Where Does All That Food Go?: How Metabolism Fuels Life [1st ed.] 9783030509675, 9783030509682

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Alicia Kowaltowski Fernando Abdulkader

Where Does All That Food Go? How Metabolism Fuels Life

Where Does All That Food Go?

Alicia Kowaltowski • Fernando Abdulkader

Where Does All That Food Go? How Metabolism Fuels Life

Alicia Kowaltowski Biochemistry Department University of São Paulo São Paulo, Brazil

Fernando Abdulkader Department of Physiology and Biophysics University of São Paulo São Paulo, Brazil

ISBN 978-3-030-50967-5    ISBN 978-3-030-50968-2 (eBook) https://doi.org/10.1007/978-3-030-50968-2 © Springer Nature Switzerland AG 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Copernicus imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

Have you ever wondered where all the food eaten by a lanky teenager goes? Questioned why you tend to like the specific foods that make you fat and unhealthy? Been baffled by contradictory information on healthy eating habits? Understanding the basics of metabolism and how it works can help you comprehend these questions and give you a better starting point to sift through all the dietary information that is out there, separating what is scientifically sound from what is simply wrong. The aim of this book is to help you understand what metabolism is, how it is organized and regulated, how our bodies handle our food, and why these processes are important. In fact, metabolism is a fascinating phenomenon and involves the very basics of what life is, and we hope this book will help you grasp its general principles. On the other hand, we give you fair warning that this is not a book on how to lose weight, get what is perceived as a perfect body, nor endorse a one-size-fits-all diet that will make you a perfect healthy human. A single unifying dietary recommendation like that, frankly, does not exist. Instead, we hope to make you aware of basic facts that will hopefully help you select the new information that you should listen to, while ignoring the miracle claims that are too good to be true. New information on diet, nutrition, and health will always appear and can, frustratingly, be different from what was accepted as the truth before. While many people interpret this as a sign that scientists don´t know what they are talking about, the fact that scientific information changes is actually a consequence of an essential characteristic of the scientific process: Science is not dogmatic (or at least should not be), and therefore the best consensus among scientists will change over time, as researchers gain new information. Accordingly, when our knowledge about metabolism increases, as it does every single day due to thousands of dedicated investigators around the globe, we gain new information and adapt prior knowledge. Within this mindset, understanding the basics that separate real science from hype could (hopefully!) help you more than learning what is specifically known today. The understanding of new information on metabolism is also confounded by the fact that scientific information is transmitted among scientists by means of scientific publications. These published papers read like gibberish to any non-specialist because they are written in technical language that requires years of training in that v

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specific area of Science to be understood. This may seem a deliberate way to conceal precious knowledge amongst the privileged group of expert scientists, but scientific papers actually allow scientists to be very precise and synthetic in what they say. In writing this book, we aimed to “translate” some of these concepts into more straightforward everyday language. The reality is that Science has accumulated too much detailed knowledge for any human individual to comprehend the full plethora of what is being investigated and discovered right now. As a result, new scientific knowledge is transmitted to a more general but well-read public by means of scientific press releases, which involve significant simplification of what the scientists originally found. These press releases are then retransmitted by very diverse media outlets, many of which are more focused on promoting a sensational title or punchline than broadcasting real information. The result is that scientists will often scratch their heads in consternation and sometimes do not even recognize their own work when it pops up in the media! There is no easy solution for this lack of precision and distortion of scientific facts and findings by the media. Indeed, this is a problem which affects many other areas of human activity. What we can recommend is to ignore any superlative or miraculous claims, and take an interest in new evidence, but wait for further news before applying anything towards your own life. Educate yourself with solid scientific sources so you can recognize dodgy ideas and stick to well-established institutions as a source of information. Above all, practice moderation – life is not just about keeping as healthy as possible. Fun is very important too, and having fun has the wonderful side effect of making you healthier! While you learn a bit about metabolism, what it is, and how it works, we hope you can grasp how fascinating this process is. We can assure you this is a very rewarding passion. The authors of this book have spent decades investigating metabolism and it has not yet ceased to amaze us. We are literally, at any given moment, transforming (metabolizing) millions of molecules within our bodies, building new ones, breaking down others, and exchanging them with the world around us. Metabolism is much more than the reason you gain weight when you overeat, it is a process that is so central for life that it defines what a living being is. São Paulo, Brazil 

Alicia Kowaltowski Fernando Abdulkader

Contents

1 What Is Metabolism?������������������������������������������������������������������������������    1 2 How Metabolism Works��������������������������������������������������������������������������    5 3 Carbohydