochondrial NAD levels in liver and skeletal muscle (Yamamoto et al., 2016). Recently, a mitochondrial NAD transporter was identified (Luongo et al., 2020; Girardi et al., 2020; Kory et al., 2020), after previous studies showed mitochondrial uptake of NAD (Davila et al., 2018) and the influence of cytosolic NAD(H) levels on the mitochondrial NAD pool (Cambronne et al., 2016). Approximately 10% of NAD is further converted by NAD kinases to NADP 1 /NADPH (Liu et al., 2018; Love et al., 2015), another pair of redox coenzymes with distinct functions. While NAD 1 /NADH is essential for catabolic reactions in energy metabolism (e.g., glycolysis, fatty acid oxidation, tricarboxylic acid cycle), one-carbon metabolism and transferring reducing equivalents to the mitochondria for oxidative phosphorylation, NADP 1 /NADPH is recognized by a different set of enzymes involved in oxidative stress defense and anabolic pathways such as fatty acid biosynthesis. Mitochondria harbor the enzyme nicotinamide nucleotide transhydrogenase (NNT) which transfers a hydride ion from NADH to NADP 1 to generate NADPH (and vice versa). Its activity is coupled to the proton gradient across

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the membrane of energized mitochondria (Rydstro¨m, 2006). NNT is essential for replenishing mitochondrial NADPH to counteract oxidative stress (AF et al., 2021; Navarro et al., 2017; TR et al., 2021).

17.3 Nicotinamide adenine dinucleotide and mitochondrial function There is an optimal amount of mitochondrial NAD1, below or above which mitochondria do not function properly. This was elegantly shown by expressing a plant NAD transporter in a mammalian cell model (HEK293 cells) which resulted in increased mitochondrial NAD 1 levels, growth retardation of the cells, and a shift of energy metabolism from oxidative phosphorylation to glycolysis (VanLinden et al., 2015). Similarly, depleting mitochondrial NAD1 by PARP targeted to mitochondria (mitoPARP) (Niere et al., 2008) led to the same cellular response, suggesting that mitochondrial NAD levels need to be tightly regulated to keep mitochondria functional. Depleting mitochondrial NAD concentrations has detrimental effects on mitochondrial function. Sterile α- and toll/interleukin-1 receptor (TIR) motifcontaining protein (SARM)1 is a mitochondrially localized TIR adapter protein (Panneerselvam et al., 2013) and mainly expressed in immune cells and neurons. Besides being a negative regulator of innate immune responses, SARM1 is a primary mediator of programmed axonal degeneration (Essuman et al., 2017). Its TIR domain has intrinsic NADase activity. The loss of NAD 1 leads to an energy deficit and injury induced axonal degeneration which can be reversed either by overexpression of NAD biosynthesis enzymes NAMPT and NMNAT1 (Gerdts et al., 2015) or by supplementation with NR (Essuman et al., 2017). NAD impacts mitochondrial biogenesis and function mainly through the activation of sirtuins. The mammalian sirtuin enzyme family consists of 7 members (Sirt1
7), which use NAD 1 to deacylate specific lysine residues in target proteins by transferring the acyl residue to ADP-ribose generating O-acyl-ADPribose and NAM. Sirt1, mainly located in the nucleus, regulates mitochondrial function via deacetylating a range of target proteins, which are comprehensively reviewed in (Nogueiras et al., 2012) and include peroxisome proliferator-activated receptor-γ co-activator (PGC)1α, a regulator of mitochondrial biogenesis (Gerhart-Hines et al., 2007) and uncoupling protein-2 (Bordone et al., 2006). Sirt1 has also been implicated in turnover of defective mitochondria by mitophagy (Jang et al., 2012). Consequently, cardiac muscle-specific knockout mice show a phenotype resembling diabetic cardiomyopathy with impaired mitochondrial biogenesis and function (Ma et al., 2017). On the other hand, Sirt1 musclespecific knockout mice showed the same response to endurance exercise as control mice, with increased PGC1α deacetylation and mitochondrial biogenesis (Philp et al., 2011). In this study, the acetyltransferase general control of amino acid synthesis 5 (GCN5) was shown to elicit the beneficial effects of exercise.

17.3 Nicotinamide adenine dinucleotide and mitochondrial function

The mitochondrial sirtuins, Sirt3, Sirt4 and Sirt5, deacylate protein targets involved in fatty acid oxidation (Hirschey et al., 2010, 2011; Schwer et al., 2006), ketogenesis (Shimazu et al., 2010), defense against oxidative stress (Someya et al., 2010), glutamate metabolism (Haigis et al., 2006), urea cycle (Nakagawa et al., 2009), as well as several other mitochondrial pathways (reviewed in (Carrico et al., 2018)). Sirt3 is considered the major mitochondrial enzyme responsible for removing acetyl residues (MJ et al., 2013; Lombard et al., 2007). When comparing acetylation in different tissues of Sirt3 KO with control mice, a set of acetylated sites emerged that were common among all tissues, with target proteins belonging to the citric acid cycle and oxidative phosphorylation machinery (Carrico et al., 2018). In addition, there were a large number of acetylation sites unique to each tissue (Carrico et al., 2018), suggesting a tissue-specific regulation of Sirt3 function. Sirt5 is a desuccinyl-, demalonyl- and deglutarylase, removing acyl residues from proteins involved in branched chain amino acid metabolism and the citric acid cycle (Carrico et al., 2018). The functional significance of Sirt4 is less well established. Sirt4 was found to regulate lipoylation and biotinylation of pyruvate dehydrogenase (RA et al., 2014), and branched-chain acylation of mitochondrial enzymes involved in leucine metabolism (Anderson et al., 2017). Sirt4 was also shown to downregulate glutamate dehydrogenase activity in pancreatic beta cells by ADP-ribosylation, which leads to a decrease of insulin secretion (Haigis et al., 2006). Considering this multitude of effectors regulated by NAD 1 -dependent sirtuins, supplementation with NAD precursors has been explored extensively in vitro and in animal models of impaired mitochondrial function. Increasing cellular NAD concentrations was shown to be beneficial in several rodent disease models [reviewed in (Canto´ et al., 2015; Yoshino et al., 2017; Mehmel et al., 2020; Kang et al., 2020)]. Augmented mitochondrial oxidative phosphorylation was reported when cellular NAD 1 levels were increased either by inhibiting degradation of NAD (Barbosa et al., 2007; Bai et al., 2011; Mukhopadhyay et al., 2016; Camacho-Pereira et al., 2016) or supplying NR (Canto´ et al., 2012) or NMN (Gomes et al., 2013) for NAD biosynthesis. Oral NR supplementation (400 mg/kg body weight/d) increased both Sirt1 and Sirt3 activity, which led to a deacetylation of target transcription factor forkhead box (Foxo)1, PGC1alpha, subunit Ndufa9 of mitochondrial complex I, and superoxide dismutase (Sod)2, and resulted in increased expression of mitochondrial proteins, higher energy expenditure of NR-supplemented mice and protection from adverse effects of a high fat diet (Canto´ et al., 2012). The efficacy of NR seems to be tissue-specific, with liver, muscle and brown adipose tissue (BAT) responding with increased cellular NAD levels, in contrast to white adipose and brain tissue, which neither showed increased NAD levels nor augmented mitochondrial function (Canto´ et al., 2012). In different mouse models of muscular dysfunction (Ryu et al., 2016), mitochondrial disease (Khan et al., 2014; Cerutti et al., 2014), and fatty liver disease (Gariani et al., 2016), oral NR induced a Sirt1 and Sirt3 dependent mitochondrial unfolded protein response, which led to increased mitochondrial protein content and enhanced activity of citric acid cycle

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enzymes, fatty acid oxidation, and oxidative phosphorylation. NR supplementation was also shown to counteract senescence of muscle, neural, and melanocyte stem cells in aged mice and a mouse model of muscular dystrophy by improving mitochondrial function (Zhang et al., 2016). In the previously mentioned studies, NR was supplemented with feed at 400 mg/kg body weight/d. NMN was reported to elicit beneficial effects on mitochondrial function in a wide range of tissues and disease models including diabetic rat brain (Chandrasekaran et al., 2020), ischemic mouse brain (Klimova et al., 2019, 2020), Alzheimer’s disease mouse brain (Long et al., 2015), skeletal muscle in a mouse model of Leigh syndrome (Lee et al., 2016), cardiac muscle in a mouse model of heart failure (Zhang et al., 2017), and livers and kidneys of a rat model of hemorrhagic shock (Sims et al., 2018). In the last study mentioned, rats were pretreated with NMN (400 mg/kg body weight/day) and were infused with 400 mg NMN/kg body weight (Sims et al., 2018), while in the other studies, NMN was administered subcutaneously at 100 mg/kg body weight/every other day (Chandrasekaran et al., 2020; Long et al., 2015), intraperitoneally at 62.5 mg/kg body weight/day (Klimova et al., 2019, 2020), at 500 mg/kg body weight once every three days (Lee et al., 2016) or at 500 mg/kg body weight/day (Zhang et al., 2017). NMN also had beneficial effects on mitochondrial function in different animal models of aging. Oral administration of NMN (300 mg/kg body weight/day) for one year led to increased mitochondrial respiratory capacity in skeletal muscle and increased physical activity in NMN-supplemented versus nonsupplemented mice (Mills et al., 2016). Interestingly, NAD levels were not significantly increased in liver, skeletal muscle, WAT, BAT, or the cortex in NMN-treated mice (Mills et al., 2016). NMN supplementation via intraperitoneal injections of 500 mg NMN/kg body weight per day increased skeletal muscle NAD levels in aged mice, counteracted the age-associated decline of mitochondrial function in skeletal muscle, reversed changes in fiber type and normalized mitochondrial homeostasis via a Sirt1-hypoxia-inducible factor (Hif)1alpha-c-myc signaling pathway (Gomes et al., 2013). In aged mouse oocytes, NMN via intraperitoneal injection (200 mg/kg body weight/day) increased oocyte NAD levels and improved reproductive outcome by restoring mitochondrial function (Miao et al., 2020). Neurodegenerative diseases are often associated with mitochondrial and mitophagic dysfunction. Mitophagy, a specialized form of autophagy, is the cellular response to dispose of damaged and dysfunctional mitochondria (Fivenson et al., 2017). In different animal models of neurodegenerative diseases, such as xeroderma pigmentosum group A (Fang et al., 2014), Werner syndrome (Fang et al., 2019) and Ataxia Telangiectasia (Fang et al., 2016), cellular NAD 1 levels were found to be decreased and could be augmented by supplementation of NR and NMN. Repleting NAD 1 led to a stimulation of neuronal DNA repair and improved mitochondrial quality via mitophagy (Fang et al., 2016). In contrast, there are a number of preclinical studies reporting on a lack of beneficial or even negative effects of supplementation with NAD precursors. In a study examining a potential interaction effect of exercise and NMN

17.3 Nicotinamide adenine dinucleotide and mitochondrial function

supplementation in a mouse model of diet-induced obesity, chronic, oral administration of NMN (400 mg/kg body weight/d) abolished the beneficial effect of treadmill exercise on HFD-induced glucose intolerance. NMN alone had no observable effect, while exercise alone significantly improved glucose tolerance compared with that observed for HFD-fed mice (Yu et al., 2021). Another study examining the effects of NR supplementation on exercise performance of healthy Wistar rats found that energy and redox metabolism was disturbed in rats receiving NR (300 mg/kg body weight/d) which lead to impaired exercise performance (Kourtzidis et al., 2016, 2018). A high dose of NR (9000 mg NR/kg diet, which corresponds to approx. 1000 mg/kg body weight/d) reduced metabolic flexibility and glucose clearance and exacerbated systemic insulin resistance in mice on a mildly obesogenic diet. Evidence of white adipose tissue dysfunction was found with an increased number of crown-like structures and macrophages, and an upregulation of pro-inflammatory gene markers, which was most likely responsible for the observed negative effects of NR (Shi et al., 2019). In two mouse models of mild obesity, NR supplementation (500 mg/kg/day) did not alter body weight, glucose tolerance, or energy expenditure, despite increasing mitochondrial oxygen consumption in soleus muscle (Cartwright et al., 2021). NR supplementation (400 mg/kg body weight/day) did not affect body composition or glucose tolerance in both hepatocyte-specific Nampt KO and control mice. Moreover, mice with hepatocyte-specific Nampt KO, despite having 50% lower hepatic NAD concentrations, did not differ from their control littermates with respect to fat mass or glucose tolerance. Primary hepatocytes isolated from the Nampt KO mice had a comparable oxygen consumption and membrane potential to Nampt wildtype hepatocytes. NR supplementation did not increase mitochondrial NAD concentrations or improve mitochondrial respiratory function in primary hepatocytes (Dall et al., 2019). When challenged with a methionine-choline deficient diet, hepatocyte-specific Nampt KO mice, however, had an increased susceptibility for nonalcoholic steatohepatitis and fibrosis. This phenotype could be reversed by supplementing either NA (75 mg/kg body weight/day) with the feed or NR at 1.9 g/L in the drinking water (Dall et al., 2021). In a mouse model of Duchenne muscular dystrophy with NAD 1 deficiency in skeletal muscle, supplementation of NR at a concentration of 12 mM ad libitum in the drinking water did not increase muscle NAD 1 levels, restore muscle contractile function, or confer protection from eccentric injury (Frederick et al., 2020). It is unclear why there are such differences in the response to supplementation with NAD precursors. There are also few studies comparing the effectiveness of different NAD precursors. A direct comparison of feeding mice with chow supplemented with NA, NR, and NMN (each 400 mg/kg body weight/d) showed that all NAD precursors were able to increase liver NAD 1 content, but muscle NAD 1 content was elevated only in mice fed with NA or NR-supplemented chow (Canto´ et al., 2012). It is likely that the type and severity of disease, timing of NAD supplementation, and type of affected tissues determine the response to NAD supplementation.

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17.4 Nicotinamide adenine dinucleotide supplementation in human diseases For human use, NA is often referred to as niacin. This term is sometimes also collectively used for NA and NAM. Both NA and NAM have been used alone or in combination with other medication for the prevention and treatment of a wide variety of diseases including lymphoma, chronic kidney disease, type 1 diabetes, headache, dyslipidemia, and cardiovascular disease, summarized in Table 17.1. For many of these disorders, the mechanism underlying the therapeutic effects is supposed to be the increase of cellular NAD 1 levels and a resulting improvement in mitochondrial function (Song et al., 2019). Doses of NAM in these studies ranged between 500
3000 mg daily. Supplementation with NA has been used extensively to treat dyslipidemia, specifically to increase circulating levels of high-density lipoprotein cholesterol (Guyton, 2004). NA supplementation, however, can lead to adverse effects, with facial flushing occurring most prominently at doses as low as 50 mg NA daily (Minto et al., 2017). This response is seen in many patients which impairs patient compliance, is not observed when supplementing with NAM, and is due to the activation of the capsaicin receptor transient receptor, potential vanilloid subfamily member 1 by NA (Ma et al., 2014). To mitigate flushing, NA is administered as an extended-release formula (Guyton, 2004) together with the prostaglandin receptor antagonist laropiprant or with prostaglandin synthesis inhibitor aspirin. Serious adverse events (new-onset diabetes, disturbances of diabetes control, bleeding, infection, and gastrointestinal upset) were reported more recently in a large clinical trial of patients at high risk of cardiovascular disease when adding extended release niacin/laropiprant to intensive statinbased low-density-lipoprotein cholesterol (LDL-C)-lowering therapy (Haynes et al., 2019; Landray et al., 2014). Importantly, the addition of extended-release niacin/laropiprant to statins did not significantly reduce the risk of major vascular events (Landray et al., 2014), which raised serious doubts about the use of niacin alone or combined with other lipid-lowering drugs to achieve cardiovascular benefits. In a recent systematic review including, 23 registered clinical trials, no evidence of beneficial effects on mortality, risk for strokes or myocardial infarctions was found both for niacin monotherapy or in combination with statin therapy (Schandelmaier et al., 2017). Several studies showed beneficial effects on circulating lipoproteins in HIVinfected patients on antiretroviral therapy, with a group of patients suffering from dyslipidemia and lipodystrophy (Chow et al., 2010; Bays et al., 2015; Dube´ et al., 2015; Balasubramanyam et al., 2011; Bays et al., 2012), although no improvement of endothelial function or circulating inflammatory biomarkers could be demonstrated (Dube´ et al., 2015). A recent long-term study showed an association of dietary intake of NA and NAM with a lower risk of death from heart failure, myocardial infarction, or sudden cardiac death. The average intake of NA and NAM was 28.9 and 26.9 mg in men and women, respectively, which is much higher than the daily-recommended intake (14
16 mg). An inverse correlation between NA and NAM intake and systolic

Table 17.1 Clinical trials testing nicotinic acid/niacin and nicotinamide in human disease. Study type

Participants

Compound/dose

Main outcome

References

Increased hazard ratio for newonset diabetes, disturbance in diabetes control, bleeding, infection and gastrointestinal upset, lack of evidence of cardiovascular benefits No reduction in overall mortality, cardiovascular mortality, fatal or non-fatal myocardial infarctions, fatal or non-fatal strokes

(Haynes et al., 2019; Landray et al., 2014)

Improved endothelial function in HIV-infected patients with low HDL-c

(Chow et al., 2010)

Significant improvements in HDLC, increased adiponectin

(Balasubramanyam et al., 2011)

Less atherogenic lipid profile independent of baseline glycemic control

(Bays et al., 2015, 2012)

Dyslipidemia and cardiovascular events Randomized, placebo-controlled

25,673 adult women and men at high risk of vascular disease

Oral NA-laropiprant (2000 mg/ 40 mg/day) for a median duration of 3.9 years

Systematic review of 23 registered clinical trials publishes between 1968 and 2015

39,195 participants

Oral NA, median dose 2000 mg/ day

(Schandelmaier et al., 2017)

Dyslipidemia and endothelial function in HIV infection Randomized, placebo-controlled

10 HIV-infected adult women and men on antiviral therapy

Randomized, placebo-controlled

21 HIV-infected adult women and men on antiviral therapy 298 Adult women and men with type 2 diabetes and antidiabetic medication

Randomized, multicenter, placebo-controlled

Oral extended release NA starting at 500 mg/night up to maximum tolerated dose (1500 mg/night) for 12 weeks Oral sustained-release NA starting at 750 mg/night up to 2000 mg for 24 weeks Oral extended release NAlaropiprant starting at 1000 mg/ 20 mg/day up to 2000 mg/ 40 mg/day for 12 weeks

(Continued)

Table 17.1 Clinical trials testing nicotinic acid/niacin and nicotinamide in human disease. Continued Study type

Participants

Compound/dose

Main outcome

References

Randomized, open-label

35 HIV-infected adult women and men on antiviral therapy

Oral extended release NA starting at 500 mg/night up to maximum tolerated dose (1500 mg/night) for 24 weeks

Significant improvements in HDLC and atherogenic lipoproteins, no improvement in endothelial function or inflammatory biomarkers

(Dubé et al., 2015)

Oral modified
release NAM 2000 mg/m2/day for 5 years

No difference in diabetes development

(Gale et al., 2004)

Oral slow release NAM 1200 mg/m2/day for 3 years

Decreased first-phase insulin secretion in response to intravenous glucose, no difference in diabetes development

(Lampeter et al., 1998)

Oral NAM 2000 mg/day for 2 weeks

23.6% decrease in insulin sensitivity

(Greenbaum et al., 1996)

Oral NAM up to 100 mg/kg body weight daily and 400 mg histone deacetylase inhibitor vorinostat twice during 21 days

Overall response rate of 24%, 57% of patients had stabilization of disease

(Amengual et al., 2013)

Type 1 diabetes mellitus (T1DM) Randomized, multicenter, placebo-controlled

randomized, placebo-controlled

Case series

552 First-degree relatives of patients with T1DM with isletcell autoantibodies $ 20 units 55 Siblings of patients with T1DM with islet-cell autoantibodies $ 20 units aged 3
12 years 8 Relatives of patients with T1DM with islet-cell autoantibodies $ 20 units aged

Lymphoma Open-label, phase 1, single-arm

25 Adult women and men with relapsed or refractory lymphoma

Chronic kidney disease Randomized, placebo-controlled

205 Adult women and men with chronic kidney disease

Oral NAM 1500 mg/day combined with phosphate binder lanthanum carbonate 3000 mg for 12 months

No significant effect on serum phosphate or FGF23 in chronic kidney disease

(Ix et al., 2019)

NA, one initial intramuscular injection followed by 6 or 8 intravenous (IV) treatments (maximum 50 mg), then regular intramuscular injections (25
50 mg) combined with 50
150 mg of oral administration IV sodium nicotinate or NA 100 mg

Positive response in 17 of 21 subjects with migraine headaches

(Prousky & Seely, 2005)

Positive response in 75 of 100 subjects with headaches of different etiologic types

(Prousky & Seely, 2005)

Positive response in 13 of 15 patients with tension headaches

(Prousky & Seely, 2005)

Positive response in 13 of 22 subjects with tension headaches Positive response of all subjects with tension headaches

(Prousky & Seely, 2005) (Prousky & Seely, 2005)

Migraine and tension-type headaches Case series

21 Patients with migraine headache

Case series

100 Patients with headaches of different etiologic types 15 Patients with tension headaches

Case series

Case series Case series

22 Patients with tension headaches 5 Patients with tension headaches

IV NA 100 mg, and additional 50
200 mg if necessary to ensure a flushing response of more than 15 minutes IV NA 100
200 mg for a total of 53 times IV NA 100 mg regularly for 12 weeks combined with graded schedule of oral dosing, beginning at 300 mg/day, increasing to 900 mg/day, tapering down to 300 mg/day

(Continued)

Table 17.1 Clinical trials testing nicotinic acid/niacin and nicotinamide in human disease. Continued Study type

Participants

Compound/dose

Main outcome

References

Case series

50 Patients with tension headaches

Positive response in 44 of 50 subjects with tension headaches

(Prousky & Seely, 2005)

Case report

1 Patient with migraine headache 2 Patients with migraine headache 1 Patient with migraine headache

IV NA 100 mg regularly for 23 weeks, continued once every 2 months and as needed, combined with graded schedule of oral dosing, beginning at 300 mg/day, increasing to 900 mg/day, tapering down to 300 mg/day Oral 300
500 mg of NA

Positive response of subject with migraine headaches Positive response of 2 subjects with migraine headaches Positive response of subject with migraine headaches

(Prousky & Seely, 2005) (Prousky & Seely, 2005) (Velling et al., 2003)

Case report Case report

Oral NA 500 mg taken at onset of acute symptoms Oral sustained-release NA 375 mg twice daily for 1 month, and 375 mg once daily for 2 months.

HDL-C, High-density lipoprotein cholesterol, IV, intravenous; NA, nicotinic acid, NAM, nicotinamide.

17.4 Nicotinamide adenine dinucleotide supplementation

blood pressure was also found (Abdellatif et al., 2021). In addition, NAD 1 levels in biopsies of donor hearts with heart failure and preserved ejection fraction (HFpEF) were found to be lower than in healthy donor hearts (Abdellatif et al., 2021), which points to a potential benefit of NAD 1 supplementation for patients with HFpEF. Based on successful preclinical studies (Reddy et al., 1990; O’Brien et al., 2000), a number of clinical trials were conducted to test NAM supplementation to prevent type 1 diabetes mellitus, which was, however, not effective in randomized, placebocontrolled clinical trials (Gale et al., 2004; Lampeter et al., 1998). Moreover, subjects receiving NAM displayed lower first-phase insulin secretion in response to IV glucose compared to the placebo, suggesting no efficacy of NAM in improving beta-cell function (Lampeter et al., 1998; Greenbaum et al., 1996). In a small, single-arm phase 1 study, oral NAM together with the histone deacetylase inhibitor vorinostat was given to patients with aggressive lymphoma, because positive synergistic effects of this drug combination was observed earlier in germinal center-derived diffuse large B-cell lymphoma (DLBCL) cell lines and a lymphoma mouse model. There was a response to the drug combination in 24% of patients and disease stabilization in 57% of patients, presenting an opportunity to use the NAM-vorinostat combination to sensitize DLBCL cancer cells to DMA-damaging agents in future clinical trials (Amengual et al., 2013). In chronic kidney disease, high circulating levels of phosphate and fibroblast growth factor-23 (FGF23) are potentially modifiable risk factors to prevent cardiovascular disease. Since NAM inhibits active intestinal phosphate transport, the efficacy of a combination of NAM and the phosphate binder lanthanum carbonate, was evaluated in patients with chronic kidney disease. Neither drug, alone or together, was able to reduce serum phosphate or FGF23 (Ix et al., 2019). There is some evidence that both oral and intravenously supplied niacin is beneficial for people suffering from migraine and tension-type headaches in a number of small case series studies, potentially because of its vasodilatory and/or its augmenting effect on mitochondrial function (Prousky & Seely, 2005). Niacin could also act positively on migraine headaches because it elevates serotonin plasma levels as a negative feedback inhibitor of the de novo NAD 1 synthesis from tryptophan. Consequently, tryptophan is shunted to serotonin biosynthesis (Velling et al., 2003). Up until now, a limited number of studies have addressed the question of whether supplementing NR (Elhassan et al., 2019; Dollerup et al., 2018; Remie et al., 2020; Dollerup et al., 2019, 2020; Martens et al., 2018; Nascimento et al., 2021) or NMN (Yoshino et al., 2021) could be advantageous for human health by exerting a positive influence on mitochondrial function. In these studies, NR was used at 1000
2000 mg daily with a duration of 3
12 weeks. For NMN, a single dose study of up to 500 mg and a 10-week trial of 250 mg daily were conducted. Results are summarized in Table 17.2. Previous reports of increased circulating LDL-C after intake of NR combined with the polyphenol pterostilbene (Dellinger et al., 2017) and of elevated plasma homocysteine levels after intake of NA and NAM (Sun et al., 2017) warranted an assessment of safety of NR and NMN supplementation.

445

Table 17.2 Clinical trials testing nicotinamide riboside and nicotinamide mononucleotide in human disease. Study type

Participants

Compound/dose

Main outcome

References

Randomized, placebo controlled, crossover Randomized, placebo controlled, parallel group As in Dollerup et al. 2018

30 healthy women and men, aged 55
79 years

NR, 2 3 500 mg/day for 6 weeks

Reduced systolic blood pressure and arterial stiffness

(Martens et al., 2018)

20 healthy, sedentary men with BMI . 30 kg/m2, aged 40
70 years As in Dollerup et al. 2018

NR, 2 3 1000 mg/ day for 12 weeks

No improvement in insulin sensitivity and whole-body glucose metabolism

(Dollerup et al., 2018)

As in Dollerup et al. 2018

(Dollerup et al., 2019)

Randomized, placebo controlled, crossover

12 Healthy men, aged 70
80 years

NR, 1 3 1000 mg/ day for 3 weeks

As in Dollerup et al. 2018 Randomized, placebocontrolled, crossover

As in Dollerup et al. 2018 11 Healthy, sedentary, postmenopausal women and men, aged 45
65 years

as in Dollerup et al. 2018 NR, 1 3 1000 mg/ day for 6 weeks

No effect on glucose tolerance, β-cell secretory capacity, α-cell function, incretin hormone secretion Elevated muscle NAD 1 metabolome, decreased circulating inflammatory cytokines, no effect on skeletal muscle mitochondrial function or whole-body energy metabolism No impact on skeletal muscle mitochondrial function Elevated muscle NAD 1 metabolome, increased body fat-free mass, increased sleeping metabolic rate, no effect on skeletal muscle mitochondrial function or whole-body energy metabolism

(Elhassan et al., 2019)

(Dollerup et al., 2020) (Remie et al., 2020)

Randomized, placebocontrolled, crossover Case report

Randomized, placebocontrolled, parallel group

8 Healthy, sedentary, postmenopausal women and men, aged 45
65 years 1 Female patient with Li-Fraumeni syndrome and severe fatigue 13 Overweight/ obese, postmenopausal women, aged

NR, 1 3 1000 mg/ day for 6 weeks

Not alteration in BAT activity or coldinduced thermogenesis

NR, gradual dosing of 250, 500, 750, 1000 mg twice a day for 12 weeks NMN, 1 3 250 mg/ day for 10 weeks

No improvement of skeletal muscle mitochondrial function

Elevated muscle NAD 1 metabolome, increased muscle insulin sensitivity, no effect on skeletal muscle mitochondrial function or whole-body energy metabolism

(Nascimento et al., 2021)

(Nicotinamide Riboside on Mitochondrial Function in LiFraumeni Syndrome—Full Text View—ClinicalTrials, 2021) (Yoshino et al., 2021)

BAT, Brown adipose tissue; BMI, body mass index; NAD, nicotinamide adenine dinucleotide; NMN, nicotinamide mononucleotide; NR, nicotinamide riboside.

448

CHAPTER 17 Nutraceuticals for targeting NAD 1 to restore

Oral supplementation of NR chloride was found to be safe up to a daily intake of 1000 mg for eight weeks with no adverse effects like flushing or an increase of circulating LDL-C or homocysteine in 140 healthy overweight men and women. Oral NR was bioavailable, since a dose-dependent increase of NAD 1 levels in peripheral blood mononuclear cells was observed after two weeks and was stable for the remainder of the study (Conze et al., 2019). For NMN, a single dose of 100, 250, and 500 mg given orally to ten healthy men, was readily metabolized as levels of NAM degradation products (MNA, N-methyl-2-pyridone-5-carboxamide, N-methyl-4-pyridone-5-carboxamide) dose-dependently increased in plasma. Oral NMN did not cause any adverse effects (Irie et al., 2020). In a 10week study, 13 overweight or obese postmenopausal women received 250 mg NMN daily. NAD 1 levels in PBMCS increased, along with plasma NAM degradation products. No adverse events were reported and no abnormalities in standard blood tests were detected (Yoshino et al., 2021). Both NR and NMN have been tested for their effects on skeletal muscle function, when given to either healthy overweight and mildly obese or elderly subjects of both sexes. NR and NMN supplementation led to higher NAD turnover in skeletal muscle as shown by increased NAM degradation products. However, neither skeletal muscle mitochondrial function, nor whole body energy metabolism was found to be altered (Elhassan et al., 2019; Dollerup et al., 2018; Remie et al., 2020; Dollerup et al., 2019, 2020; Yoshino et al., 2021). Preclinical studies pointed to an effect of NR supplementation on brown adipose tissue function (Canto´ et al., 2012). A 6-week supplementation with 1000 mg of NR given orally to middle-aged healthy volunteers did not result in altered BAT activity or cold-induced thermogenesis, although in vitro incubation of brown adipose tissue derived adipocytes induced norepinephrine-stimulated mitochondrial uncoupling (Nascimento et al., 2021). A case report on a subject with Li-Fraumeni syndrome and a history of fatigue and muscle weakness described the effect of oral NR (dose escalation to 1000 mg twice daily) for 12 weeks. Previously, a severely decreased mitochondrial function in the patient’s leg skeletal muscle was shown in vivo by 31 P nuclear magnetic resonance spectroscopy (MRS). Incubation with NR-supplemented medium was shown to rescue mitochondrial respiration in skin fibroblasts of the patient. NR treatment, however, did not improve in vivo mitochondrial function of this patient as shown by measuring phosphocreatine recovery time via MRS (Nicotinamide Riboside on Mitochondrial Function in Li-Fraumeni Syndrome— Full Text View—ClinicalTrials, 2021).

17.5 Conclusion When evaluating recent clinical trials of NAD precursor supplementation, there is little evidence of beneficial effects for humans, although supplementing NAD

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73.

CHAPTER

Curcumin for protecting mitochondria and downregulating inflammation

18

Ahmad Salimi1,2, Zhaleh Jamali3,4 and Leila Rezaie Shirmard5 1

Department of Pharmacology and Toxicology, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran 2 Traditional Medicine and Hydrotherapy Research Center, Ardabil University of Medical Sciences, Ardabil, Iran 3 Department of Addiction Studies, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran 4 Student Research Committee, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran 5 Department of Pharmaceutics, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran

18.1 Introduction Mitochondria exert a wide variety of cellular functions such as, the production of adenosine triphosphate (ATP), calcium handling, control of cell cycle and cell growth, as well as apoptosis (Modesti et al., 2021). Important cells in the body like neurons, cardiomyocytes, hepatocytes, muscle cells etc., greatly rely on optimal mitochondrial function (Glancy et al., 2020). It is therefore not surprising that mitochondrial dysfunction contributes significantly to cellular injuries and the inflammatory response in cells (Missiroli et al., 2020). Inflammation is a common outcome of many diseases and involves the synthesis and release of proinflammatory mediators, such as chemokines and cytokines (Chen, Deng, et al., 2018). The close association between inflammation and mitochondrial alterations has been reported in several diseases (Missiroli et al., 2020). It has been reported that mitochondrial dysfunction is linked to several inflammatory diseases (Missiroli et al., 2020). Furthermore, these organelles have long been proposed to play a primary role in diseases related to oxidative stress and aging (Dai et al., 2014). Due to main role of mitochondria in ATP generation through the mitochondrial respiratory chain (MRC), they are the primary source of reactive oxygen species (ROS) and are involved in disorders related to oxidative stress such as inflammatory diseases (Zorov et al., 2014). However, these reactive molecules Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00014-X © 2023 Elsevier Inc. All rights reserved.

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target mitochondria and lead to more mitochondrial dysfunctions and toxicity (Zorov et al., 2014). Under normal circumstances in the cells and tissues, about 1%
3% of molecular oxygen is incompletely reduced and this in turn leads to superoxide (O22) ion as a by-product of MRC reactions (Zhao et al., 2019). Under physiological condition, the ROS formation produced by the mitochondrial pathway is inhibited by existing antioxidant systems such as enzymatic and nonenzymatic antioxidants (Snezhkina et al., 2019). ROS play beneficial roles in important signaling pathways required for essential cellular functions in normal condition (Mittler, 2017). However, in a pathological situation, an imbalance between the generation and accumulation of ROS in cells and tissues and the inability of a biological system to detoxify these reactive products leads to a phenomenon called oxidative stress (Pizzino et al., 2017). Briefly, oxidative stress stimulates a series of cellular and metabolic disturbances, including DNA damage, protein oxidation and lipid peroxidation, eventually leading to organelle damage and cell death (Pizzino et al., 2017). In addition, oxidative stress can also promote inflammatory responses by stimulating the generation of transcription factors, cytokines, and related growth factors (Ranneh et al., 2017). Under oxidative stress condition, ROS formation leads to the oxidation of factors involved in inflammation, such as nuclear factor κB (NF-κB), the key regulator of tissue inflammation, and this in turn increases the expression of eicosanoids, chemokines, cytokines, adhesion molecules and inducible nitric oxide synthase (iNOS) (Ranneh et al., 2017). Thus, oxidative stress and inflammation are two interdependent processes (Ranneh et al., 2017). The inflammatory mediators aid in oxidative stress and simultaneously, oxidative stress can also trigger inflammation (Wadley et al., 2013). Therefore, the introduction of compounds that reduce oxidative stress by protecting mitochondria and also have antiinflammatory potential can play an effective role in reducing oxidative damage and inflammation (Forni et al., 2019). Plant-derived natural active compounds are important sources of new drugs and lead compounds (Atanasov et al., 2021). These natural compounds are beneficial in the prevention and treatment of many diseases due to their excellent biological and therapeutic activities (Leuci et al., 2021). Numerous studies have shown that plant-derived active compounds are less harmful with low toxicity (Ekor, 2014). Curcumin or diferuloylmethane (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), is a naturally occurring polyphenol found in the rhizome of Curcuma longa (turmeric) and in other Curcuma spp (Hewlings & Kalman, 2017). For a long time in Asian countries, Curcuma longa has been traditionally used as an herbal medicine due to its antiinflammatory, antioxidant, antimicrobial, antimutagenic and antitumor activities (Parham et al., 2020). Curcumin as a main polyphenol found in this plant has health-promoting impacts and a variety of pharmacological effects such as antiinflammatory, antioxidant, mitochondrial protective effects, anticancer, neuroprotective hepatoprotective, cardioprotective, etc. (Amalraj et al., 2017). These pharmacological activities of curcumin have led to its extensive application in the prevention and treatment of various diseases (Rahmani et al., 2018). Accumulating data suggests that

18.1 Introduction

curcumin is capable of targeting numerous molecular signaling pathways to apply its promising therapeutic effects (Rahmani et al., 2018). For instance, in terms of antiinflammatory activity, curcumin is able to inhibit the molecular pathways involved in ROS formation, oxidative stress, and inflammation (Xu et al., 2018). Curcumin has been indicated to have a significant effect on oxidative stress parameters including plasma activities of catalase and superoxide dismutase (SOD), as well as serum concentrations of lipid peroxides and glutathione (GSH) peroxidase (Sahebkar et al., 2015). Curcumin can scavenge different forms of free radicals, such as ROS and reactive nitrogen species (RNS) (Ghareghomi et al., 2021). This polyphenol compound can regulate the activity of SOD, catalase and GSH enzymes, and neutralize free radicals (Hasanuzzaman et al., 2020). It has been reported that curcumin also, can inhibit ROS-generating enzymes such as xanthine hydrogenase/oxidase and lipoxygenase/cyclooxygenase (Valle´e et al., 2019). As well as, due to its lipophilic properties like vitamin E, curcumin is an efficient scavenger of peroxyl radicals and is considered as a chain-breaking antioxidant (Niki, 2014). In many chronic diseases, oxidative stress plays an important role (Liguori et al., 2018). The pathological process of oxidative stress and ROS formation are closely related to inflammation induced by these chronic diseases (Biswas, 2016). Inflammation and oxidative stress are intertwined processes, which one can be easily induced by another (Biswas, 2016). At the site of inflammation, inflammatory cells release a number of free radicals which can lead to oxidative stress (Hussain et al., 2016). Therefore, the relationship between inflammation and oxidative stress is well demonstrated (Hussain et al., 2016). Moreover, a number of ROS/RNS can initiate a signaling cascade in the cell that elevates the expression of pro-inflammatory genes (Di Meo et al., 2016). Inflammation plays a key role in the development of many chronic diseases (Prasad et al., 2012). These chronic disorders include Parkinson’s disease, epilepsy, Alzheimer’s disease, multiple sclerosis, depression, cardiovascular disease, metabolic syndrome, allergies, cerebral injury, asthma, renal ischemia, psoriasis, acquired immune deficiency syndrome, colitis, bronchitis, arthritis, obesity, diabetes, fatigue, and cancer (Prasad et al., 2012). In most diseases, the major mediator of inflammation is tumor necrosis factor-alpha (TNF-α), which its role in inflammatory conditions is regulated by the activation of a transcription factor such as NF-κB (Liu et al., 2017). Whereas, the most potent NF-κB activator is TNF-α, also NF-κB regulates the expression of TNF-α (Liu et al., 2017). In addition to TNF-α, the transcription factor of NF-κB is also activated by various disease-causing viruses; gramnegative bacteria; physical, chemical, psychological, and mechanical stress; environmental pollutants; fatty acids; high glucose; cigarette smoke; ultraviolet radiation; most inflammatory cytokines and other disease-causing factors (Hewlings & Kalman, 2017). Therefore, the natural compounds such as curcumin that downregulate NF-κB and NF-κB
regulated gene products have potential efficacy against inflammation and oxidative stress-related diseases (Hewlings & Kalman, 2017). Curcumin has been shown to suppress oxidative stress through many different

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mechanisms and block NF-κB activation increased by several different inflammatory stimuli (Sharifi-Rad, Rayess, et al., 2020). In this chapter, we discuss the relationship between oxidative stress and inflammation, inflammation and mitochondria, and finally the importance of curcumin as a natural product existing in turmeric for protecting mitochondria and downregulating inflammation.

18.2 Inflammation and oxidative stress The mediators of inflammation help ROS formation and oxidative stress which can also trigger inflammation (Mittal et al., 2014). In order to manage pathogenic factors, the cells such as neutrophils and macrophages generate an excessive amount of ROS and RNS such as superoxide, hydroxyl free radical, hypochlorous acid, hydrogen peroxide, nitric oxide, and peroxynitrite (Herb & Schramm, 2021). Subsequently, during an inflammatory condition, the release of free radicals from inflammatory cells can trigger ROS and RNS formation and oxidative stress (Vona et al., 2021). It has been reported that an excessive ROS formation during the process of oxidative metabolism contributes to secretion and synthesis of inflammatory mediators by activating nuclear factor kappa B/active protein-1 (NF-κB/AP-1), as well as the generation of TNF-α (Morgan & Liu, 2011). Oxidative stress can activate transcription factors such as β-catenin/Wnt, active protein-1 (AP-1), NF-κB, p53, peroxisome proliferator-activated receptor gamma (PPAR-γ), nuclear factor erythroid 2-related factor 2 (Nrf2) and hypoxiainducible factor 1-alpha (HIF-1α) (Valle´e & Lecarpentier, 2018). The activation of these mentioned transcription factors by oxidative stress results in the expression of different genes associated with cell cycle regulatory molecules, growth factors, chemokines, inflammatory cytokines, and antiinflammatory molecules (Ahmed et al., 2017). It has been reported that oxidative stress plays a key role in the activation of NOD-like receptor protein 3 (NLRP3) inflammasome (Abais et al., 2014). The NLRP3 inflammasome as a multimeric molecular complex can initiate innate immune responses by releasing cytokines such as interleukin (IL)1β and IL-18 (Kelley et al., 2019). In addition, previous studies have been reported that the mitochondrial ROS (mtROS) plays the regulatory role in inflammatory signaling (Forrester et al., 2018). The mitochondrial dysfunction is associated with ROS formation, which results in the activation of the NLRP3 inflammasome complex and the secretion of pro-inflammatory cytokines, and finally this process leads to localized inflammation (Biasizzo & Kopitar-Jerala, 2020). Therefore, the levels and presence of antioxidant factors, both external and internal antioxidants, and mitochondrial protective agents can play an effective role in the regulation of inflammatory responses (Sharifi-Rad, Anil Kumar, et al., 2020). Natural antioxidants such as curcumin are known to have biological activates and antiinflammatory responses against inflammatory conditions induced by chemicals, drugs, and chronic diseases such cancer and neurodegenerative

18.3 Mitochondria and inflammation

disorders (He et al., 2015). Recently, it has been reported that GSH as an intracellular tripeptide is a potent antioxidant that protects the cells and tissues from oxidative stress and inflammation (Silvagno et al., 2020). Decreased GSH levels are associated with ROS production and oxidative stress, which in turn results in an imbalanced immune response, inflammation, as well as higher susceptibility to infection and other inflammatory diseases (Liguori et al., 2018). In addition, it has been suggested that DNA-based modifications caused by free radicals can trigger inflammatory responses by the activation of the NF-kB pathway (Kumar Rajendran et al., 2019).

18.3 Mitochondria and inflammation It has been reported that mitochondrial dysfunction is associated with inflammation. Mitochondrial dysfunction can lead to a noninfectious inflammatory response (Lo´pez-Armada et al., 2013). These responses are initiated and perpetuated by damage-associated molecular patterns (DAMPs), which are also called danger signals, danger-associated molecular patterns, and alarmin (Roh & Sohn, 2018). These host biomolecules originated from different sources, such as damaged or dying cells, the plasma membrane, endoplasmic reticulum (ER), extracellular matrix, granules, and damaged mitochondria (Roh & Sohn, 2018). Therefore, mitochondrial damage can lead to cytosolic exposure of several DAMPs, such as cardiolipin and mitochondrial DNA (mtDNA) (Rodrı´guezNuevo & Zorzano, 2019). These DAMPs are released from mitochondria and can activate the innate immune system by interacting with pattern recognition receptors (Rodrı´guez-Nuevo & Zorzano, 2019). In the cytosol, mitochondrial DAMPs are recognized by receptors or adapter molecules leading to an inflammatory response (Picca et al., 2021). In the cytosol, mtDNA is recognized by cyclic tolllike receptor 9 (TLR9), GMP-AMP (cGAMP) synthetase (cGAS), and the NLRP3 inflammasome, which the latter can also be activated by mtROS (Moya et al., 2021). Moreover, mtROS induced by disruption of MRC functions can promote oxidative stress and inflammation (Patergnani et al., 2021). The mitochondria-mediated apoptosis is a type cell death which is characterized by mitochondrial outer membrane permeabilization (MOMP) and subsequent release of soluble mitochondrial intermembrane space proteins such as Omi or HtrA2 (high temperature requirement factor A2), SMAC (second mitochondria-derived activator of caspase), or DIABLO (direct inhibitor of apoptosis-binding protein with low pI) and cytochrome c into the cytoplasm and activation of caspases (Saelens et al., 2004). Upon MOMP, cytosolic exposure of the inner mitochondrial membrane (IMM) and release of intermembrane space proteins occurs, which can lead to mtDAMPs exposure during mitochondria-mediated apoptosis (Vringer & Tait, 2019). Previous studies have demonstrated that the activation of apoptotic caspases has an immunosilencing

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effect during apoptosis (Van Opdenbosch & Lamkanfi, 2019; Vringer & Tait, 2019). Usually, various immunogenic pathways are activated when intermembrane space proteins are present in the cytosol (Vringer & Tait, 2019). Cyclic GMP
AMP synthase (cGAS) is one of cytosolic DNA sensors, which upon DNA binding, produces Cyclic guanosine monophosphate
adenosine monophosphate (cGAMP or cyclic GMP-AMP), from GTP and ATP. Cyclic GMP-AMP binds to the ER membrane adapter stimulator of interferon genes (STING) and acts as a secondary messenger (Collins et al., 2015; Zhong & Shu, 2021). Upon binding, the adapter protein STING is activated through a change in its conformation (Zhong & Shu, 2021). Active STING translocates from the ER to an ER-Golgi intermediate machine and the Golgi compartment (Taguchi et al., 2021). During this process, the carboxyl terminal (C-terminal tail) of the adapter protein STING recruits and activates TANK-binding kinase 1 (TBK1), which in turn phosphorylates the transcription factor, interferon regulatory factor 3 (IRF3) (Tanaka & Chen, 2012). After dimerization, the phosphorylated IRF3 translocates to the nucleus and there it initiates type I interferon responses (Ivashkiv & Donlin, 2014). The type I interferon responses act in a pleiotropic manner and activate both adaptive and innate immunity (Lei et al., 2020). Previous studies have been demonstrated that during mitochondria-mediated apoptosis under caspase-inhibited conditions, a type I interferon response is activated (Rongvaux et al., 2014; Vringer & Tait, 2019). Mouse embryonic fibroblasts and engineered mouse models with deleted caspases-3 and -7, or -9 demonstrated significant upregulation of interferon-stimulated gene response and type I interferon expression following MOMP (White et al., 2014). It has been reported that the deletion of caspase 9 in hematopoietic stem cells, enhanced basal levels of type I interferon and cellular death (Vringer & Tait, 2019). The above studies have been demonstrated that the increase of type I interferons is due to STING activation after recognition of mtDNA by cGAS (Vringer & Tait, 2019; Willemsen et al., 2021). Following MOMP, due to expanding of Bax/Bak-dependent outer membrane pores, the IMM is extruded and in turn it can release mtDNA (Riley et al., 2018). Therefore, mitochondrial toxins with mtDNA release can activate the STING pathway and lead to inflammatory responses (De Gaetano et al., 2021). In addition, other immune sensing pathways can be activated by mtDNA (De Gaetano et al., 2021). Many antigen presenting cells have toll-like receptor 9 (TLR9), which is able to recognize mtDNA by virtue of its high content of CpG (regions of DNA sequence contain a cytosine nucleotide followed by a guanine nucleotide) rich domains (Nastasi et al., 2020). Recent studies have been reported showing that the recognition of mtDNA by TLR9 can result in NF-κB translocation and a type I interferon response (Luna-Sa´nchez et al., 2021). During cell death, the released mtDNA can also activate the NLRP3 inflammasome and provoke an immune response (Shimada et al., 2012). The NLRP3 inflammasome activation leads to processing of IL-1β and IL-18, thereby activating T-cells, macrophages, monocytes, and neutrophils (Hanamsagar et al., 2011).

18.4 Mitochondria and oxidative stress

In addition, recent studies have shown that the activation of NF-κB and the expression of TNF during caspase-independent cell death (CICD) is entirely dependent on Bak and Bax proteins, indicating that MOMP is essential for initiating the inflammatory responses (Picca et al., 2021; Vringer & Tait, 2019). NF-κB plays a key role in inflammatory responses, and can be activated in some canonical and noncanonical pathways (Liu et al., 2017). It has been proven that NF-κB transcribes various inflammatory genes and can regulate the differentiation, activation, and function of inflammatory T-cells (Blanchett et al., 2021). It has also been reported that MOMP is needed to initiate nuclear translocation of NF-κB and the subsequent activation of transcription of pro-inflammatory genes by this transcription factor in caspase-inhibited conditions (Checa & Aran, 2020; RedzaDutordoir & Averill-Bates, 2016). One of the genes that is transcribed by NF-κB is TNF (Hayden & Ghosh, 2014). TNF as a pro-inflammatory cytokine can trigger necroptosis in cells (Kearney et al., 2015). It has been observed that CICD has necroptotic features and the inhibition of TNF signaling decreases cell death in this condition (CICD), indicating that TNF is needed to engage necroptosis in CICD (Vringer & Tait, 2019). However, TNF is a well-known activator of NFκB, but it has been demonstrated that TNF and the necroptotic pathway is not responsible for the activation of NF-κB during CICD (Galluzzi et al., 2017). Studies have shown that TNF expression and NF-κB activation during CICD is dependent on Bax and Bak, which indicates the role of MOMP in initiating the inflammatory responses (Giampazolias et al., 2017; Osellame et al., 2012; Tait & Green, 2013).

18.4 Mitochondria and oxidative stress In both physiological and pathological conditions, the mitochondria are widely recognized as a ROS production machinery within most mammalian cells (Zorov et al., 2014). About 1%
2% of total oxygen consumption is going to ROS formation in the cells. mtROS are generated during the process of oxidative phosphorylation (OXPHOS), as a by-product of bioenergetic metabolism (Murphy, 2009). The mtROS formation are formed by one-electron transfers to oxygen molecules, generating anion superoxide (O2•2) that can be converted to hydrogen peroxide (H2O2) by SOD enzymes (Turrens, 2003). The ROS formation in mitochondria can be produced in multiple sites including: the mitochondrial matrix by the core metabolic machinery present in the IMM and in the intermembrane space (Marchi et al., 2012). During the process of OXPHOS, the ROS production mainly generates at the electron transport chain (etc) which is located on the IMM (Zhao et al., 2019). Also, it has been suggested that other, less well described sites may take part in ROS generation including, proline dehydrogenase, dihydroorotate dehydrogenase, and the electron transferring flavoprotein/ETF:Q oxidoreductase (ETF/ETF:QOR) system of fatty acid β-oxidation (Quinlan et al., 2013). These

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different mtROS sites have distinguished signaling roles and the primary generation sites change under various physiological conditions. Indeed, the generation of ROS from the respiratory chain of mitochondria is changed by several factors, including oxygen levels, metabolic state of mitochondria, and mitochondrial membrane potential (Quinlan et al., 2013; Zorov et al., 2014). Other potential sources of ROS generation in the mitochondria are acyl-CoA dehydrogenases (ACAD), which are involved in lipid catabolism (Quinlan et al., 2013; Zorov et al., 2014). Also, adrenodoxin reductase (ADxR)-adrenodoxin (ADX)-cytochrome P450scc (CYP450) system as an enzyme in mitochondria is involved in superoxide formation and is coupled with nicotinamide adenine dinucleotide phosphate (NADPH) in the mitochondrial matrix (Patergnani et al., 2021). The imported onco-suppressor fragile histidine triad protein into mitochondria interacts with ferredoxin reductase (FDxR) and increases the intracellular superoxide generation (Druck et al., 2019). P66shc as an adapter protein may induce ROS generation and regulates the oxidative stress at different levels (Galimov, 2010). This adapter protein may induce mtROS formation by: (Modesti et al., 2021) downregulating the expression of antioxidant enzymes, such as manganese SOD (Mn-SOD) and GSH peroxidase-1 (Glancy et al., 2020), activating the enzyme of Rac-1-dependent plasma membrane NADPH oxidase (Missiroli et al., 2020), and translocating p66shc into mitochondria, after protein kinase C (PKC)-dependent phosphorylation, to transfer electrons from reduced cytochrome C to O2 (Galimov, 2010). Two forms of monoamine oxidase (MAO) isoenzymes are located in the outer membrane mitochondria (OMM) in various mammalian tissues and catalyze the oxidation of biogenic amines with the release of H2O2 (Kinemuchi et al., 1983; Sturza et al., 2019). Also, mitochondrial aconitase, an enzyme located in the matrix of the mitochondria, has an iron-sulfur cluster that can be oxidized by superoxide anion or hydrogen peroxide generating a hydroxyl radical (•OH) (Va´squez-Vivar et al., 2000). Uncoupling proteins (UCPs) as a family of mitochondrial transporters are located in the IMM and regulate the proton transport across the mitochondrial membrane (Ardalan et al., 2022). In the IMM, these proteins determine an inducible proton leak through a mild-uncoupling phenomenon (Ardalan et al., 2022; Brown et al., 2010). During the mild uncoupling process, the reduction events in the etc and the mitochondrial membrane potential (ΔΨ) were reduced, thereby resulting in a decrease in ROS generation through the etc (Patergnani et al., 2021). There are three types of UCP in the mitochondria including: UCP-1, UCP-2, and UCP-3 (Monteiro et al., 2021). Downregulation and deletion of UCP-1 is characterized by a reduced O2 consumption rate, low levels of ΔΨ, ROS formation and oxidative stress (Monteiro et al., 2021). Also, UCP-2 and UCP-3 protect the proteins of the mitochondria from endogenous ROS, and UCP-2 and UCP-3 knockout animal models show increased ROS formation (Monteiro et al., 2021). In addition, the activation of UCPs is determined by 4hydroxy-2-nonenal (4-HNE), ROS, and lipid peroxidation (Hirschenson et al., 2022). The UCPs show a feedback mechanism, where enhanced ROS formation

18.4 Mitochondria and oxidative stress

activates a reparative mechanism (uncoupling events) to decrease the ROS generation and oxidative damage (Hirschenson et al., 2022). Hexokinase (HK) 2 is another protein that maintains an efficient electron transport and regulates the ROS generation in mitochondria (Roberts & Miyamoto, 2015). HK2 is recruited to the OMM where this protein binds itself to the ADP/ATP exchange complex formed by a voltage-dependent anion channel and adenine nucleotide translocator (Roberts & Miyamoto, 2015). HK2 provides continuous ADP in order to produce the ADP/ATP recycling mechanism required to maintain the optimal respiration rate, subsequently preventing the leak of electrons of the etc, producing ROS (Roberts & Miyamoto, 2015). In line with this evidence, downregulation of HK2 expression increases ROS production and activates the mitochondrial permeability transition (MPT), while conversely, the overexpression of HK2 decreases the mtROS production in knockout animal models (Roberts & Miyamoto, 2015). Sirtuins (SIRTs) are nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases and are involved in metabolic regulation, mitochondrial functioning, protein acetylation, protein deacetylation, and cell survival (Kratz et al., 2021). All seven known SIRTs isoforms have been associated with antioxidant and redox signaling (Kratz et al., 2021). In particular, the SIRT1 has a protective role against ROS formation (Santos et al., 2016). For example, SRT1720, as a small molecule activator of SIRT1, determines the activation of SOD2 levels, accompanied by a reduction in protein carbonylation levels and in the formation of 4-HNE (Santos et al., 2016). SIRT1 is required for DNA repairing mechanisms following H2O2-induced damage and it can be directly activated by oxidative stress (Santos et al., 2016). The SIRT3 is another important SIRTs modulating ROS formation (Singh et al., 2018). SIRT3 downregulation is associated with increased ROS generation and activation of the HIF1α (Singh et al., 2018). The levels of mtROS can be reduced by the specific action of mitochondrial antioxidant systems like SODs (Wang et al., 2018). This antioxidant system converts O2•2 to H2O2 (Chen, Deng, et al., 2018). Two isoforms regulate the conversion of O2•2 into ordinary molecular oxygen and hydrogen peroxide in mitochondria including; SOD1/copper-zinc-dependent SOD (Cu, Zn-SOD) and SOD2/manganese-dependent SOD (Mn-SOD). SOD1 is widely distributed in the intermembrane space of mitochondria and parts of cells such as cytoplasm and the nucleus, whereas SOD2 is available only in the mitochondrial matrix (Wang et al., 2018). The quick conversion into hydrogen peroxide is neutralized by glutathione peroxidase (GPX) and catalase, which converts H2O2 into oxygen and H2O (Sarıkaya & Do˘gan, 2020). Catalase as an antioxidant enzyme mainly presents in the cytosol, indicating that the scavenge capability in mitochondria is left to GPX (Weydert & Cullen, 2010). The scavenge capability of GPX is dependent on the use of GSH as cofactor and electron source to negate H2O2 (Espinosa-Diez et al., 2015). To date, eight isoforms of the enzyme GSH peroxidase have been identified, where GPX1
4 and GPX6 are selenoproteins with a selenocysteine as a catalytic moiety and among them only GPX1 and GPX4 are expressed in the mitochondria (Koeberle et al., 2020).

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18.5 Mitochondrial inflammation and oxidative stress in inflammatory-related diseases Due to the supplementary role of mitochondria in inflammatory responses, today these organelles have become an important target to modulate the inflammation in related diseases (Andrieux et al., 2021). The relationship between mitochondria and inflammation has led to the definition of a new concept called mito-inflammation (Andrieux et al., 2021). Mito-inflammation is a mitochondria-related compartmentalized inflammatory response, where the mitochondria act both in front of mtDAMPs and downstream in intracellular signaling pathways triggered by exogenous pathogen-associated molecular patterns (PAMPs) (Patergnani et al., 2021). During mito-inflammation, mtROS, mitochondrial Ca21, mtDNA, cardiolipin and ATP are released in the cytosol or in the extracellular environment to enhance numerous pro-inflammatory mediators (Grazioli & Pugin, 2018). Among mtDAMPs, mtROS directly induces oxidative stress and DNA damage in mitochondria, which can lead to other mtDAMPs such as DNA damage and oxidation in other mitochondrial molecules (Nakahira et al., 2015). Also, mtROS freely move through the OMM into the cytosol and it may trigger the activation of redoxsensitive transcription factors involved in the activation of pro-inflammatory signaling pathways (Brillo et al., 2021). The redox-sensitive transcription factors such as NF-kB, AP-1 and HIF contribute to the generation of pro-inflammatory cytokines, including IL-1 and IL-8 (Brillo et al., 2021). Oxidation of mtDNA and mitochondrial proteins induced by ROS formed in the mitochondria can change the OXPHOS activity with the effect on structure and ΔΨ, in turn, generating extra mtROS (Jeˇzek et al., 2018). Oxidized mtDNA as an mtDAMPs, is released into the cytosol, mediating oxidative- and Ca21-dependent mitochondrial PTP opening, OMM permeabilization, and impaired mitophagy (Chen, Zhou, et al., 2018). The oxidized mtDNA and mtROS may also be recognized by pattern-recognition receptors (PRRs), which are localized at the cytosol and plasma membrane in tissue and immune resident cells (Faas & De Vos, 2020). The PRR receptors are categorized into four main subfamilies on the basis of their specific ligand, function, and location: C-type lectin receptors (CLRs), membrane bound Toll-like receptors (TLRs), RIG (retinoic acidinducible gene)-l-like receptors (RLRs), and the cytosolic NOD (nucleotide-binding oligomerization domain)-like receptors (NLRs) (Li & Wu, 2021). In immune cells, the binding of TLR-1, TLR-2, and TLR-4 results in the translocation of tumor necrosis factor receptor-associated factor 6 (TRAF-6) to mitochondria and induces mtROSmediated antibacterial activity (Shekhova, 2020). It engages the evolutionarily conserved signaling intermediate in Toll pathways (ECSIT), which assembles the membrane arm of mitochondrial respiratory complex I, increasing the transposition of mitochondria to the phagosomal membrane and the ECSIT-dependent oxidation (Kopp et al., 1999). Melanoma differentiation associated gene 5 (MDA5) and RIG-l-like (RIG-I) receptors as RLRs, are distributed in the cytosol and can

18.5 Mitochondrial inflammation and oxidative stress

respectively sense long and short viral dsRNA (Lazarte et al., 2019). The complex of these proteins and viral RNA interacts with the mitochondrial antiviralsignaling protein (MAVS) at the mitochondrial outer membrane (Refolo et al., 2020). This interaction results in the activation and promotion of MAVS oligomerization, which in turn leads to the activation of transcription factors NF-kB, IRF3 and IRF7 (Refolo et al., 2020). The activation of the mentioned transcription factors induces the synthesis of antiviral molecules and interferon type-I (Refolo et al., 2020). The MAVS-mediated interferon promoter and antiviral activity are regulated through the interaction of MAVS and NLR family member X1 (NLRX1), a regulator of mitochondrial antiviral immunity (Moore et al., 2008). Previous studies have enforced the strong connection between mtROS and MAVS (Moore et al., 2008; Sun et al., 2016) (Fig. 18.1). These studies indicated that there is a link between increased mtROS production and an amplification of RLR-signaling and a mtROS-dependent modulation of the biophysical properties of the mitochondrial outer membrane necessary for the oligomerization of MAVS (Meyer et al., 2018; Sun et al., 2016). The NLR proteins, as a class of cytosolic PRRs are activated by mtDAMPs and/or PAMPs and form a multiprotein complex termed “inflammasome,” after activation, which leads to the secretion of inflammatory cytokine IL-1, IL-18, and caspase-1 maturation (Yu et al., 2022). The known role of mitochondria in NLR function and activation has been acknowledged for NLR family CARD domain containing 4 (NLRC4/IPAF), NLR family pyrin domain containing 3 (NLRP3) and NLRX1 (Zhong et al., 2013). The well-characterized inflammasome NLRP3 is activated in two steps. The first step (priming), is in response to proinflammatory stimuli and the expression and posttranslational modifications of different inflammasome components such as pro-IL-1, pro-IL-18, NLRP3 and NLRC4, are induced and result in NF-kB activation. The second step (activation) is regulated by a broad range of signals and needs a physical interaction with the mitochondria for the formation of NLRP3 inflammasome and consequent autocleavage of pro-caspase-1, responsible for the generation of mature inflammatory cytokines IL-1 and IL-18 (Kelley et al., 2019). Mitochondrial localization, due to mitochondrial-anchored protein ligase (MAPL) and MAVS binding, has a central role in the modulation of the inflammasome responses because of its role scaffolding and activating NLRP3 through the release of mtROS and mtDAMPS such as mtDNA (Subramanian et al., 2013). In addition, it has been reported that both steps of the priming and the activation of NLRP3 inflammasome are linked to the new synthesis of mtDNA, which highlights the role of mitochondria in inflammasome activation in two phases (Sutterwala et al., 2014). Moreover, previous studies have suggested that the NLRC4/IPAF inflammasome may be activated by oxidized mtDNA (Zhao et al., 2014). The contribution of mtROS in inflammatory responses is strictly influenced by the status of mitochondria (Chen, Zhou, et al., 2018). mtROS formation increases mainly during the malfunction of mitochondria, which is associated with a broad range of abnormalities from excessive Ca21, OXPHOS impairment, and

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FIGURE 18.1 The role of mitochondrial ROS in inflammation. ROS formation in mitochondria causes oxidative damage to mitochondrial membrane, changes in membrane permeability, oxidation of mitochondrial molecules such lipid, proteins, and DNA, which contribute to mitochondrial impairment and exacerbation of mitochondrial ROS production. The mitochondrial ROS activates the redox-sensitive transcription factor NF-kB and this transcription factor in turn expresses inflammasome genes, such as Il1b, Nlrp3, and Nlrc4 genes. Also, the mitochondrial ROS and mitochondrial DNA promote the cytokines release mediating the inflammasome NLRP3 and NLRC4 complex activation, through the processing of pro-IL-1 and pro-IL-18 and the recruitment of pro-caspase-1. Ca21, Calcium; IL-1, Interleukin-1; IL-18, Interleukin-18; MAVS, Mitochondrial antiviral-signaling protein; mtDNA, mitochondrial deoxyribonucleic acid; NLRC4, NLR Family CARD Domain Containing 4; NLRP3, NLR Family Pyrin Domain Containing 3; NLRX1, NLR Family member X1 precursor; NF-kB, Nuclear factor kappa-light-chain-enhancer of activated B cells; ROS, Reactive oxygen species; ΔΨ, Mitochondrial membrane potential.

accumulation of unfolded proteins (Guo et al., 2013). Depending on the ROS level, the ROS can have different effects on the fate of the cell, including its role of signaling molecules at low concentrations, inflammatory response activator at moderate concentrations, and induction of oxidative stress when mitochondria ROS concentration becomes too high (Patergnani et al., 2021). Due to the importance of the ROS in the cell and also in order to avoid cellular oxidative injuries,

18.5 Mitochondrial inflammation and oxidative stress

the formation of mtROS is fine-tuned by both mitochondrial stress responses that trigger the restoration of mitochondrial homeostasis and mitochondrial antioxidant systems that reduce the free radical generation (Go¨rlach et al., 2015). Mitochondrial unfolded protein response (UPRmt), functional fusion complementation, and mitophagy intervene to preserve and recover the mitochondrial homeostasis to control innate immune response, metabolism, and cell viability (Svaguˇsa et al., 2020). Mitophagy neutralizes extra mtROS formation, oxidized mtDNA, and other mtDAMPs related to inflammation, and the consequent elimination of dysfunctional mitochondria via lysosomal degradation (Schofield & Schafer, 2021). Under stressful conditions, mitochondrial fusion compensation optimizes the functional efficiency of mitochondria and allows the exchange of materials among partially damaged mitochondria (Youle & Van Der Bliek, 2012). E3 ubiquitin ligase Parkin and mitochondrial-targeted kinase PINK1 are the main players in the mitophagic responses (Jacoupy et al., 2019). In depolarized mitochondria, parkin is recruited to the mitochondrial outer membrane by altered mitochondrial import of PINK1 (Jacoupy et al., 2019). Parkin catalyzes the ubiquitination of the mitochondrial outer membrane proteins to sequester the mitochondria in the autophagosome, while PINK1 contributes to strengthen the mitophagy, phosphorylating both ubiquitin and parkin, and recruiting the mitophagic receptors NDP-52 and optineurin to mitochondria (McLelland et al., 2018). Eventually, dysfunctional mitochondria activate the UPRmt, as a transcriptional response, where upon mitochondrial stress, activated transcription factor 5 (ATF5) fails to be imported into the mitochondria and moves to the nucleus to induce the transcription of innate immune genes, ROS detoxification protein, mitochondrial chaperones, and proteases (Melber & Haynes, 2018). In addition, the mitochondrial unfolded protein response induces the secretion and synthesis of mitochondrial-derived mediators and nuclear-encoding, named mitochondrial cytokines or mitokines, capable of transfiguring cell metabolism and viability (Yi et al., 2018). These mitokines may be considered main players in the modulation of systemic responses, such as inflammation, metabolism, and the progression of a disease (Yi et al., 2018); as well as, the extra mitochondrial Ca21 levels are crucial for inflammation and oxidative stress (Madreiter-Sokolowski et al., 2020). The disturbance of mitochondrial Ca21 signaling enhances the mtROS formation and results in the mitochondrial stress responses, inflammasome activation, and release of pro-inflammatory mediators (Madreiter-Sokolowski et al., 2020). The mtROS formation correlates with the rate of mitochondrial metabolism, which in turn, characterizes the importance of mitochondrial Ca21 signaling on mtROS levels (Zorov et al., 2014). Mitochondrial Ca21 signaling may increase mtROS formation. There are three possible pathways for the effect of mitochondrial Ca21 on ROS formation in the mitochondria (Go¨rlach et al., 2015). First, Ca21 ions can directly stimulate mitochondrial resident ROS-generating enzymes, such as glycerol-3-phosphate dehydrogenase and alpha-ketoglutarate (Go¨rlach et al., 2015). Second, Ca21 ions can also activate nitric oxide synthase and enhance nitric oxide, which in turn blocks

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FIGURE 18.2 (above) The effect of curcumin in physiological conditions and normal cells. Curcumin is able to induce cytoprotective enzymes, reactive oxygen spices (ROS) decrease, mitochondrial protection and anti-apoptotic effects. (bottom) The effect of curcumin in cancerous cells. ARE, Antioxidant response elements; ADP, adenosine diphosphate; ATP, adenosine triphosphate; CAT, catalase; CUR, curcumin; Cyt c, cytochrome c; DCm, mitochondrial membrane potential; GPx, glutathione peroxidase; GR, glutathione reductase; GST, NQO1, nicotinamide adenine dinucleotide phosphate oxidase: (Continued)

18.6 Curcumin as antioxidant and antiinflammatory agent

the mitochondrial complex IV and increases indirectly mtROS. Third, reverse electron transport induced by Ca21-dependent mitochondrial membrane depolarization increase mtROS formation (Go¨rlach et al., 2015). In turn, mtROS contribute to perturb the Ca21 signaling affect on molecules involved in Ca21 signaling pathways, Ca21-effectors and the activity of receptors, compartmentalized Ca21 signals and reshaping the intracellular space (Feissner et al., 2009). Therefore, mitochondrial dysfunction, mito-inflammation, the excessive accumulation of mtROS and the loss of mitochondrial homeostasis have been demonstrated to be linked to the development of various pathologies, such neurodegeneration, diabetes, cardiovascular diseases, and cancer (Marchi et al., 2012) (Fig. 18.2). Therefore, mitochondrial protection, reduction of oxidative stress and inflammation with mitochondrial origin can play an important role in reducing mitochondrial inflammation and oxidative stress in inflammatory-related diseases (Kowalczyk et al., 2021). Mitochondrial-friendly compounds, antiinflammatory and antioxidant agents such as curcumin can probably be very effective in the reduction of mitochondrial inflammation and oxidative stress in inflammatoryrelated diseases (Trujillo et al., 2014).

18.6 Curcumin as antioxidant and antiinflammatory agent

L

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3,5-dione), a naturally fat-soluble polyphenolic compound has antitumor, antioxidant, antiinflammatory, and other pharmacological activities (Amalraj et al., 2017). Curcumin is found in India, China, Southeast Asia and Latin America, and has a long history as a food addition. Curcumin is mainly derived from the rhizome of C. longa L. (Turmeric) of the Zingiberaceae family and the root tuber of Curcuma aromatica Salisb (Rajkumari & Sanatombi, 2017). These traditional medicines are used in removing blood stasis and promoting blood circulation, and have long been used to treat gastrointestinal inflammation, wounds, sinusitis, cough, indigestion, pain,

glutathione-S -transferase; H2O, water; HO-1, heme oxygenase 1; Mn-SOD, mitochondrial superoxide dismutase; NAD, nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; Nrf2, nuclear factor erythroid 2-related factor 2; ROS, reactive oxygen species; SOD, superoxide dismutase; quinone oxidoreductase 1; Srxn1, sulfiredoxin 1; O2, oxygen; Fe12, Fe13, iron; Ca12, calcium; BclX2, Bcl2-like 1; Bcl2, Bcell lymphoma 2; tBid, truncated BH3 interacting-domain death agonist; Bid, BH3 interacting-domain death agonist; NF-kB, nuclear factor kappa B; Bax, Bcl2-associated X protein; Apaf-1, apoptotic protease activating factor 1; casp, caspase; AIF, apoptosisinducing factor; IAP, inhibitor of apoptosis; Diablo, direct IAP-binding protein; IκBα, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha; H2O2, hydrogen peroxide; O2•2, superoxide anion; NO•, nitric oxide; OH•, hydroxyl radicals; 1O2, singlet oxygen; ROO•, peroxyl radicals; ONOO2, peroxynitrite.

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and other diseases (Rajkumari & Sanatombi, 2017). In western herbalism, the rhizome of C. longa L. of Zingiberaceae is primarily used as an antiinflammatory agent, and in India this common spice has been described in Ayurveda, as a treatment for inflammatory diseases (Rajkumari & Sanatombi, 2017). In addition to these, curcumin, the principal curcuminoid of turmeric is used for various medical purposes, for instance, as an antiinflammatory, antibacterial, antiangiogenesis, antiproliferation, antiinvasion, antioxidation, pro-apoptotic and antifungal (Sharifi-Rad, Rayess, et al., 2020). The antiinflammatory and antioxidant properties of curcumin and curcuminoid are considered to be the basis of their various pharmacological activities and play an important role in the treatment of diseases (Boroumand et al., 2018). Moreover, curcumin supplements are extremely popular today, and there are many antiinflammatory and antioxidant curcumin dietary supplements on the market (Yu et al., 2018). As an antioxidant, curcumin is capable of detoxifying oxygen and nitrogen free radicals (ROS and RNS) by enhancing the expression of antioxidant proteins through induction of upstream coding genes such as the antioxidant response element (ARE), kelch-like ECH-associated protein 1 (Keap1), and nuclear factor erythroid 2-related factor 2 (Nrf2) (Guo et al., 2021) (Fig. 18.3). Nrf2 as a transcription factor is involved in cellular stress response (He et al., 2020; Lee & Hu, 2020). Under physiological status, inside the cytoplasm, the cystine-rich zinc Keap1 metalloprotein binds Nrf2, elevating ubiquitination and the resultant proteasomal degradation and prevention of Nrf2 nuclear translocation (He et al., 2020; Lee & Hu, 2020). While under stress conditions, the phosphorylation inhibits Keap1 activity and consequently, free Nrf2 translocates from the cytosol to the nucleus, where this transcription factor binds to the antioxidant response element in the regulatory regions of cytoprotective proteins and enhances transcription of antioxidant genes such as GSH, GSH peroxidase, and SOD (He et al., 2020; Lee & Hu, 2020). As well as, Nrf2 translocation to the nucleus increases the expression of phase II detoxifying enzymes such as NAD(P)H dehydrogenase [quinone] 1 (NQO1), heme oxygenase-1 (HO-1), NADPH and GSH transferases (He et al., 2020; Lee & Hu, 2020). It has been reported that curcumin can activate Nrf2/Keap1/ARE signaling pathways through Michael reaction with thiol residues in Keap1 protein, resulting in the activation and release of Nrf2 and expressing antioxidant genes (Zhao, Qi, et al., 2021). The free radical scavenging activity of curcumin has been reported in previous studies (Ak & Gu¨lc¸in, 2008; Asouri et al., 2013). The chemical structure of curcumin scavenges various forms of free radicals such as superoxide anion, hydrogen peroxide, hydroxyl radical, nitric oxide, singlet oxygen, peroxyl and peroxynitrite radical (Ak & Gu¨lc¸in, 2008; Asouri et al., 2013). Four functional groups in the chemical structure of curcumin contribute to its free radical scavenging activity. These functional groups are: carbon
carbon double bonds, the b-diketone group, keto and enol form in aqueous solutions, and phenyl rings containing varying amounts of hydroxyl and methoxy substituents (Ak & Gu¨lc¸in, 2008; Asouri et al., 2013). Some studies have suggested that the free radical scavenging activity of

18.6 Curcumin as antioxidant and antiinflammatory agent

curcumin is mainly derived from its phenolic structure (Alisi et al., 2020; Malik & Mukherjee, 2014). Various authors concluded that presumably, the excellent antioxidant effects of curcumin are largely due to its hydrogen-atom donation from the phenolic group (Alisi et al., 2020; Malik & Mukherjee, 2014). It has been reported that curcumin is a hydrogen-atom donor by giving a hydrogenatom from the phenolic group, which is the methoxy or the central methylene group (Alisi et al., 2020). The methylene group of the b-diketone presence in the chemical structure of curcumin, is a potent anion superoxide scavenger (Priyadarsini, 2014). The only presence of the b-diketone was not sufficient for the radical-scavenging activity of curcumin (Priyadarsini, 2014). Various authors have shown that the phenolic hydroxyl group and the central hydrogen atom of the methyl is involved in the production of phenoxyl (Priyadarsini, 2014). The presence and number of phenolic hydroxyl groups in the chemical structure of

FIGURE 18.3 The antioxidant properties of curcumin and the mitochondrial protection are illustrated. H2O2, hydrogen peroxide; O2•2, superoxide anion; NO•, nitric oxide; OH•, hydroxyl radicals; 1O2, singlet oxygen; ROO•, peroxyl radicals; ONOO2, peroxynitrite; ARE, antioxidant response element; CAT, catalase; GPx, glutathione peroxidase; GST, glutathione-S -transferase; GR, glutathione reductase; HO-1, heme oxygenase 1; NQO1, nicotinamide adenine dinucleotide phosphate oxidase: quinone oxidoreductase 1; gGCL, g-glutamylcysteine ligase; SOD, superoxide dismutase; Srxn1, sulfiredoxin 1; NRF1, nuclear respiratory factor 1; Nrf2, nuclear factor erythroid 2-related factor 2. Tfam, mitochondrial transcription factor A.

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FIGURE 18.4 The potential structure of the curcumin molecule to inhibit free radicals. Functional groups of curcumin. b-Diketone group, phenyl rings with hydroxyl and methoxy, and carbon -carbon double bonds.

curcumin increases its radical scavenging activity (Priyadarsini, 2014) (Fig. 18.4). These studies show that curcumin can directly scavenge the free radicals generated in the mitochondria, and indirectly inhibit oxidative stress-related inflammation originating from the mitochondria. Previous studies have shown that curcumin has a miraculous power in antiinflammatory processes and immunomodulatory activities (Hewlings & Kalman, 2017). Various cellular, animal, and clinical studies have implicated its efficacy as an antiinflammatory compound (Boroumand et al., 2018). The antiinflammatory property of curcumin is associated with the downregulation of various signaling mediators such as Janus kinases, mitogen-activated and cyclooxygenase-2 (COX-2) activity, and inhibiting the generation of 50 -Adenosine monophosphateactivated protein kinase, TNF-α, IL-1, -2, -6, -8, and -12 (Kahkhaie et al., 2019). Curcumin is a potent blocker of inflammatory-induced NF-κB activation. NF-κB activation is suppressed by inhibition of IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) kinase and AKT (PourbagherShahri et al., 2021). It has been reported that that curcumin targets NF-κB through its antioxidant activity (Van’t Land et al., 2004). NF-κB DNA-binding activity is induced by oxidative stress, therefore, the antioxidant property of curcumin may result in a depletion of NF-κB DNA-binding activity (Lingappan, 2018). Previous studies have demonstrated that curcumin is able to decrease the expression of the NF-κB-regulated gene products such as TNF, COX-2, IL-1, IL-6, IL-8, MIP-1α, 5-LOX, CXCR-4, adhesion molecules and c-reactive protein (CRP) (Lingappan, 2018). It has been reported that one of the cell organelles showing subcellular localization of COX-2 is the mitochondria (Liou et al., 2005). This enzyme is involved in several inflammatory disorders via producing pro-inflammatory prostaglandins (Lee et al., 2020). There are contradictory and inconclusive studies regarding the effects of curcumin on COX-2 expression (Lee et al., 2020;

18.7 Mitochondrial targeting for the reduction of oxidative stress

Streyczek et al., 2022). Various studies propose that curcumin has an inducible effect, while others present that it could suppress COX-2 expression. Curcumin treatment could inhibit the expression of COX-2 at both mRNA and protein levels (Lee et al., 2020; Streyczek et al., 2022). It has been suggested that these contradictory effects may be dependent on the concentration of curcumin (Streyczek et al., 2022). In addition to COX-2 protein, iNOS is also known to be involved in inflammatory responses (Nogawa et al., 1998). Certainly, it has been reported that curcumin can target iNOS. The activation of iNOS results in NO production, a primary pro-inflammatory mediator (Boroumand et al., 2018). Various authors have shown that curcumin can act as an antiinflammatory agent via inhibiting iNOS expression at both mRNA and protein levels, and through inhibiting the NO production (Boroumand et al., 2018). In addition, curcumin shows immunomodulatory and antiinflammation activity by suppressing cytokine signaling (SOCS) expression and TLR-4/MyD88/NF-κB axis, and interacting with elements involved in inflammatory responses including the JAK/STAT pathway (Porro et al., 2019). Curcumin is bound to α,β-unsaturated carbonyl portion to residue 259 of cysteine in STAT3 and inhibits phosphorylation of JAK/STAT with subsequent activation (Hahn et al., 2018). It has been reported that curcumin is able to phosphorylate STAT3 and moderate the inflammatory response (Kahkhaie et al., 2019). The antagonist of JAK/STAT signaling is SOCS. SOCS is involved in the regulation of cytokines and inflammatory proteins (Croker et al., 2008). Previous studies have demonstrated that curcumin can restore the expression of SOCS1 and SOCS3, and inhibit expression of PGE2, TNF-α, and IL-6 (Chen et al., 2013; Porro et al., 2019). However, curcumin in vivo models, restores immunological balance by acting on JAK/STAT/ SOCS signaling, inhibiting JAK2, STAT3, and STAT6 phosphorylation and increasing SOCS1, SOCS3, and PIAS3 expression (Chen et al., 2013; Porro et al., 2019). Furthermore, curcumin can apply antiinflammatory effects via the downregulation of MyD88, TLR-4 and NF-κB signaling by inducing the release of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 (Ni et al., 2015).

18.7 Mitochondrial targeting for the reduction of oxidative stress and inflammation As briefly reviewed, mitochondrial dysfunction and mtROS formation are closely linked to oxidative stress and inflammation (Patergnani et al., 2021). Thus, therapeutic strategies targeting these organelles could be pivotal to managing oxidative stress, inflammation, and inflammatory-related diseases (Deng et al., 2021). Many studies indicate that the mitochondria are involved in the antioxidant and antiinflammatory properties of curcumin, as it can regulate many mechanisms in which mitochondria are implicated, mainly ROS formation, oxidative stress, and activation of inflammation mediators via oxidation (Bagheri et al., 2020; Gibellini

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et al., 2015; Naserzadeh et al., 2018; Sabet et al., 2020; Trujillo et al., 2014). It has been shown that curcumin reduces oxidative stress in vivo models in which ROS formation was triggered by one or more factors such as the diet quality, pathological state, chemicals, and the use of drugs (Cox et al., 2022; Gibellini et al., 2015; Naserzadeh et al., 2018; Sabet et al., 2020). Directly, curcumin can scavenge the free radicals originating from mitochondrial dysfunction (Gibellini et al., 2015). Furthermore, the antioxidant properties of curcumin are associated with nuclear translocation of Nrf2 and preservation of mitochondrial function (Tapia et al., 2012). Overwhelming evidence indicates that curcumin reduces mitochondrial alterations by decreasing oxygen consumption, calcium retention, ATP production, respiratory control rate, mitochondrial membrane potential (ΔΨm), aconitase activity, membrane integrity, and inhibiting the opening of the mitochondrial transition pore (mPT), consequently avoiding oxidative stress, inflammation, and cell death (Cox et al., 2022; Jat et al., 2013; Martı´nez-Moru´a et al., 2013; Soto-Urquieta et al., 2014; Wang et al., 2020). Therefore, mitochondria are a relevant target for the protection provided by curcumin under different pathologic scenarios such as oxidative stress, inflammation and oxidative/inflammatory-related diseases (Patergnani et al., 2021; Waseem et al., 2017). Next, we will focus on the main mechanisms by which curcumin reduces oxidative stress and inflammation.

18.8 Curcumin as a direct mitochondrial reactive oxygen species scavenger Mitochondria are the primary source of ROS generation within the cell, and these organelles are known to generate approximately 90% of cellular ROS (Patergnani et al., 2021; Quinlan et al., 2013; Zorov et al., 2014). A large amount of superoxide ions are produced as byproducts of mitochondrial metabolism in many biochemical processes including electron escape from the etc during the tricarboxylic acid (TCA) cycle and mitochondrial OXPHOS (Quinlan et al., 2013; Zorov et al., 2014). It is generally accepted that two of the respiratory chain complexes, namely complexes I and III are the prominent sites of ROS production in mitochondria (Quinlan et al., 2013; Zorov et al., 2014). Also, several matrix proteins and complexes including enzymes of the TCA cycle (e.g., α-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and aconitase) are involved in mtROS generation (Quinlan et al., 2013). Furthermore, many enzymatic reactions in mitochondria can generate ROS, including those of cytochrome b5 reductase, cytochrome P450, glycerol-3-phosphate dehydrogenase, and MAO (Quinlan et al., 2013). In fact, by their vicinity to ROS, mtDNA, proteins, and lipids are the main primary targets of these free radicals, creating a mitochondrial free radical “vicious cycle” of injury (Lagouge & Larsson, 2013) (Fig. 18.5). Previous studies have shown that curcumin can scavenge various forms of free radicals such as

18.8 Curcumin as a direct mitochondrial

FIGURE 18.5 Sources of ROS formation in mitochondria. Mitochondrial complex I and III of respiratory chain are the principal sites of superoxide anion (O2•-) production, which can be converted to hydrogen peroxide (H2O2), by superoxide dismutase (SOD) enzymes. Hydrogen peroxide is rapidly neutralized to water (H2O) and oxygen (O2) by glutathione peroxidase (GPX). Other mitochondrial proteins which are localized from OMM to matrix, may also contribute to mitochondrial ROS formation, including cytochrome (Cyt.) b5 reductase, monoamine oxidase A and B (MAO A/B), p66Shc, mitochondrial glycerolphosphate dehydrogenase (mGPDH), aconitase, adrenodoxin reductase (ADxR)adrenodoxin (ADX)-cytochrome P450scc (CYP450) system, Fhit with ferredoxin reductase (FDxR), acyl-CoA dehydrogenases (ACAD), α-ketoglutharate dehydrogenases (KGDHC).

superoxide anions, hydrogen peroxide, hydroxyl radicals, nitric oxide, singlet oxygens, peroxyl and peroxynitrite radicals originating from the mitochondria and other sources (Barzegar & Moosavi-Movahedi, 2011; Malik & Mukherjee, 2014). Some studies have suggested that the free radical scavenging activity of curcumin is mainly derived from its phenolic structure (Anjomshoa et al., 2017; Barzegar & Moosavi-Movahedi, 2011; Glancy et al., 2020). Various authors concluded that presumably, the excellent antioxidant effects of curcumin are largely due to its

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hydrogen-atom donation from the phenolic group (Anjomshoa et al., 2017; Barzegar & Moosavi-Movahedi, 2011). It has been reported that curcumin is a hydrogen-atom donor by giving a hydrogen-atom from the phenolic group, which is the methoxy or the central methylene group (Anjomshoa et al., 2017; Barzegar & Moosavi-Movahedi, 2011). The methylene group of the beta-diketone presence in the chemical structure of curcumin, is a potent anion superoxide scavenger (Anjomshoa et al., 2017; Barzegar & Moosavi-Movahedi, 2011). The only presence of the beta-diketone was not sufficient for the radical-scavenging activity of curcumin (Anjomshoa et al., 2017; Barzegar & Moosavi-Movahedi, 2011). Various authors have been shown that the phenolic hydroxyl group and the central hydrogen atom of the methyl is involved in the production of phenoxyl. The presence and number of phenolic hydroxyl group in the chemical structure of curcumin increases its radical scavenging activity (Anjomshoa et al., 2017; Barzegar & Moosavi-Movahedi, 2011).

18.9 Curcumin enhances mitochondrial antioxidants In addition to mtROS formation through the etc, TCA cycle, enzymatic reactions, matrix proteins and other complexes, homeostatic redox status in mitochondria is also controlled by mitochondria-associated antioxidant defenses (Huang et al., 2019). Accordingly, peroxiredoxin 3 (Prdx3), SOD2, thioredoxin 2 (Trx2), and glutathione reductase (GR), generally exert antioxidative roles in mitochondria (Huang et al., 2019). Under normal conditions, mtROS homeostasis is strictly maintained by free radical formation and scavenging antioxidant systems such as type-2 SOD (SOD2), GSH, GR, Trx2, and Prdx3 (Yang et al., 2020). GR is a major antioxidant enzyme essential to maintain the GSH/GSSG ratio by catalyzing recovery of reduced GSH from oxidized glutathione (GSSG) (Gu¨ller et al., 2021). It has been reported that curcumin inhibits human GR according to both in vitro and in silico studies (Gu¨ller et al., 2021). Therefore, curcumin, a potential inhibitor of human GR, can be used in drug design to target the GSH system in cellular injury (Gu¨ller et al., 2021). There are several studies that show curcumin can increase mitochondrial and cellular GSH, thereby reducing the damage caused by oxidative stress (Jat et al., 2013). Additionally, SOD2 as an antioxidative protein in the mitochondria is upregulated by curcumin (Wang et al., 2019).

18.10 Curcumin activates the Nrf2 signaling pathway and protects mitochondrial damage and oxidant generation Nrf2, as the master regulator of the cellular redox homeostasis in the cell, is well equipped to counterbalance the mtROS formation and is very important for

18.10 Curcumin activates the Nrf2 signaling pathway

maintaining the redox balance (Holmstro¨m et al., 2016). Exposure of the cells to free radicals, ROS, oxidants, and electrophiles enhance the accumulation Nrf2 in the nucleus (Ma, 2013). There, it binds to ARE in the upstream regulatory regions of genes encoding antioxidant and detoxification enzymes, leading to their enhanced transcription (Ma, 2013). Nrf2 has been involved in the regulation of over 600 target genes and has been associated with cytoprotective effect in previous studies. Nrf2 targets antioxidant enzymes and proteins involved in inhibition of inflammation, chemical/drug metabolism and clearance, repair and removal of damaged proteins, and protection against heavy metal toxicity, as well as other growth and transcription factors (Tebay et al., 2015). Nrf2 regulates the expression of GR, γ-glutamyl cysteine ligase catalytic (GCLC) and modulatory (GCLM) subunits, also the four enzymes including: 6-phosphogluconate dehydrogenase (6PGD), isocitrate dehydrogenase 1 (IDH1), malic enzyme 1 (ME1), and glucose-6-phosphate dehydrogenase (G6PD) that are responsible for the production of NADPH, all of which are involved in the maintenance and biosynthesis and of GSH (Yang et al., 2005). In turn, GSH is the principal antioxidant in the mammalian cell to neutralize free radicals (Pham-Huy et al., 2008). It has been reported that Nrf2 modulates mitochondrial function as part of its role as a master regulator of the cellular redox homeostasis and cytoprotective gene expression. It has been reported that Nrf2 regulates the mitochondrial function via two pathways (Dinkova-Kostova & Abramov, 2015). First, the Nrf2 pathway is upregulated and is involved in protection against mitochondrial toxins and second, impairment of Nrf2 function in mitochondria-related disorders, whereas Nrf2 activation has beneficial effects (Dinkova-Kostova & Abramov, 2015). A physical link between Nrf2 and mitochondria has been reported by Lo and colleagues in 2008 (Lo & Hannink, 2008). This study showed that Keap1 associates with phosphoglycerate mutase 5 (PGAM5), a protein phosphatase related to cell death, mitophagy, and mitochondria homeostasis (Lo & Hannink, 2008). Nrf2-knockout (Nrf2-KO) mice showed a significant decrease in mitochondrial respiration and ATP levels (Holmstro¨m et al., 2013). Furthermore, Nrf2 plays a pivotal role in the control of the inflammation due to its role in the expression of antioxidant genes (Saha et al., 2020). Nrf2 and Keap1 as its principal negative regulator, play a central role in the regulation of inflammation and maintenance of intracellular redox homeostasis (Saha et al., 2020). It has been reported that Nrf2 contributes to the regulation of the heme oxygenase-1 (HO-1) axis, which is a potent antiinflammatory target (Loboda et al., 2016), as well as a connection between the Nrf2/ARE system and the expression of inflammatory mediators, NF-κB pathway, and macrophage metabolism (Saha et al., 2020). As briefly reviewed, mitochondrial function, mitochondria homeostasis, mtROS, mitochondrial protection and mitoinflammation are closely linked with Nrf2 (Holmstro¨m et al., 2016). Therefore, targeting Nrf2 and Keap1 as its principal negative regulator can play an effective role in reducing mitochondrial damage, oxidative stress, and inflammation (Holmstro¨m et al., 2016). In all reported studies, it has been found that curcumin can stimulate the activation of the Nrf2 signaling pathway to apply its

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neuroprotective, cardioprotective, hepatoprotective, antiinflammatory, antioxidant, and renoprotective activities (Shahcheraghi et al., 2022). It has been reported that cardioprotective, neuroprotective, and hepatoprotective effects of curcumin are associated with the activation of the Nrf2 signaling pathway, suppressing Bax/ Bcl-2-caspase-3 pathway-mediated cell death, decreasing oxidative stress mainly through MDA, increasing antioxidant enzymes such SOD, CAT, GPx and GR, as well as diminishing fibrosis, inflammation, and hypertrophy (Ghafouri-Fard et al., 2022; Shahcheraghi et al., 2022). The activation of the Nrf2 pathway causes inhibition of the mitochondrial membrane potential loss, a reduction in oxidative stress, attenuation of cellular and mitochondrial injuries, reducing cell apoptosis, immune cell infiltration, and inflammation (Bellezza et al., 2018). Taking everything into account, it seems that curcumin can affect the Nrf2 signaling pathway in various phases, including: (1) enhancing the nuclear translocation of Nrf2, (2) inhibiting Keap1 as its principal negative regulator, (3) influencing the expression of Nrf2 and targets genes, and (4) affecting the upstream mediators of Nrf2 (Ashrafizadeh et al., 2020). Therefore, the Nrf2 signaling pathway plays a significant role in the induction of mitochondrial protective effects, antioxidant and antiinflammation activities of curcumin (Ashrafizadeh et al., 2020).

18.11 Targeting of mitochondrial uncoupling proteins by curcumin UCPs are IMM proteins that belong to the UCP family and plays an important role in dissipating metabolic energy and lowering mitochondrial membrane potential with the prevention of oxidative stress accumulation (Pierelli et al., 2017). Interestingly, the recovery and stabilization of mitochondrial function by the plant-derived active compounds are often mediated through UCP overexertion and upregulation (Salehi et al., 2020). Curcumin as a plant-derived active compound from the Curcuma longa (turmeric) plant rhizome, has many biological activities, including the amelioration of ischemic stroke, antiinflammatory, neuroprotective, and antioxidant properties (Amalraj et al., 2017). Chronic administration of curcumin orally improved cerebrovascular endothelium-dependent relaxation and reduced ROS generation in aging wild-type mice, but not in aging UCP-22/2 mice (Pu et al., 2013). Dietary administration of curcumin for one month significantly restored the impaired cerebrovascular endothelium-dependent vasorelaxation in aging Sprague Dawley rats (Pu et al., 2013). In cultured endothelial cells and cerebral arteries from the rat model, curcumin promoted endothelial nitric oxide synthase and AMP-activated protein kinase (AMPK) phosphorylation, upregulated UCPs especially UCP-2, and reduced ROS formation and oxidative stress (Pierelli et al., 2017; Pu et al., 2013). The effect of curcumin was abolished by either UCP-2 inhibition or AMPK in aging Sprague Dawley rats (Pierelli et al., 2017; Pu et al., 2013). Thus, the upregulation of

18.12 Targeting of mitochondrial sirtuins by curcumin

UCP-2 by curcumin activates AMPK in the cerebrovascular endothelium (Pierelli et al., 2017; Pu et al., 2013). This activation prevented NO reduction and antagonized superoxide anion generation in endothelial cells (Pierelli et al., 2017; Pu et al., 2013). In addition, in another study, the effect of curcumin on differentiation of adipocytes and mitochondrial oxygen consumption was studied in 3T3-L1 preadipocytes (Zhao, Pan, et al., 2021). Curcumin dose-dependently induced the intracellular fat droplet accumulation, adipogenic differentiation, and it remarkably enhanced mature adipocyte mitochondrial respiratory function and ATP production through uncoupling capacity via the regulation of PPARγ (Zhao, Pan, et al., 2021). Also, curcumin administration increased the mRNA and protein expressions of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), PPARγ, mitochondrial uncoupling protein 1 (UCP-1) and PR domain protein 16 (PRDM16) in vivo (mice) and in vitro (3T3-L1 preadipocytes) (Zhao, Pan, et al., 2021). This study demonstrated that curcumin promoted the mitochondrial oxygen consumption and adipogenic differentiation of preadipocytes in 3T3-L1 mature adipocytes by regulating UCP-1, PGC-1α, PPARγ, and PRDM16 expression (Zhao, Pan, et al., 2021). Song et al., reported that curcumin intervention reduces white adipose tissue macrophage infiltration and alters macrophage functional polarity in C57BL/6J mice (Song et al., 2018). They reported that curcumin treatment reduces M1-like macrophage markers or proinflammation cytokine expression in both adipocytes and macrophages (Song et al., 2018). In addition, this study indicated that curcumin intervention targets both white adipose tissue inflammation and brown adipose tissue UCP-1 expression (Song et al., 2018). The above studies show that curcumin can reduce the mitochondrial dysfunction induced by toxic agents and conditions, via upregulation of mitochondrial UCPs. Therefore, overexpression of UCP by curcumin can reduce mitochondrial dysfunction-mediated oxidative stress and inflammation.

18.12 Targeting of mitochondrial sirtuins by curcumin Curcumin is capable of acting on mitochondrial biogenesis and dysfunction and on apoptosis via SIRT activation by small molecules (Iside et al., 2020) (Fig. 18.6). Curcumin downregulates the expression of transcription factors, TNFs, nitric oxide synthase, growth factor receptors, and increases AMPK levels (Ghareghomi et al., 2021; Zendedel et al., 2018). Curcumin promotes the activation of AMPK, which increases ATP and superoxide production (Ghareghomi et al., 2021; Zendedel et al., 2018). This event activates SIRT1 activation and increases NAD1 levels (Ghareghomi et al., 2021; Zendedel et al., 2018). Curcumin-induced SIRT1 upregulation has beneficial effects against a range of inflammatory diseases (Zendedel et al., 2018). It has been reported that curcumin

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FIGURE 18.6 The role of sirtuins in mitochondrial functions and inflammatory responses. Sirtuins (SIRTs 1
7) have been found to be involved in modulating levels of ROS, oxidative stress, mitochondrial dysfunction, antioxidant enzymes and DNA repair through key transcription factors such as NF-κB, Nrf2, PGC-1α, FOXO and p53. After ROS production in the cells, coenzyme NAD 1 activates various sirtuins. As well as, sirtuins control the activity of the ARE, which in turn modulates the transcription of pro- and antioxidant genes to maintain redox signaling cascades. ADP, adenosine diphosphate; ARS, antioxidant and redox signaling; CAT, catalase; FOXO, class O of forkhead box transcription factors; G6PD, glucose-6-phosphate dehydrogenase; NAD, nicotinamide adenine dinucleotide; NF-κB, nuclear factor kappa B subunit; PARP1, poly (ADP-ribose) polymerase 1; PGAM2, phosphoglycerate mutase; SIRT, sirtuin.

attenuates myocardial ischemia-reperfusion (MI-R)-induced mitochondrial oxidative damage through SIRT1 action (Yang et al., 2013). Mitochondrial oxidative damage is characterized by a reduction in succinate dehydrogenase (SDH) activity, ROS formation, mitochondrial swelling, and impairment in the MRC (Sabet et al., 2020). In in vitro studies, it has been confirmed that curcumin upregulates COX, SIRT1, SDH, and downregulates Bax protein (Zendedel et al., 2018). This evidence shows that curcumin mediates cryoprotection effects via SIRT1 (Zendedel et al., 2018). Curcumin has positive effects in the inflammatory pathways by mediating the reduction of NF-κB expression. NF-κB downregulation can influence other mechanisms in which AMPK and SIRT1 are involved, such as glucose absorption in cells (Salminen et al., 2011). It has been reported that supplementation of curcumin for eight weeks in diabetic mice might promote indirect activation of SIRT1 via AMPK (Zamani & Rezagholizadeh, 2021).

18.14 Conclusion

18.13 Targeting of mitochondrial p66shc by curcumin p66Shc, the 66 Kd Shc protein, is an age-related adapter protein that has a substantial impact on mitochondrial metabolism through regulation of cellular response to oxidative stress (Pinton & Rizzuto, 2008). When the expression of p66Shc is upregulated, the generation of ROS increases and the oxidative damage to cells becomes severe, meaning that the concentration of ROS correlates positively with the expression of p66Shc (Pinton & Rizzuto, 2008). It has been reported that curcumin reduce the expression of p66Shc induced by arsenic (Wang et al., 2017). As well as, curcumin reduces the levels of ROS, increases mRNA levels of Mn-SOD and gamma-glutamyl ligase, increases GSH and protein levels of Bcl-2 and Mn-SOD, and increases the nuclear levels of Nrf2 and FOXO-3a in diabetic rats (ALTamimi et al., 2021). This study indicated that curcumin reduces the nuclear activity of NF-κB, downregulates protein kinase CβII (PKCβII), NADPH oxidase, and p66Shc, and decreases the activation of p66Shc (ALTamimi et al., 2021).

18.14 Conclusion In summary, mitochondrial functions are coordinately regulated by multiple factors, such as mitochondrial redox status, bioenergetics and biogenesis. Recently, increasing evidence has suggested that mitochondrial dysfunction plays a key role in initiating oxidative stress and inflammation through different mechanisms. Therefore, mitochondrial damage has been proposed as a promising target for the treatment of oxidative stress and inflammation. It is well documented that curcumin has antiinflammatory and antioxidant effects in animal and clinical trials. It has been suggested that curcumin restores mitochondrial redox balance, biogenesis, and bioenergetics under pathological conditions. Mitochondrial protection and mtROS reduction can have an important effect in reducing inflammatory responses. Curcumin has shown promising effects in reducing mitochondrial damage and mitochondrial-dependent oxidative stress. Previous studies have suggested that curcumin restores mitochondrial impairments by decreasing oxygen consumption, calcium retention, ATP production, and respiratory control rate, inhibiting the opening of the mitochondrial transition pore, aconitase activity and membrane integrity, thus avoiding the oxidative stress and activation of inflammatory responses in the cells. It seems that curcumin applies the mitochondrial protection effects and its subsequent antiinflammatory properties via different mechanisms such as ROS scavenging, enhancing mitochondrial antioxidants, the activation of Nrf2, targeting of SIRTs, p66Shc and UCPs, inhibition of COX-2 and MAO enzymes. However, more investigations are needed to describe the exact molecular mechanisms and relationship between curcumin and mitochondrial protection in inflammatory responses.

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Conflict of interest Authors declare that he has no conflict of interest.

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CHAPTER

19

Dihydrogen as an innovative nutraceutical for mitochondrial viability

Sergej M. Ostojic Department of Nutrition and Public Health, University of Agder, Kristiansand, Norway

19.1 Introduction Numerous beverages and food products contain molecular hydrogen or dihydrogen (H2), a diatomic gas added during the food packaging process to protect the integrity of foods and suppress unwanted chemical reactions. Dihydrogen has been traditionally considered a biologically inert gas, chemically stable and nontoxic, as the US Food and Drug Administration issued a notice of a claim that the use of dihydrogen solubilized in water is generally recognized as safe when it is added to beverages (e.g., pure drinking water, flavored beverages, soda drinks) in order to prevent oxidation (Food and Drug Administration FDA, 2014). However, an outpouring of evidence collected during the past two decades implies that molecular hydrogen might have biologically relevant effects by itself, acting as an anti-inflammatory and anti-allergic agent, antioxidant, and signaling molecule (for a detailed review see Yang et al., 2020). The fact that H2 is a natural constituent of intestinal gas, being abundantly produced by gut microbiota as an end-product of hydrogen-producing anaerobic phila (Smith et al., 2019), provides an additional argument for its plausible biological role. As a consequence, novel findings have started to remold the food industry during the past few years and push forward products focused to dihydrogen as a main active ingredient, instead of using H2 as a secondary inactive food agent. Over 200 commercial food products with dihydrogen as an active principle are now available on the international market, with many claiming favorable effects on mitochondrial performance and viability. This chapter overviews the effects of ingestion of hydrogen-enriched beverages, nutritional supplements, and food products that drive hyper-production of endogenous H2 by intestinal bacteria, on mitochondrial function in experimental and clinical nutrition.

19.2 Dietary sources of molecular hydrogen From a seminal Science report demonstrating anti-cancerous effects of H2 (Dole et al., 1975), and a game-changing paper on selective antioxidant properties of gaseous hydrogen published in Nature Medicine (Ohsawa et al., 2007), this simple Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00003-5 © 2023 Elsevier Inc. All rights reserved.

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CHAPTER 19 Dihydrogen as an innovative nutraceutical

medical gas has been evaluated in over 1000 scientific papers so far, with most studies published from the year 2000 onwards (Zhai et al., 2014). Considering other vehicles developed to deliver H2 to target tissues and organisms, beverages and dietary supplements enriched with dihydrogen appeared to be the most popular. Arguably, the most common and easy-to-administer source of dietary H2 in biomedical research is hydrogen-rich water (HRW). Also known as hydrogen-enriched water or hydrogen-infused water, HRW is produced either by a chemical reaction between water and inorganic compounds (such as magnesium hydroxide), via the electrolysis of water where H2 is generated at the cathode, and as a result of infusing gaseous hydrogen into pure water under high pressure. The amount of hydrogen gas dissolved in ready-to-drink HRW varies from 0.1 to 8.0 ppm, with most commercial HRWs having 0.5 ppm, which is generally considered a minimal amount of H2 needed to produce a biologically relevant effect. Due to a high dissipative capacity of dihydrogen, the use and storage of HRW requires specific instruction, including hydrogenconserving materials for HRW packaging, and expedited preparation or consumption (Ohta, 2012). Another dietary source of dihydrogen are nutritional supplements supplied as ready-to-ingest capsules and tablets containing various inorganic compounds (e.g., magnesium hydroxide, calcium hydride) that generate hydrogen gas from a reaction with water inside the gut, or sublingual formulations that allow for rapid production and uptake of dihydrogen into the circulation, bypassing the gut and liver. Finally, several nutritional products are designed to instigate H2 production in the gut by providing organic fuel for hydrogen-producing colonic bacteria (Holscher, 2017). These comprise non-digestible dietary fibers, such as pectin, gum arabic, and inulin. Whatever the source used, dihydrogen from the gut enters the circulation and disperses over the body, as confirmed by a rise in serum H2 levels and an increase in molecular hydrogen exhaled in the breath after ingesting hydrogen-rich food products (Shimouchi et al., 2009). In addition, dihydrogen levels in venous blood are found to be lower than in the arterial blood, implying the uptake of H2 into tissues (Ohsawa et al., 2007). Beside other possible subcellular targets, hydrogen gas could reach mitochondria due to several favorable features, including the small size of the molecule (effective diameter 2.25 3 1024 μm) that favors transport via passive diffusion, and a concentration gradient through mitochondrial membranes. In contrast, its absorption by the organelle might be hampered by a mitochondrial electrochemical gradient, high reactivity of dihydrogen, and its affinity to glycogen (Ohta, 2012; Ostojic, 2015), putting forward a complex estimation of dihydrogen net uptake by a mitochondrion.

19.3 Hydrogen-rich water and mitochondrial function A dozen interventional studies investigated the effects of drinking HRW in animal models of mitochondrial dysfunction, and humans with inherited or acquired mitochondrial disorders, with all studies published from 2011 onwards (Table 19.1). The research group from Nagoya University Graduate School of

Table 19.1 The list of interventional studies using dietary dihydrogen for mitochondrial performance and outcomes. Ref.

Species n

Model/ pathology

Ito et al. (2011) Noda et al. (2012) Li et al. (2013) Xin et al. (2014) Wang et al. (2014) Zhang et al. (2015) Hou et al. (2016) Ge et al. (2017) Korovljev et al. (2018) Feng et al. (2019) Wen et al. (2019) Xun et al. (2020)

Human

36

Rat

39

Rat

48

Mt HRW myopathies Cardiac HRW allografts Nephrotoxicity HRW

Rat

216 Hypertension

Rat

6

Neurotoxicity

Mouse

40

Rat

Source Amount

Duration

Outcomes

1 L/day

8
12 weeks

Ad libitum

50 days

1.3 6 0.2 mg/L

7 days

HRW

1.3 6 0.2 mg/L

3 months

No change in clinical symptoms Increase in lactate-to-pyruvate ratio Decrease in lactate Increase in—graft survival Reduction in intimal hyperplasia Increase in—ATP production Increase in—Mt RC enzymes Reduction in Mt ROS Increase in—ATP production Decreased Mt swelling Increase in—ATP levels Increased—integrity of Mt membrane

HRW

Ad libitum

8 weeks

No changes in Mt structure

Hepatotoxicity HRW

5 mL/kg

5 days

48

High-fat diet

CaH2

50 mg/kg/day

13 weeks

Reduction in megamitochondria number deceased Mt pyknosis Reduced Mt damage decrease in Mt ROS

Rat

90

Saline

5
6 mL/kg

6 weeks

No Mt vacuolization

Human

10

Spinal cord injury Aging

4 weeks

Rat

60

Mouse

35

Myocardial infarction Neurotoxicity

Rat

30

6 ppm per day H2 minerals H2 fluid 10 mL/kg/day

3 weeks

No change in lactate-to-pyruvate ratio No change in serum coenzyme Q10 Decrease in serum lactate Reduced Mt dysfunction Increase in mtDNA repairase

HRW

Ad libitum

7 days

Reduced Mt dysfunction Reduced neuroinflammation

Hepatotoxicity HRW

Ad libitum

8 weeks

Decreased Mt damage Increased Mt SOD2 gene

m k [ denotes an increase, decrease or no change in a specific variable, respectively; ATP, adenosine triphosphate; HRW, hydrogen-rich water; Mt, mitochondrial; RC, respiratory chain; ROS, reactive oxygen species; SOD2, superoxide dismutase 2.

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CHAPTER 19 Dihydrogen as an innovative nutraceutical

Medicine was arguably the first who evaluated the effects of HRW in mitochondrial medicine in a human trial (Ito et al., 2011). The authors performed both open-label and randomized controlled trials of drinking up to 1 L per day of hydrogen-enriched water for 8
12 weeks in patients with various mitochondrial myopathies. Although no objective improvement of clinical symptoms was observed, HRW improved several indicators of mitochondrial function (e.g., serum lactates and lactate-to-pyruvate ratio), suggesting its effectiveness for mitochondrial dysfunction in a dose-response manner. Another study evaluated the effects of HRW (dihydrogen concentration 0.5
0.6 mM) and regular water after heterotopic heart transplantation and aortic transplantation in allogeneic rat strains (Noda et al., 2012). Drinking HRW was remarkably effective in prolonging heart graft survival and reducing intimal hyperplasia in transplanted aortas, as compared to grafts treated with regular water. HRW treatment was associated with elevated levels of graft adenosine triphospate (ATP), a key high-energy molecule in the cell that is mostly produced in mitochondria, accompanied by increased activities of the enzymes in mitochondrial respiratory chain. HRW had a protective effect on mitochondrial function including ATP formation and membrane integrity in spontaneously hypertensive rats who were subjected to water rich in dihydrogen (H2 concentration 1.3 mg/L) for 3 months (Xin et al., 2014). Drinking HRW appears to be beneficial in animal models of chemical exposure and mitochondrial function. For instance, the consumption of HRW (H2 concentration 1.3 mg/L) protects against ferric nitrilotriacetate-induced nephrotoxicity and early tumor promotional events in rats, with HRW ameliorating the mitochondrial dysfunction through suppressing mitochondrial reactive oxidative species (ROS) formation, enhancing mitochondrial ATP production, and reducing mitochondrial swelling (Li et al., 2013). Drinking water rich in hydrogen (0.6 mM) during 8 weeks has shown protective effects on damaged hippocampal neurons and neuronal mitochondria in rats with chlorpyrifos-induced neurotoxicity, with most of the mitochondria in the H2 group were intact with a dark matrix, a regular distribution of cristae, and no signs of mitochondrial swelling (Wang et al., 2014). Protective effects of HRW on hepatotoxicity induced by subchronic exposure to chlorpyrifos in rats has been confirmed in a recent trial (Xun et al., 2020), with water enriched with hydrogen (0.55
0.65 mM) consumed for eight weeks alleviating mitochondrial damage and modulating the expression of several oxidative stress-related genes, including the mitochondrial superoxide dismutase 2 (SOD2) gene. HRW also protected the endoplasmic reticulum and mitochondria in the liver of mice subjected to acetaminophen-induced hepatotoxicity, by drinking water rich in dihydrogen (0.83
0.91 mmol/L) for five days which prevented megamitochondria, mitochondrial pyknosis, distension, and flocculent degeneration (Zhang et al., 2015). Finally, ad libitum drinking of HRW for seven days in mice with methamphetamine-induced neurotoxicity significantly inhibited toxininduced spatial learning impairment and memory loss, and restrained mitochondrial dysfunction, neuroinflammation, and endoplasmic reticulum stress (Wen et al., 2019).

19.4 Other dietary and complementary interventions with hydrogen

19.4 Other dietary and complementary interventions with hydrogen In addition to HRW, other hydrogen-generating nutritional approaches and enteral nutrition formulas also have an effect on mitochondrial function. An interesting study monitored six-week-old male Sprague Dawley rats fed a high-fat diet or high-fat diet with a daily oral gavage of 50 mg/kg/day calcium hydride, a new solid molecular hydrogen carrier made of coral calcium, for 13 weeks (Hou et al., 2016). The authors demonstrated that calcium hydride durably generated dihydrogen in vivo and in vitro, with the treatment effectively improving the high-fat diet-induced hepatic mitochondrial dysfunction, reducing oxidative stress, activating phase II enzymes, and reversing mitochondrial morphological damage induced by a high-fat diet. Moreover, intragastric gavage of hydrogen-rich fluid (1.6 ppm) at a dosage of 10 mL/kg weight daily for three weeks protects rats from mitochondrial dysfunction induced by an acute myocardial infarction (Feng et al., 2019). This likely happens by a hydrogen-driven upregulation of antioxidant-related proteins and mitochondrial-associated proteins, including mtDNA repairase 8-oxoguanine DNA glycosylase, translocase of the outer mitochondrial membrane 40, and translocase of inner mitochondrial membrane 23. Ge et al. (2017) reported protective effects of using a 6-week hydrogen-rich saline in rats with spinal cord hemisection-induced testicular injury. Animals who received H2 (dihydrogen levels 0.6 mM) demonstrated no significant alterations in ultrastructures of the pituitary gland and testis observed in a vehicle group, such as mitochondrial vacuolization, and reduced or absent mitochondrial cristae. Finally, orally administered H2 as a blend of hydrogen-generating minerals (supplying B 6 ppm of dihydrogen per day) tended to improve serum lactate levels, a surrogate marker of mitochondrial metabolism, in overweight women in a randomized controlled trial; however, other biomarkers of mitochondrial function (e.g., lactate-topyruvate ratio, coenzyme Q10, etc.) remained unaffected by a 4-week hydrogen consumption (Korovljev et al., 2018). To summarize, it appears that most studies documented here have used models of mitochondrial chemical toxicity in rodents, while H2 (primarily supplied as HRW) was usually consumed ad libitum, with administration typically lasting for 6
8 weeks. However, intervention protocols with H2 were highly variable in terms of duration and dihydrogen doses administered, and only two small-scale human trials evaluated the effects of dietary H2 in mitochondrial nutrition. Still, all studies revealed rather favorable effects of consuming dihydrogen, from preserving mitochondrial microstructure in stressful conditions, to upholding its role in energy metabolism and redox balance, to augment surrogate markers of mitochondrial viability. Although many issues remain undisclosed, the fact that all recorded studies are published in the past ten years signals that the association between dietary dihydrogen and mitochondrial function becomes a hot topic in experimental nutrition.

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19.5 Dihydrogen and mitochondria: molecular mechanisms H2 can affect mitochondrial function by several means: selective scavenging mitochondria-driven ROS and reactive nitrogen species, by regulating gene expression of organelle-specific proteins, and modulating mitochondrial membrane potential and other functions (Fig. 19.1). Lin et al. (2015) demonstrated that HRW directly counteracts oxidative damage by neutralizing excessive ROS and stimulates AMPactivated protein kinase (AMPK) in a sirtuin 1 (SIRT1)-dependent pathway, which upregulates forkhead box protein O3a (FoxO3a) and diminishes mitochondrial potential loss induced by amyloid β-induced cytotoxicity. Dihydrogen can also reduce neuronal loss by stimulating the expression of BCL-2, an apoptosis regulatory protein, that further suppressed the activity of voltage-dependent anion-selective channel 1 and restrained the release of cytochrome c and caspase 9 activation, resulting in

FIGURE 19.1 Possible mechanisms and target pathways for dihydrogen to affect mitochondrial function. AMPK, andeosine monophospate kinase; ATP, adenosine triphosphate; BCL-2, B-cell lymphoma 2 protein; CASP-3, caspase-3; ERC, electron transport chain; FGF21, fibroblast growth factor 21 gene; MFN2, mitofusin-2 gene; MPTO, mitochondrial permeability transition pores; Mt, mitochondrial; p38, p38 mitogen-activated protein kinases; ROS, reactive oxygen species.

19.6 Open questions and future research

ameliorated cell viability and optimization of mitochondrial functions (Mo et al., 2019). Another paper confirmed BCL-2 and caspase-3 as possible organelle-specific targets, with hydrogen-rich saline markedly increasing the antioxidant potential of mitochondria, as evidenced by elevated ATP levels, mitochondrial respiratory function, and increased levels of active BCL-2 (Liu et al., 2016) and caspase-3 activation (Li et al., 2017). Hydrogen gas also increased the mitochondrial membrane potential and the cellular ATP level, which were accompanied by a decrease in the reduced glutathione level and an increase in the superoxide level in cultured human neuroblastoma SH-SY5Y cells (Murakami et al., 2017), implying that H2 might act as a radical scavenger and a mitohormetic effector against oxidative stress. Dihydrogen exhibited substantial inhibitory activity against LPS-initiated NLRP3 inflammasome activation by scavenging mitochondrial ROS (mtROS), with mtROS elimination further inhibiting NLRP3 deubiquitination, a non-transcriptional priming signal of NLRP3 in response to the stimulation of LPS (Ren et al., 2016). Finally, Gvozdja´kova´ et al. (2020) demonstrated that exogenous dihydrogen resulted in stimulated rat cardiac mitochondrial electron respiratory chain function and increased levels of ATP production by Complex I and Complex II substrates., with H2 being a donor of both electrons and protons in the Q-cycle and stimulating coenzyme Q10 production. A possibility that dihydrogen affects mitochondrial bioenergetics has been suggested (Ostojic, 2017), with energy-modulating effects of H2 including ghrelin-related upregulation of ghrelin receptor (GHS-R1α), ghrelin- and non-ghrelin related activation of glucose transporters (GLUT1 and GLUT4), and non-ghrelin related enhanced expression of fibroblast growth factor 21 (FGF21).

19.6 Open questions and future research Several issues still remain unresolved concerning the use of dihydrogen in mitochondrial nutrition. For instance, no studies so far described intra-mitochondrial biodynamics of exogenous molecular hydrogen, from its uptake through a twolayer mitochondrial membrane, to distribution and utilization in the intermembrane space and the matrix, to biotransformation and removal from the organelle. Further, no primary molecular target for dihydrogen inside the mitochondria has been identified so far, although many potential mechanisms are proposed and discussed above, including possible crosstalk among various signaling pathways (Barancik et al., 2020). Limited information has been provided regarding the possible impact of dihydrogen on the regulation of mitochondrial biogenesis and degradation, a key feature for the interaction of the organelle with exogenous agents. If H2 affects mitochondrial function in different cells and tissues also remains unknown at the moment, with interventional studies mainly focused to myocytes, hepatocytes, and neurons while other cells rich in mitochondria (e.g., adipocytes, hormone-secreting cells) stay mainly off the research course. A dose-response effect of dihydrogen in mitochondrial nutrition remains largely unknown,

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although previous reports suggested an intriguing phenomena that the amount of administered H2 is independent of the magnitude of its effects (Ohta, 2012). Future studies should also consider a possible lack of endogenous hydrogen in the pathogenesis of primary and secondary mitochondrial diseases (Ostojic, 2017), and take into account endogenous production of H2 for a net effect of dietary hydrogen in various disease models and clinical trials (Ostojic, 2020). How dietary dihydrogen influences other mitochondria-targeted therapeutics is currently unclear but raises the prospect that H2 may act synergistically in conditions with possible mitochondrial dysfunction (Ono et al., 2011; Ishibashi et al., 2012; Xia et al., 2013).

19.7 Conclusion Dihydrogen emerges as a low-cost, convenient and safe dietary bioactive compound that demonstrates promising results in tackling mitochondrial function. From improving surrogate biomarkers of mitochondrial function to ameliorating the pathology of mitochondrial disorders, H2 shows beneficial effects in experimental and clinical nutrition, although the preliminary trials are largely preclinical, small-sampled, and short-to-medium in duration. Future studies are thus highly warranted to corroborate possible advantages of feeding mitochondria with dihydrogen, using a mechanistic approach in longitudinal well-sampled research targeting various mitochondrial disorders.

References Barancik, M., Kura, B., LeBaron, T. W., Bolli, R., Buday, J., & Slezak, J. (2020). Molecular and cellular mechanisms associated with effects of molecular hydrogen in cardiovascular and central nervous systems. Antioxidants (Basel), 9(12), E1281. Available from https://doi.org/10.3390/antiox9121281. Dole, M., Wilson, F. R., & Fife, W. P. (1975). Hyperbaric hydrogen therapy: A possible treatment for cancer. Science (New York, N.Y.), 190(4210), 152
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Fucoxantin and mitochondrial uncoupling protein 1 in obesity

20 Chunhong Yan

School of Food Science and Technology, Dalian Polytechnic University, Dalian, Liaoning, China

The global prevalence of obesity has increased dramatically in the last two decades and represents a serious public health problem. The World Health Organization (WHO) has defined obesity as “abnormal or excessive fat accumulation that may impair health.” Obesity occurs when energy assimilation exceeds energy expenditure, and the consequence is the net accumulation of energy stores as fat in the adipose tissue. A western lifestyle, which is characterized by a highcalorie low-fiber diet and the lack of physical activity, contributes to the fast growth of obesity. WHO data show that worldwide obesity has nearly tripled between 1975 and 2016, this malady continues to grow, and it is estimated that approximately 57.8% of the adult population throughout the world could be obese or overweight by 2030. Obesity is known to increase the risks for noncommunicable disease like diabetes, cardiovascular diseases, and certain types of cancer. How could we reduce obesity? It is clear that regulating either side of the energy balance equation could be of great help. Multiple lifestyle interventions, including an active lifestyle and whole grain consumption are highly recommended. Some medications have been developed for obesity. For example, orlistat is approved by the Food and Drug Administration for use in adolescent and adult obesity, for the reason that it inhibits intestinal lipase and reduces lipid absorption from gut. However, safety concerns and side effects limited its application (Yanovski & Yanovski, 2021). Food-derived bioactive components are usually considered safe to use and possess pharmaceutical properties in preventing and reducing obesity (Osuna-Prieto et al., 2021). Several mechanisms are involved for these substances to exert their function, one mechanism is through increasing mitochondrial respiration by stimulating uncoupling protein 1 (UCP1) activity in adipose tissue. This chapter will summarize the studies of UCP1-dependent thermogenesis in an antiobesity application and will be focused on fucoxanthin, a marine carotenoid, and its role in enhancing the expression and activity of UCP1 to combat obesity.

Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00018-7 © 2023 Elsevier Inc. All rights reserved.

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20.1 Three types of adipocytes Adipose tissue was historically thought to be merely a storehouse for lipid, however it has been proven to be a highly dynamic organ and has a wide range of functions, including regulating food intake, fat and glucose metabolism, and immune response. Generally, mammals possess three types of adipocytes: white adipocytes, brown adipocytes, and beige adipocytes (Sanchez-Gurmaches et al., 2016). These three kinds of adipocytes have very different morphology and physiological functions. Table 20.1 White adipocytes contain a single large lipid droplet and few mitochondria, and their main function is to store lipid/energy. On the contrary, brown adipocytes are characterized by the presence of numerous small lipid droplets and abundant mitochondria containing UCP1. Their main purpose is to burn energy to generate heat during cold exposure. Beige adipocytes are theadipocytes residing within the white adipose tissue (WAT), and they are capable of becoming “brown” under certain conditions, such as cold exposure or beta-3adrenergic receptor (ADRB3) stimulation. Like brown adipocytes, beige adipocytes also have high UCP1 expression and active mitochondria to utilize system fuel to drive thermogenesis. Thermogenic adipocytes are known to have a central role in nonshivering thermogenesis (NST), a heat-generating process that prevents hypothermia without muscle shivering and that is activated by cold exposure. These mechanisms are also known to support the maintenance of energy balance by dissipating excess energy as heat (Cohen & Kajimura, 2021). The activities of brown and beige fat cells reduce obesity and related disease in mice and humans. For example, exposing individuals to cold temperatures increased brown adipose tissue (BAT) activity, altered serum free fatty acid profiles, and improved wholebody glucose homeostasis and insulin resistance (Chondronikola et al., 2014; Iwen et al., 2017). As prolonged cold exposure is not a realistic therapy, researchers worldwide are searching for novel strategies to activate thermogenic adipocytes. Dietary components serve as external stimuli for brown and beige adipocytes activation and may be developed as a promising nutrition intervention strategy for abating obesity and other metabolic syndromes (Okla et al., 2017).

Table 20.1 Characteristics of three adipocytes. Lipid droplets Mitochondria Function Location UCP1 Developmental origin Activator

White adipocytes

Beige adipocytes

Brown adipocytes

One large 2/ 1 Energy storage WAT
Adult


Few to many 11 Thermogenesis WAT
Adult and inducible Cold

Numerous 11 1 Thermogenesis BAT 1 Embryonic Cold

20.2 The importance of uncoupling protein 1

20.2 The importance of uncoupling protein 1 in regulating energy homeostasis UCP1 is a polypeptide that is located in the inner mitochondrial membrane of brown and beige adipocytes. The well-established function of the mitochondria is to provide ATP through the process of oxidative phosphorylation. By contrast, the mitochondria in brown or beige adipocytes generate heat by uncoupling the electron transport from ATP synthesis to generate heat. UCP1 short circuits the electron transport chain, allowing protons to flow through the inner membrane, which in turn decreases ATP/ADP ratio and upregulates the rate of respiration and oxygen consumption, see Fig. 20.1. Therefore, BAT is a highly metabolically active tissue and a critical regulator of metabolic health (Bartelt & Heeren, 2014). Genetic ablation UCP1 positive cell in mice resulted in obesity, hyperlipidemia, hyperglycemia and hyperinsulinemia, indicating brown and beige adipocytes play a key role in obesity and insulin resistance (Lowell et al., 1993). Through the use of 18F-fluoro-2-deoxy-d-glucose (18F-FDG) positron emission tomography computed tomography (18F-FDG-PET) imaging, scientists found a reduced amount of BAT in obesity and old subjects. We now know more details on how brown adipocytes affect the whole-body energy homeostasis. Glucose, lipid, and branched-chain amino acid (BCAA) oxidation provide essential fuel for thermogenic reactions in BAT. BAT actively takes up glucose via glucose transporter type 1 and type 4. Stimulation of beige adipocytes formation by β3-adrenergic receptors (β3-ARs) agonist improved insulin resistance and pancreatic β cell function in obese individuals (Finlin et al., 2020). A recent study demonstrated that

FIGURE 20.1 The UCP1-dependent thermogenesis. UCP1 functions as a proton carrier that allows proton re-entry into the matrix, thus dissipates the proton gradient generated by the electron transport chain. The futile cycle reduces mitochondrial membrane potential, which in turn accelerates respiration rates and produce heat.

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BAT is the main deposit for glucose clearance in microbiota depleted mice (Li et al., 2021). Fatty acids from lipolysis of triglycerides activate and fuel UCP1mediated thermogenesis in BAT, however, these fatty acids are liberated from lipid droplets in WAT, but not BAT. Studies showed that BAT-specific loss of adipose triglyceride lipase (ATGL) did not impair thermogenesis induced by the β3-ARs or cold exposure. Instead, global ATGL knockout mice or fat-specific (both BAT and WAT) depletion of ATGL or CGI58 attenuated thermogenesis (Schreiber et al., 2017; Shin et al., 2017). Cold also stimulated mitochondrial BCAA uptake and oxidation in BAT, which contributes to the systemic clearance of BCAA. In addition, a BCAA catabolism defect in BAT impaired NST and influenced whole-body metabolism, aggregated a high-fat diet-induced weight gain, insulin resistance, and hyperlipidemia (Yoneshiro et al., 2019). Hence, thermogenetic fat acts as a “metabolic sink” for glucose, fatty acids, and amino acids, and changed the whole-body energy homeostasis (Verkerke & Kajimura, 2021).

20.3 Fucoxanthin and uncoupling protein 1 UCP1-mediated thermogenesis is activated not only by cold, but also by certain food ingredients including capsaicin, resveratrol, curcumin, berberine, fucoxanthin and dietary fatty acids like fish oil and conjugated linoleic acids (Okla et al., 2017). Fucoxanthin is a marine carotenoid with antioxidant properties, and it is abundant in edible brown seaweeds. Its chemical structure contains a polyene chain with an epoxide group and a C-8 ketone, Fig. 20.2. Like most dietary components, fucoxanthin is considered safe with no sign of toxic effect at single dose of 2000 mg/kg or repeated dose of 1000 mg/kg for 30 days in mice (Beppu et al., 2009). Fuxocanthin has been extensively studied for its antiobesity and UCP1 inducing effects in rodents. Supplementation of fuxocanthin (2% w/w) for six weeks significantly lowered a high-fat diet-induced body weight gain and visceral fat pad weight increase in mice without affecting food intake, indicating an augment of energy expenditure. Fucoxanthin increased

FIGURE 20.2 The chemical structure of fucoxanthin.

20.3 Fucoxanthin and uncoupling protein 1

ucp1 mRNA level in epididymal adipose tissue concomitantly with altered lipogenesis enzyme and fatty acid oxidation enzyme level (Maeda et al., 2005; Woo et al., 2009). Other studies using different sources confirmed that fucoxanthin can improve energy expenditure, fatty acid oxidation, and adipogenesis by upregulating UCP1, peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, peroxisome proliferator-activated receptor α, and γ (PPARγ) in diet-induced obese mice (Gille et al., 2019; Grasa-Lo´pez et al., 2016; Koo et al., 2019). In cultured 3T3-L1 cells, fucoxanthin increased UCP1 expression and inhibited adipogenesis by downregulating PPARγ. Studies also suggest fucoxanthin promotes beige adipogenesis through activating ADRB3 signaling and had a profound effect on lipid metabolism (Maeda et al., 2009). In contrast to the large amount of information on the influence of fucoxanthin on BAT and beige adipocytes (gained from experiments in rodents), there are still limited human studies focusing on fucoxanthin and obesity. Abidov et al. investigated the effect of Xanthigen, fucoxanthin dissolved in pomegranate oil, on the obese nondiabetic women. Xanthigen (600 mg/day, including fucoxanthin 2.4 mg/day for 16 weeks) resulted in body weight and body fat lose, a decrease in serum triglycerides and an increase in resting energy expenditure. Fucoxanthin supplementation also downregulated liver fat content, liver enzymes, and inflammatory marker C-reaction protein, suggesting that fucoxanthin is an encouraging food supplement for obese individuals (Abidov et al., 2010). Another clinical trial showed that fucoxanthin can reduced hemoglobin A1c level in obese adults and it is related to the thrifty allele of UCP1, indicating that fucoxanthin has positive effect on hyperglycemia (Mikami et al., 2017). However, one study focused on the browning effect in vitro cultured human adipocytes found that neither fucoxanthin nor its metabolites fucoxanthinol could alter the expression of UCP1, carnitine palmitoyltransferase 1 β, GLUT4, and PGC1a, the genes associated with browning of adipose tissue. Furthermore, there is no change in the oxygen consumption rate, indicating fucoxanthin and its metabolite did not promote mitochondrial biogenesis and stimulate human adipocyte browning (Rebello et al., 2017). However, it should be noted that the dose of fucoxnathin and fucoxanthinol utilized in this study is much lower than that used in rodent 3T3-L1 cells (1 μM vs 40 μM). Studies also indicated that there are some differences in metabolic processes between rodents and humans. Amarouciaxanthin A, the liver metabolites, is the predominant form in the blood and various organs of mice administered with fucoxanthin, which could not be detected in plasma metabolites in humans who consume brown algae or its extract (Hashimoto et al., 2012). Since 18 F-FDG-PET/CT and MRI are now available to detect the presence of human BAT depots (Chen et al., 2016), it is possible to provide reliable data on the effect of fucoxanthin on human BAT. Further studies are needed to pinpoint the mechanism by which fucoxanthin activate BAT. The effect and mechanism of fucoxanthin on WAT browning still need to be elucidated, including whether fucoxanthin acts on adipose cells directly or through stimulating sympathetic nerve system activity. Progress in this field may provide a new therapeutic avenue to promote metabolic health and reduce obesity.

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References Abidov, M., Ramazanov, Z., Seifulla, R., & Grachev, S. (2010). The effects of Xanthigen in the weight management of obese premenopausal women with non-alcoholic fatty liver disease and normal liver fat. Diabetes, Obesity & Metabolism. Available from http://www.ncbi.nlm.nih.gov/pubmed/19840063. Bartelt, A., & Heeren, J. (2014). Adipose tissue browning and metabolic health. Nature Reviews. Endocrinology. Available from http://www.ncbi.nlm.nih.gov/pubmed/ 24146030. Beppu, F., Niwano, Y., Tsukui, T., Hosokawa, M., & Miyashita, K. (2009). Single and repeated oral dose toxicity study of fucoxanthin (FX), a marine carotenoid, in mice. The Journal of Toxicological Sciences. Available from http://www.ncbi.nlm.nih.gov/ pubmed/19797858. Chen, K. Y., Cypess, A. M., Laughlin, M. R., Haft, C. R., Hu, H. H., Bredella, M. A., Enerba¨ck, S., Kinahan, P. E., Lichtenbelt, W. v M., Lin, F. I., Sunderland, J. J., Virtanen, K. A., & Wahl, R. L. (2016). Brown Adipose Reporting Criteria in Imaging STudies (BARCIST 1.0): Recommendations for standardized FDG-PET/CT experiments in humans. Cell Metabolism. Available from http://www.ncbi.nlm.nih.gov/ pubmed/27508870. Chondronikola, M., Volpi, E., Børsheim, E., Porter, C., Annamalai, P., Enerba¨ck, S., Lidell, M. E., Saraf, M. K., Labbe, S. M., Hurren, N. M., Yfanti, C., Chao, T., Andersen, C. R., Cesani, F., Hawkins, H., & Sidossis, L. S. (2014). Brown adipose tissue improves whole-body glucose homeostasis and insulin sensitivity in humans. Diabetes. Available from http://www.ncbi.nlm.nih.gov/pubmed/25056438. Cohen, P., & Kajimura, S. (2021). The cellular and functional complexity of thermogenic fat. Nature Reviews. Molecular Cell Biology. Available from http://www.ncbi.nlm.nih. gov/pubmed/33758402. Finlin, B. S., Memetimin, H., Zhu, B., Confides, A. L., Vekaria, H. J., El Khouli, R. H., Johnson, Z. R., Westgate, P. M., Chen, J., Morris, A. J., Sullivan, P. G., DupontVersteegden, E. E., & Kern, P. A. (2020). The β3-adrenergic receptor agonist mirabegron improves glucose homeostasis in obese humans. The Journal of Clinical Investigation. Available from http://www.ncbi.nlm.nih.gov/pubmed/31961829. Gille, A., Stojnic, B., Derwenskus, F., Trautmann, A., Schmid-Staiger, U., Posten, C., Briviba, K., Palou, A., Bonet, M. L., & Ribot, J. (2019). Phaeodactylum tricornutum A lipophilic fucoxanthin-rich extract ameliorates effects of diet-induced obesity in C57BL/ 6J mice. Nutrients. Available from http://www.ncbi.nlm.nih.gov/pubmed/30959933. ´ ., Quevedo-Corona, L., Paniagua-Castro, N., EscalonaGrasa-Lo´pez, A., Miliar-Garcı´a, A Cardoso, G., Reyes-Maldonado, E., & Jaramillo-Flores, M.-E. (2016). Undaria pinnatifida and fucoxanthin ameliorate lipogenesis and markers of both inflammation and cardiovascular dysfunction in an animal model of diet-induced obesity. Marine Drugs. Available from http://www.ncbi.nlm.nih.gov/pubmed/27527189. Hashimoto, T., Ozaki, Y., Mizuno, M., Yoshida, M., Nishitani, Y., Azuma, T., Komoto, A., Maoka, T., Tanino, Y., & Kanazawa, K. (2012). Pharmacokinetics of fucoxanthinol in human plasma after the oral administration of kombu extract. The British Journal of Nutrition. Available from http://www.ncbi.nlm.nih.gov/pubmed/21920061. Iwen, K. A., Backhaus, J., Cassens, M., Waltl, M., Hedesan, O. C., Merkel, M., Heeren, J., Sina, C., Rademacher, L., Windja¨ger, A., Haug, A. R., Kiefer, F. W., Lehnert, H., &

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Schmid, S. M. (2017). Cold-induced brown adipose tissue activity alters plasma fatty acids and improves glucose metabolism in men. The Journal of Clinical Endocrinology and Metabolism. Available from http://www.ncbi.nlm.nih.gov/pubmed/28945846. Koo, S. Y., Hwang, J.-H., Yang, S.-H., Um, J.-I., Hong, K. W., Kang, K., Pan, C.-H., Hwang, K. T., & Kim, S. M. (2019). Phaeodactylum tricornutumanti-obesity effect of standardized extract of microalga containing fucoxanthin. Marine Drugs. Available from http://www.ncbi.nlm.nih.gov/pubmed/31137922. Li, M., Li, L., Li, B., Hambly, C., Wang, G., Wu, Y., Jin, Z., Wang, A., Niu, C., Wolfrum, C., & Speakman, J. R. (2021). Brown adipose tissue is the key depot for glucose clearance in microbiota depleted mice. Nature Communications. Available from http://www. ncbi.nlm.nih.gov/pubmed/34354051. Lowell, B. B., S-Susulic, V., Hamann, A., Lawitts, J. A., Himms-Hagen, J., Boyer, B. B., Kozak, L. P., & Flier, J. S. (1993). Development of obesity in transgenic mice after genetic ablation of brown adipose tissue. Nature. Available from http://www.ncbi.nlm. nih.gov/pubmed/8264795. Maeda, H., Hosokawa, M., Sashima, T., Funayama, K., & Miyashita, K. (2005). Fucoxanthin from edible seaweed, Undaria pinnatifida, shows antiobesity effect through UCP1 expression in white adipose tissues. Biochemical and Biophysical Research Communications. Available from http://www.ncbi.nlm.nih.gov/pubmed/15896707. Maeda, H., Hosokawa, M., Sashima, T., Murakami-Funayama, K., & Miyashita, K. (2009). Anti-obesity and anti-diabetic effects of fucoxanthin on diet-induced obesity conditions in a murine model. Molecular Medicine Reports. Available from http://www.ncbi.nlm. nih.gov/pubmed/21475918. Mikami, N., Hosokawa, M., Miyashita, K., Sohma, H., Ito, Y. M., & Kokai, Y. (2017). Reduction of HbA1c levels by fucoxanthin-enriched akamoku oil possibly involves the thrifty allele of uncoupling protein 1 (UCP1): A randomised controlled trial in normalweight and obese Japanese adults. Journal of Nutritional Science. Available from http://www.ncbi.nlm.nih.gov/pubmed/28620480. Okla, M., Kim, J., Koehler, K., & Chung, S. (2017). Dietary factors promoting brown and beige fat development and thermogenesis. Advances in Nutrition (Bethesda, MD). Available from http://www.ncbi.nlm.nih.gov/pubmed/28507012. Osuna-Prieto, F. J., Martinez-Tellez, B., Segura-Carretero, A., & Ruiz, J. R. (2021). Activation of brown adipose tissue and promotion of white adipose tissue browning by plant-based dietary components in rodents: A systematic review. Advances in Nutrition (Bethesda, MD). Available from http://www.ncbi.nlm.nih.gov/pubmed/34265040. Rebello, C. J., Greenway, F. L., Johnson, W. D., Ribnicky, D., Poulev, A., Stadler, K., & Coulter, A. A. (2017). Fucoxanthin and its metabolite fucoxanthinol do not induce browning in human adipocytes. Journal of Agricultural and Food Chemistry. Available from http://www.ncbi.nlm.nih.gov/pubmed/29172481. Sanchez-Gurmaches, J., Hung, C.-M., & Guertin, D. A. (2016). Emerging complexities in adipocyte origins and identity. Trends in Cell Biology. Available from http://www.ncbi. nlm.nih.gov/pubmed/26874575. Schreiber, R., Diwoky, C., Schoiswohl, G., Feiler, U., Wongsiriroj, N., Abdellatif, M., Kolb, D., Hoeks, J., Kershaw, E. E., Sedej, S., Schrauwen, P., Haemmerle, G., & Zechner, R. (2017). Cold-induced thermogenesis depends on ATGL-mediated lipolysis in cardiac muscle, but not brown adipose tissue. Cell Metabolism. Available from http://www.ncbi.nlm.nih.gov/pubmed/28988821.

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Shin, H., Ma, Y., Chanturiya, T., Cao, Q., Wang, Y., Kadegowda, A. K. G., Jackson, R., Rumore, D., Xue, B., Shi, H., Gavrilova, O., & Yu, L. (2017). Lipolysis in brown adipocytes is not essential for cold-induced thermogenesis in mice. Cell Metabolism, 26 (5), 764
777. Available from https://doi.org/10.1016/j.cmet.2017.09.002, e5. Verkerke, A. R. P., & Kajimura, S. (2021). Oil does more than light the lamp: The multifaceted role of lipids in thermogenic fat. Developmental Cell. Available from http:// www.ncbi.nlm.nih.gov/pubmed/34004150. Woo, M.-N., Jeon, S.-M., Shin, Y. C., Lee, M.-K., Kang, M. A., & Choi, M.-S. (2009). Antiobese property of fucoxanthin is partly mediated by altering lipid-regulating enzymes and uncoupling proteins of visceral adipose tissue in mice. Molecular Nutrition & Food Research. Available from http://www.ncbi.nlm.nih.gov/pubmed/19842104. Yanovski, S. Z., & Yanovski, J. A. (2021). Progress in pharmacotherapy for obesity. JAMA: The Journal of the American Medical Association. Available from http://www. ncbi.nlm.nih.gov/pubmed/34160571. Yoneshiro, T., Wang, Q., Tajima, K., Matsushita, M., Maki, H., Igarashi, K., Dai, Z., White, P. J., McGarrah, R. W., Ilkayeva, O. R., Deleye, Y., Oguri, Y., Kuroda, M., Ikeda, K., Li, H., Ueno, A., Ohishi, M., Ishikawa, T., Kim, K., & Kajimura, S. (2019). BCAA catabolism in brown fat controls energy homeostasis through SLC25A44. Nature. Available from http://www.ncbi.nlm.nih.gov/pubmed/31435015.

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21

Rice bran extract for the prevention of mitochondrial dysfunction

Nancy Saji1, Boris Budiono1,2, Nidhish Francis2,3, Christopher Blanchard1,2 and Abishek Santhakumar1,2 1

School of Dentistry and Medical Sciences, Faculty of Science and Health, Charles Sturt University, Wagga Wagga, NSW, Australia 2 Gulbali Research Institute, Charles Sturt University, Wagga Wagga, NSW, Australia 3 School of Agricultural, Environmental and Veterinary Sciences, Faculty of Science and Health, Charles Sturt University, Wagga Wagga, NSW, Australia

21.1 Introduction Rice (Oryza sativa) is a cereal grain agricultural commodity with the thirdhighest worldwide production. The majority of rice is processed to remove the husk, bran, and germ layers and polished to produce the commercially preferred white rice. As a staple grain in many parts of the world, particularly in Asia, while white rice is a significant source of carbohydrates with higher glycemic index, it is nutritionally deficient compared to its whole grain form or brown rice. Furthermore, there is evidence that consumption of white rice as a possible contributor to the rise of lifestyle- or age-related chronic disease such as type 2 diabetes and metabolic syndrome (Hu et al., 2012; Krittanawong et al., 2017). Rice bran, however, is highly nutritious in chemical composition, containing 12%
17% protein, 13%
23% fat, 34%
54% carbohydrates, 6%
14% fiber and 8%
18% ash (Kalpanadevi et al., 2018). Although a wide range of nutritional components are present in rice bran, much of it is currently discarded or used as animal feed owing to postmilling handling and processing difficulties. This chapter presents results of numerous studies that utilize rice bran in the prevention and treatment of several disease models holding the potential of its utility in human clinical trials in the future. The information is presented in two ways: (1) in the context of the effects rice bran has on mitochondrial function in terms of its ability to modulate its protective mechanisms, and (2) in the context of the nutritional components of rice bran. The chapter concludes with a discussion on the current state of the research field, and how future studies can bridge the gap between experimental models to the potential of widespread utilization of rice bran for its nutritional and mitochondrial disease-preventing effects.

Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00022-9 © 2023 Elsevier Inc. All rights reserved.

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21.2 Role of mitochondrial function in disease Mitochondria are important organelles responsible for generating energy in the form of adenosine triphosphate (ATP) and heat, but also in facilitating other biosynthetic reactions and homeostatic functions. They are essential for energy metabolism, cell cycle control, cell development, regulation of cell death mechanisms and in immunity through the central role in regulating reactive oxygen species (ROS) (Mills et al., 2017; Oh et al., 2020). Since mitochondrial DNA lack the structural protection of histones (proteins that shield DNA from free radical damage) and their repair mechanisms, they are quite easily susceptible to damage (Brand & Nicholls, 2011). The mitochondria are an interesting therapeutic target as it has an integral role in both cellular and whole-organism level homeostasis. Mitochondrial dysfunction was first described in the 1960s and since then, understanding the role mitochondria play in health, disease, and aging has advanced. It is implicated in most common noncommunicable chronic diseases (Diaz-Vegas et al., 2020); and the prevalence of these diseases has increased dramatically over the past few decades, mainly due to sedentary lifestyle and increased consumption of processed and refined foods (Carrera-Bastos et al., 2011; Chapman-Kiddell et al., 2010). Previously, a wide range of seemingly unrelated disorders, such as cardiovascular disease (CVD), type 2 diabetes (T2DM), inflammatory bowel disease (IBD), Alzheimer’s and Parkinson’s disease have now been noted to have common underlying pathophysiological mechanisms which are ROS production and accumulation of mitochondrial DNA damage, resulting in mitochondrial dysfunction (Pieczenik & Neustadt, 2007). In the body, oxidative stress is normally tightly regulated by the production and/or removal of ROS, which are natural byproducts of metabolism. However, a compromised antioxidant defense system can lead to excess production of ROS resulting in cell damage.

21.3 Rice bran extracts and the mitochondria There have been numerous studies that investigate the benefits of rice bran extracts or compounds in the prevention and treatment of these conditions, such as CVD, metabolic diseases such as diabetes, neurodegenerative diseases, inflammatory conditions, and cancer (Peacenik & Neustadt, 2007). In a Caenorhabditis elegans model, rice bran peptide KF-8 improved both health- and lifespan, with the primary action of the protein acting as a scavenger of free radicals, via skn-1, an ortholog of mammalian NRF-2. NRF-2, which along with its antioxidant protection, is also controlled by the master regulator of mitochondrial biogenesis, PGC1α activating mitochondrial transcription factor A (TFAM), and inducing Mitochondrial DNA (mtDNA) replication and transcription (Cai et al., 2022, Gureev et al., 2019). Various other flavones and polyphenols (such as ferulic

21.3 Rice bran extracts and the mitochondria

acid, syringic acid) found in rice bran have shown their propensity to induce mitochondrial biogenesis, at least at a transcriptional level (Saji, Francis, et al., 2020, Perez-Ternero et al., 2017, Sabahi et al., 2020). Normally, mitochondrial size and number are dictated by the stresses of the body’s metabolic demands. For example, the increased need for oxidative capacity through stress or environmental stimuli (e.g., exercise) increases mitochondrial biogenesis (Baar et al., 2002), and this is tempered by other mechanisms such as mitochondrial fission and fusion, and mitophagy/autophagy to ensure mitochondrial and overall metabolic homeostasis (Chodari et al., 2021). Another homeostatic mechanism is the tight regulation of oxidative stress, by production and/or removal of ROS, which are natural by-products of metabolism. However, a compromised antioxidant defense system can lead to excess production of ROS resulting in cell damage. Purple rice bran has been shown to reverse the hyperglycemic effects of T2DM in a rat model, with unique proteins related to insulin sensitivity negating upregulation in proteins related to fatty acid synthesis (Hlaing et al., 2017). In a later study of diabetic rats with neuropathy, Wongmekiat et al., showed the modulation of PGC1α/SIRT3/SOD protein signaling pathway as key in preventing mitochondrial swelling, ROS production, and decreased lipid peroxidation (Wongmekiat et al., 2021). SIRT3 as a downstream effector of PGC1α activates superoxide dismutase (SOD) as a major mitochondrial antioxidant enzyme (Zhang et al., 2020). Common methods used to study mitochondrial bioenergetics will often utilize chemical permeabilizers that allow electron transport chain complexes to be individually interrogated with specific mitochondrial inhibitors and uncouplers. This allows a quantification of cellular respiration or “high resolution respirometry” of mitochondria in tissue homogenates or cultured cells (Hagl, Berressem, et al., 2015, Hagl, Grewal, et al., 2015). Dysfunctional mitochondria can result in reactive oxido/nitrosative stresses (such as the release of hydrogen peroxide (H2O2), superoxide, or peroxynitrite) that lead to damage to the structures of the organelle, the DNA, and the integrity of the cell itself. mtDNA is more susceptible to damage than nuclear DNA; but both are required for the integrity of the complexes of the electron transport chain. Accumulated damage can affect the bioenergetics of ATP production, leading to further oxidative stress and leakage. Dysfunction is not merely due to damaged mitochondria but may be the result of a “mismatch” between metabolic requirements of the cell, and activities of the organelle itself (Diaz-Vegas et al., 2020). This is best exemplified in animal cancer or ischemiareperfusion/hypoxia models where perturbations in metabolic demands result in multiple pathologies that the mitochondria are central to (Kong et al., 2009; Sun et al., 2009; Du et al., 2021). One study investigated the use of γ-Oryzanol, a bioactive component of rice bran oil (RBO), to reduce the effect of hepatic ischemia-reperfusion injury in C57BL/6 mice. Through inhibition of the HMGB1/ NLRP3 inflammasome and endoplasmic reticulum stress, the group was able to present reductions in oxidative stress, inflammation, and apoptosis with the use of the compound (Du et al., 2021). This same compound has also shown benefits in

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the reversal of pathogenic progression in a drosophila model of Parkinson’s disease (Araujo et al., 2015). Furthermore, the mitochondria are involved in cell death processes, both internally and externally, from survival mechanisms of mitophagy and autophagy, the ability to induce mitochondrial fission and fusion, and of course the induction or reduction of apoptotic mechanisms. Xiao et al., showed rice bran phenolic extract alleviated mitochondrial dysfunction in alcoholic liver disease by increasing hepatic ATP production, decreasing apoptotic markers, and augmenting mitochondrial function via the PGC-1alpha-TFAM pathway via activation of several micro-RNAs (Xiao et al., 2020). In terms of mitochondrial dynamics, fission and fusion of the organelle are important to maintain the integrity while undergoing metabolic or environmental stresses, with the former being integral to the creation of new mitochondria (biogenesis) and quality control (removal of damaged or old components), with the latter involved in the consolidation of metabolic integrity in the organelle when undergoing stress (Youle & Van Der Bliek, 2012). Rice bran extract was shown to increase mitochondrial fission in aged guinea pig brains via gene expression of Drp1 (dynamin-related protein 1) and fis1 (fission 1) to prevent age-related mitochondrial dysfunction (Hagl et al., 2013). MGN-3/Biobran, a modified arabinoxylan has been shown to sensitize human leukemia cells to apoptosis by increased activation of caspases (but not through CD95/Fas), leading to increased depolarization of the mitochondrial membrane potential (Ghoneum & Gollapudi, 2003). The same group utilized the same compound on the liver carcinogenesis rat model, equally showing a downregulation of antiapoptotic Bcl-2, but an upregulation of proapoptotic genes and proteins (p53, Bax and caspase-3) that caused mitochondrial membrane permeabilization and apoptosis in cancer cells (El-Din et al., 2020). The same compound decreased enzymatic activities of caspases to effectively attenuate radiation-induced changes in the oxidative stress of the intestinal epithelial mitochondria and improve the activity of jejunal and colonic mitochondrial respiratory chain complex activity by increasing mitochondria-encoded genes (Zhao et al., 2020). Similar effects were observed in a sporadic Alzheimer’s disease model with MGN-3 with reductions in inflammatory cytokines, and the induction of nuclear factor erythroid 2-related factor 2 (Nrf2), and antioxidant response element to provide protection against β-amyloid induced apoptosis (Ghoneum & El Sayed, 2021). Tocotrienolrich fractions from rice bran also alleviated ionizing radiation and H2O2 by preserving cell and mitochondrial morphology, ensuring the integrity of electron transport chain function (by reversing mitochondrial uncoupling), and possibly augmenting mitochondrial biogenesis (Krager et al., 2015). Antiallergic and anti-inflammatory effects of tricin, a flavone found in rice bran, was also observed on RBL-2H3 cells by preventing degranulation and attenuation of MAPK signaling (Lee et al., 2020) and preventing inflammation in mouse colon tissues through the NFκβ pathway and modulating the gut microbiota profile (Li et al., 2021). The nutraceutical benefits of rice bran are believed to prevent sensitization of oxido/nitrosative stress.

21.4 Health properties of rice bran constituents

Antioxidant capacity within the fats, proteins, and carbohydrates found in rice bran can directly bind and activate effector receptors. Another common mitochondrially-focused target of rice bran derived compounds, are peroxisome proliferator-activated receptors (PPARs), of which the primary function is to act as transcription factors regulating functions of genes (Michalik et al., 2006). PPARγ regulate fatty acid storage and glucose metabolism by stimulating lipid uptake and adipogenesis in fat cells, but in relation to the mitochondria, it results in increases in mitochondrial biogenesis, oxygen respiration, antioxidant defenses, regulation of autophagy, and reduces inflammation (Corona & Duchen, 2016). Rice bran phenolics increase PPARγ and improve gut microbiota in HFD mice (Zhao et al., 2022); and in another study, reduced LPS-induced neuroinflammation (Abd El Fattah et al., 2021). γ-Oryzanol directly binds to PPARδ to improve exercise endurance and strength, reducing inflammatory cytokines and augmenting mitochondrial biogenesis in an aged mouse model (Ahn & Cho, 2020). Most studies involving rice bran constituents and their effects on the mitochondria were observational in nature and performed in animal disease models (primarily rodents). Further work is required to: elucidate the direct and indirect pathways affected by rice bran, both as a whole extract, and when processed or distilled into specific polyphenols, peptides, or lipids. Some compounds may be difficult to be used clinically due to issues such as the expense of purification methods or low yields from rice bran, however strategies such as using a simpler distillate may reduce costs and improve yields (Zaky et al., 2022; Fabian & Ju, 2011; Krager et al., 2015).

21.4 Health properties of rice bran constituents associated with mitochondrial function 21.4.1 Proteins, nonproteogenic amino acids and derivatives Proteins present in rice bran are mostly storage proteins, that is, albumin, globulin, prolamin, and glutelin that are sequestered within the cell in dense deposits called protein bodies. They store nitrogen, carbon, and sulfur, which are essential for optimal grain growth. Besides their function as nutrients for rice production, they also have beneficial health properties, however this is tempered by the relative cost and expertise required to extract these proteins (Fabian & Ju, 2011).

21.4.2 Fats and oils Fats and oils present in rice bran also serve as a highly nutritious supplement, but its use as a food ingredient is limited due to the production of free fatty acids that lead to rancidity. In paddy rice, lipases reside in the seed coat (tegmen), with most of the oil being localized in the aleurone and germ layer. During milling, when the bran layers are removed from the endosperm, the individual cells are

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disrupted resulting in hydrolysis of fats into free fatty acids and glycerol by lipase enzymes. Within a short period of time, the bran becomes unsuitable for human consumption owing to its reduced pH, rancid flavor, and undesirable taste as the free fatty acids level increases postmilling (Yılmaz et al., 2014). However, this enzymatic degradation can be inhibited by various stabilization methods such as microwave heating, oven-drying, extrusion, and drum-drying (Saji, Schwarz, et al., 2020), which results in a longer shelf life of the product. Oxidative damage to lipids, proteins, and DNA in the central nervous system are recognized as key elements that contribute to neurological deterioration leading to diseases such as Parkinson’s and Alzheimer’s (Jiang et al., 2016). Impairment of lipids leads to damage in the membrane fluidity and permeability, whereas specific residues such as cysteine or selenocysteine are the target of oxidation, which often leads to either a loss of function or protein aggregation and DNA damage by oxidative stress. This alters its coding properties or interferes with normal metabolic function, and this damage occurs most readily at guanine bases (Jiang et al., 2016). Thus, prevention of excess ROS formation is crucial in the management of these diseases. An investigation into the consumption of γ-oryzanol from rice bran oil (RBO) in Drosophila melanogaster flies (n 5 50) for seven days was performed where the flies were concomitantly exposed to either a diet containing toxicity induced by rotenone or γ-oryzanol. The results from this study showed that γ-oryzanol consumption not only acts as an endogenous activator of the cellular antioxidant defenses, but it also ameliorates rotenone-induced mortality, oxidative stress, and mitochondrial dysfunction. This study also noted restoration of cholinergic deficits, dopamine levels, and improved motor function provided by γ-oryzanol (Araujo et al., 2015). There have also been benefits reported of RBO on both LPS-stimulated RAW 264.7 cells and mouse models demonstrate that RBO regulated inflammatory responses (decreasing COX-2 and iNOS and increasing IL-10) by increasing mitochondrial respiration (Lee et al., 2019).

21.4.3 Carbohydrates The major types of carbohydrates present in rice bran are cellulose, hemicellulose, lignin, and starch (Satter et al., 2014; Wu et al., 2021). Cellulose is composed of long cellobiose unbranched chains of D-anhydroglucose units with β-(1,4)-glycosidic bonds, that form the crystalline region of the rice bran cell wall. It is embedded in a matrix of hemicellulose and lignin. Hemicellulose is defined as a heterogeneous group of polysaccharides, characterized by β-(1 - 4)-linked backbones of sugars in an equatorial configuration whereas, lignin is an amorphous polyphenol polymer. A strong potential for using rice bran-derived cellulose in water purification against metal pollutants such as cadmium that poses high risk to humans, have been found (Wu et al., 2021). Moreover, dietary fibers are nondigestible residues of plants that cannot be digested in the small intestine but can be completely or partially fermented in the large intestine (Maani et al., 2017). These dietary fibers are rich in bound-form phenolics that have shown several

21.4 Health properties of rice bran constituents

beneficial health properties against diabetes, CVD, and cancers (Zhang et al., 2019). Investigation into the antihyperlipidemic effect of rice bran polysaccharides in a high fat diet animal model, showed a significant reduction in the body weight, liver weight, and adipose tissues of mice after ten weeks. Moreover, decreased levels of total cholesterol, triglycerides, and low density lipoprotein
cholesterol in the plasma as well as regulation of several genes associated with lipid metabolism were also detected (Nie et al., 2017).

21.4.4 Fiber Although the precise cause of IBD remains unclear, it is thought that various factors, including genetics, level of immunity, and environmental factors can cause disruption in intestinal homeostasis, leading to dysregulated inflammatory responses of the gut, resulting in IBD (Kim et al., 2012). Rice bran in the form of a new prebiotic, developed using heat-resistant amylase, protease, and a hemicellulose treatment called enzyme-treated rice fiber, has shown reduced inflammation by modulating the colonic environment and regulating immune cell differentiation in rats (Komiyama et al., 2011). Similarly, another study in dextran sodium sulfate-induced colitis in mice also showed that dietary consumption of fermented rice bran attenuates intestinal inflammation owing to elevated production of short-chain fatty acids and tryptamine, which might regulate tight junction barrier integrity and intestinal homeostasis (Islam et al., 2017).

21.4.5 Small molecule antioxidants Syringic acid is a key constituent present in rice bran (Saji, Schwarz, et al., 2020). Diabetic rats gavaged with syringic acid (25, 50, and 100 mg/kg) for six weeks were noted to have a significant attenuation in several plasma biochemical parameters including an increase in catalase activity and a reduction in the SOD activity and hepatic malondialdehyde level. Alleviation of histopathological damages in the syringic acid-treated groups were noticeably evident compared to the untreated diabetic group and therefore, treatment with syringic acid may be a suitable candidate against hepatic complications since it can reduce oxidative damages in diabetic cases and has the potential to target hepatic mitochondria in diabetes (Sabahi et al., 2020). Crohn’s disease and ulcerative colitis constitute two major subgroups of IBD, characterized by relapsing inflammation in the gastrointestinal tract with limited treatment options (Zhao & Burisch, 2019). Investigation into tricin (a constituent from rice bran) on lipopolysaccharides-activated macrophage RAW264.7 cells and colitis mouse model induced by 4.5% dextran sulfate sodium for seven days was performed. The results from the study showed that tricin (50 μM) reduced nitric oxide production in lipopolysaccharides-activated RAW264.7 cells and the anti-inflammatory activity of tricin was shown to act through the NF-κB

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pathway. Moreover, treatment with 150 mg/kg of tricin significantly reversed colon length reduction, reduced myeloperoxidase activities, and disease activity index scores, as well as restored the elevated myeloid-derived suppressive cell population in acute colitis mice. The influence from 4.5% dextran sulfate sodium on gut microbiota, such as the increased population of Proteobacteria phylum and Ruminococcaceae family, was shown to be relieved after tricin treatment (Li et al., 2021).

21.4.6 Plant-based pigments and organic compounds Colored rice bran has also been noted to be a potent inhibitor of insulin signaling molecules. Liver protein profiling of purple rice bran supplementation in normal and T2DM rats has identified the expression of five proteins in diabetic rats supplemented with purple rice bran. The unique proteins identified are correlated with the insulin signaling pathway, suggesting that purple rice bran might improve insulin sensitivity. Interestingly, 11 proteins involved in the oxidative stress response and autophagy were also noted to coexpress in both normal control rats and diabetic rats with purple rice bran supplementation, showing evidence of a molecular mechanism involved in the antidiabetic activity of purple rice (Hlaing et al., 2017).

21.4.7 Mitochondria-specific enzyme mimetics from food, administered either as monocomponent formulas or mitochondria-specific cocktails with synergetic potential Endothelial dysfunction has been identified as a primary contributor to the progression of CVD. However, treatment with rice bran phenolic extracts (25
250 μg/mL) on endothelial cells under oxidative stress has been previously noted to modulate several candidate genes associated with antioxidant and anti-inflammatory pathways (Saji et al., 2019). The vascular effects of rice bran enzymatic extract was demonstrated in a mouse model fed either a high-fat/cholesterol diet or high-fat/ cholesterol diet supplemented with 5% rice bran enzymatic extract, for 21 weeks. This study noted that the rice bran enzymatic extract diet prevented the development of atherosclerotic plaques and oxidative stress in mouse aorta and downregulated markers of mitochondrial biogenesis. Further analysis into the rice bran enzymatic extract using an endothelial cell model displayed ferulic acid as the key bioactive component responsible for the effects observed. The same study continued onto an in vivo clinical trial where ferulic acid intake was noted to reduce ROS, improve mitochondrial biogenesis, enhance resistance against hydrogen peroxide-induced apoptosis, and improve endothelial progenitor cells number and function (Perez-Ternero et al., 2017). An ex vivo study into the effect of rice bran policosanol extract in hyperlipidemic Sprague Dawley rats showed reduced

21.4 Health properties of rice bran constituents

adenosine diphosphate-induced platelet aggregation without any adverse side effects (Wong et al., 2016). Metabolic disorders such as T2DM can be identified by abdominal obesity, impaired carbohydrate metabolism, high blood pressure, and dyslipidaemia (Li & Yang, 2018). A study has shown that pancreatic cells induced with high glucose stress conditions may be treated with rice bran phenolic extracts (25
250 μg/mL). Several candidate genes investigated in this study were observed to have significantly increased their expression as well as increase insulin secretion. The effects observed may have been due to the synergistic action of polyphenols targeting signaling molecules, decreasing free radical damage (Saji, Francis, et al., 2020). A similar effect was demonstrated in vivo where 20 g of stabilized rice bran was supplemented to patients with T2DM (28 volunteers consumed either rice bran or placebo once daily for 12 weeks). At the end of the study period, consumption of stabilized rice bran was noted to lower the level of glycated hemoglobin and blood lipids and increase blood adiponectin concentrations in T2DM patients (Cheng et al., 2010). In another study, where the preventive and therapeutic effects of rice bran extract in Alzheimer’s disease in a mice model was examined, microglia, the resident immune cells of the central nervous system were noted to change from a neurotoxic microglia phenotype to a neuroprotective microglia phenotype. They also noted a significantly decreased expression of nuclear factor kappa B and the pro-inflammatory microglial marker (CD45) in parallel with increasing the expression of the anti-inflammatory microglial and phagocytic markers (arginase1, CD163, and CD36). In addition, rice bran extract also significantly increased PPAR-γ expression and reduced amino acid proteolytic product, Aβ42 deposition as well as p-tau protein levels (El-Din et al., 2021). Hence, rice bran extract may be used as a preventive and therapeutic agent in the treatment of the neuro-inflammation associated with Alzheimer’s disease. Similarly, another study where mitochondrial dysfunction was examined using a cell culture model (PC12APPsw cells) that releases very low amyloid-β (Aβ40) levels, mimicking early Alzheimer’s disease stages, treatment with rice bran extracts for 24 h increased ATP production, respiratory rates, and PGC1α protein levels in the cells, resulting in improved mitochondrial function (Hagl, Berressem, et al., 2015, Hagl, Grewal, et al., 2015). Similarly, rice bran constituents such as α-tocotrienol and γ-carboxyethyl hydroxychroman were noted to be significantly elevated in brains of animals that consumed rice bran extract, providing protective properties. In isolated mitochondria, the overall respiration and mitochondrial coupling were significantly enhanced, suggesting improved mitochondrial function. This study concluded that rice bran extract may be a potential nutraceutical for the prevention of mitochondrial dysfunction and oxidative stress in brain aging and neurodegenerative diseases (Krager et al., 2015; Hagl et al., 2013). Since diet plays a major role in disease prevention, consumption of foods rich in polyphenols and other antioxidants are essential (Visioli et al., 2000).

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21.5 Conclusion The potential health benefits of rice bran on the mitochondria are numerous. We have shown that a heterogenous selection of rice bran extracts may confer benefits against a number of chronic diseases that relate to oxidative or cellular stress by enhancing protective mechanisms within the mitochondria, namely in improvements in biogenesis, dynamics (fission/fusion), protection in apoptotic cell death, upregulation of antioxidant mechanisms, and reduction in inflammatory processes. The future challenges in this field of research will be the determination of which rice bran components confer the most benefit to prevent or treat mitochondrial dysfunction observed in chronic diseases such as diabetes, neurodegenerative, and CVDs. Is there merit to identify and isolate specific compounds or formulations, and perhaps require further processing such as fermentation, or is the bran in its entirety best capable of inducing protective mechanisms? Yields, cost of extraction, and the ability to conduct randomized controlled trials in humans will also be key to the success of bench to bedside translation. Current success of translation of antioxidant therapy in humans is mixed. For example, evidence is lacking in cancer, where, although positive results were found in observational studies, they do not seem to translate to human randomized controlled clinical trials (Bjelakovic et al., 2008). In CVD, it has been observed that certain protective mechanisms fail, or are at least blunted in at-risk populations such as the aged or comorbid (See Hoe et al., 2016). Nevertheless, the evidence so far indicates beneficial cellular signaling of rice bran to the mitochondria, namely in mitochondrial biogenesis, reduction in inflammation and ROS, and overall positive phenotype in mitochondrial dynamics and resistance to cell death or apoptosis in noncancerous cells. As rice is considered a staple food for a large proportion of the world’s population, the potential in the utilization of rice bran in its whole grain form and/or its extracted form may be a useful tool in alleviate the increasing chronic disease burden in our contemporary world.

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22

Silymarin as a vitagene modulator: effects on mitochondria integrity in stress conditions

Peter F. Surai1,2,3,4,5,6,7 1

Department of Biochemistry, Vitagene and Health Research Centre, Bristol, United Kingdom Department of Biochemistry and Physiology, Saint-Petersburg State University of Veterinary Medicine, St. Petersburg, Russia 3 Department of Microbiology and Biochemistry, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria 4 Department of Animal Nutrition, Faculty of Agricultural and Environmental Sciences, Szent Istvan University, Go¨do¨llo, Hungary 5 Veterinary Medicine Department, Sumy National Agrarian University, Sumy, Ukraine 6 Department of Grain and Compound Technology, Odessa National Academy of Food Technologies, Odessa, Ukraine 7 Agricultural Department, Russian Academy of Sciences, Moscow, Russia

2

22.1 Introduction Recent achievements in biochemistry and molecular biology, together with epidemiological data have changed our thinking about food. It has become increasingly clear that our diet plays a pivotal role in the maintenance of our health and a misbalanced diet can cause serious health-related problems. It seems likely that antioxidants are among the major regulators of many physiological processes and, therefore, a redox balance between antioxidants and pro-oxidants in the diet, gastrointestinal tract, plasma, and tissues is an important determinant of the state of our health. Plants consumed by humans and animals contain thousands of phenolic compounds. Among them, the effects of dietary polyphenols including silymarin (SM) are of great interest currently. Among the many possible molecular mechanisms of protective roles of SM in biological systems, antioxidant action is considered to be the most important one (Surai, 2015, 2020).

22.2 An integrated antioxidant defense system The antioxidant defense in biological systems in humans and animals includes several options (Surai et al., 2021b; 2021c; Surai, 2020): Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00007-2 © 2023 Elsevier Inc. All rights reserved.

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• • • • • • • • • • • •

decrease localized oxygen concentration; decrease activity of pro-oxidant enzymes; improve efficiency of electron chain in the mitochondria and decrease electron leakage leading to superoxide production; induction of various transcription factors; binding metal ions (metal-binding proteins) and metal chelating; decomposition of peroxides by converting them into nonradical, nontoxic products; chain-breaking by scavenging intermediate radicals such as peroxyl and alkoxyl radicals; repair and removal of damaged molecules; redox-signaling and vitagene activation with synthesis and increased expression of protective molecules; antioxidant recycling mechanisms, including vitamin E recycling; protein glutathionylation as a way to prevent its irreversible oxidation; apoptosis activation and removal of terminally damaged cells and restriction of mutagenesis (Fig. 22.1).

FIGURE 22.1 Antioxidant defense mechanisms. Adapted from Surai, P. F., Kochish, I. I., Fisinin, V. I., & Kidd, M. T., 2019. Antioxidant defence systems and oxidative stress in poultry biology: An update. Antioxidants, 8, 235; Surai, P. F. (2020). Vitagenes in avian biology and poultry health. Wageningen, The Netherlands: Wageningen Academic Publishers.

22.3 Mitochondria as an important source of reactive oxygen species

All antioxidants in the body work as a “team” responsible for antioxidant defense and it is called the “antioxidant system”. In this team, one member helps another, by working efficiently. In general, vitamin E and coenzyme Q are considered to be the “headquarters” of the antioxidant defenses (Surai et al., 2019a; Surai, 2020), while selenium (Se) is a “chief executive” of antioxidant defense, since from 25 known selenoproteins, more than half participate in antioxidant defences (Surai & Kochish, 2019; Surai, 2018, 2021). Furthermore, a central role in antioxidant system regulation belongs to vitagene expression and additional synthesis of protective molecules in stress conditions (“ministry of defense”) to improve adaptive ability to stress (Surai, 2020). Therefore, if relationships in this team are effective, which happens only in the case of a balanced diet and sufficient provision of dietary antioxidant nutrients, then even low doses of such antioxidants as vitamin E could be effective. On the other hand, when this team is subjected to high stress conditions, free radical production is increased dramatically. During these times, without external help, it is difficult to prevent damage to major organs and systems. This “external help” is dietary supplementation with increased concentrations of natural antioxidants. For a nutritionist or feed formulator, it is a great challenge to understand when the internal antioxidant team in the body requires help, how much of this help to provide, and what the economic return will be. Again, it is necessary to remember to keep the right balance between free radical production and antioxidant defense. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have another more attractive face participating in the regulation of a variety of physiological functions.

22.3 Mitochondria as an important source of reactive oxygen species It is generally accepted that the electron-transport chain in the mitochondria is responsible for the major part of superoxide production in the body (Halliwell & Gutteridge, 2015). Mitochondria are shown to contain up to 12 sites for ROS production associated with nutrient oxidation and respiration. Mitochondria are the primary cellular consumers of oxygen and contain numerous redox enzymes capable of transferring single electrons to oxygen, generating the ROS superoxide (O2
). It is understood that mitochondria are the main cellular consumers of oxygen and primary source of free radicals in the cell. It is proven that mitochondrial enzymes producing ROS include (Lin & Beal, 2006): • • • • • •

aconitase and α-ketoglutarate dehydrogenase, important for the tricarboxylic acid cycle enzymes; complexes I, II and III of the electron-transport chain; glycerol-3-phosphate dehydrogenase and pyruvate dehydrogenase; dihydroorotate dehydrogenase; the monoamine oxidases (MAO) A and B; cytochrome b5 reductase.

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Although energy generation in the mitochondrion is an essential and extremely important process for cell survival, excessive mitochondrial ROS production also has detrimental consequences for the cell and the whole body. In general, ubiquinone reduction site of complex I, the outer quinone-binding site of the Q-cycle in complex III, and glycerol-3-phosphate dehydrogenase are considered major contributors to ROS production (Brand, 2010). In fact, updated data indicate that there are at least 12 sites of ROS (superoxide and/or hydrogen peroxide) formation in mammalian mitochondria. Mitochondria exhibit a highly dynamic and complicated ROS release profile that varies depending on physiological conditions and carbon source, type, and availability, and cell type. However, it seems likely that complex III consistently has the highest capacity of ROS production in all tissue and cell types examined so far (Young et al., 2019). Mitochondrial electron-transport systems consume more than 85% of all oxygen used by the cell and, because the efficiency of electron transport is not 100%, about 1%
3% of electrons escape from the chain and the univalent reduction of molecular oxygen results in superoxide anion formation (Halliwell, 1994; Singal et al., 1998; Chow et al., 1999). About 1012 O2 molecules are processed by each rat cell daily, and if the leakage of partially-reduced oxygen molecules is about 2%, this will yield about 2 3 1010 molecules of ROS per cell per day (Chance et al., 1979). Recently, it has been calculated that less than 0.1% of the electrons passing through the respiratory chain could leak on to O2 to form superoxide in normal physiological conditions of electron transfer (Larosa & Remacle, 2018). Even in such conditions, about 109 molecules of ROS per cell per day could be produced. This number would depend on many factors including animal species, age, sex, tissue, etc. An interesting calculation has been made by Halliwell (1994), showing that in the human body, about 1.72 kg/year of superoxide radical is produced. In stress conditions, it would be substantially increased. Clearly, these calculations showed that free radical production in the body is substantial and many thousand biological molecules can be easily damaged if they are not protected. Furthermore, mitochondrial insults, including oxidative damage itself, can cause an imbalance between ROS production and removal, resulting in net ROS production. For example, ROS, which is an inevitable by-product of oxidative phosphorylation, induces protein modifications, lipid peroxidation, and mitochondrial DNA damage, which ultimately results in mitochondrial dysfunction (Sekine & Ichijo, 2015). For many years the detrimental effects of ROS have been a focus of research, but it is now clear that mitochondrially generated ROS have a positive side involved in the regulation of intracellular signal transduction pathways (Forrester et al., 2018; Larosa & Remacle, 2018), leading to cell adaptation to stress (Calabrese et al., 2010; Surai, 2020). Many studies have focused on the detrimental effects of ROS, but it is now clear that mitochondrially-generated ROS are also involved in regulating intracellular signal transduction pathways that result in cell adaptation to stress (Surai et al., 2019). The crosstalk of the mitochondria with various transcription factors, including Nrf2 (Tsushima et al., 2020), FOXO (Fasano et al., 2019), and NK-κB (Surai et al., 2021a) has primary

22.5 Protective effects of silymarin on mitochondria

importance, since the mitochondria are considered to be signaling hubs for stress adaptation (Surai, 2020) as well as for regulated cell death, immunity, and other important physiological functions (Tsushima et al., 2020). Therefore, polyphenolic compounds could potentially affect mitochondria functions via regulation of various transcription factors. In fact, one of the mechanisms responsible for the decrease in oxidative stress is the protective effect of SM/silibinin (SB) on mitochondrial structure and function.

22.4 Antioxidant properties of silymarin It should be noted that SM can contribute to antioxidant defenses in different ways (Surai, 2015, 2020): • • • •

•

By direct free radical scavenging By preventing free radical formation by inhibiting specific enzymes responsible for free radical production By maintaining the integrity of mitochondria and electron-transport chain of mitochondria in stress conditions By participating in the maintenance of optimal redox status of the cell by activating a range of antioxidant enzymes and nonenzymatic antioxidants, mainly via transcription factors, including Nrf2 and NF-κB By activating an array of vitagenes, responsible for the synthesis of protective molecules, including superoxide dismutase (SOD), HSPs, elements of thioredoxin (Trx) and glutathione (GSH) systems, sirtuins, and providing additional protection in stress conditions

It seems likely that direct free-radical scavenging and inhibiting specific enzymes responsible for free radical production by SM play only minor roles in antioxidant protective activity of SM (Surai, 2015, 2020). In this chapter, protective roles of SM in mitochondria integrity maintenance will be considered with a specific emphasis on some transcription factors and vitagenes.

22.5 Protective effects of silymarin on mitochondria 22.5.1 In vitro evidence Protective effects of SM on mitochondria in vitro were initially shown in 1970s. In fact, swelling effects produced in vitro by inorganic phosphate and lysolecithin on liver mitochondria was partly counteracted by SM (1 mM) added into the medium (Bocchini et al., 1973). Furthermore, SM was shown to protect both liver mitochondria and microsomes from lipid peroxide formation induced by various agents and the protective AO effect of SM in the model systems used was about 10-fold higher than that for α-tocopherol (Bindoli et al., 1977). However, those

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SM concentrations used in the aforementioned studies are not achievable in biological systems and it seems likely that direct AO activity of SM is of great importance only in the gut (Surai, 2015, 2020). It also seems likely that the regulatory effects of SM on AO enzyme expression and activity, especially in mitochondria, could contribute substantially to AO activity and the protective effects of SM. Moreoever, it was reported that SM addition (250 μM) in vitro was associated with increased SOD activity and upregulated mitochondrial membrane potential leading to the prevention of mitochondrial dysfunction and cell injury (Rolo et al., 2003). Therefore, SM demonstrated a reduction of the main parameters of mitochondrial dysfunction induced by I/R including alterations in mitochondrial respiration, reduction in membrane potential, and increased susceptibility to MPT induction. SB, at a concentration as low as 10 μM, was found to fully mitigate the rise in metabolic flow-driven ROS formation in perfused rat hepatocytes. In addition, studies on isolated liver mitochondria showed that this low dose of SB depressed ROS production linked to the electron transfer chain activity (Detaille et al., 2008). It has been reported that cold preservation and warm reperfusion of the rat liver were associated with increased lipid peroxidation and superoxide anion generation, as well as with decreased GSH, mitochondrial ATP content, and respiratory control ratio (RCR). However, preservation conducted in the presence of SB (100 μM) improved parameters affected by preservation and reperfusion. Certainly, SB promoted an increase of ATP and RCR by 39% and 16% respectively and decreased oxidative stress to values observed in livers never preserved nor perfused (Ligeret et al., 2008). It has been suggested that the uncoupler-like activity of dehydrosilybin could be the background of its ROS modulation effect in various experimental systems. In fact, dehydrosilybin uncoupled the respiration of isolated rat heart mitochondria with a very high potency in suppressing ROS production in isolated rat heart mitochondria with IC50 5 0.15 μM (Gabrielova´ et al., 2010). It is worth noting that SB in mitochondria was far more effective than in other model systems based on generating superoxide or in cells cultures, where the IC50 for dehydrosilybin exceeds 50 μM. It is important to mention that 50
300 μM of SB exerted pronounced effects on liver carbohydrate metabolism (Colturato et al., 2012). The authors suggested four different sites of metabolic action of silybin, including: • • • •

glucose 6-phosphatase activity; pyruvate carrier (monocarboxylate carrier, which operates in the plasma and mitochondrial membranes); mitochondrial respiratory chain at complex I; deviation of NADH supply for gluconeogenesis and mitochondria.

Furthermore, the authors showed that silybin at tested concentrations acted as an uncoupler of oxidative phosphorylation and inhibitor of the respiratory chain and ATPase activity in mitochondria, being responsible for an impairment of the mitochondrial energy transduction (Colturato et al., 2012). SB (10 μM) was

22.5 Protective effects of silymarin on mitochondria

shown to protect primary cultured neurons against 1-methyl-4-phenylpyridineinduced cell death and mitochondrial membrane disruption (Lee et al., 2015). In an in vitro model of nonalcoholic fatty liver disease progression based on sequential exposure of hepatocytes to high concentrations of fatty acids (FA) and TNFα, it was suggested that silybin (50 μM) was able to counteract the FAinduced mitochondrial damage by acting on the following pathways (Vecchione et al., 2017): • • • • •

increased the mitochondrial size and improved the mitochondrial cristae organization; stimulated mitochondrial FA oxidation; reduced basal and maximal respiration and ATP production in SH cells; stimulated ATP production in SS cells; rescued the FA-induced apoptotic signals and oxidative stress in SH cells.

It was demonstrated that mitochondrial abnormalities contributed to advanced glycation end products (AGE)-induced apoptosis of osteoblastic cells associated with enhanced mitochondrial oxidative stress, reduction in mitochondrial membrane potential, and ATP production, abnormal mitochondrial morphology, and altered mitochondrial dynamics. The aforementioned AGE-induced mitochondrial abnormalities were indicated to be mediated by the receptor of AGEs (RAGE). In such conditions, SB (100 μM) was able to downregulate the expression of RAGE and ameliorate RAGE-mediated mitochondrial pathways (Mao et al., 2018). In an in vitro model based on cultured hepatocytes exposed to excess fructose and FAs mimicking a severe NAFLD, silybin (50 μM) was shown to exert beneficial action by inhibiting mitochondrial respiration, which is stimulated in steatosis progression (Grasselli et al., 2019). SB (10 μM) was shown to ameliorate palmitic acid-induced mitochondrial dysfunction in pancreatic β-cells (INS-1 cells; Sun et al., 2019). In fact, the expression of cytosolic Cyt. C, mitochondrial mass, and ΔΨm were markedly affected after palmitic acid treatment, but these changes were partly reversed by SB treatment and the authors speculated that protective roles of SB are mediated by ERα (Sun et al., 2019). It was shown that the effects of SB A (Sil A) on mitochondria are condition-dependent, including its concentration, level of stress, and cell type. For example, incubation of liver HepG2 cells with 25 and 50 μM SIL A for 24 h showed a significant improvement on the basal ATP level, while 150 μM did not show any effect on ATP levels, and 500 μM significantly reduced ATP levels. However, SIL A can protect against sodium nitroprusside (SNP)-induced nitrosative stress at the level of mitochondria. In fact, under conditions of nitrosative stress, a concentration of 150 μM SIL A was shown to protect HepG2 cells from the SNP-induced drop in ATP levels, while lower concentrations, did not demonstrate any protective effects (Esselun et al., 2019). Interestingly, in PC12 cells (a neuronal model), SIL A did not affect basal ATP levels. Furthermore, in comparison to HepG2 cells, PC12 cells were more vulnerable to SIL A, since incubation with 150 μM SIL A significantly reduced basal ATP levels. PC12 cells were also more vulnerable to nitrosative stress and

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their incubation with 25 μM, 50, 100, and 150 μM SIL A prior to SNP showed a concentration-dependent, protective effect in PC12 cells (Esselun et al., 2019). The authors showed that protective effect of SIL A was also observed at the level of mitochondrial membrane potential maintenance in stress conditions. Interestingly, that SM effect on mitochondria in vitro is concentration- and condition-dependent. For example, SM at 100 μM was shown to impair the mitochondrial activity in human primary chondrocytes (Wu et al., 2021). Bovine insulin amyloid fibrils were shown to cause SH-SY5Y cell death by inducing necrosis/apoptosis and affected membrane permeability. Adding SB (250 or 500 μM) to the medium was reported to attenuate insulin fibrillation, neuronal toxicity, and mitochondria membrane damage (Katebi et al., 2018). H2O2 administration was shown to induce apoptosis in HTR-8/SVneo trophoblast cells associated with decline of mitochondrial membrane potential (Δψm) and a release of cytochrome C from mitochondria to cytoplasm. H2O2 also imposed oxidative sreess evidenced by increased ROS production, elevated levels of MDA, and reduced activity of SOD and GPx. SB (250 and 500 μM) was able to ameliorate H2O2-induced cell apoptosis and oxidative stress by activating Nrf2 signaling in trophoblast cells (Guo et al., 2020). In various in vitro model systems, SM was also shown to inhibit pro-oxidant enzymes. In fact, it was demonstrated that SB inhibits the activity of ROSgenerating MAO that catalyzes the oxidative deamination of monoamines (Mazzio et al., 1998). The formation of leukotrienes via the 5-lipoxygenase pathway was indicated to be strongly inhibited by SB. In particular, in human granulocytes IC50-values of 15 and 14.5 μM SB were detected for LTB4 and LTC4/D4/ E4/F4 formation, respectively. However, much higher SB concentrations (45
69 μM) were necessary to inhibit the cyclooxygenase pathway (Dehmlow et al., 1996). It seems likely that SM has effects on normal and cancer cells. In fact, SB (150 μM) was shown to induce a mitochondrial NOX4-mediated endoplasmic reticulum (ER) stress response and its subsequent apoptosis in human prostate cell lines, PC-3 (Kim et al., 2016). Therefore NOX4-driven mitochondrial ROS production is considered to be a potential target for development of the cancer therapy and management. SB (100 μM) was shown to induce autophagic cell death through ROS-dependent mitochondrial dysfunction and ATP depletion involving BNIP3 in human MCF7 breast cancer cells (Jiang et al., 2015). In fact, SB at higher concentrations (150
250 μM) was shown to enhance fission of mitochondria, leading to apoptosis of MCF-7 and MDA-MB-231 cells (Si et al., 2019). It is important to note that SB at lower concentrations (30
90 μM) was reported to enhance fusion of mitochondria, inhibiting migration and invasion capacity of the MDA-MB-231 cells (Si et al., 2020). SB was reported to induce apoptosis of human epidermal cancer A431 cells by activating the AMPK
eNOS pathway, leading to mitochondrial dysfunction and apoptosis (Yu et al., 2019). SB (150, 300 and 450 μM) was also shown to induce G2/M cell cycle arrest by activating dynamin-related protein 1 (Drp1)-dpendent mitochondrial fission

22.5 Protective effects of silymarin on mitochondria

dysfunction in cervical cancer cells (You et al., 2020b). It seems likely that autophagy activated by SB could contribute to glioma cell death via induction of oxidative stress-mediated BNIP3-dependent nuclear translocation of apoptosis inducing factor (Wang et al., 2020). In fact, SM and its active derivatives are shown to induce apoptosis, enhance expression of several tumor suppressors and cell cycle inhibitors, inhibit the growth and transcription factors, and mediators of cell proliferation showing strong anti-cancer effects (Boojar et al., 2020). In great contrast to cancer cells, SB can regulate mitochondrial fission in normal cells providing increased stress resistance. In particular, it was shown (You et al., 2020a) that in normal cells SB (12.5
50 μM): • • •

• • • • • • •

markedly promoted cell proliferation by facilitating G1/S transition via activating Drp1, which is responsible for mitochondrial fission in normal cells; dose-dependently increased the mitochondrial mass, mtDNA copy number, cellular ATP production and mitochondrial membrane potential; dose-dependently increased the expression of Drp1; The aforementioned data obtained in various in vitro systems can be summarized as follows: SM/SB has regulatory effects on AO enzyme expression and activity in mitochondria; SM/SB demonstrate a reduction of the main parameters of mitochondrial dysfunction induced by I/R; At a concentration as low as 10 μM (achievable in the liver in vivo), SB was found to depress ROS production linked to the electron transfer chain activity; At 10 μM SB was effective in protection of primary cultured neurons against toxicant-induced cell death and mitochondrial membrane disruption; At 10 μM SB ameliorated palmitic acid-induced mitochondrial dysfunction in pancreatic β-cells; At 12.5
50μM SB can regulate mitochondrial fission in normal cells providing increased stress resistance; effects of SB on mitochondria are condition-dependent, including its concentration, level of stress, and cell type.

22.5.2 In vivo evidence In rats, hepatic accumulation of MDA-protein adducts and formation of mitochondrial MDA after iron overload were significantly decreased by treatment with silybin (100 mg/kg body wt/day for 4 months): mitochondrial energy wasting and tissue ATP depletion induced by iron overload were successfully counteracted by silybin (Pietrangelo et al., 1995). An iron chelating ability of silybin was, at least partly, responsible for protective effects of silybin. In a later study it was confirmed that the protective action of silybin against iron-induced oxidative injury strictly depended on its capability to counteract the rise of mitochondrial chelatable iron level (Masini et al., 2000). By using in situ detection of oxidative stress gene

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response, membrane oxidation by-products and electron microscopy, it was clearly demonstrated that, in acutely iron-dosed gerbil, iron-induced oxidant stress was accompanied by irreversible mitochondrial dysfunctions. In vivo administration of silybin (100 mg/kg body wt/day for 8 weeks) was shown to be able to prevent oxidative stress and mitochondrial defects (Pietrangelo et al., 2002). Silybin (0.4 g/kg) in a complex with phospholipid (SILIPHOS) was effective in decreasing severe oxidative stress and preserving hepatic mitochondrial bioenergetics, mitochondrial proton leak, and ATP reduction in nonalcoholic steatohepatitis induced by the methionine- and choline-deficient (MCD) diet (Serviddio et al., 2010). In a rat model of nonalcoholic steatohepatitis, a control group of animals was fed a standard liquid diet for 12 weeks, while the experimental groups were fed a high-fat liquid diet for 12 weeks without (NASH) or with simultaneous daily supplement with SB
phosphatidylcholine complex (SB 200 mg/kg) for the last 5 weeks. Treatment with SB was demonstrated to improve liver steatosis and inflammation and decreased NASH-induced lipid peroxidation, plasma insulin, and TNF-α and returned GSH concentration back to normal. However, SB was not able to correct the cellular energetic imbalance associated, indicated by a significant decrease in hepatic mitochondrial ATP production (Haddad et al., 2011). In a fatty liver model based on feeding a high-fat diet to rats for six weeks, silybin treatment (26.25 mg/kg/day), was shown to significantly protect against fatty liver by stabilizing mitochondrial membrane fluidity (Yao et al., 2011). In a model of nonalcoholic steatohepatitis, a four-week daily dose of SB (20 mg/kg i.p.) was administrated to db/db mice fed a methionine
choline deficient diet. SB was shown to decrease levels of isoprostanes, 8-deoxyguanosine, and nitrites/nitrates and restored GSH levels in the liver and in the heart of the experimental animals. Furthermore, liver mitochondrial respiratory chain activity was shown to be dramatically impaired in untreated mice, while it was completely restored in SB-treated animals (Salamone et al., 2012). Changes in mitochondrial respiratory complexes in fatty hepatocytes were attenuated by SB-vitamin E complex (15 mg vitamin E and 47 mg silybin) fed to rats with a major protective effect on Complex II subunit CII-30 (Grattagliano et al., 2013). It was shown that chronic cholestasis was associated with cardiolipin oxidation leading to impairment of mitochondrial function and increased ROS production. Interestingly, silybin (0.4 g/kg diet) was shown to limit mitochondrial failure. For example, cirrhosis was shown to induce a significant reduction in ΔΨ from complex I that was completely prevented by treatment with silybin. In addition, silybin was reported to positively affect complex V activity, strongly reduced in cirrhotic liver, and also limited proton leak and ameliorated the decrease in ATP synthesis. Furthermore, silybin restored the normal expression of PGC-1α (a key transcription regulator of cellular energy metabolism and mitochondrial biogenesis), and mtDNA copy number in cirrhotic liver (Serviddio et al., 2014). SM oil (10 mL/kg/BW) significantly increased levels of membrane fluidity and membrane potential of liver mitochondria (Zhu et al., 2014). It has been suggested that the protective mechanism of action of SB (50
200 μM) in intrastriatal MPP 1 -injected rats was associated with

22.5 Protective effects of silymarin on mitochondria

the maintenance of mitochondrial bioenergetics and integrity (Geed et al., 2014). Rats exposed to a carcinogen (1,2-di-methylhydrazine DMH) showed increased activities of phase I enzymes (cytochrome b5, cytochrome b5 reductase, cytochrome P450, cytochrome P450 reductase, cytochrom P4502E1) in the liver and colonic mucosa as compared to control rats. SB supplementation (50 mg/kg/BW) modulates the xenobiotic metabolizing enzymes including decreasing activity of ROS-producing cytochrome b5 reductase (Sangeetha et al., 2012). SM was shown to protect mitochondria from pathological events by triggering pro-survival cell signaling. For example, SB supplementation is reported to optimize the electron-transport chain, decreasing electron leakage and ROS formation and directly reducing activities of ROS-producing enzymes in the mitochondria. The administration of SM (30 mg/kg body weight/d for 4 weeks) was reported to significantly ameliorate a high fat diet (HFD)-induced glucose metabolic disorders, oxidative stress, mitochondrial biogenesis, and pathological alterations in the kidney. SM (12.5
100 μM) also significantly mitigated renal mitochondrial biogenesis and partly restored mitochondrial membrane potential in palmitic acid treated HK2 cells (Feng et al., 2017). In a model system based on streptozotocin-induced diabetes in mice, SM (104 mg/kg) was shown to increase/restore oxygen flux through NADH oxidase and succinate oxidase in the liver mitochondria that were compromised in the diabetic group (Stolf et al., 2018). The authors showed that SM not only restored oxidase activity, but also significantly increased it in comparison to a control nondiabetic animal, reflecting an improvement in AO defences in general. In a stress model of rat cardiac hypertrophy induced by partial abdominal aortic constriction (PAAC), a significant reduction in cardiac mtDNA concentration as compared to normal control rats was observed. Treatment with SM (100 mg/kg/day, p.o) was shown to significantly prevent oxidative stress (restoration of SOD activity and GSH concentration) and partly restore mtDNA concentration in PAAC-treated rats (Sharma et al., 2019). In the mice model of myocardial I/R SB was shown to reduce cardiomyocytes apoptosis, attenuate mitochondrial impairment and ER stress, and alleviate ROS generation, neutrophil infiltration, and cytokines release (Chen et al., 2020). Clinical applications of SM and its constituents are quite diverse. In particular, SB was demonstrated to be effective in the control of neuropathy, retinopathy, impaired healing, hepatopathy, cardiomyopathy, and osteoporosis in diabetic patients by regulating insulin sensitivity, oxidative stress, inflammation, apoptosis, proliferation, differentiation, and ECM remodeling (Chu et al., 2018). Therefore, in a variety of in vivo studies, a range of stress-related model systems were used including iron overload, nonalcoholic steatohepatitis induced by the MCD diet or by a high-fat liquid diet, chronic cholestasis and cirrhosis, exposure to carcinogens, HFD-induced metabolic disorders, streptozotocin-induced diabetes, cardiac hypertrophy induced by PAAC, myocardial I/R, and others. SM supplementation was associated with: • •

prevention of oxidative stress and mitochondrial defects; preservation of hepatic mitochondrial bioenergetics, proton leak and ATP content;

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• • • •

stabilizing mitochondrial integrity, membrane fluidity, and membrane potential; restoring mitochondrial respiratory chain activity, cellular energy metabolism, and mitochondrial biogenesis; triggering pro-survival cell signaling; inhibiting cardiomyocytes apoptosis

22.6 Effect of SM on vitagene expression Emerging findings suggest a large number of potential mechanisms of action of SM (polyphenols) in preventing disease, which may be beyond their direct conventional antioxidant activities. It seems likely that SM, similar to other polyphenols, can affect the vitagene network in various stress models. The relevant recent findings are reviewed next. First of all, SM/silybin affects HO-1 activity in different model systems. For example, silychristin A, the major flavonolignans from SM, was shown to activate Nrf2-HO-1/SOD2 pathway to reduce ROS-induced apoptosis and improve GLP-1 production through upregulation of estrogen receptor α (Erα) in GLUTag cells (Wang et al., 2020). Protective antioxidant effects of SM (25 mg/kg) on D-galactosamine (Ga1N)-induced oxidative stress and liver injury in experimental rats was associated with a partial restoration of AO enzyme (SOD, GPx and CAT) activities in the livers of Ga1N-treated animals (Jo et al., 2020). SM (25 or 50 mg/kg body weight orally for seven consecutive days) was reported to significantly upregulate the expression of Nrf2, HO-1 and Bcl-2 and downregulate the expression of Bcl-2 associated X protein (Bax) in the brain and sciatic nerve tissues of Docetaxel-treated rats (Yardım et al., 2021). Protective effect of SM (400 mg/kg, p.o.) against Thioacetamide (TAA, single dose 50 mg/kg, i.p) toxicity in rats was found to be associated with increased GSH content, SOD and HO-1 activity, and Nrf2 expression, decreased Keap1 and TNF-α expression in the liver tissues compared to the TAA group (Hussein et al., 2021). Supernatant of monocytes from preeclamptic women was shown to induce oxidative stress in primary human umbilical vein endothelial cells and SB in these conditions increased levels of nitrite, reduced lipid peroxidation, and increased HO-1 activity (Gomes et al., 2021). It was suggested that Erα could be involved in the activation of Nrf2-antioxidant pathways in pancreatic β-cells by SB. In particular, it was demonstrated that SB (10 μM) increased the viability of high glucose/palmitate-treated INS-1 cells accompanied by elevated expression of ERα, Nrf2, and HO-1 as well as reduced ROS production in vitro. In fact, treatment of INS-1 cells by ERα antagonist (MPP) or silencing ERα expression in the cells with siRNA was shown to abolish the protective effects of SB (Chu et al., 2020). In a nonalcoholic steatohepatitis model, SB treatment (10 and 20 mg/kg BW, once a day for 6 weeks) of mice was shown to increase the expression of NRF2 and the activities of CAT, GSH-Px and HO-1 in the liver (Liu et al., 2019a).

22.7 Application of silymarin in poultry

From the data presented above, it is clear that SM/SB can upregulate HO-1 and improve antioxidant defences. There is also evidence that SM can affect other vitagenes’ expression and activity, including HSP70 (Bongiovanni et al., 2007; Oskoueian et al., 2014; Khamisabadi, 2020; Lu et al., 2020), sirtuins (Zhang et al., 2018; Sarubbo et al., 2018; Wu et al., 2021), and Trx (Grattagliano et al., 2013; Liu et al., 2018). Therefore, the vitagene network represents the major cellular pathway involved in the so called “programmed cell life” as an opposite to apoptosis, providing an effective protection against oxidative stress and toxic products of ROS metabolism (Calabrese et al., 2009, 2009a, 2010). In particular, nutritional antioxidants can be neuroprotective through the activation of hormetic pathways under the control of the Vitagene protein network (Calabrese et al., 2014). Recently, the modulatory effect of SM on Nrf2-regulated regulated redox status and NF-κBmediated inflammation in experimental gastric ulcers have been evaluated (Keshk et al., 2017). In general, in stress conditions, SM can prevent a decrease in expression of Nrf2 (Vargas-Mendoza et al., 2020) and its target AO (SOD, GPx; Surai, 2020), and ameliorate increased NF-κB, TNFα, MDA and activity of myeloperoxidase (MPx; Surai, 2020; Surai et al., 2021a). The aforementioned results clearly indicate that, similar to other polyphenols, SM, or its active ingredient SB, affect the vitagene network, decreasing detrimental consequences of stressors. Further research is needed to fully understand the molecular mechanisms involved in the vitagene network activation by SM/silybin.

22.7 Application of silymarin in poultry Based on multiple protective effects of SM shown above in various model systems one can expect its beneficial effect in commercial poultry production associated with stress conditions. Data on protective effects of SM and milk thistle seeds (MTS) on poultry are shown in Table 22.1. Data on SM effects on poultry are very limited and obtained mainly in the last 7 years. In normal physiological conditions SM can decrease oxidative stress imposed by commercial conditions of chicken housing (Schiavone et al., 2007; Ahmad et al., 2020). More pronounced protective effects of MTS or SM were observed in various stress models, including heat stress (Morovat et al., 2016), a high energy diet (C ¸ eribaşı et al., 2020), AF (Amiridumari et al., 2013; Jahanian et al., 2017) or ATA (Khatoon et al., 2013) dietary contamination, or CCl4 treatment (Baradaran et al., 2019). In general, several important aspects of the beneficial properties of milk thistle on poultry growth performance in experimentally-induced aflatoxicosis (Alhidary et al., 2017; Zaker-Esteghamati et al., 2020; El-Sheshtawy et al., 2021) and SM hepatoprotective properties, (Saeed et al., 2017) and its usage as a protective agent against natural or chemical toxicities (Fanoudi et al., 2020) have recently been reviewed. Protective effects of

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Table 22.1 Protective effects of SM on poultry. No.

Experimental design

Effects of SM

References

1

180 male chicks (ROSS 508), basal diet (BD), BD 140 or 80 ppm SM, 60 days

Schiavone et al. (2007)

2

120 broiler chicks, BD, BD 1 milk thistle seeds (MTS): 5, 10 or 15 g/kg, 42 days

3

240 chicks (ROSS 308), BD and BD 1 100ppm SM, 42 days, days 7
28, increased by 4 C temperature for 4 h 120 male 15-day-old Japanese quail chicks, 4 groups, BD, high energy diet (HD), Milk thistle seeds (MTS, 1%), HD 1 MTS for 35 days

Reduced lipid content of breast and thigh muscles, increased muscles resistance to oxidative stress. MTS at 15 g/kg decreased the negative effects of natural summer stressed broilers: Serum MDA, antibody titer against ND, improved FCR for weeks 2, 3, 4 and 5. Improved performance, immunity and carcass characteristics and decreased the adverse effects of the heat stress HD imposed oxidative stress in testes (MDA, GPx) and disturbed spermatogenesis; almost all the disturbances were ameliorated by MTS. AFB1 disturbed metabolism of Ca, glucose and creatinine, increased AST activity (liver damage). MTS significantly reduced the effect of AFB1 on the above parameters SM improved performance of AF-challenged birds by suppressing ileal bacteria and enhancing absorptive surface area SM and Vit. E alone or in combination ameliorated the immunotoxic effects induced by 1.0 mg OTA/kg, no protection against 2.0 mg OTA/kg. CCl4 damaged liver and imposed oxidative stress by downregulating CAT, GPx, and MnSOD. SM showed protective effects by ameliorating those changes Silymarin (SM) strengthened the AO system (elevation of free SHgroups concentration), but no protection against lipid peroxidation

4

5

216 one-d-old Ross 308 male broiler chicks, 9 groups, 250
500 ppb AFB1, 0.5 and 1% MTS.

6

430 (7-day-old) Ross broiler chicks. BD, AF mix -0.5 or 2 ppm, SM 0.5 or 1 g/kg for 21 days.

7

240 one-day-old birds, 12 groups. BD, OTA (1or 2 mg/kg), SM (10 g/kg), Vit.E (200 mg/kg) for 42 days

8

240-day-old broilers, 4 groups. BD, SM 100 mg/kg BW, CCl4 1 mL/kg and SM 1 CCl4

9

18 white, female, Hungarian ducks. Normal diet (ND) from day 14 to day 47, Experimental diet ED 5 ND 1 DON (4.9 mg/kg) and ZEA (0.66 mg/kg), ED 1 milk thistle seed (0.5%)

Ahmad et al. (2020)

Morovat et al. (2016)

Çeribaşı et al. (2020)

Amiridumari et al. (2013)

Jahanian et al. (2017)

Khatoon et al. (2013)

Baradaran et al. (2019)

Egresi et al. (2020)

22.8 Conclusions

MTS or SM were related to the prevention of oxidative stress and to maintenance of productive performance under stressful conditions. Based on the multiple protective actions of SM in various stress models in vitro and in vivo presented above, we can expect more research in this area related to a design of protective nutrient compositions to deal with commercially relevant stresses in egg and meat production.

22.8 Conclusions SM and its active constituents are shown to have various antioxidant actions and their mitochondria-stabilizing effects play major roles in stress protection. In fact, accumulating evidence related to in vitro effects of SM/SB on mitochondria include their regulatory effects on AO enzyme expression and activity in mitochondria; a reduction of the main parameters of mitochondrial dysfunction induced by I/R; at a concentration as low as 10 μM (achievable in the liver in vivo), inhibition of ROS production linked to the electron transfer chain activity; at 10 μM effective protection of primary cultured neurons against toxicant-induced cell death and mitochondrial membrane disruption; at 10 μM ameliorating palmitic acid-induced mitochondrial dysfunction in pancreatic β-cells; at 12.5
50 μM SB can regulate mitochondrial fission in normal cells providing increased stress resistance. Interestingly, effects of SB on mitochondria are condition-dependent, including its concentration, level of stress, and cell type. In a variety of in vivo studies, a range of stress-related model systems were used including iron overload, nonalcoholic steatohepatitis induced by the MCD diet or by a high-fat liquid diet, chronic cholestasis and cirrhosis, exposure to carcinogens, a HFD-induced metabolic disorders, streptozotocin-induced diabetes, cardiac hypertrophy induced by PAAC, myocardial I/R, and others. SM supplementation was associated with: prevention of oxidative stress and mitochondrial defects; preservation of hepatic mitochondrial bioenergetics, proton leak and ATP content; stabilizing mitochondrial integrity, membrane fluidity and membrane potential; restoring mitochondrial respiratory chain activity, cellular energy metabolism and mitochondrial biogenesis; triggering pro-survival cell signaling; inhibiting cardiomyocyte apoptosis. Furthermore, in stress conditions, SM can prevent a decrease in expression of Nrf2 and its target AO, ameliorate increased NF-κB, TNFα, MDA and activity of myeloperoxidase. In addition, similar to other polyphenols, SM, or its active ingredient SB, affect vitagene network to decrease detrimental consequences of stresses. Data on SM effects on poultry are very limited and obtained mainly in the last seven years. In general, SM can decrease oxidative stress imposed by commercial conditions of chicken housing. However, more pronounced protective effects of MTS or SM were observed in various stress models, including heat stress, high energy diet, mycotoxin contamination, or CCl4 treatment. Protective effects of MTS or SM were related to prevention of oxidative stress and to maintenance of productive performance under

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stressful conditions. Based on the multiple protective actions of SM in various stress models in vitro and in vivo, more research in this area related to a design of protective nutrient compositions to manage commercially-relevant stresses in egg and meat production is expected. Various phytochemicals, including flavonoids/polyphenols, are an essential part of the animal/human diet, responsible for turning on and maintaining an optimal status of the antioxidant defenses. Since flavonoids/polyphenols are not well absorbed in the gut, their active concentration in the plasma and target tissues are comparatively low, but most likely sufficient for Nrf2 activation and NF-κB suppression as well as vitagene activation (Surai, 2020). Indeed, it seems very likely that activation of the Keap1/Nrf2/ARE pathway and inhibition of the NF-κB pathway, rather than direct free radical scavenging activity, may be the main mechanisms of the health benefits of phytochemicals (Calabrese et al., 2012), including SM (Surai, 2020). Therefore, consumption of phytochemicals, including SM, could have a preconditioning effect on the antioxidant system of the body. This could explain the beneficial health-promoting effects of a diet rich in fruits and vegetables as important sources of the aforementioned chemicals (polyphenols and other phytochemicals) maintaining the body’s ability to be highly adaptive to various stresses. SM and its main component SB are part of the phytochemical mixture consumed regularly by animals/human with feed/food and it is responsible for regulation of the antioxidant defenses in the gut and in the whole body. It could be that some dietary constituents which are not well-absorbed could have healthpromoting properties by maintaining redox balance in the large intestine, where concentration of other antioxidants (vitamin E, carotenoids, ascorbate) could be low, but pro-oxidants (iron, oxidized PUFAs, etc.) and substrates of oxidation are still present (Surai et al., 2003, 2004; Surai & Fisinin, 2015; Surai, 2014, 2020). This protective effect in the large intestine could be responsible, for example, for bowel cancer prevention. Therefore, there could be a biological reason for some nutrients not being absorbed, but still being involved in antioxidant protection in the lower gut. Taking into account high concentrations of phytochemicals in the gut, it could be that they play an essential part in maintaining an optimal antioxidant-pro-oxidant/redox balance in the digestive tract responsible for additional health effects of phytochemicals including SM. In conclusion, there are many possible mechanisms by which SM can improve the antioxidant defense mechanisms in the body. They include direct and indirect SM actions (Fig. 22.2). Firstly, a direct scavenging free radicals and chealating free Fe and Cu (mainly effective in the gut). Secondly, preventing free radical formation by inhibiting specific ROS-producing enzymes, or improving the integrity of mitochondria in stress conditions as a result of SM consumption, is of great importance. Thirdly, maintaining an optimal redox balance in the cell by activating a range of antioxidant enzymes and nonenzymatic antioxidants, mainly via Nrf2 activation, is probably the main driving force of AO action of SM. Fourthly, decreasing

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FIGURE 22.2 Protective actions of silymarin (Surai, 2020).

inflammatory responses in the gut and other tissues by inhibiting NF-κB pathways is an emerging mechanism of SM protective effects on liver toxicity and diseases. Finally, activating vitagenes, responsible for synthesis of protective molecules, including HSP, Trx, sirtuins, etc., and providing additional protection in stress conditions deserves more attention in future research. In addition, effects on the microenvironment of the gut, including SM-bacteria interactions, await future investigation. Finally, mitochondria-stabilizing effects of SM/SB are proven in in vitro and in vivo model systems and play a central role in their protective activity in various stress conditions. Animal/poultry nutrition and a disease prevention strategy SM alone, or in combination with other hepato-active compounds (carnitine, betaine, vitamin B12, Mn, Zn, etc.), are shown to have hepatoprotective health-promoting effects as described in humans with similar mechanisms of protective action (Surai, 2020).

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372). London, UK: Elsevier, Academic Press. Surai, P. F., Kochish, I. I., & Kidd, M. D. (2021c). Natural antioxidants in redox balance maintenance and animal health protection. In M. D. Kidd (Ed.), Branched-chain amino acids: Metabolism, benefits and role in disease (pp. 31
76). Nova Science Publishers, Inc.

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Surai, P. F., Kochish, I. I., Romanov, M. N., & Griffin, D. K. (2019a). Nutritional modulation of the antioxidant capacities in poultry: The case of vitamin E. Poultry Science, 98, 4030
4041. Tsushima, M., Liu, J., Hirao, W., Yamazaki, H., Tomita, H., & Itoh, K. (2020). Emerging evidence for crosstalk between Nrf2 and mitochondria in physiological homeostasis and in heart disease. Archives of Pharmacal Research, 43, 286
296. ´ ., Morales-Martı´nez, M., Soriano-Ursu´a, M. A., Vargas-Mendoza, N., Morales-Gonza´lez, A ´ lvarezDelgado-Olivares, L., Sandoval-Gallegos, E. M., Madrigal-Bujaidar, E., A Gonza´lez, I., Madrigal-Santilla´n, E., & Morales-Gonzalez, J. A. (2020). Flavolignans from Silymarin as Nrf2 bioactivators and their therapeutic applications. Biomedicines, 8, E122. Vecchione, G., Grasselli, E., Cioffi, F., Baldini, F., Oliveira, P. J., Sarda˜o, V. A., Cortese, K., Lanni, A., Voci, A., Portincasa, P., & Vergani, L. (2017). The nutraceutic silybin counteracts excess lipid accumulation and ongoing oxidative stress in an in vitro model of non-alcoholic fatty liver disease progression. Frontiers in Nutrition, 4, 42. Wang, C., He, C., Lu, S., Wang, X., Wang, L., Liang, S., Wang, X., Piao, M., Cui, J., Chi, G., & Ge, P. (2020). Autophagy activated by silibinin contributes to glioma cell death via induction of oxidative stress-mediated BNIP3-dependent nuclear translocation of AIF. Cell Death & Disease, 11, 1
16. Wu, W. T., Chen, Y. R., Lu, D. H., Senatov, F. S., Yang, K. C., & Wang, C. C. (2021). Silymarin modulates catabolic cytokine expression through Sirt1 and SOX9 in human articular chondrocytes. Journal of Orthopaedic Surgery and Research, 16, 147. Yao, J., Zhi, M., & Minhu, C. (2011). Effect of silybin on high-fat-induced fatty liver in rats. Brazilian Journal of Medical and Biological Research, 44, 652
659. ¨ zdemir, S., C¸omaklı, S., Caglayan, C., Kandemir, F. M., & Yardım, A., Kucukler, S., O C¸elik, H. (2021). Silymarin alleviates docetaxel-induced central and peripheral neurotoxicity by reducing oxidative stress, inflammation and apoptosis in rats. Gene, 769, 145239. You, Y., Chen, L., Wu, Y., Wang, M., Lu, H., Zhou, X., Liu, H., Fu, Z., He, Q., Ou, J., & Fu, X. (2020a). Silibinin promotes cell proliferation through facilitating G1/S transitions by activating Drp1-mediated mitochondrial fission in cells. Cell Transplantation, 29, 963689720950213. You, Y., He, Q., Lu, H., Zhou, X., Chen, L., Liu, H., Lu, Z., Liu, D., Liu, Y., Zuo, D., & Fu, X. (2020b). Silibinin induces G2/M cell cycle arrest by activating Drp1dependent mitochondrial fission in cervical cancer. Frontiers in Pharmacology, 11, 271. Young, A., Gill, R., & Mailloux, R. J. (2019). Protein S-glutathionylation: The linchpin for the transmission of regulatory information on redox buffering capacity in mitochondria. Chemico-biological Interactions, 299, 151
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767. Zhu, S. Y., Dong, Y., Tu, J., Zhou, Y., Zhou, X. H., & Xu, B. (2014). Silybum marianum oil attenuates oxidative stress and ameliorates mitochondrial dysfunction in mice treated with D-galactose. Pharmacognosy Magazine, 10, S92
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CHAPTER

Buckwheat trypsin inhibitors: novel nutraceuticals for mitochondrial homeostasis

23 Si-Quan Li

Michael Foods, Inc./Post Holdings, Hopkins, MN, United States

23.1 Introduction Trypsin is a widely present and critical proteolytic enzyme for living organisms including humans. Its inactive precursor, trypsinogen, is produced in exocrine cells of the pancreas then secreted in the lumen of intestine. Trypsinogen is activated in the duodenum by an autocatalytic hydrolysis process driven by duodenal mucosal enterokinase. This activation step is so fast that all the trypsinogens secreted into the duodenum are almost immediately activated and, as a result, all the detectable trypsin is in active format. Trypsin activity is regulated by many factors, ranging from genetic expression, feedback control on amount of production, activation rate of trypsinogen, substrate structures, environmental factors, and presence of inhibiting factors. Trypsin is highly selective and more efficient clipping at peptide bonds formed by the carbonyl groups of lysine or arginine. Its proteolytic activity is determined by the environmental conditions it is exposed to. Trypsin is a serine protease. The active site contains a serine residue and its hydroxyl group plays a critical role for the hydrolytic function of this protease. The hydroxyl group of the serine residue is crucial for forming the appropriate structure and thus the electromagnetic environment for the active site to perform its normal proteolytic activity. Modification of the side-groups within this active site, particularly the hydroxyl group of this serine residue, will result in changes, even the termination of its proteolytical activity. Its activity can be inhibited by trypsin inhibitors such as buckwheat trypsin inhibitors (BTIs). Deliberate regulation of trypsin activity is critical to maintain the normal functionality of organs, such as the pancreas, deep at cellular and mitochondrial levels. Misregulated trypsin activity can be a physiological cause for some acute inflammation diseases, such as acute pancreatitis. For instance, although many factors can lead to acute pancreatitis, such as alcohol abuse and gallstones, all patients with an onset of acute pancreatitis showed significantly elevated trypsin activity levels. The main mechanism for pancreatitis onset is long believed to be autodigestion of pancreatic structural cells by trypsin (and others) due to misregulated proteolytic activity Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00009-6 © 2023 Elsevier Inc. All rights reserved.

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in the organ. It is generally believed that this misregulation of trypsin activity is through a responding mechanism to the ectopic (intrapancreatic) activation of trypsinogen. Gene mutations in the cationic trypsinogen gene, known as protease serine type 1 gene, were shown to be directly linked to the onset of pancreatitis as early as 1996 (Whitecomb et al., 1996). Attention has also been solicited to the downregulating capacity by trypsin inhibitors, including those commonly encountered from food intake, such as buckwheat, for potential health benefits and for chronic disease mediation applications. The reasons behind this are complicated but mainly driven by the demands of health-conscious consumers looking for a “natural” way to mitigate chronic and age-related diseases. Trypsin activity regulation is one of the important areas worthwhile for further investigation. Due to the aged and heavily age-skewed global population (WHO, 2018), chronic diseases, particularly those that are agerelated, demand more than ever before, so efforts from scientific communities are ongoing to discover more effective cures. Life is a process of aging and healthy aging is and will continue to be a substantial topic for scientific studies. Among many, mitochondrial homeostasis is one of the critical subsystems for maintaining aging process on the right track. Food choice is an important factor affecting the overall bioavailability of critical micronutrients. Numerous food and medicine dual-use food materials have been long used in ancient herbal medicine practices. In the proven traditional medicine practices, buckwheat is an important medicine while also an important food material, even food staple in some regions. Buckwheat is a pseudo-grain and a traditional food in Far East region and widely spret to a much bigger population around the globe for health benefits. Buckwheat is rich in many nutraceutical components which play critical roles in maintaining desired health status (Li & Zhang, 2001). Its health benefits, including those from BTIs, have attracted increasing interests from scientists and health professionals. In this chapter, we will focus on discussion on BTIs (natural and recombinant rBTI) as a novel nutraceutical, their roles and mechanisms in trypsin inhibition and mitochondrial homeostasis regulation, and further effects in managing chronic diseases, including age-related diseases such as Alzheimer’s disease (AD) and neurodegenerative diseases such as diabetic retinopathy. Further research trends in the area will also be briefly discussed.

23.2 Roles of mitochondrial proteases in maintaining mitochondrial homeostasis and deliberate regulation by protease inhibitors 23.2.1 Mitochondrial metabolisms and homeostasis Normal functionality and survival of human cells largely depends on those essential processes that maintaining the deliberate balances between mitochondrial biogenesis and mitophagy. Cells constantly adapt their metabolisms to meet their

23.2 Maintaining mitochondrial homeostasis

energy needs and respond to varying nutrient availability in the ever-changing environment where they are exposed to. Eukaryotes have evolved a very sophisticated system to detect changes in ATP levels via the serine/threonine AMP-activated protein kinase (AMPK) complex (Herzig & Shaw, 2018). Under conditions of low energy, AMPK phosphorylates specific enzymes and growth control nodes to increase ATP production while reduce ATP consumption. This energy switch controls cell growth and several other cellular processes, including lipid and glucose metabolism and cell autophagy. AMPK, as the master mediator, is involved in multiple cell processes and coordinates multiple mechanisms of mitochondrial biology including autophagy thus maintaining its normal function is critical to mitochondrial homeostasis (Shaw, 2018). In higher eukaryotes, when energy level is low, by sensing the changed ATP-to-ADP or ATP-to-AMP ratio, AMPK is activated and promotes kinase activity accordingly thus further regulates the biologic mechanisms to increase catabolism and decrease anabolism by phosphorylation of key proteins in multiple pathways, including mTOR complex, lipid homeostasis, glycolysis and mitochondrial homeostasis. The potent of AMPK, as the master mediator, to regulate cellular function and particularly mitochondrial homeostasis through autophagy energy regulation, has caught lots of interests in research and therapeutic applications such as in cancer and type-2 diabetes. Metformin, a long used drug for treating type-2 diabetes, is an example using AMPK function to treat targeted diseases, in this case type 2 diabetes. In general, Metformin activates AMPK in hepatocytes via induced energy stress by inhibition of complex I of the respiratory chain thus alters the ATP-to-AMP ratio in hepatocyte mitochondria. The success of metformin application provided an example and evidenced the possibility to manipulate mitochondrial mechanisms so to achieve desired intervention and alter the otherwise natural aging process encoded in human genes. This may provide researchers and health professionals a tool to alter, regulate or improve or extend the lifespan of mitochondria by sustaining its energy production level and a well-maintained homeostasis. Mitochondria are small but important organelles critical to sustain cell life cycles and functions. Mitochondrial proteases regulate the healthy biogenesis while function as tools for efficient and complete degradation of misfunction proteins. Mitochondrial proteases can be classified into different groups by different criteria—by their functions or by their locations. Based on their functionality, mitochondrial proteases are assigned into three groups: ATP-dependent proteases, processing peptidases and oligopeptidases. ATP-dependent proteases function either as regulators or quality control proteases to degrade those nonnative polypeptides into much smaller peptides and remove the undesired functionalities associated with the target proteins. The function of the processing peptidase group includes modifying the targeted mitochondrial precursors into needed proteins with regulatory functions—they are critical to supply the needed building blocks for mitochondrial biogenesis. Oligopeptidases degrade the excess small peptides, generated from activities of ATP-dependent proteases and processing proteases, into amino acids for recycling or expelled outside of mitochondria.

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The mitochondrial proteases can also be categorized into four different groups based on where are located within the mitochondria structures. MIM proteases, MOM proteases, matrix proteases and those can be transferred cross the membranes upon needed. Mitochondria are enveloped by outer (MOM) and inner mitochondrial membranes. There is an aqueous intermembrane space (IMS) between the MOM and MIM. Both energy dependent and independent proteases are located across organelle, operating in both polar and nonpolar environment (Koppen & Langer, 2007). Two AAA 1 family members (i-AAA and m-AAA), collectively called the mitochondrial AAA proteases, are attached to either face of the MIM and catalyze substrates from either side of the MIM—substrates either from the IMS or from matrix of the mitochondria. Glynn (2017) provided a comprehensive review on mitochondrial proteases regarding their structural locations and mechanistic principles that allow these enzymes to recognize, engage and process protein substrates, with lots of focuses on detailed discussions on AAA proteases. Mitochondria perform critical functions necessary for survival and activity sustainability of eukaryotic cells. These activities are coordinated by a vast number of proteins encoded both in nucleosomes and in mitochondrial genomes. Mitochondrial proteolytic activity must be precisely regulated and coordinated subtly among all the involved proteases in order to maintain normal functionality of the mitochondria and the cells hosting them. Otherwise, mitochondrial functions would be impaired and result in development severe diseases and altered aging process.

23.2.2 Proteases and their inhibitors are critical for health and mitochondrial homeostasis Mitochondrial proteins are building blocks and effectors for many critical functionalities, such as ATP generation and production of those mitochondrial genome encoded proteins, of the organelle. Maintaining the optimal and delicate dynamic balance between protein synthesis (and transfer) and degradation so to sustain the optimal concentration of each key protein components, is crucial to maintain normal mitochondrial functions. Furthermore, the master mediator for this orchestra is a complex of enzymes, including numerous proteases, particularly multiple serine peptidases which proteolytic activities are prone to the presence of trypsin inhibitors. While their proteolytic activities are critical to maintain mitochondrial homeostasis, mitochondrial proteases themselves are of protein nature and often the substrates of other proteases that they encounter. Therefore, maintaining appropriate proteolytic activities of mitochondrial proteases is crucial to sustain the normal functionalities of healthy mitochondria. While pH and ionic strength are important factors affecting mitochondrial protease activity. Through millions of years of evolution human biosystems have established a complicated mechanism to maintain body temperature and tissue pH at optimal levels almost all the time even under significant variation of intake levels in diets. Nevertheless,

23.2 Maintaining mitochondrial homeostasis

protease activity is also significantly impacted by presence protease inhibitors, such as trypsin inhibitors—among them are BTIs or rBTI (BTI I). BTIs are heat stable—BTI I and BTI II do not loss of activity at all after 95 C
30 min heat treatment while BTI III only loss about 20% of its native activity (Ikeda & Kusano, 1983). Most food cook methods, such as baking and steaming, are unable to inactivate BTIs so significant amount of residual BTIs are likely advanced into GI track, survive the GI tract proteolysis then potentially transmitted crossing the epithelial barrier into blood stream for whole body distribution. Proteases including trypsin, chymotrypsin, elastase, and their likes, are heavily present in human GI tract, both in the lumen and deep in the tissues. Pancreatic proteases (trypsin, chymotrypsin, and elastase, etc.) are released into the lumen of upper GI tract, where they are activated and execute proteolytic functions. Another major source, along with intestinal epithelial tissues, of GI tract proteases is microbiota, which produce not only serine peptidases like trypsin and chymotrypsin but also cysteine, aspartic and metalloproteases as well. These proteases together execute the proteolytic function in the GI tract and significantly affect the metabolisms and composition of the GI content (Vergnolle, 2016). Meanwhile, GI tract is also heavily presented by protease inhibitors, including those originated from food intake (such as buckwheat as mentioned earlier), and circulating inhibitors produced at distance from GI tract, that is liver, produced at the intestinal epithelial cells and inflammatory cells. The unbalanced or dysregulated proteolytic homeostasis in GI tract can result in severe inflammatory bowel diseases (IBDs), such as Crohn’s disease and UC, which are suggested to be due to the overly elevated protease level in GI tract (Motta et al., 2011; Vergnolle, 2016). Trypsin activity was increased in the GI tissues from patients with Crohn’s disease and UC (Cenac et al., 2007). A study further showed that the increased matrix metallopeptidase activity in the tissues from IBD patients was restored to the same level of healthy controls after treatment with infliximab, a chimeric monoclonal antibody to tumor necrosis factor alpha and later used for treatment of fistulas from complications of Crohn’s disease patients (de Bruyn et al., 2014). This suggested that elevated protease activity is associated with IBDs and downregulation of the protease activity helps to improve and restore the health of GI tract. Trypsin inhibitors are involved in many physiological mechanisms and the pathway how they function are mostly still exclusive. For instance, tumorassociated trypsin inhibitor (TATI) has been long used as a tumor marker for diagnosis of ovarian cancer and other malignancies including cancers from the urogenital tract (Meria et al., 1995). However, its presence through healthy pregnancy increases significantly from only 12 μg/L when at 6
9 weeks of amenorrhea to 26.8 μg/L when measured at the 35
40 weeks of amenorrhea (Schlageter et al., 1998). The 95th percentile value was 25 μg/L when tested with serum samples from 100 healthy blood donors. At the very early stage of pregnancy, TATI was very low and below 50% level of normal cutoff value from healthy individuals. Then concentration gradually increased to doubled value, high but still below cutoff value, at the late stage of pregnancy. It is believed that the trypsin

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inhibitor plays important roles from beginning of the pregnancy and its roles may also change at different stage of healthy pregnancy. Nevertheless, little is known how trypsin inhibitor and TATI execute their functions and how they impact the overall progression of a healthy pregnancy. Proteasome inhibition was confirmed to be able to alter neural mitochondrial homeostasis and mitochondrial turnover (Sullivan et al., 2004). Inhibition of proteasome activity occurs in an otherwise normal healthy aging process and in many neurodegenerative conditions including AD and Parkinson’s disease. The neurodegenerative conditions are associated closely with mitochondrial dysfunction that is regulated by proteasome activity inhibition. When tested with C6, a clonal line of human SH-SY5Y cells, the authors noted that mitochondria, when exposed to low level proteasome inhibition, displayed similar morphological features and similar rates of electron transport chain activity to those untreated neural cultures with equal passage number. However, maximal complex I and complex II activities were dramatically reduced in the neural cells that were subjected to the low-level proteasome activity inhibition. It was also observed, that proteasome activity inhibition increased mitochondrial reactive oxygen species (ROS) production, reduced intramitochondrial protein translation, and increased the cellular dependency on glycolysis. Proteasome inhibition altered mitochondrial bioenergetics (mitochondria oxygen consumption rate dropped from the control’s almost 450 nmols O2/min/mg to less than 150 nmols O2/min/mg) and mitochondrial ROS was elevated significantly in C6 line, even with a low level of proteasome inhibition. With the significant reduction in mitochondrial bioenergetics and proteasomal activity, the overall mitochondrial protein synthesis was decreased by 64.8% in the C6 cell line, compared to the untreated control. Further focused pulse-chase experiments showed a startling 81.4% reduction in mtDNA-encoded proteins in C6. The authors further noted the increased level of lipofuscin that may suggest impairments in mitochondrial turnover caused by even low-level proteasome inhibition. The study demonstrated the fact that proteasome activity inhibition dramatically alters specific aspects of neural mitochondrial homeostasis and further alters lysosomal-mediated degradation of mitochondria. It was stated that both alterations in mitochondrial homeostasis and in lysosomal-mediated degradation of mitochondria, contribute to an altered aging process and age-related disease in the nervous system. The reduced bioenergetics reduced ATP production and the overall energy supply to the functional systems. The aging-related mitochondrial homeostasis alteration, and its resultant bioenergetics reduction contribute to the age-related symptoms such as reduced organ functions, fatigue, reduction in cognitive capability, reduction in organoleptic sensitivity, reduction in mobility and, gradually and eventually, reduced desires for involvement in energetic activities and the social activities which they used to be active with. The reduced protein synthesis rate may also result in a lowered mitochondrial regeneration, along with increased mitophagy. It is reasonable to infer that the dual impacts of aging-related proteasomal activity inhibition, causing both lowered mitochondrial regeneration and increased mitophagy, likely could

23.3 Buckwheat, health benefits and presence of trypsin inhibitors

cause an exponential acceleration of the aging process through one’s lifespan. This observation may help to explain why the aging process is not a linear course but fits better in an exponential descriptive model.

23.3 Buckwheat, health benefits and presence of trypsin inhibitors 23.3.1 Buckwheat as a food staple in some regions and its global presence as a functional food Buckwheat is a pseudo-grain, originating from the far east region and then spread globally. It is highly adaptive to adverse environments so it can grow well at high-altitude cold, dry mountainous terrains such as the Tibetan Plateau including Tibet, west Sichuan, Qinghai, Gansu and Inner Mongolia and other mountainous regions such as Liang San Yi Autonomous Region in China’s Sichuan Province and Jing Zhou in China’s Guizhou Province and many other regions where the land is so infertile and most other crops do not grow well, however buckwheat thrives. Nowadays buckwheat grows in many different countries and regions, including China, Russia, United States, Germany, Italy, Japan, Korea, Poland, France and Canada, to list a few. Its production has been significantly improved and a 3400 kg per hectare was reported in Russia in 1990 (Alekseeva, 1993). In the United States, buckwheat grows in North Dakota, South Dakota, Minnesota, Montana, Washington, California, Pennsylvania, Ohio, and New York. The total buckwheat output in US was estimated to be 17,000
20,000 tons per year (Peterson et al., 1992). According to North Dakota Consolidated Farm Service Agency, buckwheat production in North Dakota were 11,228, 15,389, 8477, and 10,627 tons in 1991, 1992, 1993, and 1994, respectively (Edwardson, 1996): Per a report released by Statista in January 2022, buckwheat production in the United States reached 85,594 and 86,397 metric tons in 2019 and 2020, respectively (online access: statista.com/statistics/486495/us-buckwheat-production/). It is used as staple foods for some remote regions in many different formats, such as griddle cakes, noodles, bread, and cookies. But globally it is mainly used as a supplemental food for its potential health benefits such as hypotension, cholesterol-lowering, and blood sugar downregulation effect (Li & Zhang, 2001).

23.3.2 Potential health benefits from consuming buckwheat foods As summarized in a brief review (Li & Zhang, 2001), buckwheat is rich in many bioactive components including flavones, fagopyrin, phytosterols, thiamin-binding proteins, trypsin inhibitors, and unique buckwheat storage proteins that have significantly lower lysine/arginine ratio and methionine/glycine ratio than most of other food protein sources. There were substantial reports suggesting that proteins

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with lysine/arginine ratio and methionine/glycine ratio provide potent cholesterollowering effects, or hypocholesterolemic effects (Carroll & Kurowska, 1995; Huff & Carroll, 1980; Sugiyama et al., 1985). Rich flavonoids in the buckwheat products are beneficial to improve the flexibility of blood vessels and the overall health status of the cardiovascular system via their potent antioxidant activity, reducing stresses from free peroxide radicals and meliorating chronic inflammation. It was previously reported that flavonoids showed strong antioxidant effects, reducing blood sugar, maintaining blood vessels strong and flexible, assisting in hypotension effects, and prevented human lymphocyte DNA from oxidative damage (Noroozi, 1998; Waterman, 1986). The addition of vitamin C further strengthened the flavonoid’s antioxidative effects (Noroozi, 1998). Diet supplementary studies also suggested quercetin and apigenin, the two main flavonoids in buckwheat, may improve hemostasis both in vitro and vivo (Janssen & Karin, 1998). The authors argued that supplementing flavonoids in the human diet may prevent the onset of some cardiovascular diseases and improve those already initiated. Flavonoid-rich herbs, such as buckwheat, have been long used as “cold” or “calm” components in Chinese traditional medicines to improve cardiovascular system health and chronic inflammation in patients. Phytosterols and buckwheat proteins are reported to be the effectors for buckwheat’s hypocholesterolemic effects, while blood sugar reducing effect from consuming buckwheat products was likely attributed to buckwheat’s proteins and flavonoids. A clinical study with 84 subjects at Tong-Ren hospital, Beijing, suggested buckwheat may be effective at improving diabetes and arteriosclerosis with a potent double down effect (reducing both blood sugar and blood lipid content) (Lu, 1993). Lee et al. (2012) reported that buckwheat flavonoids, quercetin, in buckwheat extract were effective to inhibit blood glucose level and insulin levels of the tested mice model, suggesting buckwheat can be used for treatment of type II diabetes mellitus. Many research interests reside in the bioactive components of buckwheat such as trypsin inhibitors, flavonoids, phytosterols, fagopyrins, thiamin-binding proteins, vitamins, dietary fiber, and its unique high arginine and low sulfur amino acid content proteins, while some are also attracted by buckwheat’s strong capacity to adapt to adverse environments from a sustainability point of view. Its pseudocereal nature also helps buckwheat products carry gluten-free claims and provide celiac patients with new relief options (Jin et al., 2020).

23.3.3 Presence of buckwheat trypsin inhibitors, characteristics and physiological roles Buckwheat contains high content of BTIs, which have long been considered as major antinutritional factors affecting buckwheat protein’s biological value compared to other major crops. Seven BTIs had been characterized in the early 1980s (Kiyohara & Iwasaki, 1985), including four so-called permanent inhibitors, BTI I,

23.3 Buckwheat, health benefits and presence of trypsin inhibitors

IIa, IIb and IIIa, and three so-called temporary inhibitors, BTI IIc, BTI IIIb1 and IIIb2. Permanent inhibitors have a small molecular weight at about 6000
7000 Daltons while the temporary ones are slightly bigger at 10,000 to 11,500 Daltons. The amino-terminal residue is leucine and carboxyl-terminal residue is alanine for the permanent BTIs, and serine at the amino-terminal and leucine at the carboxylterminal for the temporary BTIs. The two groups were well differentiated from their distinct primary structures. The temporary character was judged based on the observation that BTI IIc, IIIb1, and IIIb2 showed instable inhibitory effects against trypsin upon prolonged incubation at neutral and alkaline pH’s. Ikeda and Kusano (1983) also reported similar results on the molecular weights of permanent BTIs. High content of BTIs in buckwheat products were suggested to be the factors causing their low digestibility in animals (FAO, 1970; Javornik et al., 1981) while Farrell’s feeding experimental results with rats, pigs, chickens, and laying hens (Farrell, 1976; Farrell, 1978) demonstrated that the generally poor biological availability of buckwheat proteins (against all wheat diets) is due to the presence of high BTI content, although different animal species responded differently with the all-buckwheat diet. However, besides their fundamental protease inhibitory effects, recent studies suggested that trypsin inhibitors may possess many potential health benefits via their crucial roles played through systematic proteolytic activity regulation in cellular and subcellular levels, such as that in the subtle regulation of serine peptidase activity in mitochondria, where a wellmaintained homeostasis is critical to sustain the normal function of this organelle. Due to the broad inhibiting spectrum and potent serine-protease inhibiting power, potential health benefits of BTIs, including potentially effective medicines for anticancers, antiinflammation, antiaging, antimicroorganism, and antivirus applications, have drawn substantial interests of many researchers globally. Furthermore, BTIs, particularly BTI I (rBTI) and BTI II, are highly heat stable, with no significant inhibiting activity loss after 30 min heat treatment at 95 C and pH 8.3, while BTI III, which are the most abundant, are slightly less heat stable, losing about 20% of inhibiting activity after 95 C for 30 min (Ikeda & Kusano, 1983). Research suggested that the ingested trypsin inhibitors may be able to transfer across the intestinal lumen barrier and infuse into blood stream. A clinical study concluded that the absence of trypsin inhibitors (from corn) significantly increased the thrombin generation in several of the samples from a pancreatic cancer group (Hellum et al., 2017), suggesting inhibitory effects of trypsin inhibitors on thrombin generation in pancreatic cancer patients. Park and Ohba (2004) confirmed that the tested BTIs significantly suppressed the growth of T-ALL cells, tested using lab purified BTI 1a and BTI 2a and human tumor T-acute lymphoblastic leukemia (T-ALL) cell lines, JURKAT and CCRF-CEM. BTIs triggered programmed cell death (apoptosis) of the cell strains with DNA fragmentation. Modification of the arginine residue in BTIs inactivated BTIs’ suppression capability. It was also reported that trypsin inhibitors prevent pancreatic injuries induced by pancreaticobiliary duct obstruction with cerulein stimulation

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and systemic hypotension in rats (Hirano & Manabe, 1993). After ingestion of BTI-rich buckwheat food products, remaining BTI residues could be transferred into the blood stream, and further transferred into cells, and subsequently mitochondria, further executing their proteolysis regulating function, depending on the environments they are exposed to. BTIs’ physiological functions and potential impacts on human health are worth further detailed investigations.

23.4 Roles of mitochondrial homeostasis in healthy aging and improvement by presence of recombinant buckwheat trypsin inhibitor 23.4.1 Roles of mitochondrial homeostasis in healthy aging Mitochondrial homeostasis is critical to healthy aging and its maintenance is an important determinant of lifespan (Princz et al., 2020). Mitochondria are highly dynamic organelles. They provide energy and necessary regulations for the maintenance of cellular homeostasis in eukaryotic cells. Mitochondria also play critical roles in calcium storage, signaling, metabolite synthesis, and apoptosis (Palikaras & Tavernarakis, 2014). Due to its vital role in maintaining normal functionality of animal physiology, mitochondria have long been a topic of studies and results have well supported the claim that mitochondrial metabolism plays vital roles in regulating the aging process and directly determines quality of the health of aging. Substantial evidence suggested that mitochondrial dysfunction is a major hallmark of aging—highlighting the significance of proper mitochondrial activity for survival (Lopez-Otin et al., 2013; Princz et al., 2020). Mitochondria, like many other organs and organelles, also undergo both autophagy and biogenesis. To maintain normal functionality, mitochondria need to replenish the supply of necessary nutrients as building blocks for replacing the damaged parts with new ones, so as to sustain the desired efficiency of the overall organelle. Meanwhile, the spent or damaged organelles and parts need to be digested and removed in a timely manner, to prevent cellular toxicity from accumulating. Due to the limited life cycles of the organic biosystems such as mitochondria, both biogenesis and mitophagy are continuous tasks throughout the lifetime. More importantly, it is critical to maintain the deliberate balance between mitophagy and biogenesis in order to maintain the appropriate mitochondrial function for a healthy aging process. Ideally, rates between mitophagy and biogenesis can be maintained precisely the same, so any dysfunctional parts/mitochondria can be replaced by fresh, fully functional ones immediately as they are detected. Nevertheless, the processes involved for both mitophagy and biogenesis are extremely complicated, including signaling, detection, regulation, digestion, biogenesis, and activation. There is also a long process between the time when a mitochondrion starts showing minor malfunction, and when it is being detected, then mitophagy starts. Through this

23.4 Healthy aging and improvement

long process, the mitochondria try to self-regulate by correcting the dysfunctional parts and systems with certain downregulating or upregulating of the relevant activities, such as protease activity. From an energy efficiency point of view, mitochondria also try to repair themselves in an effort to restore the dysfunction detected, rather than simply replace all the dysfunctional parts with new ones, until the dysfunctional parts or the whole mitochondria have accumulated errors so large, preventing them from meeting the basic functional requirements. As the results suggest, there are almost always many mitochondria in one’s body dysfunctioning to some extent, progressing toward gradually getting less functional over the time, while internal systems try to correct the errors by adjusting the regulation levels and restoring the normal functions, to maintain the vital balance between mitophagy and biogenesis. These battles between dysfunction worsening and function correcting are continuing through one’s life. How to slow down the dysfunction worsening while improving the function correcting and maintain a deliberate balance between mitophagy and biogenesis are crucial to maintain a healthy aging process, better yet, to slow down the aging process and allow one to live longer, healthily. When tested with Caenorhabditis elegans, Princz et al. (2020) noted that, by controlling the activity of nodal transcription factors, the insulin/IGF signaling pathway impacted lifespan across distant taxa. The transcription regulators dauer formation abnormal-16 (DAF-16)/FOXO and SKN-1/Nrf functioned to promote longevity under conditions of low insulin/IGF signaling and stress. The authors claimed that aging causes significant increases of USMO levels in C. elegans. In turn, SUMO fine-tunes DAF-16 and SKN-1 activity in C. elegans tissue to improve stress resistance. Through SUMOylation of DAF-16 SUMO mitochondrial homeostasis is modulated. This interruption of SUMO resulting in improved homeostasis of mitochondria, changed the course of mitochondrial dynamics and mitophagy. The improved mitochondrial homeostasis status, as the result, increased the longevity and lifespan of the tested C. elegans. This research provided evidence that mitochondrial homeostasis status, the deliberate equilibrium between mitochondrial genesis and mitophagy, is a critical determinant of longevity, and healthy aging. The heart, as the master driver of the body’s logistic system, is an organ that requires consistent and continuous energy/ATP supply throughout life to sustain its contractile function. Cardiomyocytes contain a large number of mitochondria that attribute more than 95% of the total ATP production derived from oxidative phosphorylation (Barth et al., 1992). Even minor changes in mitochondrial function and mechanisms may cause significant interruption to the consistent and uninterruptable ATP supply, resulting in substantial impacts on cardiac function. A reduced capability of the heart contractile and thus a reduced blood pumping capacity, due to either medical conditions or by natural courses of aging, would result in reduced capability in mobile, cognitive, organoleptic, digestive, and other bodily functions and further result in the mental feeling of “aged” (or “old”) with a lack of interest for being active. Another example demonstrating the crucial

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roles mitochondria play, is the reduced myocardial mitochondrial ATP generation and supply due to aging. This reduced ATP supply in the myocardial cells resulted in fatigue, reduced mobility, significantly less activity than youth, in social words often described as nonenergetic or aged. It happens in young patients with certain cardiovascular diseases and elderly persons whose aging process take on an accelerated pace. In an animal trial, adult mice with cardiac-specific Klf4 deficiency developed cardiac dysfunction with aging in response to pressure overload (Liao et al., 2015). These cardiac dysfunctions were characterized as reduced myocardial ATP levels, elevated ROS, and significant alteration of mitochondrial morphology—shape, size, ultrastructure, and alignment. Studies on the isolated mitochondria from Klf4-deficient heart showed reduced respiration rate likely due to defects in mitochondrial electron transport chain complex. Deletion of embryonic cardiac-specific Klf4 resulted in postnatal premature mortality, impaired mitochondrial biogenesis, and altered mitochondrial maturation. While this study provided evidence that Klf4 may be a critical transcriptional regulator of mitochondrial homeostasis, it also reaffirmed the critical importance of mitochondrial homeostasis to sustain the normal functions of heart and energetic daily activities that require the heart to deploy the needed nutrients, messengers, and effectors throughout the whole body. Diabetic retinopathy is also closely associated with impaired mitochondrial functions and mechanisms. Increased leakage of cytochrome C from damaged mitochondria accelerates retinal capillary cell apoptosis, which precedes the formation of acellular capillaries and pericyte ghosts (Kowluru & Abbas, 2003; Mizutani et al., 1996). In diabetic retinopathy, mitochondrial dynamics and biogenesis are compromised, with which mitochondrial fusion protein, mitofusin 2 (Mfn2) is decreased and fission protein dynamin 1-like protein (Drp1) is increased, leading to smaller mitochondria with increased mtDNA instability (Zhang & Kowluru, 2011). The mtDNA in impaired mitochondria also are damaged with increased severity at its D-loop. The mtDNA transcription is impaired, thus the electron transport system is compromised and further compromises the overall stability of the whole mitochondria (Madsen-Bouterse et al., 2010). In diabetic retinopathy, DNA methyl transferases and activated Ten-Eleven translocases are responsible for maintaining retinal mitochondrial DNA methylation status. Within the impaired retinal mitochondria, mtDNA’s promoter DNA of Mfn2 and Mlh1 are highly methylated and 5-methyl cytosine level is significantly increased. To make things worse, retinopathy continues worsening and DNA methylation machinery continues to function aberrantly even after the hyperglycemic conditions are removed (Mishra & Kowluru, 2019) by managing blood glucose levels in an otherwise healthy level. With the increasing incident rate, particularly with elders, diabetes and its associated complications may have a direct linkage with conditionally compromised structural and functional alteration of impacted mitochondria. With an increasingly aged world population, it is worthwhile to closely investigate mitochondrial structures, functionality, integrity and the mechanisms to maintain them forvoptimal homeostasis status for prolonged time. It would also

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be likely that therapeutic drugs can be developed to effectively intervene in mitochondrial homeostasis and/or make corrections to errors and mismatches in the transcriptional pathway and restore the healthy equilibrium between fission and fusion and between biogenesis and autophagy. Kowluru and Mohammad (2020) reported their studies on epigenetics and mitochondrial stability in the metabolic memory phenomenon associated with continued progression of diabetic retinopathy. The authors claimed that supplement of Dnmts inhibitor, 5-aza-2’-deoxycytidine (Aza), during reversal of high glucose insult, restored normal methylation in mtDNA and prevented a decrease in the transcription level of mtDNA-encoded genes. They further investigated the effects of inhibition of Dnmts on restoration of mitochondrial dynamics and DNA stability. They observed that the decrease (due to induced diabetic retinopathy) in Mfn2 gene transcription was also restored. They observed increased Mfn2-CoxIV colocalization, and a higher person’s correlation in HG/Aza and HG-NG/Aza groups compared to HG and HG-NG groups and accordingly stated that Aza supplementation significantly improved mtDNA transcription and mitochondrial stability through improved Mfn2 mitochondrial localization. Mitochondrial homeostasis plays critical roles in skeletal muscle wasting in patients following severe burn trauma. Dramatically increased skeletal muscle wasting was observed with severe burn trauma patients (Ogunbileje et al., 2018). Skeletal muscle wasting is a hallmark of the long-term pathophysiological stress response to severe burn trauma. Patients with a $ 30% TBSA burn can lose up to 25% of their body mass in the first month post injury (Ogunbileje et al., 2016). Although muscle synthesis rate was also increased, the rate for muscle breakdown increased so dramatically that resulted in a severe net muscle loss and a fast skeletal muscle wasting, even when aggressive nutritional support was provided. Hypermetabolism was reported as one of the major symptoms following severe burn trauma—this was to serve the purpose for wound healing, fighting infection, and maintaining core temperature. The authors argued that burn trauma induces mitochondrial stress in skeletal muscle and results in the hypermetabolism, which further caused skeletal muscle wasting. In burn patients, elevated respiration rate stresses tissue mitochondria such as those in skeletal muscles, then further increases protein oxidation and activates mitochondrial unfolded protein stress response, further resulting in increased production of oxygen superoxide anions. As a result, hydrogen peroxide production is increased and reducing iron is accumulated (Cantu et al., 2009) due to inactivation or inhibition of ROS sensitive mitochondrial aconitase by the elevated concentration of oxygen superoxide anions. Continuous production of O2 2 and  OH damages both mitochondrial proteins and other structures within the cells. The stressed and damaged mitochondria proteins trigger the hypermetabolism for the mitochondria to intend to repair the damaged structures. The majority of ROS in a cell is produced in its mitochondria. Extended mitochondrial hypermetabolism following burn trauma results in the elevated production of ROS and further releases ROS into the cellular cytosol, causing damage and rupture of cellular structures and functions.

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Treating the affected cells with mitochondrial-targeted antioxidants and antioxidant enzymes attenuated cell damage and protected cellular integrity (Maharjan et al., 2014). The studies by these authors evidenced the critical roles mitochondria play and that altered mitochondrial homeostasis status could cause severe health and medical complication, including but not limited to skeletal muscle wasting in burn trauma patients. The author’s work also demonstrated the feasibility to artificially intervene in the mitochondrial mechanisms and purposely alter their homeostasis status. In addition to the application of antioxidant enzymes to repair damaged mitochondrial homeostasis status, there might be other means that could be employed to effectively improve mechanisms, function, and the overall mitochondrial homeostasis status. This purposely altered mitochondrial homeostasis status could be used not only for acute disease mitigation, but also for correcting age-associated derail in mitochondrial homeostasis.

23.4.2 Buckwheat trypsin inhibitor and recombinant buckwheat trypsin inhibitors: properties, functionality and their potential roles in maintaining stability of mitochondrial homeostasis Over one-third of known proteolytic enzymes are serine proteases. Among them, trypsins underwent the most predominant genetic expression yielding enzymes responsible for digestion, blood coagulation, fibrinolysis, development of fertilization, apoptosis, and immunity (Cera, 2009). Of 699 proteases in man, 178 are serine proteases and 138 of them belong to PA Clan’s S1 family, where human trypsin also belongs. S1A family proteases are trypsins that mediate a variety of biological processes including protein turnovers. Trypsin-like serine proteases are the largest group of homologous proteases in the human genome. These trypsinlike proteases play critical roles regulating and executing a vast number of biological reactions, providing homeostasis for many mechanisms involved, including cellular and mitochondrial homeostasis. Their activities, their inhibition (such as that by BTIs), and stimulation, are of great importance to disease fighting and health maintenance including healthy aging. BTIs’ basic properties, thermal stability, and fundamental functionalities were discussed earlier in this chapter. Other than their potential health benefits as a functional and bioactive component for functional foods (Li & Zhang, 2001), buckwheat products are rich in BTIs. In addition to their traditionally believed “antinutritional” function via inhibiting trypsin activities in the GI tract, BTIs may also play critical roles for maintaining health from deep in the cytologic and mitochondrial level. Buckwheat seeds contain seven different types of trypsin inhibitors as described earlier, four permanent inhibitors (BTI I, IIa, IIb and IIIa), and three temporary inhibitors (BTI IIc, IIIb1 and IIIb2). All BTIs can effectively inhibit trypsin activity, but some of them, that is BTI I, have a much broader inhibitory spectrum and can also effectively inhibit other serine endopeptidases such as chymotrypsin, subtilisin, and nucleoporin.

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Due to challenges and low efficiency in the isolation of BTIs from buckwheat seeds directly, tartary buckwheat BTI I genes are engineered into other vectors, such as E. coli (Zhang et al., 2007) for production through controlled fermentation followed by separation and purification steps. The resultant rBTI is BTI I, belonging to the potato trypsin inhibitor family I. Compared with natural plantbased trypsin inhibitors such as that from soybean, rBTI has approximately the same inhibition efficiency (as indicated by inhibition constant Ki)— 7.41 3 1029M for rBTI versus 6.52 3 1029M for soybean trypsin inhibitor (Li et al., 2021). Trypsin inhibitory activity of both rBTI and SBTI were reported stable over a broad pH range from pH 2.0 to pH 12.0, while rBTI showed a higher thermal stability than SBTI. Nevertheless, their sensitivity to the presence of zinc, KSCN, vitamin C, and urea were quite different. While the natural soybean trypsin inhibitor showed no changes with the presence of these compounds, rBTI showed significant inhibitory activity loss. Caution is needed when suggesting dose levels for rBTI if inferred based on experimental data collected from natural BTIs, considering concerned dietary items, particularly diet supplement items such as zinc, multimineral supplement, and vitamin C. The inhibitory activity of both rBTI and soybean trypsin inhibitor did not show sensitivity to the presence of Mg11 and Cu11. The rBTIs have the same function inhibiting broad spectrum serine proteases including that of BTI I, but potentially contain less impurities and are free from the allergens and photosensitizers from buckwheat. rBTI may be safer to buckwheat-sensitive patients, while potentially having a lower cost compared to BTIs. Furthermore, rBTI may potentially be enhanced in inhibiting efficacy through genetic engineering tools. Nevertheless, the cheaper price and more efficient production of rBTIs should not exclude the health benefit values of natural BTIs, particularly as a means for prehealth condition management and for healthy aging. While rBTIs could potentially be used for acute and chronic disease treatment with targeting medical conditions, BTIs can be taken via daily buckwheat food consumption for the benefits of supporting healthy aging and improving age-related chronic diseases. Similar to other protease inhibitors, rBTI conducts conformational changes upon binding with substrate trypsin (Wang et al., 2011), or other target serine proteases. Functionality and trypsin inhibiting efficacy of rBTI can be manipulated by modifying its critical amino acid residues such as P2 (Pro44) and P8’ (Trp53) residues via bioengineering editing tools. One of the mutants, P44T, which the P2 Proline residue is replaced with a threonine residue, showed significantly higher potent inhibiting bovine trypsin activity than its wild-type rBTI sister (dissociation constant of P8’ and P2 hydrophobic force was reported only 5.24 3 1027 s21 with the P44T mutant while this dissociation constant within wild-type rBTI was 1.25 3 1023 s21). Other examples of rBTI mutants include W53F—replacing P8’ tryptophane residue with a phenylalanine residue, W53R—replacing P8’ residue with an arginine residue, and W53R/P44T mutant—replacing both P8’ residue with an arginine residual and P2 residue with a threonine residue. As previously reported, BTI-1 showed significant suppressive activity against human T-acute

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lymphoblastic leukemia cell lines (Park & Ohba, 2004). rBTI also showed activity inhibiting the proliferation of IM-9 human B lymphoblastoid cells from a patient with multiple myeloma in a dose-dependent manner (Zhang et al., 2007), and antitumoral activity when tested with several human solid tumor cell lines (EC907, Hep G2 and HeLa) via inducing tumor cell apoptosis (Li et al., 2009). The realizability of intentional manipulation of rBTI function and potency, may provide useful tools for designing medicines for more effective treatments of different chronic medical conditions such as cancers. The anticancer activity of rBTI was proposed through its upregulating mitophagy, thus inhibiting the proliferation of tumor cells. Recombinant BTIs were reported effective in preventing fat accumulation in C. elegans line under both high glucose and normal glucose conditions (Li, Ning, et al., 2019). This preventing of fat accumulation effect resulted in a significantly narrower body width of the test C. elegans, without affecting their feeding behaviors. The authors argued that rBTI altered expression, transcription, and activities of the key enzymes involved in lipolysis and fat biosynthesis. The authors claimed that the fat-fighting effects of rBTI are through the insulin/insulin-like growth factor pathway; and further went on stating that rBTI can be used for treatment of metabolic diseases including obesity, hyperglycemia, and hyperlipidemia. rBTIs have been reported improving the mitochondrial homeostasis. In a vitro study tested with C. elegans cell model, rBTIs were found to significantly promote autophagy and alleviate the age-related functional decline via DAF-16 (Li, Cui, et al., 2019). During the study, the authors cultured the Day ten N2 C. ellegans, as aging model, and Day six AM140 C. ellegans, as age-related disease model, at 25 C. ATP production capacity and damaged mitochondrial DNA were measured to compare impacts of rBTI enrichment. Soluble protein content, autophagy marker protein Igg-1, and lysosomal content were measured to quantify autophagy development. They used chloroquine, an autophagy inhibitor, a DAF16 mutant, and RNA interference to determine the roles autophagy played in rBTI-mediated effects. Motor function was also assessed as an aging dependent function indicator. It was found that the enriched rBTI significantly reduced insoluble proteins and damaged mitochondria and further reduced the motility rate of both models. It was noted that rBTI activated mitochondrial autophagy, and inhibited autophagy by adding chloroquine, which significantly reduced the observed benefits from rBTI. Further genetic analyses suggested rBTI increased DAF-16 transcriptional activity. The authors argued that rBTI promotes mitochondrial autophagy to alleviate the age-related function loss through DAF-16 pathway. rBTI was reported to directly target mitochondria and induces mitochondrial fragmentation and mitophagy (Wang et al., 2015). rBTI increased mitochondrial membrane potential and ROS generation due to increased superoxide dismutase and catalase activity in the mitochondria affected. Enrichment of rBTI also increased the glutathione peroxidase concentration and activity thus changing the ratio between GSH and oxidized glutathione and systemic redox condition.

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Compared to untreated cell controls, rBTI treated cells had significantly more colocalization. rBTI promoted mitochondrial fragmentation. The elevated level of ROS induced by rBTI in the test cells was believed to be the signaling molecule responsible for the observed Hep G2 cell mitophagy and removal of the dysfunctional mitochondria. In the test Hep G2 cell line, rBTI directly targeted at a subunit of the translocase of the outer membrane (Tom20) of the mitochondria and formed a rBTI 1 Tom20 complex. This rBTI 1 Tom20 complex was proposed to initiate the downstream cascade for ROS production enhancement and other responses. Mitophagy was a critical aspect of overall mitochondrial homeostasis, removing damaged and dysfunctional mitochondria timely to prevent dysfunctional mitochondria from accumulation and become overwhelmed in the host cells. In theory, timely removal of the dysfunctional mitochondria would clear out the needed room in the affected cells and further stimulate the biosynthesis of fresh mitochondria with normal functionalities. However, more direct evidence is needed to confirm rBTI’s potential indirect upregulation of mitochondrial biosynthesis. Should rBTI’s upregulation on mitogenesis be confirmed, rBTI may not only promote mitophagy upon detection of elevated level of dysfunctional mitochondria in the cells but also autonomously upregulate mitogenesis and thus promote the overall mitochondrial homeostasis under adverse health conditions such as those induced by aging, ROS stress, AD, and other complications. When tested with C. elegans AD model of β-amyloid peptide toxicity, rBTIs showed clear protective effects and delayed amyloid beta (Aβ) peptide-triggered body paralysis without increasing transactivity of HSF-1 or solubility of proteins in aged AD worms (Li et al., 2017). The rBTI treatment showed significant effect in increasing transcriptional activity of DAF-16. Disruption of DAF-16 removed this rBTI-mediated protective effect. The authors claimed that rBTI treatment activated the autophagy-lysosomal degradation pathway and reduced the accumulation of Aβ in the AD model, thus further stating that rBTI may be a potential tool to protect people from AD. AD is an age-related cognitive disease that affects millions of patients and their families. The adverse impacts on patients and their loved ones by AD is so desperate and their quality of life plummets. As one of the major concerned age-related diseases, AD causes a poor quality and accelerated aging process. Although tremendous efforts have been undertaken, unfortunately, there is still no proven effective cure, even effective enough to significantly slow down the aggression of AD. rBTI and natural BTIs may be the light at the end of the tunnel and provide, hopefully, an effective cure or improvement to AD. Improvement of AD symptoms will improve the aging process of the patients, and to those around them as well. Furthermore, it was reported that, in a different study, rBTI can prolong the lifespan of the test model C. elegans (Li, Cui, et al., 2019). Nevertheless, one should be aware that current studies on AD protection effects and longevity effects were performed with a worm AD model. There is still a long way to go before solidly proving rBTI’s protective effects in humans. Tremendous research work is needed both in clinical studies to validate its physiological effects, and in medicine labs for developing appropriate

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formats and dosing to deliver the effects. As for the food industry, it is foreseeable that buckwheat foods will continue to thrive and gain more and more interest from health-conscious consumers for their health benefits, including potentially AD protection and aging improvement. The significant antitumoral effects of rBTI, both in vitro and in vivo (with mice, abdominal injection daily for eight days, four test groups each at 0.125, 0.25, 0.50 and 1.0 mg/kg dose levels), suggested that rBTI reduces tumor cell viability by inducing apoptosis (Bai et al., 2015). Formation of apoptotic bodies and DNA fragmentation was observed in the test H22 hepatic cancer cells treated when with rBTI. Growth inhibition rate of rBTI was 17.8%, 27.3%, 43.6%, and 62.7% at rBTI concentration levels of 6.25, 12.5, 25.0, and 50.0 μg/mL (in ascites fluid), respectively. The cancer cell inhibition effects of rBTI markedly increased along with the increase of rBTI concentration. Under microscope, the rBTItreated H22 hepatic cancer cells showed clear and dramatic development of apoptotic bodies and ruptures after only 24 h of treatment. The rBTI induced apoptosis caused mitochondrial dysfunction in the test H22 hepatic cancer cells and further caused release of cytochrome C from the affected mitochondria into cytosol. Further activation of caspase-3, -8 and -9, suggested rBTI’s apoptosisinducing effect was through its capacity mediating mitochondrial pathway via capase-9. The antitumoral effects of rBTI was reported dose- and time-dependent. It was also noted that this apoptosis inducing effect of rBTI had no effects on normal liver cell line H7702. This suggested that some elevated and unique marker factor(s) in the cancer cells may serve as the trigger to initiate BTI’s apoptosisinducing cascade, further suggesting rBTI (or BTIs) could potentially be a potent and safe anticancer treatment with very minimal side effects. It also confirmed rBTI’s capacity to alter mitochondrial function thus the overall homeostasis through different pathway in different cells, such as in this reported hepatic cancer H22 cell line. In summary, the mechanisms responding for rBTI’s mitophagy promoting effects in disease altered cells could be highly complex and more studies are needed to elucidate the relevant questions. It was noted that rBTI promotes mitophagy in tested cancer cells such as G2 and H22 lines but not in the normal control hepatic cells. While it is good news to affirm the efficacy of rBTI and support the safe applications, it is still not well understood which of the hallmark components in the cancer cells serves as the triggering or signaling messenger for rBTI to promote mitophagy and cancer cell apoptosis. The potential mechanisms that rBTI involves for molecular communication and mitophagy promoting effects in the affected mitochondria are illustrated in Fig. 23.1. As shown in Fig. 23.1, there are four (A, B, C and D) different pathways for mitochondrial molecular communication without presence of rBTI and 1 pathway, E, or potentially more, demonstrating how rBTI’s presence to change the otherwise “normal” mitochondria in cancer cells or, possibly, other disease-altered cells and to promote mitophagy. The elevated mitophagy in these disease altered cells further causes reduced ATP production in mitochondria and eventually cellular apoptosis.

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FIGURE 23.1 rBTI promotes mitophagy via increasing ROS and formation of Daf-16 and overall molecular communication between mitochondrial biogenesis and mitophagy.

A. Increased cytoplasmic calcium level stimulates the activation of CaMK, and the later further phosphorates PGC-1α, which promotes mitochondrial biogenesis. Further, derivative CaMKKβ can activate AMPK, promote ULK-1/2 and then induce mitophagy. B. Nutrient deprivation or other stressors triggers AMPK activation, then AMPK initiates a response that promotes both mitophagy and mitochondrial biogenesis to offset the impacts from the stressors and rebalance the mitochondrial homeostasis. This partially explains the potential health benefits from intermittent fasting. C. AMPK promotes SIRT1 activity by increasing cellular NAD1 levels. Then SIRT1 deacetylates PGC-1α and resultant deacetylated PGC-1α activates and promotes mitochondrial biogenesis. D. Dysfunctional mitochondria are observed with enhanced ROS production. PKD is activated during the process in response to detected dysfunctional mitochondria and elevated levels of ROS production. Elevated ROS production promotes mitophagy while activation of elevated PKD activity promotes mitochondrial biogenesis. Pathway D is vital for host cells to remove dysfunctional mitochondria and replenish with fresh ones to sustain a health mitochondrial homeostasis status. E. In hepatic cancer cell Hep G2, rBTI directly targets at Tom20 and forms rBTI 1 Tom20 and further initiates elevated ROS production, with observed reduction in transmembrane potential and elevated SOD, CAT and GSH levels (Wang et al., 2015). These resultant shifts in cancer cell mitochondria further cause reduced ATP production, increased mitochondrial fragmentation, dysfunctional mitochondria, enhanced mitophagy and, finally, death of the cancer cells due to potentially short supply of

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energy (ATP) via alteration of desired mitochondrial homeostasis. Reduced transmembrane potential may also weaken the integrity of the mitochondrial membrane and increase the probability of mitochondrial structural disruption. More importantly, rBTI’s mitophagy and cancer cell apoptosis promotion was not observed with control cells. Some unique hallmark compound(s) in the test cancer cells likely serves as the trigger or sensitizer for stimulating the formation of rBTI 1 Tom20 complex and enhanced DAF-16 transcriptional activity then further causing the downstream cascade reactions which eventually result in the death of the test cancer cells—but not to normal control cells, which are absent of such a sensitizer or trigger.

23.4.3 Potential future trends in research and studies Due to its potent efficacy in inducing mitophagy upon detecting dysfunctional mitochondrial mechanisms and critical roles in maintaining health mitochondrial homeostasis and potential to be an effective cure for targeting age-related chronic diseases, such as cancers, diabetes, AD, heart failure, rBTI (and BTIs) is attracting more and more interests in more detailed research and development work globally. Below are some brief projections outlining research and development trends related to BTIs and rBTI in the foreseeable future. •

•

• • •

• •

Studies in the roles and mechanisms defining how rBTI is involved in inducing and promoting mitophagy upon detection of dysfunctional mitochondria and cells. Investigation of rBTI’s indirect upregulating effects to mitogenesis while promoting mitophagy to orchestrate a dynamic homeostasis in mitochondria when exposed to adverse physiological conditions. Investigation of rBTI potential side effects for treatment of patients with different target diseases and health conditions. Development of more efficient isolation and purification means of BTI from buckwheat seeds and the plants. Improvement of potent of rBTI trypsin inhibiting activity through bioengineering tools while improving rBTI tolerance to sensitive components such as vitamin C and zinc. Development of clinic-forward drugs from rBTI for clinic validations. Development of food supplements and functional foods from BTIs for promotion of health aging and for prevention of target chronic diseases.

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5334. Kowluru, R. A., & Mohammad, G. (2020). Epigenetics and mitochondrial stability in metabolic memory phenomenon associated with continued progression of diabetic retinopathy. Nature, 10, 6655. Available from https://doi.org/10.1038/s41598-020-63527-1. Li, C., Li, W. J., Zhang, Y., & Simpson, B. K. (2021). Comparison of physysicochemical properties of recombinant buckwheat trypsin inhibitor (rBTI) and soybean trypsin inhibitor (SBTI). Protein Expression and Purification, 171(1), 105614, -105614. Li, C., Ning, L., Cui, X. D., Ma, X. L., Li, J., & Wang, Z. H. (2019). Recombinant buckwheat trypsin inhibitor decrease fat accumulation via the IIS pathway in Caenorhabditis elegans. Experimental Gerontology, 128110753. Lee, C. C., Hsu, W. H., Shen, S. R., Cheng, Y. H., & Wu, S. C. (2012). Fagopyrum tartaricum (buckwheat) improved high-glucose-induced insulin resistance in mouse heptocytes and diabetes in fructose-rich diet-induced mice. Exp Diabetes Res, 2012, 235673. Available from https://doi.org/10.1155/2012/375673, In this issue. Li, J., Cui, X., Ma, X., Li, C., & Wang, Z. (2019). Recombinant buckwheat trypsin inhibitor improves the protein and mitochondria homeostasis in Caenorhabditis elegans model of aging and age-related disease. Gerontology, 65, 513
523. Li, J., Cui, X. D., Ma, X. L., & Wang, Z. H. (2017). rBTI reduced β-amyloid-induced toxicity by promoting autophagy-lysomal degradation via DAF-16 in Caenorhabditis elegans. Experimental Gerontology, 89(3), 78
86. Li, Y. Y., Zhang, Z., Wang, Z. H., Wang, H. W., & Zhang, I. (2009). rBTI induces apoptosis in human solid tumor cell lines by loss in mitochondrial transmembrane potential and caspase activation. Toxicology Letters, 189, 166
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464. Liao, X., Zhang, R., Lu, Y., Prosdocimo, D. A., Sangwung, P., Zhang, L., Zhou, G., Anand, P., Lai, L., Leone, T. C., Fujioka, H., Ye, F., Rosca, M., Hoppel, C. L., Schulze, P. C., Abel, E. D., Stamler, J. S., Kelly, D. P., & Jain, M. K. (2015). Kruppellike factor 4 is critical for transcriptional control of cardiac mitochondrial homeostasis. Journal of Clinical Investigation, 125(9), 3461
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707.

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Whole-diet interventions and mitochondrial function

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Diet restriction-induced mitochondrial signaling and healthy aging

24

Meredith Pinkerton1,2 and Antoni Barrientos1,3 1

Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, United States 2 Neuroscience Graduate Program, University of Miami Miller School of Medicine, Miami, FL, United States 3 Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL, United States

24.1 Mitochondrial pathways induced by caloric restriction Dietary restriction with adequate nutrition is the most studied, nongenetic, nonpharmacological intervention used to extend health span and lifespan. Its beneficial effects have been observed experimentally on many organisms, including yeast, fly, mouse, rat, and nonhuman primate model systems (Lee & Longo, 2016; Mattison et al., 2012; Roth et al., 2002). The overwhelmingly positive results have encouraged the interest in utilizing the potential therapeutic value of dietary restrictions to assist treatment to a multitude of diseases, including neurological and neurodegenerative disorders, metabolic disorders, cardiovascular diseases, and cancer (Amigo et al., 2017; Cava & Fontana, 2013; Cerqueira et al., 2012; Lane et al., 1999; Shinmura et al., 2011). Among the different types of dietary intervention, caloric restriction (CR) is defined as a sustained reduction of regular energy intake, usually by 20%
50%, maintaining adequate micronutrient intake (Masoro, 2005, 2010). The general mechanisms by which CR operates in all organisms, from yeast to mammals, involve, in part, inhibition of nutrient-responsive kinases, general enhancement of stress-resistance mechanisms, and metabolic remodeling, including the modulation of mitochondrial and antioxidant activities in cells and tissues. In mammals, the pro-health and antiaging effect of CR is mediated by an array of signaling pathways, including the insulin and insulin-like growth factor-1 (IGF-1), mTOR, NAD1/NADH-dependent sirtuin deacetylases, AMP-activated protein kinase (AMPK), PGC-1α, and retrograde mitochondrial pathways (Fontana et al., 2010). These pathways crosstalk extensively, forming a complex network (Fig. 24.1). Among the myriad of CR-induced changes, mitochondrial adaptation represents a key component of the response to CR. The activated pathways impact several aspects of mitochondrial physiology and turnover, including Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00027-8 © 2023 Elsevier Inc. All rights reserved.

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FIGURE 24.1 Evolutionary conserved mammalian energy-sensing pathways underlying the beneficial effects of caloric restriction and fasting dietary interventions. Nutrient and energy-sensing pathways interacting with each other include the evolutionary conserved key regulators mammalian target of rapamycin (mTOR), AMP-activated protein kinase (AMPK), insulin/insulin-like growth factor 1 pathway (IIS), and sirtuins. mTOR is a serine/threonine protein kinase that, when nutrients are available, is involved in activating protein synthesis, cellular growth and proliferation, and inhibiting cell maintenance pathways such as autophagy. Mammalian mTOR exists in two complexes, mTORC1, implicated in most actions exerted by this kinase, and mTORC2. mTORC1 receives and integrates many hormonal stimuli coming from the IIS pathway and signals from specific nutrients, in particular amino acids, ATP, and glycolysis metabolites. TORC2 is also activated through the IIS pathway by insulin and epidermal growth factor (EGF) and can activate TORC1 through AKT. Scarcity of nutrients, such under dietary restriction or reduced signaling of the IIS pathway, leads to decreased kinase PI3K-AKT pathway, which causes the inhibition of mTOR and activation of forkhead box O transcription factor (FOXO), which, in turn, suppresses mTOR. When the energy status of the cell is low in these conditions, AMPK is induced and inhibits TORC1. Simultaneously, AMPK contributes to activating sirtuins such as nuclear SIRT1 and mitochondrial SIRT3, which through a positive loop, further favor activation of AMPK itself. SIRT1 and AMPK activate FOXO and the cotranscriptional factor PGC-1α. PGC-1α activation induces the transcription of many important genes involved in mitochondrial biogenesis via nuclear respiratory factors NRF1/2 coactivation, which induces transcriptional self-activation, and the expression of TFAM, mitochondrial biogenesis factors, and OXPHOS complex (Continued)

24.1 Mitochondrial pathways induced by caloric restriction

mitochondrial aerobic energy production and ROS generation, mitochondrial biogenesis and mitophagy, and the communication of mitochondria with other organelles. This section will summarize the mechanisms involved and their effect on regulating health span and lifespan in animal models, most extensively in mammals.

24.1.1 Caloric restriction, inhibition of insulin/insulin-like growth factor-1 signaling insulin-like growth factor 1 pathway, and mitochondria

L

In animals, CR counteracts the effects of insulin and IGF-1, whose levels increase in the presence of nutrients and activate pro-aging pathways, contributing to the diseases of aging. In mammals, when nutrients are available, insulin/insulin-like molecules, or IGF-I bind to their respective receptors triggering a cascade of events (Fig. 24.1): activation of phosphatidylinositol 3-kinase (PI3K) and serine/ threonine protein kinases [AKT-1/AKT-2/protein kinase B (PKB)], and phosphorylation of a stress-resistance factor, the forkhead transcription factor, FoxO. Phosphorylated FoxO factors are exported from the nucleus. During CR, insulin signaling is decreased, leading to enhanced FoxO expression and nuclear compartmentalization, thus promoting the buildup of robust antistress defenses. Mutations in mammalian genes that attenuate IIS extend the lifespan by mechanisms similar to CR (Bonkowski et al., 2006), as it occurs in dwarf mice, which carry mutations in the pituitary growth hormone (GH) receptor that regulates IGF-I release from the liver [reviewed in (Longo & Finch, 2003)]. Like TOR, IIS activates the downstream kinase effector S6K, which regulates aging in worms and mammals (Selman et al., 2009). S6K1 knockout mice display ameliorated age-related pathology and lifespan extension similar to the effects of CR. The absence of S6K1 enhances AMPK activity, which further regulates the TOR

subunits. PGC-1α also induces the transcription of many important genes involved in complete fatty acid oxidation and stress-resistance pathways as well as the mitochondrial sirtuins such as SIRT3. SIRT3 activates FOXO3, which induces the transcription of key ROS antioxidants such as the mitochondrial superoxide dismutase (SOD2) and catalase and key genes involved in autophagy. SIRT1 further activates autophagic machinery, which also leads to activation of mitophagy, leading to selective clearance of damaged or unfit mitochondria. In the mitochondria, SIRT3 deacetylates electron transport chain proteins, SOD2, and the transition pore (MPTP) component cyclophilin D, resulting in greater respiratory efficiency, antioxidant defense, and apoptotic resistance. In general, AMPK, sirtuins, FOXO, and PGC-1α can engage in a positive feedback loop, partially depicted here, that connects these nutrient sensors into a unified response which in many instances improves health span and may have an effect on lifespan. The model presented here is simplified and does not consider tissue-specific responses. Arrows indicate activation, and T bars indicate suppression.

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pathway (Selman et al., 2009). TOR kinase and AMPK are major upstream regulators of mitochondrial metabolism and contribute to mediating CR-induced longevity (Bratic & Larsson, 2013). Dwarf mice have increased mitochondrial respiration and increased metabolism per body weight, indicating that decreased GH signaling may beneficially affect mitochondrial flexibility by increasing the capacity for fat oxidation (Westbrook et al., 2009, 2014). Similarly, long-lived fat-specific insulin receptor knockout mice have increased expression of the peroxisome proliferator-activated receptor-γ coactivators, PGC-1α and PGC-1β, leading to enhanced mitochondrial gene expression, and boosting oxidative and lipid metabolism in white adipose tissue (Katic et al., 2007). Studies in the worm Caenorhabditis elegans have shown that the longevity effects of reduced IIS are contributed by the oxidative stress response transcription factor Nrf2 or nuclear factor erythroid 2 (worm SKN-1) (Tullet et al., 2008). Worm SKN-1 promotes the activation of detoxification and stress response genes (An et al., 2005), and its overexpression extends the lifespan independently of DAF-16/FoxO (Tullet et al., 2008), probably resulting from enhanced ROS clearance in the mitochondria, which has also been observed in human cells. However, although metabolic and protein homeostasis and activation of tissue-specific cytoprotective proteins are dependent on Nrf2 expression in mice, knockout of Nrf2 resulted in shortened lifespan but only under ad libitum conditions. CR-mediated lifespan extension and physical performance improvements did not require Nrf2 (Pomatto et al., 2020).

24.1.2 Caloric restriction, inhibition of target of rapamycin signaling, and mitochondria The amino acid (AA) sensing TOR kinase pathway mediates CR-induced longevity from yeast to mammals (Kaeberlein et al., 2005; Zid et al., 2009). The TOR kinase exists in two separate multiprotein complexes, designated TORC1 and TORC2, which have different biological functions (Fig. 24.1). In mammals, a single TOR gene functions in both the TORC1 and TORC2 complexes (Huang & Fingar, 2014). TORC1 induces protein synthesis and becomes active only when an adequate supply of AAs is available within the cell, particularly leucine (Avruch et al., 2009). TORC1 activity is stimulated by signals of energy availability, including ATP, insulin, and growth factor signaling, and is inhibited by cellular stress signals, including ROS, ER stress, and hypoxia (Huang & Fingar, 2014). Dysregulation of TORC1 signaling plays a significant role in many cancers and age-associated diseases (Johnson et al., 2013a). Like TORC1, TORC2 is stimulated by the availability of nutrients and growth factors, although through lesserknown mechanisms. TORC2 functions as a key regulator of pathways involved in cytoskeletal maintenance and growth as well as cellular metabolism (Huang & Fingar, 2014). Both TORC1 and TORC2 inhibit autophagy, with TORC1 known to specifically interact with the autophagy initiation complex (Huang & Fingar,

24.1 Mitochondrial pathways induced by caloric restriction

2014; Johnson et al., 2013a). Thus, it may be assumed TOR signaling may also inhibit the selective degradation of damaged mitochondria through mitophagy since both processes share the same initiation mechanisms. Upstream activators of TORC1 include the insulin/PI3K/AKT-signaling pathway, stimulated when circulating insulin increases in response to high glucose availability. Activation of the pathway leads to the phosphorylation and activation of AKT, and finally leads to an enhancement in TORC1 activity. TORC1 can additionally be activated by other growth factors, such as epidermal growth factor, which signal through the Ras/MAPK/ERK pathway (Huang & Fingar, 2014). Downstream targets of TORC1 include eukaryotic translation initiation factor 4E-binding protein (4EBP) and S6K, both of which function to increase global protein synthesis. TORC2 activates Akt, further stimulating TORC1 activity and inhibiting the activity of FoxO3 (Johnson et al., 2013a). The intricacies of TOR signaling have been extensively reviewed (Huang & Fingar, 2014; Johnson et al., 2013a). The TOR kinase regulates mitochondrial mass and function. In Drosophila, 4EBP1 extends lifespan upon dietary restriction by translationally upregulating mitochondrial oxidative phosphorylation (OXPHOS) components specifically, and enhancing mitochondrial activity, as measured in whole animal homogenates (Zid et al., 2009). Similar to what has been observed in yeast tor1 knockouts (Bonawitz et al., 2007), increased mitochondrial respiration upon TORC1 ablation (raptor KO in adipose tissue) has also been seen in mice (Polak et al., 2008). However, in mammalian cells, TORC1 (mTORC1) inhibition reduces mitochondrial function by acting at multiple levels. mTORC1 controls mitochondrial biogenesis through a yin-yang 1/PGC-1α transcriptional complex to balance energy metabolism through transcriptional control of mitochondrial oxidative function (Cunningham et al., 2007). mTORC1 also directly regulates mitochondrial activity via the phosphorylation of specific proteins, the uptake and utilization of carbohydrates to balance glycolytic flux with mitochondrial respiration, and the elimination of damaged or dysfunctional mitochondria via mitophagy, a specialized form of autophagy (Groenewoud & Zwartkruis, 2013). In mammals, the mitophagy pathway involves PTEN-induced putative protein kinase 1 (PINK1), which becomes stabilized in the mitochondrial membrane upon its depolarization leading to the recruitment of the E3 ubiquitin ligase Parkin (Chen et al., 2020). Through the inhibition of the TOR pathway, CR induces the expression of PINK1 and PARKIN, and genes promoting mitochondrial fission (FIS1, DRP1), a process required to facilitate mitophagy (Chen et al., 2020). Maintenance of mitochondrial homeostasis involves feedback of mitochondrial function on TORC1, to adjust mitochondrial biogenesis to nutrient availability. In this respect, TORC1 activity is regulated by ROS. Although many effects of ROS on TORC1 activity are cell-type specific, they seem to be concentration-dependent: TORC1 is induced by low levels of ROS, while mid-range and high levels of ROS inhibit TORC1 activity (Groenewoud & Zwartkruis, 2013). Although its mechanism of action is still unclear, TORC2 could also play a distinct role in mitochondria. Upon activation, it can localize to specialized areas

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of mitochondria-associated endoplasmic reticulum (ER) membranes, contributing to the regulation of mitochondrial and ER calcium release/uptake and therefore modulation of OXPHOS performance and resistance to apoptosis (Betz et al., 2013).

24.1.3 Caloric restriction, sirtuin activation, and mitochondria Sirtuins are directly linked to nutrient and metabolic signaling through their requirement of NAD1 as a cofactor (Fig. 24.1). Sirtuins regulate many proteinposttranslational modifications in multiple cellular pathways, thereby conveying widespread functional changes with altered nutrient availability (Baur et al., 2012). Pioneering studies in yeast linked Sir2, the lone yeast sirtuin gene, to lifespan extension by CR (Lin et al., 2000), and overexpression of Sir2 orthologs was found to convey lifespan extension in yeast, worms, flies, and mice (Baur et al., 2012; Chang et al., 2015). Mammals have seven sirtuin proteins (SIRT1
7), each with distinct cellular localizations and functions. Of these, nuclear- and cytoplasmic-localized SIRT1 and mitochondrial-localized SIRT3 deacetylases have been most extensively studied for their possible roles in the benefits of CR (Baur et al., 2012). For example, SIRT1 expression increases during CR in mice and humans and declines under a high-fat diet or obesity [25]. SIRT1 overexpression in mice promotes CR-like beneficial health effects and protects various markers of health upon diet-, and disease-related stressors but not lifespan extension (Baur et al., 2012). Protein pathways activated by SIRT1 deacetylation include PGC-1α-regulated mitochondrial biogenesis, AMPK signaling, autophagy [reviewed in (Baur et al., 2012)], and perhaps activation of the hypoxia-inducing factor-1 alpha (HIF-1α) to promote mitochondrial biogenesis indirectly (Gomes et al., 2013). SIRT3, SIRT4, and SIRT5, are the mitochondrial sirtuins (Figs. 24.1 and 24.2) due to their prominent localization in the mitochondrial matrix, have been proposed to function as a link between aging and metabolism (Ji et al., 2021; van de Ven et al., 2017). Like SIRT1, also SIRT3 and SIRT5 activation is increased, and SIRT4 expression decreased after multiple CR regimens and decreased under high-fat diets in rodents (Ma et al., 2020; Nakagawa et al., 2009; Palacios et al., 2009). While CR induces deacetylation of a wide range of mitochondrial proteins, deletion of SIRT3 leads to robust mitochondrial protein hyperacetylation, particularly of the CR-targeted proteins (Hebert et al., 2013). This suggested that SIRT3 is a key mediator of CR effects on mitochondria (Hebert et al., 2013). The primary function of SIRT3 in mitochondria is maintaining basal ATP levels through regulating the respiratory chain (MRC). SIRT3 directly deacetylates MRC complex I and II, enhancing their activity, and thereby stimulating ATP production (Ahn et al., 2008). In the absence of SIRT3, MRC complex IV activity is also attenuated, and ROS levels are enhanced (Kong et al., 2010). SIRT3 overexpression results in reduced ROS levels (Kong et al., 2010) mediated in part by direct deacetylation of mitochondrial SOD2, leading to enhancement of its enzymatic

24.1 Mitochondrial pathways induced by caloric restriction

FIGURE 24.2 Network of mitochondrial sirtuins in standard and caloric restriction conditions. Mitochondria are energy transducer organelles, which can metabolize fuels, such as fatty acids, amino acids, and pyruvate, derived from glucose and transduce the energy extracted into the chemical form of ATP. Electrons extracted from nutrients are transported through the respiratory chain complexes (I
IV) in a process coupled to the generation of a proton gradient across the inner membrane that is used to drive ATPase to generate ATP through the process of oxidative phosphorylation (OXPHOS). Three sirtuins (SIRT3/4/5) are localized in the mitochondrial matrix. SIRT3 is an NAD1-dependent deacetylase that can regulate apoptosis (Fig. 24.1), binds to respiratory complexes I and II, regulating cellular energy levels. Furthermore, SIRT3 deacetylates and activates acetylCoA synthetase 2 (AceCS2), glutamate dehydrogenase (GDH), and long-chain acyl-CoA dehydrogenase (LCAD). SIRT4 can transfer the ADP-ribose group from NAD1 onto acceptor proteins, acting opposite to SIRT3 and SIRT5. SIRT3 and SIRT5 are induced in CR conditions, and SIRT4 expression is inhibited.

activity (Qiu et al., 2010; Tao et al., 2010). In cardiac hypertrophy mouse models, SIRT1 and SIRT3 were found to regulate deacetylation of the transcription factor FOXO3A, stimulating its relocation to the nucleus and expression of antioxidant enzymes, as an example of nucleus-mitochondria crosstalk (Sundaresan et al., 2009). SIRT3 also deacetylates cyclophilin D, a key component of the mitochondrial permeability transition pore, inhibiting mitochondrial apoptosis (Hafner et al., 2010). SIRT4 also has deacetylase activity but primarily functions as an NAD1-dependent ADP-ribosyltransferase and regulates multiple metabolic pathways, including glutamine catabolism, fatty acid oxidation, and AA catabolism acting in the opposite direction to SIRT3 and SIRT5 (Kumar et al., 2015). SIRT5 has weak deacetylase activity but catalyzes demalonylation, desuccinylation, and

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deglutarylation of mitochondrial enzymes related to metabolic pathways such as glycolysis, the urea cycle, and fatty acid oxidation (Fig. 24.2) (Ji et al., 2021). A detailed description of the regulation of the nutrition intervention-related pathways by mitochondrial sirtuins can be found elsewhere (Ji et al., 2021).

24.1.4 Caloric restriction, AMP-activated protein kinase activation, and mitochondria When nutrients are scarce, cells preferentially initiate signaling pathways driving catabolism. The adenosine monophosphate-AMPK senses intracellular ATP levels (the ATP:AMP/ADP ratio), and when these are low, phosphorylates mTOR to contribute to rapidly suppressing anabolic reactions and restore metabolic homeostasis (Inoki et al., 2003) (Fig. 24.1). In addition, AMPK promotes catabolism by enhancing glycolysis, including enhancement of glucose uptake and activating glycolysis enzymes (Burkewitz et al., 2014). Moreover, AMPK promotes lipid usage by stimulating lipase to release fatty acids for mitochondrial oxidation and implements a long-term metabolic switch by increasing mitochondrial content and the use of mitochondrial substrates as an energy source (Mihaylova & Shaw, 2011). The switch to mitochondrial OXPHOS is achieved by phosphorylation of the transcriptional coactivator PGC-1α (Ja¨ger et al., 2007), which leads to enhanced mitochondrial biogenesis. In C. elegans and Drosophila, overexpression of wild-type, constitutively active AMPK, or AMPK upstream activators consistently extends lifespan (Burkewitz et al., 2014). AMPK promotes healthy aging in part from its ability to integrate multiple signaling and transcriptional pathways known to promote longevity. However, frequent feedback regulation within the network creates a challenge in defining important linear pathway components. In accordance with the contrasting roles of activation, there is considerable crosstalk between the AMPK and the two TOR pathways. Activated AMPK acts to inhibit TORC1 at multiple levels through direct and upstream inhibition (Huang & Fingar, 2014). Additionally, S6K, a downstream target of TORC1, phosphorylates and inactivates AMPK (Burkewitz et al., 2014). Therefore, TORC1 inhibition can further enhance the activation of AMPK. Furthermore, AMPK activation also leads to increased autophagy, while TORC1 and TORC2 suppress autophagy. On the other hand, AMPK has been shown to act in a positive feedback loop with both SIRT1 and SIRT3 by increasing the NAD1/NADH ratio (Canto et al., 2009), leading to sirtuin activation. Activation of both SIRT1 and SIRT3 then leads to the deacetylation and activation of the upstream activator of AMPK (LKB1), resulting in increased AMPK activation (Palacios et al., 2009). SIRT1 and AMPK further act together to activate PGC-1α through respective deacetylation and phosphorylation (Canto et al., 2009; Ja¨ger et al., 2007), therefore regulating mitochondrial biogenesis and cellular bioenergetics. For a review of AMPK signaling, see (Burkewitz et al., 2014).

24.1 Mitochondrial pathways induced by caloric restriction

24.1.5 Caloric restriction, PGC-1α activation, and mitochondria The transcriptional coactivator PGC-1α is the master regulator of mitochondrial biogenesis (Martin-Montalvo & Cabo, 2013; Puigserver & Spiegelman, 2003). PGC-1α interacts with several transcription factors to induce and coordinate gene expression leading to stimulation of mitochondrial oxidative metabolism in many tissues, fiber-type switching in skeletal muscle, and multiple aspects of the fasting response in the liver, among other functions (Puigserver & Spiegelman, 2003). To regulate mitochondrial biogenesis, PGC-1α interacts with the nuclear respiratory factors nuclear factor 1 (NRF1) and NRF2 to induce the expression of genes involved in OXPHOS, mitochondrial DNA (mtDNA) expression, and the ROS scavenging program (Spiegelman, 2007) (Fig. 24.1). The role of PGC-1α in CR and other models of longevity has been extensively reviewed (Corton & Brown-Borg, 2005) since it is a target of the sirtuin pathway (SIRT1 interacts with and deacetylates PGC-1α to enhance its activity), TOR pathway (mTORC1 regulates PGC-1α gene transcription and translation), and AMPK pathway (its activation suppresses mTORC1 signaling, and phosphorylates FoxO to modulate PGC-1α gene expression). Studies in flies showed that enhancement of mitochondrial biogenesis mediated by overexpression of PGC-1α in intestinal stem cells enhanced coupled mitochondrial OXPHOS and reduced ROS generation leading to lifespan extension (Rera et al., 2011). However, ubiquitous PGC-1α overexpression also enhanced mitochondrial biogenesis and induced glycogen accumulation in most tissues but moderately shortened lifespan, suggesting that these outcomes might have unfavorable effects in select tissues (Rera et al., 2011). In mammals, PGC-1α levels decline with age and are restored by CR (Lopez-Lluch et al., 2006). Although some effects of CR on mammalian metabolism remain controversial, it is accepted that CR increases mitochondrial biogenesis (Lopez-Lluch et al., 2006; Nisoli et al., 2005), which has been associated with an accumulation of partially-uncoupled mitochondria that maintain cellular ATP synthesis with lower ROS production (Lopez-Lluch et al., 2006). Overexpression of PGC-1α to restore normal mitochondrial biogenesis and function is also beneficial in some mouse models of Huntington’s disease and other neuromuscular degenerative diseases (Cui et al., 2006), although it has not been shown to extend the lifespan of control mice. However, excessive PGC-1α levels can have adverse effects in tissues such as skeletal muscle or the heart, suggesting that mitochondrial biogenesis needs to be fine-tuned carefully, as it occurs during CR, to achieve the desired health benefits (Wenz, 2009).

24.1.6 Caloric restriction and mitochondrial signaling to the cell CR, inhibition of IGF-1 signaling, and inhibition of TOR signaling promote longevity at least in part by eliciting mitochondrial retrograde signaling processes, broadly defined as signaling from the mitochondria to the rest of the cell [reviewed in (Long et al., 2014)]. For example, by enhancing mitochondrial

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respiration, these interventions increase ROS production, which act as adaptive signaling molecules by activating pathways that promote cellular resistance to various stresses. This phenomenon is known as mitochondrial preconditioning or “mitohormesis” (Ristow & Schmeisser, 2011). Mitochondrial stress and mito-nuclear protein imbalance also trigger the mitochondria-specific unfolded protein response (UPRmt) (Haynes et al., 2013; Pellegrino et al., 2013), a mitochondria-nuclear signaling pathway that induces the expression of mitochondria-protective molecular chaperones and other regulators of mitochondria homeostasis. Interventions such as CR and inhibition of TOR signaling generate mito-nuclear imbalance and thereby also elicit the UPRmt, which contributes to longevity mechanisms that have been extensively reviewed (Houtkooper et al., 2013; Schulz & Haynes, 2015). Recent studies have identified microRNAs (miRNAs) as contributors to mediate CR-induced mitochondrial proteostasis (Zhang et al., 2019). miRNAs are evolutionarily conserved RNAs of 21
22 nucleotides that constitute an important layer of gene regulation in eukaryotes by promoting mRNA degradation or inhibiting translation in the cytoplasm (Hausser & Zavolan, 2014). A few studies in mice and rhesus monkeys have suggested that CR impacts the expression levels of miRNAs in several tissues (Mercken et al., 2013; Schneider et al., 2017). In addition to the cytosol, miRNAs have also been identified in the mitochondria (Jagannathan et al., 2015; Schneider et al., 2017). Recent data have suggested that, in skeletal muscle, miR1 coordinates the myogenic program and that miR-21 in cardiac tissues lowers blood pressure by upregulating mitochondrial translation and biogenesis (Zhang et al., 2014). In mouse models, CR globally induced miRNAs in the liver to induce the UPRmt and enhancement of OXPHOS function in mouse livers, and those miRNAs are required for the CR-induced improvements even in the overall metabolism and physical activity of mice (Zhang et al., 2019). Specifically, either knockdown of Drosha or knockout of miR-122 attenuated the effect of CR on the activation of mitochondrial translation and the improvement of mitochondrial proteostasis in vivo (Zhang et al., 2019). Therefore, CR-induced miRNA biogenesis and the increased miRNA levels in mitochondria may be physiologically relevant in the upregulation of mitochondrial gene expression and mitochondrial biogenesis. Another class of mitochondrial signals capable of regulating aging and lifespan comprises the mitochondrial-derived peptides (MDPs), encoded by functional short open reading frames in the mtDNA (Alis et al., 2015). Among them, Humanin (HN) was the first identified and most studied MDP, reported to play critical roles in aging as well as multiple conditions, including metabolic, cardiovascular, and autoimmune diseases. HN is found in tissues and plasma and declines with age in both rats and humans, supporting a potential role in longevity (C Lee et al., 2013; Yen et al., 2013). HN has been shown to modulate multiple biological processes, including autophagy and mitophagy, ER stress, cellular metabolism, oxidative stress, and inflammation (Hashimoto et al., 2001; Yen et al., 2020), and can act as a caloric-restriction mimetic by suppressing IGF-1

24.1 Mitochondrial pathways induced by caloric restriction

levels in mice (Lue et al., 2015). MOTS-c is another MDP capable of altering metabolism through downstream effects on the AMPK signaling pathway leading to PGC-1α expression, attenuated insulin resistance, and enhanced glucose metabolism in mice (Lee et al., 2015; Yang et al., 2021).

24.1.7 Mitochondria-mediated tissue-specific effects of caloric restriction Despite the overwhelmingly positive effects of CR reported throughout literature, controversy on the systemic impact of CR arose due to multiple variables: age of dietary onset, age of the individuals, tissue specificity, and percent of calorie restriction. In this section, to establish fair comparisons among studies, we have included studies reporting tissue-specific effects on mitochondrial function and signaling following mild (16%
25% decrease) or severe (40%
60%) CR regimes in mammals.

24.1.7.1 Adipose tissue Because of the potential benefit of CR on obese individuals, the response of adipose tissue to CR regimes was one of the first to be extensively characterized. Among multiple reports, studies in 6-week-old mice fed at 40% CR showed induced expression in both RNA and protein levels of the mitochondrial biogenesis gene PGC1α, the mitochondrial fission genes DRP1 and MFF, and the mitochondrial fusion genes OPA1 and MFN1/2 (Mooli et al., 2020). Studying the effect on adipocyte mitochondria from mice fed a 30%
40% CR diet compared with ad libitum fed mice disclosed an increase in mitochondrial biogenesis and gene expression, including mRNA levels of PGC-1α, mitochondrial transcription factor A (TFAM), NRF1, MFN1/2, COX-IV, cytochrome c, and eNOS (Cohen et al., 2004). Because these results were not seen in eNOS-KO mice, the researchers concluded that the CR effects are partially due to an increase in nitric oxide (NO) levels (Cohen et al., 2004), which acts through enhancing the transcription levels of PGC-1α. Other studies showed that CR enhanced mitochondrial biogenesis in white but not brown, adipose tissue (Linford et al., 2007; Okita et al., 2012). Some of these studies have shown that rather than SIRT1, the sterol regulatory element-binding protein 1c (SREBP1c) and fibroblast growth factor 21 (FGF21) act as white adipose tissue-specific mediators of CR-induced mitochondrial biogenesis, with SREBP-1c likely increasing PGC-1α expression not only directly, but indirectly, via FGF21 (Kobayashi et al., 2018, 2021).

24.1.7.2 Skeletal muscle Skeletal muscle fiber survival and contraction relies on healthy mitochondria, whose functions decline with age. This, together with increased oxidative stress, deficient satellite cell recruitment, and motor unit decline, result in the ageassociated loss of skeletal muscle mass, or “sarcopenia”. CR has been shown to

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attenuate age-associated mitochondrial dysfunction in skeletal muscle and prevent sarcopenia in rodents, nonhuman primates, and humans (Rhoads et al., 2020; Serna et al., 2020). Some specific studies exploring mitochondrial functions are listed next. In a clinical trial, 25% of CR individuals had enhanced expression of mitochondrial genes PGC-1α, TFAM, eNOS, SIRT1, and PARL, accompanied by decreased oxidative DNA damage (Civitarese et al., 2007b). This suggested that CR might be acting through PGC-1α-mediated regulation of free radical production and oxidative stress. This observation was corroborated in a mouse model comparing WT with skeletal muscle PGC-1α-KO mice, finding that mitochondrial genes (TFAM, ATP5O, CYCs, MCAD, and SOD2) that were stunted in the skeletal muscle in the absence of PGC-1α, were increased in heart and liver tissue, in the presence of PGC-1α (Finley et al., 2012). Individuals given a 25% CR diet experienced a significant increase in skeletal muscle SIRT1 expression while simultaneously showing a decrease in NO-related genes (Sparks et al., 2017). The sirtuin activator resveratrol had effects similar to CR (see the section on CR mimetics), causing an increased expression of SIRT1, which led to increased PGC-1α activity and enhancement of mitochondrial biogenesis (Lagouge et al., 2006). In rat muscle, 60% CR increased UCP3 content, suggesting that, along with PGC-1α-mediated induction of antioxidant defenses, uncoupling proteins (UCPs) might also play a pivotal role in decreasing mitochondrial ROS (Bevilacqua et al., 2005).

24.1.7.3 Liver Multiple studies have reported an age-linked decline in liver functions associated with mitochondrial alterations and enhanced oxidative damage. Studies in murine models have shown that CR induces significant beneficial changes in liver mitochondrial biogenesis, OXPHOS efficiency and antioxidative stress defenses. Rats fed with a 40% CR diet had a significant decrease in H2O2 production in the liver, resulting from decreased electron transport chain complex I activity (Hagopian et al., 2005). A similar study showed increased expression of PGC-1α and PPAR and decreased ROS levels (Lo´ Pez-Lluch et al., 2005). Also in mice, a 40% CR diet resulted in enhanced mRNA levels of PGC-1α, TFAM, NRF1, MFN1/2, COX-IV, cytochrome c, and eNOS (Cohen et al., 2004). The results were reproduced in rats fed with a more severe 60% CR diet, although in this case, the levels of SIRT1 were also increased in the brain, adipose, kidney, and liver tissues. The effect involved SIRT1-mediated downregulation of Bax, a protein involved in cellular apoptosis, thus promoting cell survival (Cohen et al., 2004). Another study in rats sought to test the existence of boundaries to the CRs efficacy in a large set of mitochondrial markers in aged (28-month-old) and extremely aged (32-month-old) rats fed 25% CR during the first four months of life and 40% CR for the rest of their lives. The analysis of the results revealed that CR was able to convey beneficial effects as it prevented most of the agerelated alterations of mitochondrial biogenesis and dynamics, and mtDNA

24.1 Mitochondrial pathways induced by caloric restriction

damage in the liver of 28-month-old rats. However, the effects were not significant in 32-month-old rats (Chimienti et al., 2021). This suggests an age limitation of the CR effect, or that although CR extended health span, it could not increase longevity.

24.1.7.4 Brain Metabolic and mitochondrial abnormalities, as well as genetic variability, epigenetic changes, oxidative/nitrosative stress, and DNA damage greatly contribute to age-associated neurodegenerative disorders. The importance of a healthy diet, balanced in macro- and micronutrients, in preventing neurodegeneration and slowing down disease progression is well documented. Preclinical studies have shown that CR supports brain health by mitigating age-related declines in mitochondrial biogenesis, dynamics and function, neuronal activity (Lin et al., 2014), and attenuating oxidative stress (Hyun et al., 2006). Studies in mice fed a 20%
40% CR diet showed prominent effects in cerebral tissue. Those included elevated deacetylation of mitochondrial proteins, primarily through SIRT3 activation, increased SOD2 activity, enhanced activities of electron transport chain complexes I, III, and IV, and a significant increase in Drp-1 and Mfn-2 that regulate mitochondrial dynamics and mitochondrial axonal transport (Amigo et al., 2017). Similar results were obtained in a mouse model fed 60% CR, which also showed increased eNOS and nNOS (Cerqueira et al., 2012). When looking only at H2O2 production and oxidative damage, a study subjecting rats to 40% CR showed a B25% decrease in H2O2 production and oxidative damage, attributed to a decrease in electron leak at electron transport chain complex 1 (Sanz et al., 2005). These CR effects may be transduced through the classical nutrient-sensing pathways. As other organs and tissues, the brain is also sensitive to changes in nutrient levels, and CR inhibits the mTOR pathway, which for example, influences the formation of memory in the hippocampus, and activates the AMPK, sirtuin, and PGC-1α pathways to preserve neuronal integrity, metabolism, and function (Hadem et al., 2019).

24.1.7.5 Heart and cardiovascular system Nutrition is fundamental for maintaining cardiac function by regulating insulin and mitochondrial efficiency, which are essential to support energy production for contractility. CR diets could minimize the age-associated decline in myocardial efficiency and prevent heart failure by maintaining mitochondrial functions. Studies in old mice fed with a 40% CR diet showed increased levels of PGC1α, enhanced SIRT3-mediated levels of deacetylated electron transport chain complexes I and III, and decreased H2O2 production (Shinmura et al., 2011), along with increased mitochondrial fusion and decreased mitochondrial fission factors, suggesting optimized mitochondrial dynamics (Palee et al., 2019). Another study found that, with the enhancement of mitochondrial dynamics, the phosphorylation of mitochondrial signaling proteins AMPK and Akt, were

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increased (Niemann et al., 2021), which would lead to the cascade of events described earlier in this chapter. In general, the cardiometabolic benefits of CR in animal models and humans involve decreases in body weight and fat mass, improvement of insulin sensitivity, decrease in blood pressure, decrease in circulating lipids, and decreased levels of serum inflammatory markers (Brandhorst & Valter, 2019; Johnson et al., 2016). Mechanistically, all these beneficial effects result from several metabolic adaptations, including enhancement of mitochondrial biogenesis, decreased ROS production, and the resulting improvement in cardiac and vascular function (Savencu et al., 2021).

24.1.8 Effects of calorie restriction in mitochondrial biogenesis and energy metabolism in nonhuman primates and healthy humans While in short-lived organisms, including rodents, CR regimes consistently decrease the biological rate of aging and extend lifespan, the effects of CR on long-lived species remain less explored. Nonetheless, results reported so far from several nonhuman primate colonies suggest that CR might have a similar impact in longer-lived species (Colman et al., 2014; Mattison et al., 2017). These studies, performed in rhesus monkeys, yielded apparently conflicting results due to differing control group diets (Mattison et al., 2017). However, while lifespan data remains inconclusive (Ingram et al., 2006), monkeys undergoing CR displayed a substantially attenuated age-related morbidity (Bodkin et al., 2003; Lane et al., 1995). These data indicate that moderate CR is enough to provide health span benefits in primates but may not be enough to confer longevity benefits compared to more severe CR (Colman et al., 2014). One study reported that CR delays aging-induced cellular phenotypes in rhesus monkey skeletal muscle (McKiernan et al., 2011). Despite the muscle fiber preservation and delay of sarcopenia by CR, the proportion of fibers with mitochondrial OXPHOS enzyme abnormalities and their load of mtDNA deleted molecules was not attenuated. This indicated that CR may have contributed to the maintenance of affected fibers by pathways other than preventing the common stochastic mtDNA deletion mutations in muscle fibers (McKiernan et al., 2011). A more recent study reported that CR enhanced fiber-Type I mitochondrial OXPHOS activity and contractile content, specifically in Type I-fibers, while Type II were unaffected (Rhoads et al., 2020). CR maintained contractile content at the muscle level and decreased age-related metabolic shifts among individual fiber types with higher mitochondrial OXPHOS activity, altered redox metabolism, and smaller lipid droplet size, suggesting that CR-induced reprogramming of metabolism contributes to delaying skeletal muscle aging in rhesus monkeys (Rhoads et al., 2020). Health span data from controlled CR trials in humans are slowly becoming available (Heilbronn et al., 2006). The Comprehensive Assessment of Long-term

24.2 Mitochondrial mechanisms underlying health span extension

Effects of Reducing Intake of Energy organization is applying a multicenter effort focused on the effects of short-term CR on physiology, body composition, and risk factors for age-related diseases. CR regimes implemented for 6
12 months on moderately overweight individuals resulted in weight loss, enhanced glucose tolerance and insulin sensitivity, decreased metabolic rate, and improved serum indicators of disease risk (Heilbronn et al., 2006), including some oxidative stress markers (Meydani et al., 2011). A specific study tested the hypothesis that shortterm CR (25%), with or without exercise, increases the efficiency of mitochondria in human muscle (Civitarese et al., 2007a), as it was observed in mice and rhesus monkeys. Participants in the CR groups had significantly increased expression of genes encoding proteins involved in mitochondrial function such as PGC-1α, TFAM, eNOS, and SIRT1, mtDNA content increased by 35%, and mtDNA oxidative damage was attenuated, although enzymatic markers of mitochondrial proliferation remained unchanged (Civitarese et al., 2007a). Beneficial effects of long-term CR on disease risk in humans have also been reported in studies involving members of the Caloric Restriction Society who voluntarily engage in CR regimes. In these individuals, insulin sensitivity was enhanced, and adiponectin levels were increased (Hamdy, 2005), suggesting an increase in mitochondrial fatty acid oxidation. However, because the study was performed in obese individuals, it remained unclear whether these benefits were a consequence of modulating the aging process itself or just reducing obesity. A recent review has evaluated the “fact and fiction” of CR interventions in humans (Lee et al., 2021). The authors argued that the interpretation of CR studies in humans has been difficult in some cases, due to the lack of control for reduced caloric intake in the diet group. Also, although less studied, the CR effects on lifespan are not uniform and highly dependent on genotype (Lee et al., 2021). Despite their limitations, these studies provide invaluable information to understanding the cellular response to low nutrient availability. The authors proposed that one unifying concept may be convergence on the mTOR signaling pathway (Lee et al., 2021), which has vast implications for the regulation of mitochondrial turnover and physiology. Another conclusion was that the efficacy and safety of these diets for humans largely remain to be established (Lee et al., 2021).

24.2 Mitochondrial mechanisms underlying health span extension by popular restrictive diet regimes in mammals With the beneficial effects of a CR diet and the promising results as a potential therapeutic, a more realistic, dietary restriction regime that is easier to commit to, has been sought. Here, we will reveal the most standard and promising dietary

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restrictions: ketogenic diet (KD), essential AA restriction, and intermittent fasting (IF); and their effects on mitochondrial signaling reported in the current literature.

24.2.1 Ketogenic diet A popular form of dietary restriction dating back to 1925 is the KD. A KD is comprised of 55%
60% fat, 30%
35% protein, and 5%
10% carbohydrates (Dhamija et al., 2021). This high fat, low carbohydrate diet became popular following the discovery of its extraordinary regulation of seizures on children with epilepsy (Peterman, 1925; Wheless, 2008). In the last 40 years, KD research has expanded beyond epilepsy, to cancer, mitochondrial diseases, neurodegenerative diseases, obesity, and others (Branco et al., 2016; Lange et al., 2017; Schugar & Crawford, 2012; Włodarek, 2019). Although there are seen benefits of amelioration of diseases, the systemic impact of KD is unclear and controversial since too many ketones in the blood may have harmful health effects. The results of a high-fat diet in Zuker rats, showed an increase in calcium/calmodulin-dependent protein kinase (CaMK) phosphorylation, which supported the idea of an altered calcium leak from the sarcoplasmic reticulum (SR), which increased overall skeletal muscle mitochondrial biogenesis. This increase would be produced through enhanced mitochondrial ROS generation, which would induce SR Ca21 release (Jain et al., 2014). Although the underlying mechanism is unknown, the increase in cytosolic Ca21 in muscle fibers increased expression levels of mitochondrial biogenesis transcriptional regulators PGC-1α (Wright et al., 2007) and the respiratory factors NRF1/2 (Ojuka et al., 2003). Additional activation of the erythroid 2-related factor 2 (Nrf2) signaling pathway induces the expression of antioxidant defenses under KD (Milder et al., 2010). In a study of aging mice, short-term KD induced an increased expression of mitochondrial biogenetic markers, TFAM, SIRT1, SIRT3, and PGC-1α. Also, the long-term KD increased PGC-1α-TFAM levels (Wallace et al., 2021). A KD or supplemented β-OHB diet led to increased expression of mitochondrial coupling dynamics and biogenesis genes, UCP2, PGC-1α, Drp1, and Mfn1 in hippocampal tissue (Hasan-Olive et al., 2019). The increased mRNA levels of UCP2, PGC-1α, and SIRT1 (Ahn et al., 2008), led to the hypothesis that there is a PGC-1αSIRT3-UCP2 coordination accounting for the KD-dependent reduction of oxidative stress and decreased mitochondrial dysfunction. In a fetal brain, UPC2 expression was overexpressed, which was shown to diminish mitochondrial membrane potential, ultimately decreasing mitochondrial ROS production through a similar mechanism that a KD induces (Sullivan et al., 2003). This concept is corroborated in brown adipose tissue, showing an increased expression level of UPC1, PGC-1α, PPARy, and SIRT1 (Srivastava et al., 2013). Along with the PGC-1α-SIRT3-UCP2 axis, a key regulatory pathway decreased during the KD is the mTORC1 pathway, which is attributed to the increased expression of DNA damage-inducible transcript protein 4 (DDIT4) and p53 acetylation, which are both negative regulators of mTORC1 (Roberts et al.,

24.2 Mitochondrial mechanisms underlying health span extension

2017). It was also proposed that the decreased activity of the mTORC1 pathway is due to an increased expression of AMPK (McDaniel et al., 2011). However, the KD does not seem to be as advantageous in certain tissues. Recent publications on prolonged KD identify a decrease in mitochondrial biogenesis in cardiac tissue. The circulating levels of β-hydroxybutyrate (β-OHB), a ketone body that is increased during KD, promotes SIRT7 activation, which inhibits transcription of genes encoding for mitochondrial biogenesis (Xu et al., 2021).

24.2.2 Macronutrient restriction Some studies focused on identifying key nutrients that would induce CR-like effects when restricted. A 2014 National Health and Nutrition Examination Survey identified a reduction in mortality with only the restriction of protein intake (Levine et al., 2014). Specifically, a study in Drosophila found that the supplementation of essential AA retarded the increased longevity of CR flies (Grandison et al., 2009), leading scientists to believe that simply a reduction in specific AA could have the same effects as mild CR. Among several AA analyzed, restriction of methionine had pronounced beneficial effects in mice (Caro et al., 2009; Lee & Longo, 2016; Lo´pez-Torres & Barja, 2008). Methionine restriction (MR) decreases mitochondrial ROS levels in mice (Caro et al., 2008; Sanz et al., 2006) through an unclear mechanism. MR decreased several levels of oxidative damage markers systemically, including 8OHdG and erythrocyte protein-bound glutathione (Maddineni et al., 2013). It is argued that the lower mitochondrial ROS is not due to an increase in antioxidants but rather an enhancement of proton leak, which would reduce the generation of mitochondria ROS (Tamanna et al., 2019; Ying et al., 2015). To further support the enhancement in proton leak hypothesis, UCP1 expression was found to increase following MR-induction (Wanders et al., 2015), which would reduce mitochondrial membrane potential through uncoupling and increase mitochondrial proton leak (Diano et al., 2003). Like other dietary restriction regimens, MR was also shown to be tissuespecific. In adipose tissue, MR increased the expression of PGC-1α and UCP1, decreased expression of TFAM, and had no change in NRF1/2. However, in liver and skeletal muscle, PCG1α showed no significant difference. These findings led to the hypothesis that, during a MR diet, mitochondrial biogenesis is induced in adipose tissue, while in the liver and skeletal muscle, mitochondrial metabolism favors fatty acid oxidation (Perrone et al., 2010). The results were supported by the observation that MR diet in old rats decreased steady-state levels of SIRT1 and TFAM, without affecting the levels of PCG-1α, NRF2, or SOD2. However, while there were no changes in mitochondrial biogenesis proteins, there was a decrease in mitochondrial ROS, identified by the oxidative stress marker 8-OHdG in the liver (Sanchez-Roman et al., 2012), heart (Sanchez-Roman et al., 2011), and serum (Maddineni et al., 2013). These observations lead to the hypothesis

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that MR attenuates mitochondrial ROS production by decreasing the activity of complexes I and III, the two major ROS producers in the electron transport chain (Caro et al., 2008).

24.2.3 Intermittent fasting IF is currently one of the most popular dietary regimens. IF is a form of dietary restriction that cycles from a period of ad-libitum eating to a period of total fasting (Caramoci et al., 2016). Although IF is becoming a more popular form of CR, the long-term impacts are unknown. IF in young animals has shown controversial results. In a short-term intermittent fast, an increase in protein carbonyls, with no significant change in respiration activity or ROS production were seen in skeletal muscle tissue (Chausse et al., 2015) and nerve terminals (Carteri et al., 2021). However, IF provided an age-related rescue of mitochondrial metabolism parameters, including an increase in mitochondrial biogenesis, a recovery of the OXPHOS function, and a decrease in ROS generation (Savencu et al., 2021). In older rats, the age-related increase in protein carbonyl level (Castello et al., 2010) and ROS production (Singh et al., 2012) were significantly decreased, which could be attributed to increased electron transport chain complex IV activity (Singh et al., 2012), and increased SOD2 expression (Ooi et al., 2020). SIRT1 was also shown to increase following IF treatment in a mouse model (Tajes et al., 2010), suggesting that there might be additional factors involved with the preservation of mitochondrial energy metabolism and the enhancement of antioxidant defense mechanisms underlying IF. Other benefits associated with IF relate to its impact on the inflammasome response. IF induced a decrease in NF-kB (Castello et al., 2010) and MAPK signaling pathways (Fann et al., 2014), which decreased the overall transcription of pro-inflammatory cytokines, associated with age-related inflammatory diseases. IF is also known to decrease circulating IGF-1 (Lee et al., 2012), a key regulator of mTOR, that in turn decreases the mTOR pathway activity in the cancer cells (Di Biase & Longo, 2016) and neurons (Alirezaei et al., 2010).

24.3 Mitochondrial pathways activated by caloric restriction mimetics Although dietary restriction has proven successful in extending the health span and/or lifespan of multiple model organisms, from yeast to nonhuman primates, humans may encounter difficulties adhering to strict regimes. Reducing calorie intake from 20% (mild CR) to 50% (severe CR) without incurring malnutrition (Nikolai et al., 2015) is not achieved without effort. Therefore, the demand is high for compounds, dietary supplements or drugs that would mimic the positive antiaging effects that CR has. Effective CR mimetics (CRMs) would alter key

24.3 Mitochondrial pathways activated by caloric restriction mimetics

metabolic pathways as CR does without the need to reduce food intake. They target at least one of the several genes and pathways involved in the positive actions of CR in model organisms. Several functionally and chemically diverse CRM candidates have been identified, such as sirtuin activators, AMPK activators, polyamines, and polyphenols targeting multiple pathways, NAD1 precursors, mTOR inhibitors, or mitochondrial uncouplers, among others (Fig. 24.3). This section will review potential CR mimetics in these groups known to modulate mitochondrial functions.

24.3.1 Multifunctional compounds: polyphenols and polyamines 24.3.1.1 Polyphenols Polyphenols, ubiquitously present in fruits and vegetables, are characterized by great chemical diversity (Vogt, 2010) and constitute an unavoidable component in the human diet. They have emerged as well-tolerated CRMs that target mitochondrial turnover, oxidative stress, and other aspects of mitochondrial physiology (Davinelli et al., 2020). The more than 800 different polyphenols so far identified are classified into flavonoids (e.g., flavanones, and flavonols) and nonflavonoids (e.g., phenolic acids, and stilbenes) and accumulate in many plant foods and beverages, including berries, cereals, coffee, tea, and cacao (Haytowitz & Wu, 2018). Relevant examples of polyphenols that may act as CRMs in humans include resveratrol, curcumin, epicatechin, epigallocatechin-3-gallate (EGCG), gallic acid, and quercetin. Although the effects of polyphenols on health span often vary significantly between studies, current information indicates that polyphenol-rich diets may decrease the risk of developing high-prevalence diseases characterized by age-associated inflammation, which contributes to the onset of age-related diseases (Hofer et al., 2021). Mechanistically, the effects of polyphenols are frequently associated with the modulation of oxidative stress. However, like CR, their mechanism of action involves the modulation of various enzymes and transcription factors that regulate the stress response, cellular survival, and mitochondrial function (Madeo et al., 2019). Resveratrol is a stilbene enriched in the skin of red grapes (B3.00 mg/100 g fresh weight) and red wine (B3.02 mg/100 mL). Initially identified in yeast as a sirtuin (Sir2) activator capable of extending replicative lifespan (Howitz et al., 2003), resveratrol gained momentum with studies in mice and monkeys fed a high-fat diet, showing that the compound could mediate multiple beneficial health effects such a reduced risk for cancer and cardiovascular disease and improvements in metabolic fitness (Timmers et al., 2012). However, studies in humans have reported promising but mixed results that have been reviewed elsewhere (Ramı´rez-Garza et al., 2018). Mechanistically, it has been suggested that SIRT1 and alternative targets could mediate the physiological effects of resveratrol in mammals in a dose-dependent manner. Resveratrol may act through SIRT1 at low doses. However, it also directly or indirectly activates AMPK across

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FIGURE 24.3 Effects of CR mimetics on nutrient-sensing pathways. CR mimetics are compounds that produce CR effects by targeting metabolic and stress response pathways regulated by CR but without restricting caloric intake. This figure depicts the action of some of these compounds. Metformin decreases mitochondrial respiratory function through complex I inhibition, increasing AMP:ATP ratios and levels of NAD1. By increasing NAD1 levels, Metformin promotes SIRT1 activity and all downstream pathways, including the SIRT1-PGC-1α-TFAM mitochondrial biogenesis pathway (see Fig. 24.1). Similarly, NAD1 precursors (e.g., Nicotinamide riboside, Nicotinamide, and Niacin) elevate tissue NAD1 levels resulting in improved cellular energetics and activation of NAD1 dependent enzymes such as sirtuins. Metformin-induced elevated AMP:ATP ratio leads to activation of AMPK, which is phosphorylated and activated by LKB1 and SIRT1-mediated deacetylation, generating a feedback loop. AMPK inhibits glycogen synthesis, fatty acid oxidation, HMG-Co-A Reductase, and mTOR function (see Fig. 24.1). Decreased mTORC1 function results in increased autophagy and mitophagy, decreased S6K activity and protein translation, and inhibition of HIF1α, resulting in reduced cell cycle progression and glucose metabolism. Rapamycin blocks mTORC1 kinase activity, mirroring some of the effects of Metformin. Polyphenols (e.g., Resveratrol, Catechins, and Curcumin) and Polyamines (e.g., Spermidine) increase SIRT1 activity and downstream pathways, including inhibition of NF-κB-mediated pro-inflammatory gene expression. AMPK and SIRT1 activation by the different CR mimetics result in activation of PGC-1α and enhancement of mitochondrial biogenesis, fatty acid oxidation, and stressresistance pathways.

concentrations to produce CR-like beneficial effects on mitochondrial function through activation of PGC-1α, either SIRT1-mediated deacetylation or additional mechanisms, and subsequent enhancement of mitochondrial biogenesis, OXPHOS, and fatty acid

24.3 Mitochondrial pathways activated by caloric restriction mimetics

oxidation (Price et al., 2012). Some studies in human cultured cells have shown that resveratrol can also induce mitochondrial biogenesis by a mechanism dependent on the erythroid 2-related factor 2 (Nrf2) signaling pathway crosstalking with AMPK to enhance cellular redox maintenance and mitochondrial biogenesis, and regulate mitochondrial turnover (Cao et al., 2014). As such, resveratrol has been found to activate mitophagy and attenuate myocardial infarction and mitophagy disturbance in rodents via the acetylation of sirtuins SIRT1 and SIRT3, which then deacetylate and activate FOXO3 (transcription factor forkhead box protein O), leading to the activation of the PINK1/Parkin pathway, inducing mitochondrial mitophagy (Das et al., 2014). Curcumin is a polyphenolic compound isolated from the rhizomes of turmeric (Curcuma longa), frequently powdered and used as a spice and food coloring agent. It has been estimated that the average curcumin content in dried turmeric is 2,213.57 mg/100 g fresh weight (Hofer et al., 2021). Epidemiological studies have shown that long-term interventions with curcumin may reduce total body fat and visceral fat, but mixed data was reported regarding significant effects on body weight and BMI (Akbari et al., 2019; Hariri & Haghighatdoost, 2018). Although the lipid-lowering effects of curcumin remain inconclusive, positive effects have been observed in lowering cholesterol in patients at risk of cardiovascular disease and lowered blood glucose concentrations of individuals with dysglycemia (Qin et al., 2017), and risk of developing type 2 diabetes (Chuengsamarn et al., 2012). The beneficial effects of curcumin have been associated with enhanced mitochondrial biogenesis via upregulation of PGC-1α and enhanced AMPK signaling in mouse adipocytes and skeletal muscle (Ray Hamidie et al., 2015), and downstream activation of NRF1 that controls the expression of multiple genes coding for mitochondrial proteins including the TFAm (Liu et al., 2014). Some data have indicated that curcumin may also act as a mild mitochondrial uncoupler to potentially help minimize ROS production (Lim et al., 2009). Epicatechin and EGCG belong to the flavan-3-ol subclass of flavonoids. Epicatechin is found abundantly in different fruits and legumes, such as apples, pears, berries, cocoa, and broad beans. Likewise, EGCG is the most biologically active and abundant flavan-3-ol in green tea. Considering a large variability among reports, the values of flavan-3-ols ranged from 3 to 544 mg/100 g in apples, dark chocolate, and green tea (Rothwell et al., 2013). The consumption of epicatechin-rich dark chocolate has been associated with improved insulin sensitivity and drops in the incidence of cardiovascular diseases, including heart failure (Raman et al., 2019; Tan et al., 2021). Mechanistically, EGCG has been found to promote mitochondrial biogenesis in mouse neurons by acting through the modulation of AMPK- and sirtuin-dependent pathways (Ray Hamidie et al., 2015), and to upregulate uncoupling protein 3 (UCP3) in murine pancreatic beta-cells to protect against apoptosis (Jia et al., 2020). Epicatechin was found to stimulate mitochondrial biogenesis in the heart and skeletal muscle of mice, leading to improved exercise performance (Nogueira et al., 2011). Furthermore, a study in humans reported that the administration of epicatechin-rich cocoa to individuals

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with type 2 diabetes stimulated mitochondrial biogenesis by enhancing the levels of NO, SIRT1, PGC-1α, and mTFA in skeletal muscle biopsies (Taub et al., 2012). Gallic acid is a well-known polyphenol belonging to the class of phenolic acids, which can be found in berries, citrus fruits, leafy vegetables, soy products, and black tea. Its beneficial effects include antioxidant, antiinflammatory, and antineoplastic properties (Kahkeshani et al., 2019). Gallic acid (GA) conjugated to a mitochondrial targeting sequence has been developed to deliver the molecule to the organelle and found to exhibit antioxidant activity for cardiovascular disease therapy and cytoprotection against anticancer treatments (Bae et al., 2022). It also has the ability to manage obesity, type 2 diabetes, and metabolic syndrome (Afzal & Gortmaker, 2015). In vitro and in vivo studies have shown that gallic acid regulates mitochondrial physiology and apoptotic intrinsic pathway under stress. Like other polyphenols, gallic acid was found to increase NAD1 levels and modulate mitochondrial biogenesis through a SIRT1/PGC-1α/NRF-1/TFAMdependent pathway in human fibroblasts and lymphoblastoid cells (Valenti et al., 2013). The flavanol quercetin, mainly found in onions, apples, and berries, is one of the most extensively studied polyphenols for its anticancer, antiaging, and antiinflammatory activities (Huang et al., 2020). Several clinical trials have shown conflicting results on indices of lipid profile after quercetin treatment but a clear result in the reduction of BP and management of glucose-related parameters (reviewed in (Hofer et al., 2021)). Mechanistically, the treatment of rats with quercetin increased the mRNA and protein levels of nuclear respiratory factors NRF1 and NRF2, and mTFA through the induction of PGC-1α (Sharma et al., 2015), in some models by activating the Nrf2 pathway (Li et al., 2016). However, quercetin administration in humans did not increase mitochondrial biogenesis significantly, although the individuals improved their exercise performance (Nieman et al., 2010). Like resveratrol, quercetin was also reported to activate FOXO3mediated mitophagy in an AMPK-dependent pathway that also involved parkin activation (Yu et al., 2016).

24.3.1.2 Polyamines Polyamines (putrescine, spermidine, and spermine) are naturally-occurring organic cations found in microbes, plants, and animals, formed by the enzymatic decarboxylation of the AAs arginine or ornithine. They become available to the body via the diet, production by the microbiome, and endogenous biosynthesis. They play relevant biological roles by interacting with nucleic acids and proteins, including regulation of protein synthesis, autophagy, cell survival, and proliferation (Madeo et al., 2018; Minois et al., 2011). Although they play roles in stress and disease resistance, high levels of polyamines are found in some cancer cells due to their fast proliferation (Gerner & Meyskens, 2004). It has been observed that polyamine synthesis decreases in senescent cells and that at least induction of autophagy by spermidine promotes longevity in yeast and human cells (Eisenberg

24.3 Mitochondrial pathways activated by caloric restriction mimetics

et al., 2009; Minois et al., 2011). Furthermore, dietary spermidine intake induces cardioprotective and neuroprotective effects in mice and rats, activates autophagy, and extends health- and lifespan (Eisenberg et al., 2009, 2016; Madeo et al., 2018; Schroeder et al., 2021; Singh et al., 2021; Wirth et al., 2021). Therefore, spermidine is considered as a potential CRM, with antiinflammatory and antioxidant properties, enhances mitochondrial biogenesis and function through the SIRT1-PGC-1α-NRF1-TFAm pathway, induces mitophagy through ATM (ataxia telangiectasia mutated)-dependent activation of the PINK1/Parkin pathway, promotes chaperone activity and improves proteostasis (Bhukel et al., 2017; Qi et al., 2016).

24.3.2 NAD1 precursors A century after the identification of the coenzymatic activity of NAD1, interest in NAD1 metabolism heightened due to the therapeutic relevance of NAD1 precursors, biosynthetic enzymes, and NAD1-regulated enzymes (Houtkooper et al., 2010). NAD1 is an essential cofactor for redox reactions and energy metabolism, and for NAD1 consuming enzymes such as sirtuins, poly(ADP-ribose) polymerase (PARP), and CD38. NAD1 thus directly or indirectly regulates many key cellular functions, including energy metabolism, redox, DNA repair, cellular senescence, and immune regulation, which are essential for maintaining metabolic homeostasis and health (Verdin, 2015). NAD1 levels decline with age primarily through its degradation by the NADase CD38 (Camacho-Pereira et al., 2016; Schultz & Sinclair, 2016), and restoring these levels harbors therapeutic potential in humans against multiple age-related diseases, including neurodegenerative diseases, cardiovascular diseases, diabetes, and cancer (Katsyuba et al., 2020; Rajman et al., 2018). Also, in mice, increasing intracellular NAD1 can prevent age-related metabolic decline and improve the function of mitochondria and stem cells (Zhang et al., 2016), maintain skeletal muscle function and exercise capacity (Frederick et al., 2016), and slow down or even reverse the progression of many aging-related diseases to extend their lifespan (Zhang et al., 2016). The primary de novo biosynthesis of NAD1 starts with the essential AA Ltryptophan, taken up from the diet, following the kynurenine pathway. However, the main source of NAD1 is from salvage pathways, which require the uptake of other NAD1 precursors from the diet, such as nicotinic acid (NA, niacin, or vitamin B3), nicotinamide (Nam), and nicotinamide riboside (NR). These precursors are used as effective CRMs to treat all kinds of age-related and metabolic disorders. A search in www.clinicaltrials.gov yielded hundreds of registered clinical trials that include niacin or nicotinamide in diverse clinical settings and cohorts. NR and NAM reduce inflammation, and NAM and Niacin are effective against hypercholesteremia (reviewed in (Hofer et al., 2021)). A connection between NAD1 biosynthesis and mitochondria has been recently established by showing that NAD1 precursors such as niacin and NR have beneficial effects in preclinical

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settings and patients suffering from mitochondrial myopathy and neurodegeneration (Elhassan et al., 2019; Pirinen et al., 2020). Mechanistically, NAD1 precursors elevate tissue NAD1 levels resulting in improved cellular energetics and activation of NAD1 dependent enzymes such as sirtuins and PARPs. For example, administration of NR or niacin increases NAD1 levels and SIRT1 activity, thereby activating the SIRT1-PGC1α-TFAM mitochondrial biogenesis pathway. Cytosolic NAD1 is essential for glycolysis, whereas NAD1 reduced to NADH serves as an electron carrier. NAD1 can also be phosphorylated to NADP1 via NAD1 kinases and, with its NADPH pair, provides the reducing power for anabolic reactions and redox balance (Chu & Raju, 2022).

24.3.3 AMP-activated protein kinase agonists AMPK agonists activate the enzyme by altering the AMP/ADP:ATP balance in the cell and inducing an array of pathways that mimic CR. AICAR (5-aminoimidazole-4-carboxamide-1-D-ribo-furanoside) and metformin are among the beststudied AMPK agonists, capable of modulating mitochondrial biogenesis, mitophagy, OXPHOS, and autophagy, and can even promote longevity in some models, particularly in C. elegans and mice. AICAR is transported into the cell by adenosine transporters and converted to an AMP analog (ZMP). AICAR has been found to promote mitochondrial biogenesis in fibroblasts from patients suffering from mitochondrial disorders (Golubitzky et al., 2011). In mice, AICAR induced a PGC-1α-mediated increase in mitochondrial biogenesis, and a 1-month treatment of AICAR conferred comparable benefits as endurance training to sedentary mice in terms of both muscle mitochondrial profiles and treadmill endurance (reviewed in (Burkewitz et al., 2014)). Metformin is widely used to treat type 2 diabetes since it decreases hyperglycemia and circulating lipids without affecting insulin secretion. It also has neuroprotective, cardio-protective, and tumor-suppressive potential (Wang et al., 2017). Metformin administration to mice for 2 months reproduced up to 85% of the gene expression pattern induced by long-term CR (Dhahbi et al., 2005) and therefore has been considered a potentially optimal CRM. As such, preclinical investigations in animal models have suggested that metformin can slow aging and prolong lifespan (Onken & Driscoll, 2010; Wu et al., 2016). However, its mode of action in distinct cell types remains to be fully identified. Metformin is regarded as a bioenergetic disruptor targeting mitochondria that specifically inhibits the respiratory chain complex I and thereby attenuates aerobic synthesis of ATP (Cameron et al., 2018; Wheaton et al., 2014). The consequent shift in ATP/AMP ratio would lead to a compensatory increase in glycolysis and to LKB1 (Live kinase B1)mediated activation of the energy sensor AMPK. AMPK-mediated phosphorylation would then activate pathways required to adapt to energy stress conditions, including the SIRT1-PGC-1α-NRF1/2 mitochondrial biogenesis pathway, the

24.3 Mitochondrial pathways activated by caloric restriction mimetics

activation of autophagy by inhibiting the TOR pathway, and mitophagy by p53mediated induction of the PINK/PARKIN pathway (Burkewitz et al., 2014; Tulipano, 2021).

24.3.4 Mammalian target of rapamycin inhibitors mTOR (mechanistic target of rapamycin) is a conserved threonine and serine protein kinase that was identified as the target of the immunosuppressive drug rapamycin. mTOR plays a pivotal role in governing cell growth and proliferation, hence making mTOR a therapeutic target for disease conditions such as cancer, caused by deregulated cell proliferation. Multiple clinical trials have used first- or second-generation mTOR inhibitors, alone or in combination with other approaches, to treat cancer with positive outcomes only for selected types of cancer (Zheng & Jiang, 2015). Inhibition of TOR signaling by rapamycin is a robust CR mimetic, shown to consistently increase lifespan in many model organisms from yeast to mammals [reviewed in (Johnson et al., 2013b)]. Rapamycin increases mitochondrial oxidative capacity and resistance to oxidative stress in yeast and fly models, like what is seen in CR (Pan et al., 2011; Ruetenik & Barrientos, 2015; Villa-Cuesta et al., 2014). In mammals, inhibition of mTORC1 by rapamycin induces stress responses, including attenuation of protein synthesis, and induction of autophagy and mitophagy, which are protective mechanisms for the cells to survive under stress conditions. In many types of cancer cells, inhibition of mTORC1 turns off an S6K-dependent negative feedback loop that downregulates upstream signaling of oncoproteins PI3K/AKT, resulting in enhanced PI3K/AKT activity that promotes cell survival (Manning, 2004). One of the most significant effects of the mTOR pathway is adjusting protein synthesis and proteome content, according to available nutrients and growth factors, to balance cellular proliferation with resistance to stress (Bjedov & Rallis, 2020). Like AA starvation, rapamycin treatment leads to TORC1 activity repression and lifespan extension. At the transcriptional level, TORC1 inactivation promotes the expression of ATF4 (activating transcription factor 4) in mammalian cells to induce AA transporters, metabolic enzymes, as well as autophagy factors (reviewed in (Bjedov & Rallis, 2020)). Chronic rapamycin treatment prevented the detrimental effects of both a high-protein and high-fat diet in mice, including the loss of mitochondrial mass and function (Mitsuishi et al., 2013). Furthermore, mTOR inhibition by rapamycin also activates selective degradation of unfit mitochondria through PINK/Parkin-driven mitophagy and mitofusin enhancement of mitochondrial dynamics, which have been shown to positively shift mtDNA heteroplasmy in cellular models of mitochondrial disorders due to mtDNA mutations (Dai et al., 2014) and to restore mitochondrial defects in glioblastoma cells (Lenzi et al., 2021). Some second-generation mTOR inhibitors have already been tested in the context of aging. Dual mTORC1/C2 inhibitors such as Torin1 and Torin2 were shown to suppress hypertrophy, senescent morphology, and extend the

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chronological lifespan of human fibroblasts even better than rapamycin, suggesting that at doses lower than anticancer concentrations, pan-mTOR inhibitors could be used as antiaging interventions (Bjedov & Rallis, 2020; Leontieva & Blagosklonny, 2016). Like rapamycin, they induce CR mitochondrial mechanisms, as seen in nicotine-exposed rat cardiomyocytes in which mitophagy impairment and decreased mitochondria-derived superoxide production were prevented by treatment with Torin1 (Meng et al., 2021).

24.3.5 Mitochondrial uncouplers Mitochondrial uncoupling occurs when a fraction of the proton gradient generated during mitochondrial respiration by the electron transport chain is not used to drive ATP synthesis by OXPHOS but dissipated in the form of heat. This process occurs naturally as part of body temperature regulation in mammals. Significantly, it contributes to modulating the generation of ROS from mitochondria, which is a feature of interventions aiming to extend the lifespan in model organisms (Mookerjee et al., 2010). ROS can act as signaling molecules to precondition against age-associated oxidative stress and as damaging molecules. Therefore, the regulation of ROS generation by mitochondrial uncoupling would appear to be of utmost relevance during the aging process (Mookerjee et al., 2010). Although it would be expected that under CR, bioenergetics would become more efficient to save energy, it decreases the coupling of respiration and OXPHOS by, for example, increasing the levels of UCP, particularly UCP2 and UCP3, as shown in muscle mitochondria in the mice (Krauss et al., 2005). Because CR also induces mitochondrial biogenesis and enhances respiration, which could lead to mitochondrial hyperpolarization and enhanced ROS generation, the increase in UCP proteins is expected to prevent damaging oxidative stress. For example, a study in primary hepatocytes from 12-month-old rats fed 40% CR since weaning, showed that they had increased SIRT1 leading to boosted stress resistance, and enhanced PGC-1α activation creating increased mitochondrial biogenesis and improved bioenergetics based on UCP-rich low-potential mitochondria that sustain reduced respiration and maintain cellular ATP levels while reducing ROS production (Lopez-Lluch et al., 2006). Pharmacological interventions mimicking mitochondrial uncoupling bear a high risk since, in excess, it could induce bodily overheating, aerobic energy deprivation, or eliminate ROS required for stress signaling. Therefore, uncoupling interventions must be tightly controlled. Mild/weak uncoupling achieved by low doses of the protonophore compound FCCP (p-triflouromethoxyphenylhydrazone) has been found to significantly decrease ROS generation and increase the NAD1/ NADH ratio without affecting ATP concentrations in mouse neurons. Among the known uncoupler molecules, the anionic compound DNP (dinitrophenol) is the best studied for its effects on mammalian physiology. It was used on humans as a treatment for weight loss in the 1930s prior to its discontinuation due to toxic

References

side effects (Colman, 2007). Although the therapeutic range of DNP is small and its optimal dose may be variable, it has been shown to increase lifespan, accompanied by decreases in oxidative damage in flies and mice, supporting the benefit of mitochondrial uncoupling (Caldeira da Silva et al., 2008). A potentially more suitable family of chemical compounds with uncoupling activity are the lipophilic penetrating cations, such as C12TPP (dodecyltriphenylphosphonium). Their mitochondrial accumulation is proportional to the mitochondrial transmembrane potential, and it would therefore be preferentially targeted to highly polarized segments of the mitochondrial network, thus sparing mitochondria with relatively low potential levels whose ATP synthesis could be affected (Severin et al., 2010).

24.4 Concluding remarks Studies in model organisms have allowed identifying conserved pathways that promote health- and lifespan in response to environmental nutrient availability. This knowledge facilitates the development of targeted interventions that mimic molecular responses to reduced calorie intake. The key signaling pathways involved include IIS, mTOR, NAD1/NADH-dependent sirtuin deacetylases, AMPK, PGC-1α, and retrograde mitochondrial pathways, all of which form a complex network of interactions. Through all these pathways, a key component of the response to CR is represented by mitochondrial adaptation in the form of enhanced mitochondrial biogenesis, improved mitochondrial quality control, increased OXPHOS efficiency, and reduced oxidative stress. It is becoming apparent that DR-mediated preservation of mitochondrial functions is fundamental to delay the onset of age-related diseases and maintain healthier longevity, particularly in individuals with preexisting conditions such as obesity or age-associated disorder. As recently stated by Lee and colleagues, given our genetic variability, further investigations in human populations are required before broadly recommending CR diets, or mimetics, for otherwise healthy individuals (Lee et al., 2021).

Funding Our work is supported by a Merit Award from the Veterans Administration (VA) Biomedical Laboratory Research and Development [1I01BX003303
01 (to A.B.)].

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33. Westbrook, R., Bonkowski, M. S., Strader, A. D., & Bartke, A. (2009). Alterations in oxygen consumption, respiratory quotient, and heat production in long-lived GHRKO and Ames dwarf mice, and short-lived BGH transgenic mice. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences, 64(4), 443
451. Wheaton, W. W., Weinberg, S. E., Hamanaka, R. B., Soberanes, S., Sullivan, L. B., Anso, E., Glasauer, A., Dufour, E., Mutlu, G. M., Budigner, G. S., & Chandel, N. S. (2014). Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis. Elife, 3, e02242. Wheless, J. W. (2008). History of the ketogenic diet. Epilepsia, 49(Suppl. 8), 3
5. Available from https://onlinelibrary.wiley.com/doi/full/10.1111/j.1528-1167.2008.01821.x. (January 13, 2022). Wirth, A., Wolf, B., Huang, C. K., Glage, S., Hofer, S. J., Bankstahl, M., Ba¨r, C., Thum, T., Kahl, K. G., Sigrist, S. J., Madeo, F., Bankstahl, J. P., & Ponimaskin, E. (2021). Novel aspects of age-protection by spermidine supplementation are associated with preserved telomere length. Geroscience, 43(2), 673
690. Włodarek, D. (2019). Role of ketogenic diets in neurodegenerative diseases (Alzheimer’s disease and Parkinson’s disease). Nutrients, 11(1), 169. Available from https://www. mdpi.com/2072-6643/11/1/169/htm. Wright, D. C., Geiger, P. C., Han, D. H., Jones, T. E., & Holloszy, J. O. (2007). Calcium induces increases in peroxisome proliferator-activated receptor γ Coactivator-1α and mitochondrial biogenesis by a pathway leading to P38 mitogen-activated protein kinase activation. Journal of Biological Chemistry, 282(26), 18793
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CHAPTER

Rejuvenation of mitochondrial function by time-controlled fasting

25 Michael N. Sack

Laboratory of Mitochondrial Biology and Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, United States

25.1 Introduction The concept that caloric restriction (CR) or intermittent fasting (IF) confer health benefits has been evident for centuries as epitomized by the celebrated Venetian centenarian Luigi Cornaro in his 16th-century treatise on “The Art of Living Long.” He postulated that living temperately extends one’s life and conversely, that man’s weak indulgence of his appetite shortens his life. This concept has been scientifically validated in multiple eukaryotic species, although in primates’ life extension has not been firmly established (Fontana & Partridge, 2015). However, CR has been shown to markedly extend health span in both primates and humans, with most data supporting a reduction in cardiometabolic risk/disease in long-term clinical studies (Fontana & Partridge, 2015; Fontana et al., 2004) and in reduced cardiometabolic risk, brain atrophy and cancer in primates (Colman et al., 2009; Mattison et al., 2012). Over the last few decades, a concerted effort has been focused on the mechanisms whereby time-controlled fasting (TCF) confers these health and lifespan benefits. These effects have been explored in the context of numerous organs and diseases employing multiple model systems. The underlying evolutionary theory underlying the salutary effects of TCF, are linked to the concept termed “hormesis.” This refers to adaptive responses of biological systems to transient environmental challenges that evoke functional and/or tolerance to subsequent environmental challenges (Calabrese & Mattson, 2017). The underlying mechanisms postulated to support hormesis in TCF include neuro-hormonal effects, modulation of paracrine and intracellular signaling, chromatin remodeling, gene transcription, posttranslational modifications, and regulation of intracellular organelle homeostasis (Mattson, 2008a,b). As mitochondria are regulated by and respond to hormetic triggers (Yun & Finkel, 2014), this review will focus on the mitochondria as a pivotal organelle in this biology and will define the role of TCF on mitochondrial biology in different organ systems and disease processes.

Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00017-5 2023 Published by Elsevier Inc.

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25.2 Strategies employed to study the effects of timecontrolled fasting Multiple interventions have been evaluated for efficacy of TCF with a myriad of biological readouts. The most common strategies include IF, chronic CR, and time-restricted feeding (Mattson et al., 2014). Moreover, an IF-restriction mimetic diet has been developed (Brandhorst et al., 2015; Choi et al., 2016; Wei et al., 2017) and investigators have directly targeted TCF pathways with pharmacologic (Bitto et al., 2016) or nutrient supplements (Elhassan et al., 2019; Zhang et al., 2016) to partially reconstitute the effects of fasting. As might be expected, pharmacologic and/or nutritional supplements do not recapitulate all the effects of TCF (Birkisdottir et al., 2021), most likely reflecting that TCF affects multiple organs, has cell autonomous and paracrine effects (Han et al., 2021a,b; Jordan et al., 2019), and modifies the gut microbiome due to the nutrient-load effect on gut flora (Cignarella et al., 2018). On reviewing these data, and the practicalities of these dietary interventions, the two strategies emerging as the most feasible for the management of human health include IF, or chronic time-restricted feeding (de Cabo & Mattson, 2019).

25.3 Time-controlled fasting and health The most ubiquitous effect of TCF interventions is a reduction in cardiometabolic risk including reduced blood glucose, cholesterol, and blood pressure levels and improved insulin sensitivity (Fontana et al., 2004; Wilkinson et al., 2020). At the same time, subjects lose weight (Fontana et al., 2004; Racette et al., 2006), have modifications in their gut microbiome (Maifeld et al., 2021), and exhibit a reduced core temperature (Heilbronn et al., 2006). Hence, the ability to dissociate the direct effects of reducing caloric intake vs. the multisystemic consequences of these interventions, add a level of complexity that needs to be acknowledged. Nevertheless, in humans in addition to the reduction in cardiometabolic risk factors (Fontana et al., 2004; Maifeld et al., 2021; Wilkinson et al., 2020), TCF and mimetic interventions have shown a multitude of beneficial effects on numerous diseases. These include: a reduction in innate and adaptive inflammatory immune responses (Han et al., 2021b; Traba et al., 2015) without impairing cellmediated immunity (Meydani et al., 2016); a reduction in inflammation in asthma and rheumatoid arthritis (Fraser et al., 2000; Han et al., 2018; Johnson et al., 2007a); an improvement in vasodilatory function (Raitakari et al., 2004) and a delay in aging-linked decline in diastolic function (Meyer et al., 2006). Animal models have replicated the findings exhibited in human subjects, and additionally, show further beneficial effects in models of disease. Examples of this include the blunting central nervous system autoimmunity (Cignarella et al., 2018); delayed aging in progeroid mice (Vermeij et al., 2016); enhanced efficacy

25.5 Temporal caloric restriction effects on mitochondrial biogenesis

of chemotherapy (Lee et al., 2012) with the induction of the activity of tumorinfiltrating lymphocytes (Di, 2016); and in blunting age-linked hearing loss (Someya et al., 2010). Overall, these investigations of TCF in human disease and animal models have defined the role of hormonal and extracellular signaling molecules, intracellular signaling networks, and modifications in organelle homeostasis to confer these to overall health span and lifespan beneficial effects. These have been recently reviewed (de Cabo & Mattson, 2019; Longo & Mattson, 2014) whereas, the mitochondrial-directed regulatory control nodes will be expanded upon further in this review.

25.4 Effects of time-controlled fasting on mitochondrial function Mitochondria function to sustain numerous homeostatic processes within cells and organs including energy generation, control of calcium, iron and redox homeostasis, biosynthetic roles, for example, for cholesterol synthesis and in retrograde signal transduction and transcriptional regulation. Mitochondria also play a role in regulating susceptibility to injury, to injury-initiated programmed cell death, and to resilience of these programs through the modulation of mitochondrial quality control (Sack et al., 2017). At the same time, mitochondria are specifically programmed for distinct functions in different cell types and organs (Johnson et al., 2007b), and their function, in part, reflect the underlying functions and energetic demands of their tissue of residence (Glancy et al., 2015; Graham et al., 2017). Given this myriad of functions, and differing biology in different organs systems, it may be expected that the nutrient-load reductive interventions may confer organ-specific effects on mitochondria. In the first instance, the mitochondrial consequence to nutrient-load restriction will be reviewed and categorized according to the effect on mitochondrial function, rather than classified by organ system or disease. Additionally, as one may expect, given the ability to genetically modulate pathways to validate their roles, mechanistic insight into the regulatory control of mitochondrial function in response to different TCF and/or CR interventions has been delineated in greater depth in animal models and here the investigations uncover effects in specific organs, which will be highlighted. The studies highlighting these effects are also summarized in Table 25.1.

25.5 Temporal caloric restriction effects on mitochondrial biogenesis The regulatory program to increase mitochondrial content has been well described and is predominantly operational to increase mitochondrial capacity in response

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Table 25.1 Summary of the effects of time-controlled fasting (TCF) interventions on mitochondrial biology. Mitochondrial phenotype

Acute/ subacute TCF interventions

Mitochondrial biogenesis Mitochondrial dynamics Mitophagy

Chronic TCF interventions

References

Or unchanged

Civitarese et al. (2007); Lanza et al. (2012); Nisoli et al. (2005) Faitg et al. (2019)

or

Deter and De (1967); Hailey et al. (2010); Kim and Lemasters (2011); Oost et al. (2019); Price et al. (2012); Shirakabe et al. (2016); Springer et al. (2021) Bevilacqua et al. (2005); Traba et al. (2015)

Oxygen consumption (VO2) Proton leak ROS production

ATP generation

Oxidative phosphorylation Substrate utilization

Cholesterol synthesis Purine recycling

Heart— unchanged Skeletal muscle

Bevilacqua et al. (2004) Davis et al. (2008); Descamps et al. (2005); Drew et al. (2003); Traba et al. (2015); Traba et al. (2017) Drew et al. (2003)

Habegger et al. (2010) Fatty acid oxidation ketone oxidation

Kuhla et al. (2014); Puchalska and Crawford (2017) Seok et al. (2018)

Puchalska and Crawford (2017) Sonanez-Organis et al. (2012)

to increasing demands for example, in skeletal muscle following endurance training and/or in response to browning of adipose tissue for heat generation. A gene transcriptional component is central to driving mitochondrial biogenesis which involves the transcriptional coactivator peroxisome proliferator activated receptor gamma-coactivator 1 alpha (PGC1a) (Wenz, 2013) and transcription factor A of mitochondria (TFAM) (Jornayvaz & Shulman, 2010), which are both mediated, in part, via CR activation of the deacetylase enzyme Sirt1 and through eNOS signaling (Nisoli et al., 2005). Given these regulatory programs, the role of TCF interventions in regulating mitochondrial biogenesis has been explored. In murine

25.6 Fasting effects on mitochondrial dynamics and turnover

models, up to 12 months of CR is associated with increased Sirt1, PGC1a, and TFAM-linked mitochondrial biogenesis in multiple tissue types, including skeletal muscle, and adipose tissue (Nisoli et al., 2005). In this model, this increase in mitochondrial content was found to be genetically-dependent on eNOS signaling (Nisoli et al., 2005). In parallel, in young overweight individuals, six months of CR has a greater effect on augmenting skeletal muscle mitochondrial mass compared to a less robust CR regimen combined with a structured exercise program, although both interventions increased PGC1a, TFAM, Sirt1, and eNOS levels (Civitarese et al., 2007). A question arises whether this induction of mitochondrial mass is sustained, given that the cessation of CR in mice after six weeks of CR, reverses mitochondrial biogenesis in adipose tissue (Mooli et al., 2020). Additionally, whether this regulatory program is a transient adaptation to a restricted caloric load has been posited, given that lifelong CR in mice sustains skeletal muscle mitochondrial energetic function without evidence of increased mitochondrial biogenesis or increased steady-state mitochondrial mass (Lanza et al., 2012). Taken together, these studies suggest that the induction of the mitochondrial biogenesis program may be an early response to CR, but that the integrity and function of mitochondria may be sustained by nutrient deprivation without the long-term requirement of increased mitochondrial density.

25.6 Fasting effects on mitochondrial dynamics and turnover Mitochondrial fusion, fission, and recycling through the mitophagy program function as integrated components in the maintenance of mitochondrial quality control. In parallel, the interconnected vs. fragmented architecture of mitochondria also play an important role in bioenergetic capacity and the ability to respond to metabolic demand. The molecular motor proteins and signaling molecules that orchestrate these programs have been extensively investigated (Twig & Shirihai, 2011), although how they are regulated by TCF interventions has not been as thoroughly explored. Mitochondrial morphology and biochemical signatures of mitochondrial dynamics have been delineated in different muscle fiber types in response to 13 months of CR in aged rats. This intervention resulted in the prevention of sarcopenia compared to aged ad-libitum fed rats and concurrently appeared to promote mitochondrial dynamics in oxidative fibers, promoting mitochondrial branching in glycolytic fibers (Faitg et al., 2019). This study explored steady-state levels though and could not determine dynamic changes in mitochondrial fission/fusion, nor did they determine if the changes observed had any functional effect on mitochondrial function. As macroautophagy senses the nutritional state of cells, it would be expected that this program and possibly the targeted recycling of mitochondria through

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mitophagy would be modulated by TCF. An interesting concept that should be highlighted here, as a prelude to exploring findings to date, is that TCF and CR would not result in the uniform depletion of nutrient availability to different tissue types or organs, but rather result in metabolic reprograming with induction of catalytic programs within the liver, adipose and skeletal muscle beds, to supply glucose, ketones, fatty acids, and amino acids to sustain requirements for energetic demand in most organs. Hence, the induction of mitophagy may be restricted to distinct tissue types as opposed to being a generalized response to nutrientrestricted signaling. In addition, the role of nutrient deprivation on mitophagy may be dynamic, much like the signaling of mitochondrial biogenesis, with distinct responses to acute vs. chronic dietary interventions and to the composition of the macronutrient content of the caloric-restricted diet. These concepts are borne out by the data spanning from cell culture studies to the effects of fasting in mouse models. First, time-lapsed imaging in starved cells demonstrates that mitophagy is induced and that the outer mitochondrial membrane contributes to the autophagosome membrane formation (Hailey et al., 2010) and that the glucagon, a pancreatic hormone induced by fasting, promotes mitophagy, including the degradation of mitochondrial DNA (mtDNA) in primary murine hepatocytes (Kim & Lemasters, 2011). This acute induction of mitochondrial turnover is also evident in the in vivo murine liver, in response to glucagon (Deter et al., 1967) and combined with overnight fasting, shows the distinct induction of the mitochondrial-bound mitophagy receptor BNIP3 (Springer et al., 2021). Interestingly, this acute induction of mitophagy during fasting was also evident in skeletal muscle and in the heart (Oost et al., 2019; Shirakabe et al., 2016). In contrast to these acute effects, metabolic 2H2O-labeling in chronic caloricallyrestricted mice, showed reduced hepatic mitochondrial protein turnover, suggesting that a component of prolonged CR-induced cellular fitness includes reduced mitochondrial protein turnover (Price et al., 2012). In parallel, mice subjected to 40% CR for six months showed evidence of higher expression of hepatic proteins linked to mitochondrial fission (Fis1 and Drp1) with no changes in proteins linked with mitochondrial fusion (Khraiwesh et al., 2013). Interestingly, in this same mouse model, the lipid content of the diet also affected the ability to retain skeletal muscle mitochondrial ultrastructure and dynamics is response to CR (Gutierrez-Aguilar et al., 2018). Concepts uncovered by these studies suggest that dynamic regulation of these mitochondrial quality control programs may be responsive to macronutrient content (Villalba et al., 2015) and whether the TCF intervention is IF, vs. acute or chronic CR.

25.7 Effects on mitochondrial energy metabolism The density, organization, and metabolic capacity of mitochondria vary by orders of magnitude in different tissues which reflects in both the overall energetic

25.7 Effects on mitochondrial energy metabolism

demands of specific tissue types (Johnson et al., 2007b) and spatiotemporal energetic requirements in specialized areas of a cell, for example, the neuronal synaptic junctions (Graham et al., 2017) or in skeletal muscle (Glancy et al., 2015). Given this diversity of both morphology, subcellular distribution, and distinct tissue-type energetic demands, the effects of TCF on mitochondrial energy metabolism would be expected to be both complex and would need to be explored in different organs. At the same time, mitochondrial energetics is intricately linked with mitochondrial proton leak and with the production of mitochondrial reactive oxygen species (ROS). These parameters were measured concurrently in rats exposed to 6 and 12 months of a 40% CR diet. Interestingly, the whole body Vo2 was reduced by 1/3 by CR and skeletal muscle mitochondrial proton leak and ROS production were diminished. This skeletal muscle phenotype was accompanied by no significant change in mitochondrial proton leak, although the levels of uncoupling protein 3 (UCP3) were induced (Bevilacqua et al., 2005). At the same time, mice, subjected to a similar degree of CR for six months, despite changes in markers of mitochondrial dynamics, showed that the ability of hepatic mitochondria to generate ATP was unchanged (Khraiwesh et al., 2013). The tissue specificity of bioenergetic demands was clearly delineated where the effects of aging with and without lifelong CR on mitochondrial ATP content was compared in rat hearts and skeletal muscle (Drew et al., 2003). Interestingly, given the sustained metabolic demands on the heart, cardiac ATP production rate and mitochondrial content was not affected by aging or CR (Drew et al., 2003). In contrast, skeletal muscle which were subjected to sarcopenia with aging, resulted in reduced mitochondrial ATP production and content that was independent of CR; however, CR attenuated oxidative damage in skeletal muscle mtDNA with aging (Drew et al., 2003). In contrast, the oxidative capacity in response to a 24-hour fast has been shown to be induced, in parallel with enhanced antioxidant defenses in peripheral blood mononuclear cells, compared to these parameters in the re-fed state (Traba et al., 2015). Whether TCF interventions modulate mitochondrial fuel substrate utilization is another question arising with the possibility of modulating mitochondrial energetic efficiency. Nevertheless, it is well established that during early fasting, the liver stimulates glycogen breakdown to increase glucose levels and that with progressive fasting following glycogen depletion, the liver activates mitochondrial fatty acid b-oxidation (FAO) to produce energy for gluconeogenesis and to generate intermediates for ketogenesis (Habegger et al., 2010). In addition to the role of glucagon in this metabolic remodeling, emerging evidence supports epigenetic regulatory events in orchestrating fasting-induced hepatic FAO-encoding genes through the Sirt1 deacetylase and the Jumonji D3 histone demethylase (Seok et al., 2018). In addition, ketogenesis occurs exclusively in hepatic mitochondria and following ketone body secretion from the liver, these intermediates are used as a substrate for ATP production in peripheral tissues (Puchalska & Crawford, 2017). At the same time, TCF interventions increase circulating fatty acids from adipocyte lipolysis (Nielsen et al., 2014) and AMPK-initiated autophagy and

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proteolysis in skeletal muscle for amino acid release (Bujak et al., 2015) as substrates for energy metabolism. In nondiabetic obese subjects subjected to six weeks of an extreme caloricrestricted diet (550 calories per day), cardiac fuel uptake was measured by positron emission tomography. The study subjects showed an improvement in insulin-sensitivity, without a change in myocardial glucose uptake, although they did show a parallel reduction in cardiac fatty acid uptake and in cardiac triglyceride content (Viljanen et al., 2009). In mice, lifelong CR also reprograms hepatic fat metabolism with transcript signatures and substrate analysis supporting decreased lipogenesis and hepatic lipid accumulation and circulating evidence of enhanced ketogenesis (Kuhla et al., 2014). Although these studies do not measure mitochondrial metabolic flux per se, they do support that mitochondrial fuel substrates are altered, acutely in response to fasting by increasing fatty acid oxidation and following CR due to reduced systemic lipid storage and the increased availability of ketones for oxidation. This remodeling would not necessarily alter energetic outputs but would result in altered energetic production and utilization in response to the effects of TCF interventions in response to specific organ energetic demands.

25.8 Effects on reactive oxygen species handling The temporal and quantitative production of mitochondrial ROS determines their role as either signal transducers or in promoting oxidative damage and adverse cellular effects. The mechanisms underlying how TCF may modulate mitochondrial ROS production and scavenging could potentially be modulated, in part, by the effects on mitochondrial fidelity and oxidative phosphorylation as described above. One such mechanism includes the TCF effect on the production of ketone bodies. Here, b-hydroxybutyrate (bOHB) and high concentrations of its intermediate acetoacetate (AcAc), via inhibition of class I histone deacetylases (HDACs), promote acetylation and activation of antioxidant encoding genes including the transcription factor FOXO3A and the Metallothionein 2 (Mt2) with the subsequent blunting of oxidative stress (Shimazu et al., 2013). Another mechanism in response to a single prolonged fast, is via SIRT3mediated deacetylation and activation of the mitochondrial superoxide dismutase SOD2 (Traba et al., 2015, 2017). The activation of SOD2 is also evident in response to long-term alternate-day fasting in mice, which is associated with a blunting of the development of murine lymphomas (Descamps et al., 2005). Interestingly, both 24-hour fasting, and ketone supplementation conferred neuroprotection from a subsequent traumatic brain injury, linked with reduced oxidative stress and a sustenance of mitochondrial oxidative phosphorylation, although the mechanisms underpinning these effects were not delineated further (Davis et al., 2008). As discussed earlier, rat exposure to prolonged CR showed

25.10 Fasting-mediated modulation of mitochondrial signaling

a reduction in skeletal muscle mitochondrial proton leak and ROS production in parallel with the induction of UCP3 levels (Bevilacqua et al., 2005). Furthermore, lifelong CR has also been shown to reduce oxidative modification of mtDNA in skeletal muscle, independent of any effect on skeletal muscle ATP levels (Drew et al., 2003). The extent of TCF is important with respect to the mitochondrial response, in that rats subjected to a prolonged 72-hour fast, indicative of starvation rather than a hormetic effect, exhibited increased liver mitochondrial proton leak, with higher ROS generation and evidence of increased hepatic membrane susceptibility to lipid peroxidation (Sorensen et al., 2006). Taken together, these data show that the duration and degree of TCF interventions can have different effects on mitochondrial ROS production and that these effects are regulated at various levels spanning from transcriptional control to the posttranslational mediated activity of ROS scavenging enzymes and to the control of mitochondrial proton leak. Numerous other mechanisms are operational in mitochondrial ROS production (reviewed (Andreyev et al., 2015)) but have not been assessed in the context of TCF.

25.9 Effects on mitochondrial synthetic function In addition to its role in generating energy, in calcium homeostasis, and in controlling ROS production and levels, mitochondria have multiple synthetic functions, which may also be modified in the mitochondrial response to TCF. This is most evident where increased FAO and ketogenesis during fasting generates excess acetoacetate (AcAc) as a metabolic intermediate. AcAc in turn, contributes to extra-mitochondrial lipid and cholesterol synthesis (Puchalska & Crawford, 2017). Although experimental strategies need to be devised to evaluate whether these synthetic pathways play adaptive or maladaptive roles in response to fasting (Puchalska & Crawford, 2017). Mitochondria also play a role in the nucleotide synthesis salvage pathways especially in postmitotic tissues (Wang, 2016). Also, extensive investigation into the effect of fasting on these pathways has not been performed. However, prolonged fasting increases purine recycling in postweaned elephant seal pups to putatively enhance the supply of ATP (Sonanez-Organis et al., 2012). Whether TCF modulates other mitochondrial synthetic pathways remains to be investigated.

25.10 Fasting-mediated modulation of mitochondrial signaling Signaling from mitochondria can initiate intracellular signaling via: ROS and calcium handling, energy-sensing signaling, posttranslational modification of

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proteins by metabolic intermediates (Chandel, 2015), the direct inhibition of enzymes, and even by the release of mtDNA to initiate intracellular innate immune sensing (Traba & Sack, 2017). In mice and humans, a single prolonged fast blunts ROS production via the activation of SIRT3 and SOD2 to blunt activation of the NLRP3 inflammasome in myeloid cells (Traba et al., 2015, 2017). Prolonged fasting in lean and obese individuals also showed differential effects on systemic mitochondrial metabolism and skeletal muscle AMPK signaling. Interestingly, both lean and obese individuals showed a switch from glucose to lipid oxidation, although only the lean subjects showed a concomitant reduction in skeletal muscle AMPK activity (Wijngaarden et al., 2013). These data again point to the complexity of characterizing the effects of fasting and highlight that tissue-specific and disease-linked perturbations remain to be characterized. Changes in nutrient load and metabolic pathway flux also give rise to increased metabolic intermediates such as acyl groups that can bind to and modify protein function. Fasting and CR have been shown to modify proteins, for example via acetylation (Nakamura et al., 2013), succinylation, and malonylation (Newman et al., 2012). These modifications have been shown to regulate individual protein activities, with both salutary and adverse effects (Lu et al., 2011, 2015), although the relative stoichiometric occupancy of target amino acid residues on these specific proteins appear to be low (Baeza et al., 2016; Weinert et al., 2015).; how these posttranslational modifications exert their intrinsic biological effects remains controversial (Scott & Sack, 2020). In response to fasting, hepatic mitochondrial ketogenesis can increase circulating ketones by around 400% within 24 hours of fasting with further increases due to a longer duration fasting or starvation (Cahill, 2006; Puchalska & Crawford, 2017). Different ketone bodies have now begun to be explored as signaling intermediates. This can result from their oxidation, via the inhibition of class I HDACs or via functioning as ligands that signal through G-protein coupled receptors (GPRs), (Puchalska & Crawford, 2017) and more recently to modulate posttranslational modifications via protein lysine b-hydroxybutyrylation (Koronowski et al., 2021). As described previously, bOHB-mediated inhibition of HDACs results in the upregulation of antioxidant genes with increased resilience to oxidative stress (Shimazu et al., 2013). bOHB also signals via the G-protein coupled receptor, GPR109A to inhibit adipocyte lipolysis via the inhibition of adenylyl cyclase (Puchalska & Crawford, 2017; Tunaru et al., 2003). The role of AcAc as a signaling intermediate is also shown where AcAc, via activation of MEK1-ERK1/2cyclin D1 plays a role in muscle regeneration in a murine model of muscular dystrophy (Zou et al., 2016). Collectively, these data show that fastinginduced mitochondrial orchestrating signaling may play multiple roles in the biological effects of fasting in different organs and possibly with different effects dependent on the disease state. The direct mitochondrial-mediated signaling events are schematized in Fig. 25.1.

25.12 Time-controlled fasting strategies

FIGURE 25.1 Mitochondrial signaling events in response to TCF interventions. The major signaling events linked to mitochondrial perturbations in response to TCF interventions. Additional mitochondrial linked signaling pathways have to date, not been explored in response to TCF interventions. AMPK, Adenosine monophosphate kinase; eNOS, endothelial nitric oxide synthase; ERK, extracellular-signal-regulated-kinase; GPR, G Protein
coupled receptor signaling; HDAC, histone deacetylase; MEK, mitogen-activated protein kinase kinase; PTMs, posttranslational modifications; TCF, time-controlled fasting.

25.11 Adverse effects on mitochondrial function in response to fasting As described, the duration and frequency of TCF interventions play a myriad of roles in their effects on mitochondrial, cellular, and organismal function. At the same time, the effect of a single prolonged fast (72 h) showed increased mitochondrial electron leak, elevated ROS generation, and increased susceptibility to membrane oxidative damage (Sorensen et al., 2006). Interestingly, CR and fasting also enhance the risk of acetaminophen toxicity (Whitcomb & Block, 1994). Here, fasting via the activation of SIRT3 and deacetylation of the mitochondrial enzyme, aldehyde dehydrogenase 2 is operational in increasing this toxic susceptibility (Lu et al., 2011). It is quite plausible that TCF effects on mitochondrial function could have other yet unidentified adverse effects.

25.12 Time-controlled fasting strategies to boost mitochondrial fidelity and disease amelioration Although the preponderance of evidence supports that intermittent or modest TCF interventions have salutary effects on health, the direct link of these effects to

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changes in mitochondrial function are less well characterized. Nevertheless, the effects of fasting and CR on blunting mitochondrial ROS have been linked to the prevention of hearing loss (Someya et al., 2010), to the blunting of the NLRP3 inflammasome (Traba et al., 2017), and to attenuating hepatic ischemiareperfusion injury (Miyauchi et al., 2019). In addition, the beneficial effects of TCF-initiated hepatic ketogenesis similarly protects against hepatic ischemiareperfusion injury (Miyauchi et al., 2019) and protects against myeloid linked inflammation through GPR109A-linked signaling with ameliorative effects against atherosclerosis, inflammatory bowel disease, and neurodegeneration (Graff et al., 2016).

25.13 Fasting and other organelles Mitochondria do not function in isolation within the cell, but rather rely on interactions with other organelles as evident by the contact sites between mitochondria and the ER (Rieusset, 2018), between mitochondria and lysosomes (Wong et al., 2019), and mitochondrial reliance on the autophagosome machinery for turnover and quality control. These organelles are similarly affected by TCF (Fu et al., 2012; Mani et al., 2018; Miyauchi et al., 2019; Webster et al., 2014), although how the coordinated regulation of these intracellular organelles function in concert to regulate the salutary effects of these nutritional interventions remain to be determined.

25.14 Conclusion Little did Luigi Cornaro know when he wrote his treatise, that modern science would carefully dissect out the mechanisms whereby living temperately could extend health span and lifespan. The complexity and interrelated mechanisms and their effects on different organ systems and on the microbiome requires additional work. This review begins to highlight the effects of nutrient-load restriction interventions on one organelle, the mitochondria, with the subsequent consequences on health and disease. This review also highlights that the consequences of the extent of caloric deprivation can span from adaptive to maladaptive effects and points out the numerous gaps in our knowledge as to how TCF interventions modulate mitochondrial function. Nevertheless, a most promising and emerging theme is that the ameliorative effects of optimized TCF and their effects on mitochondrial function appear to be operational in both animal models and in humans. Our further understanding of the mechanisms underlying these effects may uncover new avenues to prevent or alleviate disease.

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Dietary modulation and mitochondrial DNA damage

26

Thiago de Souza Freire and Nadja C. de Souza-Pinto Departmento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil

26.1 Introduction Food is an essential basic need for all heterotrophic organisms, as it provides substrates and energy for all cellular reactions and functions. In addition, micronutrients and essential nutrients (compounds that the organism is not capable of synthesizing) are also obtained through diet. Although it is intuitive to think that diet is an important factor in maintaining organismal health, only relatively recently has substantial experimental evidence linked unbalanced diet with disease (Mozaffarian, 2016). A diet considered healthy or balanced occurs when the types and the amount of food consumed can provide adequate amounts of all macro- and micronutrients needed by the individual who ingests them (de Ridder et al., 2017). It is now clear that a lifestyle that adopts a healthy diet and regular physical activity plays a fundamental role in reducing the risk of disease and improving overall quality of life (Atallah et al., 2018). Nonetheless, the molecular mechanisms mediating the effects of a healthy diet in protecting against disease or improving overall health are still unclear. Several cellular targets and signaling pathways have been implicated in the metabolic effects of dietary modulation (Martı´n & Ramos, 2021; Yang et al., 2021), but the effects that nutrients have on body functioning and how they could produce, on one hand a healthy physiological state, or a metabolic imbalance that contributes to disease development of diseases, are still under investigation (Sales et al., 2014). In eukaryotic cells, mitochondria play a central role in energy metabolism, as the main cellular site for ATP production through oxidative phosphorylation (OXPHOS), oxidizing substrates obtained from the diet (Benard et al., 2010). In addition to energy production, the mitochondrial metabolism generates essential intermediates for both mitochondrial and cellular function, and functions as a hub for several essential metabolic pathways (Frezza, 2017). The OXPHOS pathway is intrinsic to most mitochondrial functions such as intramitochondrial redox balance, ion transport, and reactive oxygen species (ROS) generation that are all linked to the electron transport chain, the proton motive force, and the inner Molecular Nutrition and Mitochondria. DOI: https://doi.org/10.1016/B978-0-323-90256-4.00020-5 © 2023 Elsevier Inc. All rights reserved.

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mitochondrial membrane integrity (for a comprehensive review, see Batalha et al., 2022). OXPHOS is carried out by five protein complexes (complexes I
V) located in the inner mitochondrial membrane, which carry out electron transport from reduced coenzymes (NADH and FADH2) to O2 (Complex I
IV) proton pumping, and the ATP synthase (Complex V), which uses the proton motive force to phosphorylate ADP (reviewed in Kowaltowski et al., 2009). Out of the five OXPHOS complexes, four contain protein subunits encoded in the mitochondrial DNA (mtDNA), with Complex II (succinate dehydrogenase) being exclusively nuclearencoded. While most mitochondrial proteins are encoded in the nuclear DNA (nDNA), the mtDNA encodes 13 protein subunits of OXPHOS complexes, which are seven subunits of Complex I, one of Complex III, three of Complex IV and two of Complex V (Anderson et al., 1981). Although the mtDNA-encoded subunits are numerically fewer than the nDNA encoded OXPHOS components, mutations in the mtDNA have a severe impact on mitochondrial function and can cause maternally inherited human diseases or contribute to complex diseases of great public health impact, such as cancer, metabolic diseases, and neurodegeneration, and also to normal aging (for review, see Wallace, 2018). Somatic mtDNA mutations can arise from replication error, unrepaired or incorrectly repaired DNA lesions (Szczepanowska & Trifunovic, 2017). Thus, understanding the mechanisms by which mtDNA accumulate damage and the mtDNA repair pathways is essential to gain insight into the pathways that lead to mutation accumulation and mitochondrial dysfunction and disease. In this context, understanding how dietary components can impact DNA damage levels and mtDNA maintenance mechanisms may help to develop interventions aimed at maintaining mtDNA integrity, potentially reducing the risk of disease development and favoring a healthier life.

26.2 Mitochondrial DNA damage accumulation and maintenance of the mitochondrial DNA The mtDNA is a circular DNA molecule, of about 16.5 Kb in humans, physically associated with the inner side of the inner mitochondrial membrane. The mtDNA is found in a proteinaceous complex known as the mitochondrial nucleoid. The nucleoid contains several different proteins associated with the mtDNA, but the mitochondrial transcription factor A (TFAM) is a major component and essential for nucleoid stability (Farge & Falkenberg, 2019). TFAM binding to mtDNA imposes a turn in the molecule, and with its dimerization and cooperative binding the result is a significant packing of the mtDNA, which is virtually entirely covered by TFAM (Ngo et al., 2011). This structure is believed to provide protection to mtDNA against damaging agents (Chew & Zhao, 2021), while it may also

26.3 Caloric restriction and dietary restriction

impair DNA repair activities (Canugovi et al., 2010), implying that nucleoid remodeling is an essential component of the mtDNA maintenance pathways. The mtDNA is subjected to damage by several types of endogenous and exogenous agents (Muftuoglu et al., 2014; Roubicek & Souza-Pinto, 2017). Moreover, its association with the inner mitochondrial membrane, where ROS are constantly generated as by-products of the OXPHOS, renders the mtDNA more susceptible to oxidative damage than the nDNA. In fact, several groups, including ours, have demonstrated that the mtDNA accumulates more oxidized bases than the nDNA (for review, see Muftuoglu et al., 2014). Nonetheless, despite initial observations that mammalian mitochondria do not repair UV-induced DNA damage (Clayton et al., 1974), it is now clear that mitochondria are quite proficient in DNA repair pathways, efficiently removing several types of DNA lesions. For a comprehensive review of mtDNA repair pathways refer to Alencar et al., 2019. DNA damage can impair mitochondrial function directly or through mutagenesis. Damaged templates can lead to incomplete replication and result in mtDNA depletion, while incomplete, or faulty DNA repair can also lead to mtDNA deletion accumulation (Fontana & Gahlon, 2020). In either case, mitochondrial function is impaired due to respiratory defects, highlighting the relevance of mtDNA maintenance pathways such as DNA repair. In addition, mitochondrial fusion and fission contribute to maintain a pool of viable mtDNA molecules, as mitochondrial nucleoids can be exchanged during fusion/fission events. In cases where the repair and tolerance mechanisms are not sufficient, mitophagy, the selective degradation of dysfunctional mitochondria, also contributes to maintaining the mitochondrial pool and, consequently, cellular homeostasis (Carelli et al., 2015). In the following sections, we will review current knowledge on how nutrients and nutritional interventions may affect mtDNA stability through modulation of damage, repair, or signaling pathways.

26.3 Caloric restriction and dietary restriction Caloric restriction (CR) and dietary restriction (Dr) are nutritional interventions widely described in the literature as capable of promoting beneficial physiological responses. CR is a dietary intervention that consists of decreasing, generally between 30%
40% of the calorie intake compared to consumption ad libitum (30% CR means that the animal will consume 70% of calories in relation to what is consumed by the animal that can consume food freely). CR without malnutrition was one of the first interventions shown to increase longevity in animal models. The effect on lifespan was initially demonstrated in rats (McCay et al., 1935) and later confirmed in several species, but not all (Roth & Polotsky, 2012). The protective effect of CR on mtDNA damage accumulation promoted by CR was clearly demonstrated in mice deficient in Ercc5 (component of the nucleotide excision repair pathway), which showed a significant decrease in mtDNA damage when maintained on 30% CR (Vermeij et al., 2016).

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In Dr intervention, instead, the focus is not on reducing total calorie consumption but rather limiting the consumption of specific nutrients such as carbohydrates, lipids, proteins, or amino acids (Piper et al., 2005). Protein restriction (PR) or restriction of the essential amino acid methionine (MetR) proved to be able to decrease the production of mitochondrial ROS, which in turn resulted in the reduction of oxidative damage in mtDNA (Sanz et al., 2006a). On the other hand, similar effects were not observed when lipids or carbohydrates were restricted (Sanz et al., 2006b). Both CR and Dr appear to exert their effects by regulating various nutrient sensing signaling pathways such as insulin/IGF-1 (IIS), mechanistic target of rapamycin (mTOR) and sirtuins. The IIS signaling pathway regulates the metabolism of carbohydrates, proteins, and lipids. Insulin is stimulated mainly by foods rich in carbohydrates, while IGF-1 is stimulated by foods rich in proteins. Both are peptide hormones that bind to their respective receptors and activate a signaling cascade that results in a series of physiological responses related to anabolic processes (Rincon et al., 2004). Decreased stimulation of the IIS pathway by reducing insulin/IGF-1 levels, reduced activity of the IGF-1 receptor (IGF-1R), or decreased activation of downstream factors in the IIS pathway such as insulin receptor substrate 1 (IRS-1) can induce adaptive physiological responses that result in increased longevity in several model organisms (worms, flies, and mice) (Piper et al., 2008). The observation that mtDNA haplotypes modulate insulin sensitivity and glucose metabolism (Sammy et al., 2021) directly links mtDNA stability and the IIS signaling pathway and metabolic diseases. CR and Dr (particularly PR) also decrease plasma insulin/IGF-1 concentrations, leading to reduced IIS signaling and decreased PI3K/AKT kinase activity (Lee and Longo, 2016). As a result, the forkhead box O (FOXO) transcription factor activates a transcriptional stress response program which includes antioxidant defenses, favoring the reduction of damage caused by ROS (Kim et al., 2015), such as the mtDNA damage accumulated with aging. The mTOR is another central protein in nutrient sensing signaling, activated mainly by amino acids and growth factors (Wullschleger et al., 2006). When activated, mTOR positively modulates mRNA translation, ribosome biogenesis, and nutrient metabolism, and negatively regulates autophagy thus favoring cell growth (Sarbassov et al., 2005). Data from the literature data support that mTOR inhibition is an important component of CR/Dr effects on longevity (Emran et al., 2014), mostly through induction of autophagy (Jung et al., 2010). In fact, TORC1, one of two protein complexes regulated by mTOR, directly regulates mitophagy in yeast (Liu & Okamoto, 2018). Although mitophagy deregulation has been proposed to play a role in disease processes (Li et al., 2021), CR/Drinduced mitophagy could contribute to preferentially eliminating damaged mtDNA molecules. Sirtuins are a group of NAD1-dependent deacetylases, highly conserved in eukaryotes, that also respond to nutrients and control cellular metabolism in response to stressful conditions (Vassilopoulos et al., 2011). Several lines of

26.4 Dietary components with the potential to activate the nutrient

evidence show sirtuin activation by CR, particularly SIRT1 and SIRT3, and with CR-induced longevity in mammals (Satoh & Imai, 2014). There is evidence that SIRT1 modulates mitochondrial functions by increasing mitochondrial biogenesis and attenuating oxidative stress (Bordone & Guarente, 2005). In addition, SIRT1 also activates the transcriptional coactivator PGC1-α, which is an important regulatory factor in mitochondrial biogenesis and essential for the maintenance of mtDNA (Gerhart-Hines et al., 2007), such that CR-induced SIRT1 activation results increased in mitochondrial biogenesis, which attenuates the mtDNA depletion observed in old rats (Picca et al., 2013). On the other hand, the mitochondrial sirtuin SIRT3 promotes its effect by direct deacetylation of mitochondrial proteins (He et al., 2012), and the lifespan extension effects of CR are believed to be mediated, at least in part, by SIRT3-mediated SOD2 activation (Qiu et al., 2010). The sirtuin pathway also regulates FOXO activity (Mouchiroud et al., 2013) and, consequently, the antioxidant stress response pathway that could protect mtDNA from oxidant-induced damage (Houtkooper et al., 2012). In addition to packing the mtDNA into nucleoids, TFAM is also required for mtDNA replication and protects the mtDNA from damage and regulates mtDNA repair (Canugovi et al., 2010). Thus, the observation that SIRT1 regulates TFAM expression via PGC1-α (Chandrasekaran et al., 2019) suggests another mechanism by which CR could modulate mtDNA stability. The effects of CR/Dr on DNA repair activities have, so far, been inconclusive (Radak et al., 2013). In particular, the effect of CR on the mitochondrial BER pathway was shown to be tissue-specific, with some tissues showing increased activity and others decreased mtBER activity in CR-mice when compared to ad libitum (Stuart et al., 2004). Thus, one can speculate that the protective effects of CR/Dr on mtDNA result from the combination of several adaptive responses that lead to reduced ROS generation, increased antioxidant defenses, and regulation of repair pathways in conjunction with mitochondrial turnover mechanisms.

26.4 Dietary components with the potential to activate the nutrient sensing pathways Several compounds present in food have been studied for their ability to regulate the nutrient sensing and stress response pathways. Some of those seem to exert effects like those induced by CR/Dr, thereby classifying them as calorie restriction mimetics. (Madeo et al., 2014). Understanding the mechanisms and potential benefits of these compounds may help to devise strategies to get the benefits of CR/Dr without having to undergo restrictive diets that are often unfeasible to practice. We review here some of these compounds, specifically the one with the most robust experimental support. Resveratrol is a polyphenol present in plant sources (red grapes, berries, and nuts) that potentially has several biological effects. Clinical trials suggest that

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resveratrol positively influences biomarkers of neurological disorders, cardiovascular diseases, and diabetes (Berman et al., 2017). Regarding its mitochondrial effect, resveratrol treatment upregulated expression of SIRT1 in cardiomyocytes, resulting in induced expression of several stress response genes, including the mitochondrial isoform of SOD (Mn-SOD), resulting in increased mitochondrial biogenesis and lower oxidative stress (Li et al., 2013). In addition, resveratrol was also shown to promote mitochondrial biogenesis and maintain mtDNA levels in hippocampal neurons in a seizure model via PGC1-α (Chuang et al., 2019), suggesting that it helps to prevent mtDNA damage accumulation. Curcumin is a polyphenolic compound found in turmeric, which has been suggested to regulate multiple signaling pathways that result in beneficial health responses to inflammation and metabolic dysfunction (Hewlings and Kalman, 2017). Its main mechanism of action seems to be as an epigenetic modulator, regulating DNA methyltransferases, histone acetyltransferases, histone deacetylases, and microRNAs (Hassan et al., 2019). The protective potential that curcumin confers to mtDNA was evidenced in a study showing that supplementation of curcumin in broilers reduced ROS production, increased Mn-SOD activity, mtDNA copy number, and stimulated the mitochondrial thioredoxin system (Zhang et al., 2018). Observational data have suggested that the consumption of green tea (Camellia sinensis L.) has a protective effect under several pathological conditions such as cancer, heart disease, liver disease, type 2 diabetes, obesity and metabolic syndrome (Chacko et al., 2010), all conditions that have been associated with mitochondrial dysfunction and mtDNA instability (reviewed in Wallace, 2018). In a study with rats where liver mtDNA damage and mitochondrial redox imbalance was induced by alcohol consumption, supplementation with green tea extract restored normal levels of antioxidant defense enzymes and glutathione content and reduced oxidative damage and deletions in mtDNA (Reddyvari, et al., 2017). In addition, several studies have shown that epigallocatechin gallate (EGCG), the most abundant flavonoid found in green tea, has antioxidant and antiinflammatory activities. These properties are considered responsible for the benefits attributed to the consumption of green tea in pathologies related to metabolic syndrome (Legeay et al., 2015). Supplementation with EGCG in mice submitted to a high-fat diet lessened body weight gain and lipid content in the plasma and liver. In addition, mice supplemented with EGCG exhibited higher body temperature and mtDNA content in brown adipose tissue (BAT). These results suggest that EGCG may modulate the activity of adenosine monophosphate-activated protein kinase (AMPK), resulting in increased expression of genes related to thermogenesis and mitochondrial biogenesis in BAT (Lee et al., 2017). Spermidine is a natural polyamine found in large quantities in some important diet components like soybeans and wheatgerm. Studies suggest that spermidine is a strong inducer of autophagy and has beneficial effects on the cardiovascular system, neuroprotective effects, and antitumorigenic properties (Madeo et al., 2018).

26.5 Impact of high-fat diets on mitochondrial DNA

These effects have been partially attributed to spermidine-induced activation of the PINK/PARKIN pathway that leads to an increase in mitophagy and clearance of dysfunctional mitochondria (Qi et al., 2016). In addition, spermidine appears to exert its effects via the activation of the SIRT1/PGC-1α/NRF-1 and NRF-2 signaling axis, as spermidine supplementation in senescent cardiomyocytes previously treated with H2O2 increased the expression of SIRT1, PGC-1α, NRF-1, NRF-2, and TFAM, and decreased ROS production resulting in improved OXPHOS (Wang et al., 2020).

26.5 Impact of high-fat diets on mitochondrial DNA Diets high in fat (HFD) and the Western diet (WD), which consists of a diet rich in fats and sugars, are widely studied due to their deleterious effects that include promoting obesity, heart disease, hypertension, type 2 diabetes, autoimmune disease, and some types of cancer (Moszak et al., 2020). In mice fed either HFD or WD, mitochondrial dysfunction preceded liver steatosis, and this dysfunction was associated with impaired mtDNA copy number and replication and altered expression of both mitochondrial and nuclear OXPHOS genes (Malik et al., 2019). This study suggested that HFD inhibited the turnover mechanisms of damaged mtDNA copies, which could explain the increase in mtDNA copy number associated with decreased expression of mitochondrial genes. In support of this hypothesis, Chen et al. showed that rats fed HFD long-term have fewer copies of mtDNA in myocardial tissue and dysregulated expression of genes related to mitochondrial fission and fusion (Chen et al., 2018), which are essential for eliminating dysfunctional mitochondria. There is evidence that defects in mtDNA repair can contribute to the harmful effects of HFD. NEIL2/2 mice, deficient in the Nei-like DNA glycosylase 1which participates in the nuclear and mitochondrial base excision repair, submitted to HFD, gain more weight and accumulate more body and liver fat when compared to NEIL1/1 mice. In addition, the NEIL2/2 mice had reduced oxygen consumption, increased expression of proinflammatory genes, reduced mtDNA and mitochondria proteins, directly implicating mitochondria in the pathogenic mechanism (Sampath et al., 2011). In addition to the amount, the types of dietary lipids can also differently affect mitochondrial functions. A study comparing the consumption of monounsaturated fatty acids (MUFAs) with that of n-6 polyunsaturated fatty acids (PUFAs) in mice showed that the MUFA enriched diet decreased lipoperoxidation markers and prevented the mtDNA deletion accumulation in old animals (Ochoa et al., 2011), suggesting that consumption of MUFAs, like those found in virgin olive oil, seems to have a mitochondrial protective effect when compared to the consumption of n-6 PUFAs, like those found in sunflower oil. Accordingly, the Mediterranean diet, a diet rich in virgin olive oil, has been associated with

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decreased risk of cardiovascular disease, type 2 diabetes, some types of cancer, and cognitive disorders (Schwingshackl et al., 2020). Although still controversial, the ketogenic diet (KD), a diet rich in fats and very restrictive in carbohydrates, has been shown to exert some beneficial metabolic effects (Caprio et al., 2019). In vitro studies showed that an heteroplasmic cell line, containing a mixture of wild-type mtDNA and deleted mtDNA molecules, maintained in a ketogenic culture medium selectively increases wild-type mtDNA levels, from 13% to approximately 22%. This shift in wt/deleted mtDNA ratio was accompanied by an increase in the expression of mitochondrial proteins (Santra et al., 2004). KD has also been used for many decades in the treatment of epilepsy, but its mechanism of action in controlling the clinical manifestations of the disease is not well understood. (Paoli et al., 2013). Interestingly, rats fed KD showed a twofold increase in glutathione levels in hippocampal mitochondria, with reduced mitochondrial H2O2 production and mtDNA oxidative damage (Jarrett et al., 2008), suggesting that the therapeutic effect of KD may be due, at least in part, to regulation of mitochondrial antioxidant defenses and lower mtDNA damage.

26.6 Fructose and ethanol as potential metabolic toxins Fructose is a monosaccharide component of sucrose, the disaccharide that makes up most of the sugar consumed in the world. There is evidence suggesting that excessive fructose consumption is responsible for part of the metabolic disorders related to WD (Stanhope et al., 2013). Interestingly, high-fructose diet fed rats showed mtDNA damage accumulation, mtDNA depletion and a reduction in the expression of genes related to mitochondrial biogenesis in the liver (Cioffi et al., 2017), suggesting that mtDNA instability is a causally linked to the metabolic disorders induced by high fructose consumption. Considering the extremely high average sucrose consumption in the world, 20 kg per capita/year (Organization for Economic Co-operation and Development (OECD) Library, 2022), it is reasonable to consider that fructose-induced mtDNA damage may contribute to sugar-related health problems. Alcohol (ethanol) is also an important dietary component widely consumed. Average alcohol consumption was estimated to be 6.2 liters of pure alcohol per person 15 years and older in 2018, and unrecorded consumption may account for up to 26% of total consumption (World Health Organization (WHO), 2022). Although significant amounts of data in the literature suggest that moderate alcohol consumption can promote beneficial health effects, this hypothesis is still under debated. On the other hand, there is a consensus that excessive alcohol consumption promotes disease, and several studies have linked that to metabolic and mitochondrial dysfunction (Zhong & Lemasters, 2018). In animal models, alcohol consumption causes damage accumulation and depletion of mtDNA, decreases

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26.7 Conclusion Mitochondria are central cellular sites for energy and anaplerotic metabolism. Mitochondrial function is essential to cellular homeostasis and mitochondrial dysfunction is a common feature to several and seemingly diverse pathologies like cancer, metabolic diseases, neurodegeneration, and aging. Here we reviewed how different dietary components may directly, or indirectly via alterations in signaling pathways, affect mtDNA stability and, consequently, mitochondrial functions. As mitochondria are also at the center of the cellular redox homeostasis, it is not surprising that the nutritional modulations that seem to have the most influence on mtDNA act through alterations in the redox balance, either by regulating mitochondrial oxidant production or the mitochondrial antioxidant defenses. Although there is still a lot to be learned about the mechanisms responsible for diet regulation of mtDNA stability, it is clear that nutritional manipulations are an important strategy to promote mitochondrial function and to prevent and treat diseases.

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Index Note: Page numbers followed by “f” and “t” refers to figures and tables respectively.

A Acetate, 244 Acetoacetate (AcAc), 640
641 Acetyl-coenzyme A (acetyl-CoA), 73, 79 carboxylase, 171
172 pool, 76 reaction, 81
82 Acetylation reaction, 81
82 Activated transcription factor 5 (ATF5), 471 Activating transcription factor 4 (ATF4), 611 Active DNA demethylation, 72
73 Active protein-1 (AP-1), 464
465 Adenosine monophosphate-activated protein kinase (AMPK), 656 50 -adenosine monophosphate-activated protein kinase (AMPK), 27 Adenosine triphosphate (ATP), 3, 226
227, 383, 461
462, 502
504, 522 ATP-dependent proteases function, 563 synthesis via oxygen-dependent pathways, 214
215 Adherent cells evaluation with crystal violet staining assay, 200
201 Adipocytes adipocyte-specific functions of mitochondria, 401
404 characteristics, 514t types of, 514 Adipose tissue, 514, 597 creatine metabolism in, 402
403 Adipose triglyceride lipase (ATGL), 515
516 Adrenodoxin (ADX), 467
468 Adrenodoxin reductase (ADxR), 467
468 Adsorption Distribution Metabolism Excretion (ADME), 45 Adult mitochondrial diseases patients, nutritional assessment in, 96
98 Advanced glycation end products (AGE products), 40, 541
542 Aerobic capacity, 214
215 Age-related Macular Degeneration (AMD), 36 Aging, 131
132, 589
590 mitochondria, reactive oxygen species and free radical theory of aging, 133
135 mitochondria and “inflammaging”, 142
144 mitochondrial dynamics, mitophagy and aging, 136
140 mitocondrial DNA and aging, 135
136

necessity for alternative theory, 149
151 gradual ROS response hypothesis, 151 ROS signaling, aging, and lifespan, 150
151 retrograde signaling, 140
142 theory, 139
140 vitamin E, mitochondria, and aging, 147
149 Alcohol consumption, 658
659 Alpha-ketoglutarate (α-KG), 168, 182 α-carotene, 35
36 α-isoform of tocopherols (α-T), 144
145 α-ketoglutarate, 76 α-linolenic acid (ALA), 34, 213
214 α-lipoic acid, 32
33, 51 α-tocopherol, 46
51 α-tocotrienol, 529 Alternative oxidases (AOXs), 105, 107
108 Alzheimer’s disease (AD), 4, 195
197, 227, 270, 395
396, 522, 526, 562 Amino acids (AA), 383, 590
591 5-aminoimidazole-4-carboxamide-1-D-ribofuranoside (AICAR), 610 AMP-activated protein kinase (AMPK), 304
305, 351, 484
485, 506
507, 562
563, 587
589 activation and mitochondria, 594 agonists, 610
611 Amyloid beta (Aβ), 577
578 Amyloid lateral sclerosis (ALS), 397 Amyotrophic lateral sclerosis, 398
399 Anthocyanidins, 316 Anthocyanins, 343 Antioxidant activity of CoQ, 258 Antioxidant response element (ARE), 27, 476 Antioxidants (AOX), 3, 82, 132 defense in biological systems, 535
537 system, 350, 461
462, 537 AO enzyme, 539
540, 546
547 Apoptosis in cardiomyocytes, 308
309 Arabidopsis thaliana, 112 Arachidonic acid (AA), 213
214 Areacyl-CoA dehydrogenases (ACAD), 467
468 Arginines, 183 clinical study of L-arginine in MELAS, 421
422 contraindication in treatment of MELAS, 425
426 endothelial dysfunction in MELAS, 419 mitochondrial angiopathy in MELAS, 418
419

667

668

Index

Arginines (Continued) neuroimaging of stroke-like episodes in MELAS, 420
421 superacute intervention by L-arginine, 422 plasma L-arginine levels at SLE in clinical trial, 423f therapeutic regimen of L-arginine for MELAS, 422
425 Ascorbate, 225
226 Ascorbic acid, 225
226, 234
235 Asisobutyric acid, 244 Aspergillus niger, 108
109 Associated adenovirus (AAVs), 115 Asymmetrical dimethyl-arginine (ADMA), 419 Ataxia telangiectasia mutated-dependent activation (ATM-dependent activation), 608
609 Atherosclerosis, 306 Athletes cardiac and skeletal muscles of, 389
393 creatine monohydrate on cardiac muscle mitochondria, 393 on skeletal muscle mitochondria, 390
392 Autism Spectrum Disorders (ASD), 247 Autophagy, 140, 369 mitochondrial enzymes in, 309
310 Autophagy-related proteins (ATG), 309 Aza-stilbenes (AZA-ST), 197
198 classification of polyphenols, 196f flow cytometric measurement of mitochondrial ROS production with MitoSOX-Red, 201 quantification of cells with depolarized mitochondria with DiOC6(3), 201 material and methods, 198
202 cell culture and treatments, 198
199 evaluation of adherent cells with crystal violet staining assay, 200
201 measurement of cell viability with fluorescein diacetate assay, 200 statistical analysis, 202 synthesis of aza-stilbenes I to VII, 198, 199t results, 202
206

B B cell lymphoma 2 (BCL-2), 308 B vitamins, 30
31 deficiencies, 167
168 and mitochondrial metabolism, 168
173 vitamin B1, 168 vitamin B2, 168
169 vitamin B3, 170 vitamin B5, 170
171 vitamin B6, 171

vitamin B8/B7, 171
172 vitamin B11/B9, 172 vitamin B12, 173 mitochondrial signaling metabolites, 181
185 B vitamin impacts on methylation of histone and DNA, 182
183 regulator of histone acetylation, 184
185 vitamins and HIF1 signaling, 181
182 oxidative stress and mitochondrial toxicity, 173
178 role as mitochondrial nutrients, 178
181 B-hydroxybutyrate (bOHB), 640
641 Bacterial endotoxins, 319 Bcl-2 associated X protein (Bax), 39
40, 546
547 Beige adipogenesis, 516
517 Benzoic acid, 364
365 Beta-3-adrenergic receptor (ADRB3), 514 β-amyloid proteins, 197 β-carotene, 35
36, 51 β-catenin/Wnt, 464
465 BGP-15, 279 Bioenergetics of mitochondria, 334
337 Biogenesis, 31 Biotin. See Vitamin B7 Blood-brain barrier (BBB), 45, 246, 385 BMI, 95 Body composition, 94, 94f, 97 Borreria hispida, 322 Brain, 599 disorders, 246 SCFAs influencing appetitive function on mitochondria in, 248
249 influencing cognitive and psychological function on mitochondria in, 247
248 Brain-derived neurotrophic factor (BDNF), 41
42 Branched-chain amino acids (BCAAs), 173, 515
516 Brown adipocytes (BAT), 402, 514 Brown adipose tissue (BAT), 514, 656 “Browning” process, 402 Buckwheat trypsin inhibitors (BTIs), 561
562 health benefits and presence of trypsin inhibitors, 567
570 characteristics and physiological roles, 568
570 food staple in some regions and global presence as functional food, 567 potential health benefits from consuming buckwheat foods, 567
568 roles of mitochondrial homeostasis in healthy aging, 570
574

Index

and improvement by presence of recombinant, 570
580 potential future trends in research and studies, 580 recombinant BTIs, 574
580 roles of mitochondrial proteases, 562
567 Butyrate, 244 Bypasses complex I-deficiency, 260
261

C C oxidase (COX), 418
419 c-reactive protein (CRP), 478
479 C-type lectin receptors (CLRs), 470
471 Cadmium, 526
527 Caenorhabditis elegans, 27, 522
523, 571, 590 Caloric restriction (CR), 117, 587, 633, 653 mitochondrial pathways induced by, 587
601 AMP-activated protein kinase activation, and mitochondria, 594 evolutionary conserved mammalian energysensing pathways, 588f inhibition of insulin/IGF-1signaling IGF1pathway, and mitochondria, 589
590 inhibition of target of rapamycin signaling, and mitochondria, 590
592 mitochondria-mediated tissue-specific effects of, 597
600 and mitochondrial signaling to cell, 595
597 PGC-1α activation, and mitochondria, 595 sirtuin activation, and mitochondria, 592
594 Camellia sinensis, 344, 656 cAMP-response element binding protein (CREB), 39
40 Cancer, 131
132 therapy, 364 Carbohydrates, 526
527 Carbon dioxide (CO2), 171
172 Cardiac muscle mitochondria, creatine monohydrate on, 393 Cardiac-specific Klf4 deficiency, 571
572 Cardiolipin (CL), 270, 465 Cardiometabolic diseases, 333
334. See also Mitochondrial diseases cytoprotective actions of green tea polyphenols, 346
347 mitochondria and metabolic stress, 339
340 and role in metabolism, 337
339 mitochondrial fission and fusion, 340
341 molecular mechanisms of action of tea polyphenols, 350
353 molecular mechanisms of flavonoids in, 349
350

nutraceuticals effects on, 348
349 polyphenols as functional food, 341
343 structure and bioenergetics of mitochondria, 334
337 tea and health benefits, 344
346 Cardiomyocytes, 571
572 apoptosis, 545 mitochondrial enzymes, 306
310 for apoptosis in, 308
309 Cardioprotection, biological action of flavonoids in, 316
323, 317t Cardioprotective activity, structure activity relationship of flavonoids for, 314
316 Cardiovascular damage, 306 Cardiovascular diseases (CVD), 4, 26, 79
80, 266, 333, 347, 363, 522 therapeutic potential of MitoQ in treatment of, 275
276 Cardiovascular disorders, 131
132 Cardiovascular health, mitochondria as essential organelle for, 305
306 Cardiovascular ischemia-reperfusion injury, nanoquercetin effects in, 367
368 Cardiovascular risks, 333 Cardiovascular system, 334, 656
657 Carnitine, 33
34 Carotenoids, 35
36 Caspase-independent cell death (CICD), 467 Catabolism, 386 Catechin, 315, 334, 345
346 Cationic trypsinogen gene, 561
562 Cells, 464
465 culture and treatments, aza-stilbenes, 198
199 culture model, 529 with depolarized mitochondria with DiOC6(3), 201 measurement of cell viability with fluorescein diacetate assay, 200 phosphocreatine “shuttle” system in cell energy homeostasis, 389 signaling pathways, 350
351 Cellular antioxidant enzymes, 173
175 Cellular apoptosis, 369
370 Cellular bioenergetics, creatine in, 387
389 Cellular metabolism, 303 Cellulose, 526
527 Central nervous system (CNS), 243, 395 creatine and central nervous system mitochondria, 395
401 creatine, mitochondrial bioenergetics, and neurodegenerative disorders, 397
398 creatine, neuronal mitochondrial dysfunction, and amyotrophic lateral sclerosis, 398
399

669

670

Index

Central nervous system (CNS) (Continued) creatine, neuronal mitochondrial dysfunction, and multiple sclerosis, 399
400 creatine treatment and mitochondria, 400
401 devoted energy provider for neuronal mitochondria, 395
397 gut microbiota and SCFAs, 243
244 SCFAs effects on modulating CNS function, 246
249 regulating peripheral organizational activities, 244
245 Chemokines, 461
462 Chitosan (CS), 375 Chromatin remodeling, 73 Chronic hepatitis C (HCV), 268
269 Ciona AOX, 109
110, 112 Ciona intestinalis, 109
110, 113 Cobalamin. See Vitamin B12 Coenzyme A (CoA), 170 Coenzyme Q importance of coenzyme Q in mitochondria, 256
257 structure of, 258
260 Coenzyme Q-cytochrome c (cyt c), 5 Coenzyme Q10 (CoQ10), 5, 46, 235 prevents oxidative damage, 257
258 Colored rice bran, 528 Complex V (CV), 109
110 Congestive heart failure (CHF), 230
231 Core OXPHOS system, 105 CR mimetics (CRMs), 604
605 Creatine, 33 creatine/CK system, 394 mitochondrial bioenergetics, and neurodegenerative disorders, 397
398 neuronal mitochondrial dysfunction, and amyotrophic lateral sclerosis, 398
399 neuronal mitochondrial dysfunction, and multiple sclerosis, 399
400 treatment and mitochondria, 400
401 Creatine (Cr), 383 Creatine kinase (CK), 386 isoenzymes, 388
389 Creatine monohydrate, 383
386 on cardiac muscle mitochondria, 393 catabolism, 386 in cellular and mitochondrial bioenergetics, 387
389 creatine kinase isoenzymes, 388
389 phosphocreatine “shuttle” system in cell energy homeostasis, 389 creatine/mitochondrial creatine kinase system in health and disease, 389
404

and adipocyte-specific functions of mitochondria, 401
404 cardiac and skeletal muscles of athletes, 389
393 and central nervous system mitochondria, 395
401 in muscle disorders, 393
394 pregnancy and gestation, 394
395 de novo synthesis of, 383
385 on skeletal muscle mitochondria, 390
392 structure, 383 supplementation form, 385 tissue distribution of, 385
386 Creatine transporter (CRT), 383 Creatinine (Crn), 385 Crohn’s disease, 527
528, 564
565 Crystal violet assay, 197 Crystal violet staining assay evaluation of adherent cells with, 200
201 Curcuma longa (turmeric), 41, 462
463, 484
485, 607 Curcuma spp, 462
463 Curcumin, 462
465, 475
476, 607, 656 activating Nrf2 signaling pathway and protecting mitochondrial damage and oxidant generation, 482
484 as antioxidant and antiinflammatory agent, 475
479 as direct mitochondrial reactive oxygen species scavenger, 480
482 enhancing mitochondrial antioxidants, 482 inflammation and oxidative stress, 464
465 mitochondria and inflammation, 465
467 and oxidative stress, 467
469 mitochondrial inflammation and oxidative stress in inflammatory-related diseases, 470
475 mitochondrial targeting for reduction of oxidative stress and inflammation, 479
480 targeting of mitochondrial p66shc by, 487 targeting of mitochondrial sirtuins by, 485
486 targeting of mitochondrial uncoupling proteins by, 484
485 Curcuminoids, 41
42 Cyanamide, 385 Cyclic GMP
AMP synthase (cGAS), 142
144, 465
466 Cyclic guanosine monophosphate
adenosine monophosphate (cGAMP), 465
466 Cyclopia subternata, 318 Cymbopogon citratus (DC), 320 Cysteine, 526 Cytochrome C (Cyt c), 133
134, 304
305, 309

Index

Cytochrome P450 (CYP), 146 Cytokines, 461
462, 464
465

D Damage-associated molecular patterns (DAMPs), 142
144, 465 Dauer formation abnormal-16 (DAF-16), 571 Dehydroascorbic acid (DHA), 29 Dehydrogenase complex (OGDH complex), 168 Delayed on-set muscle soreness (DOMS), 232
233 Deoxythymidine (dT), 172 Deoxythymidine monophosphate (dTMP), 172 Diabetes, 131
132, 274
275 Diet, 529 modulates epigenome, 78
80 Diet-related metabolic connections between mitochondria and cytoplasm to affect epigenome, 80
82 acetyl-coA and acetylation reactions, 81
82 antioxidants, 82 methyl donors, one-carbon cycle and methylation reactions, 80
81 Dietary and complementary interventions with hydrogen, 505 Dietary fatty acids, 34
35 omega-3 polyunsaturated fatty acids, 34
35 Dietary fibers, 243
244 Dietary flavonoids flavonoids and bioactivity, 312t structure and function, 310
311 Dietary interventions, mitochondrial disease, 99 Dietary modulation caloric restriction and dietary restriction, 653
655 dietary components with potential to activate nutrient sensing pathways, 655
657 fructose and ethanol as potential metabolic toxins, 658
659 Dietary restriction (Dr), 587, 653 caloric restriction and, 653
655 Dietary sources of molecular hydrogen, 501
502 Dietary Supplements Health and Education Act, 383 Diets high in fat (HFD), 657 Diferuloylmethane, 462
463 Diffuse large B-cell lymphoma (DLBCL), 445 3,30 -dihexyloxacarbocyanine iodide (DiOC6(3)), 197, 201 cells with depolarized mitochondria with, 201 Dihydrogen (H2), 501 as innovative nutraceutical for mitochondrial viability

dietary and complementary interventions with hydrogen, 505 dietary sources of molecular hydrogen, 501
502 hydrogen-rich water and mitochondrial function, 502
504 and mitochondria, 506
507 Diiron core, 107 Dilated cardiomyopathy, 306 Dimethyl sulfoxide (DMSO), 198 Dimyristoyl phosphatidylcholine (DMPC), 375 Dinitrophenol (DNP), 612
613 Diosmin, 320 Direct inhibitor of apoptosis-binding protein with low pI (DIABLO), 465
466 Direct mitochondrial reactive oxygen species scavenger, curcumin as, 480
482 Disease, role of mitochondrial function in, 522 DMD long-term IDE study (DELOS), 266 DNA methylation, 72
73 DNA methyltransferases (DNMTs), 72
73 Docosahexaenoicacid (DHA), 213
214 Dodecyltriphenylphosphonium (C12TPP), 612
613 Dopamine (DA), 246 Dopaminergic neurons (DA neurons), 27 Double-stranded DNA (dsDNA), 142
144 Drosophila, 117
118, 591 Drosophila melanogaster, 112
113, 526 Duchenne muscular dystrophy (DMD), 266 Dynamin 1-like protein (Drp1), 572
573 Dynamin 2 (Dnm-2), 39
40 Dynamin-related protein 1 (Drp1), 136
137, 215
216, 524, 542
543 Dysfunctional mitochondria, 523
524

E 4E-binding protein (4E-BP), 590
591 Eicosapentaenoic acid (EPA), 213
214 Electron transferring flavoprotein/ETF:Q oxidoreductase (ETF/ETF:QOR), 467
468 Electron transport chain (ETC), 76
77, 229
230 Encephalopathy, 417 Endogenous antioxidants, 31
33 glutathione, 31
32 lipoic acid, 32
33 NAC, 32 Endogenous metabolites and transporters, 33
34 carnitine, 33
34 creatine, 33 Endoplasmic reticulum (ER), 306, 465, 542
543, 591

671

672

Index

Endoplasmic/sarcoplasmic reticulum (ER/SR), 216
217 Endothelial cell (EC), 230
231 Endothelial dysfunction, 528
529 in MELAS, 419 Endothelial NO synthase (eNOS), 419 Energy homeostasis, importance of uncoupling protein 1 in regulating, 515
516 Energy metabolism, 178, 433 Energy requirements, 96
97 Enzymatic antioxidants, 461
462 Enzyme-treated rice fiber, 527 Epicatechin, 315, 607
608 Epicatechin-3-gallate (ECG), 345
346 Epigallocatechin (EGC), 345
346 Epigallocatechin-3-gallate (EGCG), 40, 315, 345
346, 605, 656 Epigenetic modifications, 72
74, 78
79 DNA methylation, 72
73 histone modifications and chromatin remodeling, 73 noncoding RNA, 73
74 Epigenome diet impact on, 78
84 diet modulates epigenome, 78
80 focus on diet-related metabolic connections between mitochondria and cytoplasm, 80
82 nutrients and diet effects on mitochondrial epigenetics and mito-epigenetics, 80
81 Estrogen receptor α (Erα), 546
547 Ethanol as potential metabolic toxins, fructose and, 658
659 Eukaryotic cells, 651 European Medicine Agency (EMA), 26 European Society of Clinical Nutrition and Metabolism (ESPEN), 98 Evolutionarily conserved signaling intermediate in Toll pathways (ECSIT), 470
471 Exercise, 229
230 vitamin C and, 231
234

F 18

F-fluoro-2-deoxy-d-glucose positron emission tomography computed tomography (18FFDG-PET), 515
516 18 F-fluoro-2-deoxy-d-glucose (18F-FDG), 515
516 FAD synthase (FADS), 169 Fats, 525
526 Fatty acid b-oxidation (FAO), 639
640 Fatty acids (FAs), 31, 213, 541 Fermentable component (FCs), 248
249 Ferredoxin reductase (FDxR), 467
468

Fetal bovine serum (FBS), 198 Fiber, 527 Fibroblast growth factor-23 (FGF23), 445 Fibroblast growth factor21 (FGF21), 506
507, 597 Fission 1 (Fis1), 136
137, 524 Fission process, 215
216 Flavan-3-ols, 321 Flavanones, 315
316 Flavin adenine dinucleotide (FAD), 30, 168
169 Flavin mononucleotide (FMN), 30 Flavoenzymes, 178
179 Flavones, 315
316, 522
523 Flavonoids, 39
40, 51
53, 195
197, 303, 310
311, 322, 343 biological action of flavonoids in cardioprotection, 316
323, 317t antiatherogenic activity, 321 antihypertensive activity, 320
321 anti-inflammatory activity, 319
320 antioxidant activity, 318
319 antiplatelet activity, 318 hypoxia, necrotic and apoptotic activity, 321
323 mitophagy, 323 flavonoid-rich diet, 321 flavonoid-rich plant foods, 311 molecular mechanisms of flavonoids in cardiometabolic diseases, 349
350 structure activity relationship of flavonoids for cardioprotective activity, 314
316 Flavonols, 315 Flow-mediated dilation (FMD), 275
276 Fluorescein diacetate assay (FDA assay), 197, 202 measurement of cell viability with, 200 Fluorescent photoactivated protein technology, 217
218 Folate deficiency, 177
178, 182
183 Folic acid, 177 Food, 651 choice, 562 cook methods, 564
565 intake, 95 mitochondria-specific enzyme mimetics from, 528
529 packaging process, 501 Forkhead box O transcription factor (FOXO transcription factor), 654 Forkhead box protein O3a (FoxO3a), 506
507 Forkhead transcription factor (FOXO1), 39
40 Frataxin, 323 Free fatty acid receptors (FFARs), 246 Free radical theory of aging, 131
135, 255 Free radicals, 476
478, 480
482

Index

Friedreich’s ataxia, 4, 263 therapeutic use of idebenone in, 264
265 Fructose as potential metabolic toxins, 658
659 Fucoxantin, 516
517 Fusion, 215
216, 335
337

G G-protein coupled receptors (GPRs), 243, 641
642 Gallic acid (GA), 608 Gallocatechin, 315 Gallocatechin gallate (GCG), 345
346 γ-carboxyethylhydroxychroman, 529 γ-glutamyl cysteine ligase catalytic (GCLC), 482
484 Gastric ulcers, nanoquercetin effects in prevention of, 368 Gastro intestinal problems, 95 Gene expression, 71 Gene mutations, 561
562 Genistein, 316, 322 Ginsenosides, 36
37 GLIM criteria, 98 Glucose, 245 Glucose-6-phosphate (G6P), 351 Glucose-6-phosphate dehydrogenase (G6PD), 482
484 Glutaryl-CoA, 79 Glutathione (GSH), 5
26, 75
76, 169, 179
180, 306
308, 462
463, 539 peroxidase-1, 467
468 Glutathione, 31
32 Glutathione peroxidase (GPx), 46, 173
175, 255
256 Glutathione reductase (GR), 32, 169, 306
308, 482 Glutathione S-transferase (GST), 350 Glycine, 75
76 Glycitein, 316 Glycogen synthase kinase-3 (GSK3), 41
42 Green tea polypenols, 334 cytoprotective actions of, 346
347 Growth hormone (GH), 589
590 Guanidinoacetic acid (GAA), 384
385 Gum arabic, 501
502 Gut microbiota, 243
244 gut microbiota-derived B vitamins, 167 Gut-brain axis, 243

H Heart and cardiovascular system, 599
600 failure, 276, 306

Heat shock protein, 60 (HSP60), 177 HEK293T cells, 110 Heliotropium taltalense, 320 Heme oxygenase-1 (HO-1), 350, 476, 482
484 Hemicellulose, 526
527 Hepatic cancer cells, 578 Hesperidin, 320 Hexokinase II (HK2), 351, 467
468 HIF1 signaling, vitamins and, 181
182 High fat diet-induced glucose metabolic disorders (HFD-induced glucose metabolic disorders), 545 High-fat diets on mitochondrial DNA, impact of, 657
658 Histidines, 183 Histone acetylation, regulator of, 184
185 Histone acetyltransferases (HATs), 73, 184 Histone and DNA, B vitamin impacts on methylation of, 182
183 Histone deacetylases (HDACs), 73, 640
641 Histone methyltransferases, 73 Histone modifications, 73 Histone phosphoriylases, 73 Histone tails, 73 Histonedeacetylase (HDAC), 243 Homeostatic mechanism, 523 Hormesis, 232 Human diseases, 131
132 Human endothelial cells (HUVECs), 138 Human muscle cells, 218
219 Humanin (HN), 596 Huntington’s disease (HD), 4, 267, 595 Hydrogen peroxide (H2O2), 134
135, 306
308, 464
465, 467
468, 476
478 Hydrogen-enriched water, 501
502 Hydrogen-infused water, 501
502 Hydrogen-rich water (HRW), 501
502 interventional studies using dietary dihydrogen for mitochondrial performance, 503t and mitochondrial function, 502
504 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL), 278
279 Hydroxybenzoic acids, 38 Hydroxycinnamic acids, 38 8-hydroxydeoxyguanosine (8-OHdG), 177
178 Hydroxyl free radical, 464
465 Hydroxyl radical (•OH), 134
135, 476
478 Hydroxylated flavonoids, 319
320 5-hydroxymethylcytosine (5-hmc), 72
73 4-hydroxynonenal (4-HNE), 151 Hypertension, 306, 351 Hypertrophic cardiomyopathy (HCM), 306 Hypochlorous acid, 464
465 Hypoxia-Inducible Factor 1 (HIF1), 181

673

674

Index

Hypoxia-inducible factor-1α (HIF-1α), 150
151, 351, 464
465, 592

I Idebenone (IDE), 258
259 reduces ROS levels, 260
261 therapeutic use of, 264
267 in Friedreich ataxia, 264
265 of Leber hereditary optic neuropathy and neuropathic diseases, 265
266 therapeutic use of idebenone in oxidativedamage related diseases, 266
267 IKβ kinase (IKK), 369
370 Immune response, MitoQ use in, 267
269 In vitro evidence, 539
543 model systems, 542 In vivo evidence, 543
546 Inducible nitric oxide synthase (iNOS), 461
462 Inflammaging process, 142
144 Inflammation, 461
464 curcumin, 464
465 mitochondria and, 465
467 mitochondrial targeting for reduction of, 479
480 Inflammation response, MitoQ use in, 267
269 Inflammatory bowel diseases (IBDs), 522, 564
565 Inflammatory cytokines, 319 Inflammatory responses, 464
465 Inflammatory-related diseases, oxidative stress in, 470
475 Inhibition of insulin/IGF-1, 589
590 of target of rapamycin signaling, and mitochondria, 590
592 Inner mitochondrial membrane (IMM), 5, 465
466 Insulin receptor substrate 1 (IRS-1), 654 Insulin-like growth factor-1 (IGF-1), 587
589 pathway, and mitochondria, 589
590 Insulin/IGF-1 (IIS), 654 Integrated antioxidant defense system, 535
537 Interferon (IFN), 269 Interferon regulatory factor 3 (IRF3), 465
466 Interleukin-18 (IL-18), 464
465 Interleukin-1β (IL-1β), 464
465 Intermembrane space (IMS), 564 Intermittent fasting (IF), 633 International cooperative ataxic rating scale (ICARS), 264
265 Intervertebral disk (IVD), 4 Inulin, 501
502

Iron-sulfur (Fe-S), 171 Ischemia-reperfusion injury, 306 Ischemia/infarction disorder, 393
394 Ischemia/reperfusion and organ transplantation, MitoQ, 273 Isocitrate dehydrogenase 1 (IDH1), 482
484 Isoflavones, 316 Isothiocyanates, 42
43 SFN, 42
43 Isovaleris acid, 244

J Jumonji Cdomain-containing protein (JMJDs), 76

K Kaempferol, 315 Kawasaki disease (KD), 272 Kelch-like ECH-associated protein 1 (Keap1), 36, 476 Ketogenic diet (KD), 658 Kidney dysfunction, 275 Krebs cycle, 75, 226
227, 339, 372
374 Kru¨pple like factor-2 expression, 314
315

L L-arginine, 384
385 clinical study of L-arginine in MELAS, 421
422 L-arginine for MELAS, 422
425 L-ascorbic acid (AA), 225 L-Carnitine, 33
34, 44 Lactase phlorizin hydrolase (LPH), 311
314 Lactobacillus casei cell wall extract (LCWE), 272 Leber Hereditary Optic Neuropathy (LHON), 264 idebenone treatment of, 265
266 Leigh syndrome (LS), 266 Lifespan ROS signaling, aging and, 150
151 Linoleic acid (LA), 34, 213
214 Lipoic acid, 32
33 Lipopolysaccharide (LPS), 245 Lipoxygenase/cyclooxygenase, 462
463 Live kinase B1-mediatedactivation (LKB1mediatedactivation), 610
611 Liver, 598
599 fibrosis, 273 Long noncoding RNAs (lncRNAs), 73
74 Low-density lipoproteins (LDLs), 257 Low-density-lipoprotein cholesterol (LDL-C), 440
445 Lycopene, 35
36 Lysines, 183 Lysosomes, 140
141

Index

M Macro-nutrients, 651 Macroautophagy, 637
638 Macrophages, 464
465 Magnetic resonance spectroscopy (MRS), 420
421, 448 Magnetite nanoparticles (MNPs), 374
375 Malic enzyme 1 (ME1), 482
484 Malignant neoplastic disease, 363 Malnutrition, 93 prevalence of malnutrition in mitochondrial diseases, 95
96 Malonyl-CoA, 79 Mammalian cells creatine de novo synthesis in, 384
385 drosophila melanogaster, 112
113 models, 109
112 TORC1 inhibition, 591 Mammals AMP-activated protein kinase agonists, 610
611 effects of CR mimetics on nutrient-sensing pathways, 606f mammalian target of rapamycin inhibitors, 611
612 mitochondrial mechanisms underlying health span extension by restrictive diet regimes in, 601
604 mitochondrial uncouplers, 612
613 multifunctional compounds, 605
609 NAD1 precursors, 609
610 polyphenols and polyamines, 605
609 Manganese SOD (Mn-SOD), 467
468 Mechanistic target of rapamycin (mTOR), 27, 611, 654 Mediating brain health, 243 Membrane-bound organelles, 303 Mesoporous silica NPs (MSNs), 375 Metabolic disorders, 346, 529 Metabolic stress, mitochondria and, 339
340 Metabolic syndrome (Ms), 333, 339, 521 and related diseases, 273
275 Metabolism, 111 mitochondria and role in, 337
339 Metallothionein 2 (Mt2), 640
641 Metformin, 562
563, 610
611 Methionine (MetR), 653 Methionine-and choline-deficient diet (MCD diet), 544 Methyl donors, 80
81 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 5
26 Methyl-malonic acid (MMA), 173

Methylation reactions, 80
81 Methylcobalamin, 173 Methylcrotonyl-CoA carboxylase (MCCC), 171
172 5-methylcytosine (5mC), 72
73, 82 Methylmalonyl-CoA, 173 Methylmalonyl-CoA mutase (MUT), 184
185 5-methyltetrahydrofolate-homocysteine, 173 Methyltransferase (MTR), 173 Mexico CVD, 363 MGN-3/Biobran, 524 Micro-RNA (miRNAs), 73
74, 78, 596 Micronutrients, 83, 651 Milk thistle seeds (MTS), 547 Mito-epigenetics, 74
78 epigenetic regulations in mitochondrial genome, 76
78 mitochondrial transcription factor A and mitochromosome structure, 77
78 mitoMIRs, 78 mtDNA methylation, 77 nutrients and diet effects on, 80
81 Mito-inflammation, 470 Mitochondria, 3
4, 74, 82, 110, 131
136, 142
144, 168, 178
179, 215
216, 226, 303, 308, 335
337, 433
434, 461
462, 522, 524, 563, 592
594, 651 affecting epigenetic pathways, 74
76 biogenesis, 140 in brain SCFAs influencing appetitive function on mitochondria in brain, 248
249 SCFAs influencing cognitive and psychological function on, 247
248 creatine and adipocyte-specific functions of, 401
404 creatine and obesity, 403
404 creatine metabolism in adipose tissue, 402
403 creatine treatment and, 400
401 dihydrogen and, 505 as essential organelle for cardiovascular health, 305
306 as important source of reactive oxygen species, 537
539 and inflammation, 465
467 mediation of, 78
84 and metabolic stress, 339
340 mitochondria-bound proteins, 136
137 and mitochondrial enzymes in cellular functions, 304
305 and oxidative stress, 467
469 per cell, 226
227 protective effects of silymarin on, 539
546

675

676

Index

Mitochondria (Continued) and retrograde signaling, 140
142 rice bran extracts and, 522
525 and role in metabolism, 337
339 structure and bioenergetics of, 334
337 structure and roles, 226
227 vitamin C and, 227
228 vitamin E, mitochondria, and aging, 147
149 vitamin E functions in, 146
147 Mitochondria DNA (mtDNA), 71, 226 methylation, 77 Mitochondria-mediated apoptosis, 465
466 Mitochondria-mediated tissue-specific effects of caloric restriction, 597
600 adipose tissue, 597 brain, 599 effects of calorie restriction in mitochondrial biogenesis and energy metabolism, 600
601 heart and cardiovascular system, 599
600 liver, 598
599 skeletal muscle, 597
598 Mitochondria-related diseases, therapeutic potential of alternative enzymes in, 120 Mitochondria-specific cocktails with synergetic potential, 528
529 Mitochondria-specific enzyme mimetics from food, 528
529 Mitochondria-specific unfolded protein response (UPRmt), 596 Mitochondria-targeted antioxidants CoQ10 prevents oxidative damage, 257
258 idebenone reduces ROS levels and bypasses complex I-deficiency, 260
261 importance of coenzyme Q in mitochondria, 256
257 mitochondria-targeted compounds, 277
279 MitoQ strong antioxidant that protects against apoptosis and induces mitophagy, 261
262 pharmacokinetics of mitochondrial-targeted antioxidant, 262
264 structure of coenzyme Q and mitochondrialtargeted coenzyme Q-related compounds, 258
260 therapeutic activity of MitoQ, 267
277 therapeutic use of idebenone, 264
267 Mitochondria-targeted compounds, 277
279 Mitochondria-targeted plastoquinone SKQ1, 277
278 Mitochondrial angiopathy in MELAS, 418
419 muscle pathology in MELAS, 418f Mitochondrial antioxidants, curcumin, 482 Mitochondrial Antiviral-Signaling Protein (MAVS), 269, 470
471

Mitochondrial bioenergetics, 397
398 creatine in, 387
389 Mitochondrial biogenesis, 214
218, 257, 522
523 and energy metabolism in non human primates and healthy humans, 600
601 n-3 polyunsaturated fatty acids effect on, 218
220 temporal caloric restriction effects on, 635
637 Mitochondrial Ca21 signaling, 473
475 Mitochondrial creatine kinase system in health and disease, 389
404 Mitochondrial deoxyribonucleic acid (mtDNA), 3
4 Mitochondrial diseases. See also Cardiometabolic diseases food intake, 95 gastro intestinal problems and BMI, 95 nutritional assessment and dietary interventions, 99 optimal method for nutritional assessment in adult mitochondrial diseases patients, 96
98 prevalence of malnutrition in mitochondrial diseases, 95
96 sex differences, 99 vitamin C role in, 229
231 Mitochondrial DNA (mtDNA), 215, 303, 335, 465, 522
523, 595, 637
638, 652 damage accumulation and maintenance of, 652
653 fructose and ethanol as potential metabolic toxins, 658
659 impact of high-fat diets on, 657
658 Mitochondrial dynamics, 136
140, 214
218, 524 fasting effects on turnover and, 637
638 n-3 polyunsaturated fatty acids effect on, 218
220 Mitochondrial dynamics protein of 49 kDa (MiD49), 136
137 Mitochondrial dynamics protein of 51 kDa (MiD51), 136
137 Mitochondrial dysfunction, 3, 229, 255, 522 challenges and limitations of using nutrients to target, 43
45 diseases involving, 3
4 targeting mitochondrial dysfunction with nutrients, 5
43 topical use of nutrients for dermo-cosmetic applications, 45
53 Mitochondrial electron transport chain (mETC), 255
256 Mitochondrial electron-transport systems, 538
539

Index

Mitochondrial elongation factor 1 (MIEF1), 136
137 Mitochondrial energy metabolism, effects on TCF, 638
640 Mitochondrial enzymes, 304
305 in cellular functions, 304
305 role in cardiomyocytes, 306
310 for apoptosis in cardiomyocytes, 308
309 in autophagy, 309
310 for scavenging reactive oxygen species, 306
308 Mitochondrial epigenetics, 74
78 epigenetic regulations in nucleus affect mitochondrial functions, 74
75 mitochondria affect epigenetic pathways, 74
76 mitochondrial functions impact nuclear epigenome, 75
76 nutrients and diet effects on, 80
81 Mitochondrial fatty acid oxidation, 305
306 Mitochondrial fission, 321
322, 340
341, 341t, 523 Mitochondrial fission 1 protein (FIS1 protein), 215
216 Mitochondrial fission factor (MFF), 136
137 Mitochondrial free radical theory of aging (MFRTA), 131
132 Mitochondrial function, 243, 247
248, 334, 436
439 in disease, 522 effects of time-controlled fasting on, 635 health properties of rice bran constituents associated with, 525
529 hydrogen-rich water and, 502
504 impact nuclear epigenome, 75
76 in response to fasting, adverse effects on, 643 Mitochondrial fusion, 136
137, 340
341, 341t, 523 Mitochondrial genome, 77, 172 epigenetic regulations in, 76
78 Mitochondrial homeostasis and regulation by protease inhibitors, 562
567 mitochondrial metabolisms and homeostasis, 562
564 proteases and inhibitors critical for health and mitochondrial homeostasis, 564
567 roles in healthy aging, 570
574 and improvement by presence of recombinant BTIs, 570
580 Mitochondrial inflammation, 470
475 Mitochondrial intermediates, 335 Mitochondrial isoform of SOD (Mn-SOD), 655
656 Mitochondrial matrix, 179
180 Mitochondrial membranes, 215
216, 335
337

Mitochondrial metabolic stress, 338 Mitochondrial metabolism, 74, 107
108 B vitamins and, 168
173 and homeostasis, 562
564 Mitochondrial morphology, 637 Mitochondrial myopathy, 393, 417 Mitochondrial myopathy, encephalopathy, Lactic acidosis, and stroke-like episodes (MELAS), 417
419 Mitochondrial nucleoid, 652
653 Mitochondrial nutrients, B vitamins role as, 178
181 Mitochondrial outer membrane permeabilization (MOMP), 465
466 Mitochondrial OXPHOS, 304 Mitochondrial p66shc by curcumin targeting of, 487 Mitochondrial permeability transition (MPT), 467
468 Mitochondrial permeability transition pore (mPTP), 33 Mitochondrial proteases, 562
567 Mitochondrial proteins, 136, 564
565 Mitochondrial reactive oxygen species production with MitoSOX-Red, 201 Mitochondrial ROS (mtROS), 34
35, 117, 464
465, 506
507 Mitochondrial signaling to cell, caloric restriction and, 595
597 fasting-mediated modulation of, 641
642 mitochondrial signaling events in response to TCF interventions, 643f metabolites, 181
185 B vitamin impacts on methylation of histone and DNA, 182
183 regulator of histone acetylation, 184
185 vitamins and HIF1 signaling, 181
182 mitochondrial mechanisms underlying health span extension, 601
604 Mitochondrial sirtuins by curcumin, targeting of, 485
486 Mitochondrial SOD, 134
135 Mitochondrial stress, 596 Mitochondrial superoxide, 118
119 Mitochondrial toxicity, B vitamins, 173
178 Mitochondrial transcription factor A (TFAM), 77
78, 522
523, 597 Mitochondrial transmembrane potential (ΔΨm), 201 Mitochondrial uncouplers, 612
613 Mitochondrial uncoupling proteins by curcumin, targeting of, 484
485 Mitochondrial unfolded protein response (UPRmt), 141, 471
473

677

678

Index

Mitochondrial vacuolization, 505 Mitochondrial-anchored protein ligase (MAPL), 471 Mitochondrial-derived peptides (MDPs), 132, 596 Mitochondrial-targeted antioxidant, pharmacokinetics of, 262
264 Mitochondrial-targeted coenzyme Q-related compounds, structure of, 258
260 Mitochondrion, 3, 334
335, 538
539 Mitochondriopathies, 228
229 Mitochromosome structure, 77
78 Mitocondrial DNA and aging, 135
136 Mitofusin 1 (MFN1), 39
40, 136
137 Mitofusin-2 (Mfn-2), 27, 39
40, 136
137, 572
573 Mfn-2-dependent mitochondrial fusion, 27 Mitofusins, 310 Mitogen-activated protein kinase (MAPKs), 246 MitoMIRs, 78 Mitophagy, 323, 340, 403, 587
589 and aging, 136
140 mitophagy/autophagy, 523 Mitoquinone (MitoQ), 26
27, 259 strong antioxidant that protects against apoptosis and induces mitophagy, 261
262 therapeutic activity of, 267
277 ischemia/reperfusion and organ transplantation, 273 liver fibrosis, 273 metabolic syndrome and related diseases, 273
275 rare diseases, 272 therapeutic potential of MitoQ in treatment of cardiovascular diseases, 275
276 as treatment in neurodegenerative diseases, 269
272 use in inflammation and immune response, 267
269 uses of, 276
277 MitoSOX-Red, mitochondrial reactive oxygen species production with, 201 Molecular compounds, 132 Molecular hydrogen, 501 dietary sources of, 501
502 Molecular mechanisms, 506
507 Molecular oxygen, 255
256 Monoamine oxidases (MAO), 467
468, 537 Monocomponent formulas, 528
529 Monounsaturated fatty acids (MUFAs), 34, 657
658 Multidrug resistant type breast cancer cells (MCF-7), 372 Multiple sclerosis (Ms), 399 Murine model of ubiquitous NDH2 expression, 115

Muscle disorders, 393
394 ischemia/infarction, 393
394 mitochondrial myopathy, 393 sarcoma and chemotherapy, 394 Myocardial function, 305
306

N n-3 polyunsaturated fatty acids, 34
36, 219 effect on mitochondrial biogenesis and dynamics, 218
220 n-6 Polyunsaturated fatty acids (n-6 PUFAs), 657
658 N-acetylcysteine (NAC), 5
26, 31
32 Na 1 -dependent glucose cotransporter (SGLT1), 311
314 NAD(P)H dehydrogenase [quinone] 1 (NQO1), 476 NADH dehydrogenases (NDH2s), 105, 108 alternative, 108
109 Nanoliposomes (NL), 368
369 Nanomaterials for quercetin encapsulation, 372
376 Nanoparticles (NPS), 363 Nanoquercetin effect on sperm quality and fertility, 368
369 effects in cardiovascular ischemia-reperfusion injury, 367
368 effects in prevention of gastric ulcers, 368 against tumor cells, 371
372, 373t Nanostructured lipid carrier (NLC), 368
369 Naringin, 323 Natural antioxidants, 464
465 NDI1, 108
109, 111
112 Neuro-2a cells (N2a cells), 198 Neurodegenerative diseases (NDs), 131
132, 367, 397
398, 438, 562 MitoQ as treatment in, 269
272 Neuroimaging of stroke-like episodes in MELAS, 420
421 Neuroinflammation, 504 Neuromuscular disorders, 94 Neuronal mitochondria, devoted energy provider for, 395
397 Neuronal mitochondrial dysfunction and amyotrophic lateral sclerosis, 398
399 and multiple sclerosis, 399
400 Neuropathic diseases, idebenone treatment of, 265
266 Neutraceuticals, 346
347 Neutrophil extracellular traps (NETs), 269 Neutrophils, 464
465 Niacin. See Vitamin B3

Index

Nicotinamide (NAM), 31, 170, 179, 433, 609 Nicotinamide adenine dinucleotide (NAD), 433 biosynthesis pathways, 435 NAD1 precursors, 609
610 Nicotinamide adenine dinucleotide (NAD), 433 biosynthesis, 433
434 cellular and mitochondrial nicotinamide adenine dinucleotide metabolism, 434
436 and mitochondrial function, 436
439 as redox cofactor and signaling molecule in mitochondria, 433
434 supplementation in human diseases, 440
448 Nicotinamide adenine dinucleotide (NAD/NADH), 5, 179 Nicotinamide adenine dinucleotide phosphate (NADP/NADPH), 179, 467
468 Nicotinamide mononucleotide (NMN), 31, 170, 433 Nicotinamide phosphoribosyltransferase (NAMPT), 435 Nicotinamide riboside (NR), 170, 433, 609 Nicotinic acid (NA), 31, 170, 433 Nicotinic acid, 179 Nicotinic acid riboside (NAR), 433 Nitric oxide (NO), 26, 244
245, 308, 348
349, 419, 464
465, 476
478 levels, 597 Nitric oxide synthase (NOS), 308, 419, 658
659 NIX-dependent mitophagy, 309
310 NLR proteins, 471 NMN adenylyltransferases (NMNAT), 434
435 NOD-like receptor protein3 inflammasome (NLRP3 inflammasome), 464
465 Non-digestible dietary fibers, 501
502 Nonalcoholic steatohepatitis, 544 Noncoding RNAs (ncRNAs), 72
74 Noncommunicable diseases, 333, 363, 365
372 Nonenzymatic antioxidants, 461
462 Nonflavonoids, 195
197 Nonproteogenic amino acids and derivatives, 525 Nonshivering thermogenesis (NST), 514 Noradrenaline (NA), 246 Nrf2-knockout (Nrf2-KO), 482
484 NRS
2002 screening tool, 98 Nuclear DNA (Ndna), 226, 652 Nuclear factor erythroid 2-related factor 2 (Nrf2), 27, 464
465, 476, 524 and curcumin, 482
484 Nuclear factor kappa B (NF-κB), 27, 246, 350, 461
462, 464
465 Nuclear factor kappa B/active protein-1 (NF-κB/ AP-1), 464
465 Nuclear genomes, 172 Nuclear mitochondrial proteins, 334
335

Nuclear respiratory factors (NRF), 39
40 NRF1, 595 NRF2, 595 Nucleus affect mitochondrial functions, epigenetic regulations in, 74
75 Nutraceuticals, 348 components, 562 effects on cardiometabolic disorders, 348
349 Nutricosmetics, 51
53 Nutrients for dermo-cosmetic applications, 45
53, 47t to target mitochondrial dysfunction, 43
45 targeting mitochondrial dysfunction with, 5
43 carotenoids, 35
36 dietary fatty acids, 34
35 endogenous antioxidants, 31
33 endogenous metabolites and transporters, 33
34 ginsenosides, 36
37 isothiocyanates, 42
43 nurients described for beneficial role in, 6t polyphenols, 37
42 vitamins and cofactors, 5
31 Nutrition, 71, 115
118, 339
340, 599 Nutritional assessment, 96 in adult mitochondrial diseases patients, 96
98 mitochondrial disease, 99

O Obesity, 513 creatine and, 403
404 Odd-chain fatty acids, 173 Oils, 525
526 Oligopeptidases, 563 Omega-3 (n-3), 213
214 polyunsaturated fatty acids, 34
35 Omega-6 (Omega n-6), 213
214 polyunsaturated fatty acids, 34 One-carbon cycle, 80
81 Optic atrophy gene 1 (OPA1), 136
137, 215
216 Optimal method for nutritional assessment in adult mitochondrial diseases patients, 96
98 body composition, 97 energy requirements, 96
97 functional parameters, 97 GLIM criteria, 98 NRS
2002 screening tool, 98 nutritional assessment, 96 PG SGA, 97
98 sarcopenia, 98 Ornithine, 384
385 Outer membrane mitochondria (OMM), 467
468

679

680

Index

Oxidative metabolism, 82 Oxidative phosphorylation (OXPHOS), 3, 76
77, 105, 133
134, 170, 217, 304, 335, 467
468, 591, 651 Oxidative stress, 108
109, 146
147, 178, 256, 258, 461
464 of B vitamins, 173
178 curcumin, 464
465 in inflammatory-related diseases, 470
475 mitochondria and, 467
469 mitochondrial targeting for reduction of, 479
480 oxidative stress-related genes, 504 Oxidative-damage related diseases, idebenone in, 266
267 Oxidized glutathione (GSSG), 169 Oxygen isotope, 107, 110

P P-triflouromethoxyphenylhydrazone (FCCP), 612
613 p53, 464
465 Panax ginseng, 36
37 Pantothenic acid, 176 Paraoxonase 2 (PON2), 367 Parkin, 400 Parkinson’s disease (PD), 4, 195
197, 247, 267, 310, 397, 522, 526 Partial abdominal aortic constriction (PAAC), 545 Pathogen-associated molecular patterns (PAMP), 470 Pectin, 501
502 Percutaneous coronary intervention (PCI), 232
233 Peroxiredoxin 3 (Prdx3), 482 Peroxisome proliferator-activated receptor gamma (PPAR-γ), 464
465, 516
517 Peroxisome proliferator-activated receptor α, 516
517 Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), 366
367, 516
517, 635
637 Peroxisome proliferator-activated receptors (PPARs), 525 Peroxyl radical, 476
478 Peroxynitrite, 464
465 Peroxynitrite radical, 476
478 PG SGA, 97
98 Pharmacokinetic profile of flavonoids (ADME of flavonoids), 311
314 Phenolic acids, 38 Phosphatase and tensin homolog (PTEN), 136 Phosphate buffered saline (PBS), 198

Phosphatidylinositol-3 kinase (PI3K), 40, 589
590 Phosphocreatine (PCr), 383 PCr-Shuttle, 386 shuttle system in cell energy homeostasis, 389 6-phosphogluconate dehydrogenase (6PGD), 482
484 Phosphoglycerate mutase 5 (PGAM5), 482
484 Phospholipase A2 (PLA2), 246 Phosphorylated forms (PCr), 385
386 PCr-shuttle system, 387
388, 395 Physical activity level (PAL), 96
97 Physiology, 78
79 Phytochemicals, 36, 38, 43, 53
54 Phytosterols, 568 Piwi-interacting RNAs (piRNAs), 73
74 Plant polyphenols, 51 Plant-based organic compounds, 528 Plant-based pigments, 528 Plant-derived natural active compounds, 462
463 (10-(6-plastoquinonyl) decyltriphenylphosphonium bromide), 259 Poly-ADP ribose polymerase (PARPs), 170 Poly(acrylic acid) (PAA), 375 Poly(ADP-ribose)polymerases (PARPs), 433
434 Poly(lactic-co-glycolic acid) (PLGA), 367, 372
374 Polyamines, 608
609 Polyethylene glycol (PEG), 374
375 Polyphenols, 37
42, 195
197, 319, 321, 343, 347, 522
523, 605
608 curcuminoids, 41
42 flavonoids, 39
40 as functional food, 341
343 phenolic acids, 38 stilbenoids, 40
41 Polyunsaturated FAs (PUFAs), 34, 213 Potassium channels, 320
321 Potential health benefits from consuming buckwheat foods, 567
568 Poultry, application of silymarin in, 547
549 protective effects of SM on poultry, 548t PPAR γ coactivator 1α (PGC1α), 139
140 Pregnancy and gestation, 394
395 Primary mitochondrial disorders (PMD), 3
4 Pro-inflammatory mediators, 461
462 Proanthocyanidin, 315, 343 Procyanidins, 315 Programmed cell life, 547 Progressive optic neuropathy (PoAG), 271 Proliferator-activated receptor γ coactivator 1α (PGC-1α), 27, 215, 339 Prolyl hydroxylase enzymes, 181 Propionate, 244

Index

Protease inhibitors critical for health and mitochondrial homeostasis, 564
567 mitochondrial proteases in maintaining mitochondrial homeostasis and deliberate regulation by, 562
567 Protease serine type 1 gene, 561
562 Protein bodies, 525 Protein kinase B (PKB), 370, 589
590 PKC-dependent phosphorylation, 467
468 Protein kinase C-dependent phosphorylation (PKC-dependent phosphorylation), 467
468 Protein lysine b-hydroxybutyrylation, 641
642 Protein restriction (PR), 653 Proteinkinase B (Akt), 40 Proteins, 134
135, 303, 525 Proteolytic enzyme, 574
575 Protopanaxadiols, 36
37 Pseudo-grain, 567 PTEN-induced putative kinase 1 (PINK1), 136, 310, 591 Puerarin, 323 Pyridoxal (PL), 171 Pyridoxal 50 phosphate (PLP), 171, 179
180 Pyridoxal phosphate. See Vitamin B6 Pyridoxamine, 171 Pyridoxin. See Vitamin B6 Pyrimidines, 82 Pyruvate carboxylase catalyzes, 171
172 Pyruvate dehydrogenase (PDH), 168

Q Quality control (QC), 339 Quercetin (QCT), 315, 363 as anticancer agent, 369
372 effects of nanoquercetin against tumor cells, 371
372 as antioxidant compound, 365
369 nanoquercetin effect on sperm quality and fertility, 368
369 nanoquercetin effects in cardiovascular ischemia-reperfusion injury, 367
368 nanoquercetin effects in prevention of gastric ulcers, 368 metabolism, biodistribution and pharmacokinetics, 364
365 nanomaterials for quercetin encapsulation, 372
376 in noncommunicable diseases, mechanism of protection of, 365
372 Quinone-based vitamins and coenzymes, 5
28

R Rapamycin inhibitors, mammalian target of, 611
612 Reactive nitrogen species (RNS), 462
463 Reactive oxygen species (ROS), 3, 82, 118
119, 131
135, 149, 170, 227, 244
245, 255, 303, 334, 366
367, 393
394, 461
462, 504, 522, 537, 566
567, 638
639, 651 effects on TCF ROS handling, 640
641 gradual ROS response hypothesis, 151 mitochondria as important source of, 537
539 mitochondrial enzymes for scavenging, 306
308 ROS signaling, aging, and lifespan, 150
151 ROS superoxide (O2
), 537 Reactive oxygen/nitrate species (R/NOS), 365
366 Receptor of AGEs (RAGE), 541
542 Redox pathways, 303 Regenerated silk fibroin (RSF), 371 Research with paraoxonase1 (rePON1), 315 Respiratory chain (RC), 3 RC enzyme, 105 alternative NADH dehydrogenase, 108
109 alternative respiratory chain pathways, 106f AOXs, 107
108 mammalian cell models, 109
112 metabolic impact of, 115
119 in mitochondria-related diseases, therapeutic potential of, 120 nutrition, 115
118 reactive oxygen species, 118
119 rodent models, 113
115 transgenic models of, 109
115 Respiratory control ratio (RCR), 540 Respiratory syncytial virus (RSV), 269 Resting energy expenditure, 96
97 Resveratrol (RSV), 40
41, 195
197, 605
607, 655
656 Retinoic acid inducible gene (RIG), 470
471 Retrograde signaling, 140
142 Reverse electron transfer (RET), 118 Riboflavin. See Vitamin B2 Riboflavin transporter deficiency (RTD), 278 Ribosomal RNA (rRNA), 76
77, 334
335 Rice (Oryza sativa), 521 Rice bran constituents, 529 Rice bran extract constituents associated with mitochondrial function, health properties of, 525
529 carbohydrates, 526
527 fats and oils, 525
526 fiber, 527

681

682

Index

Rice bran extract (Continued) mitochondria-specific enzyme mimetics from food, 528
529 plant-based pigments and organic compounds, 528 proteins, nonproteogenic amino acids and derivatives, 525 small molecule antioxidants, 527
528 and mitochondria, 522
525 role of mitochondrial function in disease, 522 Rice bran oil (RBO), 523
524 Rodent models, 113
115

S S-adenosylmethionine (SAM), 75
76, 172 Saccharomyces cerevisiae, 108
109 Sarcoma and chemotherapy, 394 Sarcopenia, 98, 195
197 Satellite cells, 138 Saturated fatty acids (SFAs), 213 Scavenging antioxidant systems, 482 Scutellaria baicalensis, 318 Second mitochondria-derived activator of caspase (SMAC), 465
466 Selenocysteine, 526 Serine, 75
76 Serotonin (5-HT), 246 Sex differences, 99 Short-chain fatty acids (SCFAs), 243
244 effects on modulating central nervous system function, 246
249 influencing appetitive function on mitochondria in brain, 248
249 influencing cognitive and psychological function on mitochondria in brain, 247
248 regulating peripheral organizational activities, 244
245 Silibinin (SB), 538
539 Silybin, 544 Silymarin (SM), 535 antioxidant properties of, 539 application in poultry, 547
549 effect on vitagene expression, 546
547 integrated antioxidant defense system, 535
537 mitochondria as important source of reactive oxygen species, 537
539 protective effects on mitochondria, 539
546 in vitro evidence, 539
543 in vivo evidence, 543
546 Single photon emission tomography (SPECT), 420
421 Singlet oxygen, 476
478

Sirtuin1 (SIRT1), 27, 81
82, 366
367, 433
434, 467
468 activation, 592
594 network of mitochondrial sirtuins, 593f dependent pathway, 506
507 SIRT1
7, 140, 592 Skeletal muscle, 213
214, 597
598 cells, 219
220 creatine monohydrate on skeletal muscle mitochondria, 390
392 phenotype, 638
639 SLC6A8, 386 Small interfering RNAs (siRNAs), 73
74 Small molecule antioxidants, 527
528 Sodium dodecyl sulfate (SDS), 198 Sodium nitroprusside-induced nitrosative stress (SNP-induced nitrosative stress), 541
542 Sodium/chloride dependent CRT, 386 Soluble cytosolic protein, 136
137 Soluble mitochondrial intermembrane space proteins, 465
466 Somatic mtDNA mutations, 652 Spermidine, 656
657 Spinocerebellar ataxia type 1 (SCA1), 27, 272 Standard deviation (SD), 202 Sterile α-and toll/TIR motif-containing protein 1 (SARM 1), 436 Sterol regulatory element-binding protein 1c (SREBP1c), 597 Stilbenoids, 40
41 Stroke-like episodes (SLEs), 417 Substantia nigra compacta (SNc), 27 Succinate dehydrogenase (SDH), 169, 417 Succinyl-CoA, 79 Suffering subarachnoid hemorrhage (SAH), 271 Sulforaphane (SFN), 42
43 Superoxide, 464
465 Superoxide anion, 476
478 Superoxide dismutase (SOD), 110, 134
135, 255
256, 365
366, 523 Superoxide dismutase 2 (SOD2), 482, 504, 640
641 Suppressing cytokine signaling (SOCS), 479 Symplocarpus renifolius, 111 Syringic acid, 527

T T-acute lymphoblastic leukemia cell (T-ALL cell), 569
570 TANK-binding kinase 1 (TBK1), 465
466 Tea and health benefits, 344
346 Tea polyphenols, molecular mechanisms of action of, 350
353

Index

Temozolomide (TMZ), 371 Ten-eleven translocation (TET), 183 TET proteins, 72
73 Tetrahydrofolate, 173 Thermogenic adipocytes, 514 Thiamin, 178 Thiamin pyrophosphate (TPP), 178 Thiamine. See Vitamin B1 Thiamine diphosphate, 168 Thiamine pyrophosphate (TPP), 30 Thioredoxin (Trx), 306
308, 539 Thioredoxin 2 (Trx2), 482 Thioredoxin-reductase (TrxR), 306
308 Tigriopus californicus, 108 Time-controlled fasting (TCF), 633 adverse effects on mitochondrial function in response to fasting, 643 fasting and organelles, 644 fasting effects on mitochondrial dynamics and turnover, 637
638 fasting-mediated modulation of mitochondrial signaling, 641
642 and health, 634
635 mitochondrial energy metabolism, effects on, 638
640 mitochondrial synthetic function, effects on, 641 reactive oxygen species handling, effects on, 640
641 strategies employed to study effects of, 634 strategies to boost mitochondrial fidelity and disease amelioration, 643
644 TCF on mitochondrial function, effects of, 635, 636t temporal caloric restriction effects on mitochondrial biogenesis, 635
637 Tissue distribution of creatine, 385
386 Tocotrienol-rich fractions, 524 Tocotrienols, 144
145 Toll-like receptor 9 (TLR9), 142
144, 465
466 Toll-like receptors (TLRs), 470
471 Toll/interleukin-1 receptor (TIR), 436 Topotecan (TPT), 371 Trans-resveratrol, 197 Transcription factor A (TFAM), 215 Transcription factor A of mitochondria (TFAM), 635
637 Transfer RNA (tRNA), 75, 334
335 Translocase of outer membrane (Tom20), 576
577 Traumatic brain injury (TBI), 27, 271 Triboxycyclic acid cycle (TCA cycle), 108
109, 142
144, 304, 480
482 Triphenylphosphonium cation (TPP 1 ), 26
27, 375

Triplenegative breast cancer (TNBC), 372 Trypanosoma brucei, 107 Trypsin, 561 health benefits and presence of trypsin inhibitors, 567
570 Trypsinogen, 561 Tumor necrosis factor receptor-associated factor 6 (TRAF-6), 470
471 Tumor necrosis factor-alpha (TNF-α), 463
464 Tumor-associated trypsin inhibitor (TATI), 565
566 Type 2 diabetes (T2DM), 521
522, 529

U (10-(6’-ubiquinyl)decyl triphenyl phosphonium), 259 Ubiquitous mitochondrial CK (umtCK), 394
395 Ulcerative colitis, 527
528 Ultraviolet radiation (UVR), 45 Uncoupling protein 1 (UCP1), 402, 513, 516
517 importance in regulating energy homeostasis, 515
516 three types of adipocytes, 514 UCP1-mediated thermogenesis, 516 Uncoupling protein 3 (UCP3), 607
608, 638
639 Uncoupling proteins (UCPs), 467
468, 598 Unified Parkinson’s Disease Rating Scale (UPDRS), 401 University of Wisconsin (UW), 273 US Food and Drug Administration, 501

V Valeric acid, 244 Vasodilatory function, 634 Vatiquinone, 278 Vitagene expression, effect of SM on, 546
547 Vitagene network, 547 Vitamin B1, 30, 168, 173
175 Vitamin B2, 30, 168
169, 173
175, 235 Vitamin B3, 31, 170, 176, 184, 433, 440 Vitamin B5, 170
171, 179 Vitamin B6, 171, 176
177 Vitamin B7, 177, 180 Vitamin B8, 31 Vitamin B8/B7, 171
172 Vitamin B9, 31 Vitamin B11/B9, 172 Vitamin B12, 173, 178, 181 Vitamin C, 5
26, 29, 132, 225
226 as ergogenic factor, 234
236 and exercise, 231
234 and mitochondria, 227
228 role in mitochondrial disease, 229
231 safety of, 231

683

684

Index

Vitamin E, 28
29, 46, 132, 144
149 and aging, 147
149 and antioxidant capacity, 145 functions in mitochondria, 146
147 uptake and cellular distribution of, 145
146 Vitamin K, 28 Vitamin PP. See Vitamin B3 Vitamins, 167 and cofactors, 5
31

W Water-soluble homologs of CoQ and vitamin E, 260 Water-soluble vitamins, 167

Western diet (WD), 657 White adipose tissue (WAT), 402, 514 White rice, 521 Whole-body maximal oxygen uptake (VO2max), 214
215 Wild-type males (WT males), 112
113 World Health Organization (WHO), 513

X X-linked adrenoleukodystrophy (X-ALD), 272 Xanthine hydrogenase/oxidase, 462
463 Xanthone, 343 Xanthophylls, 35
36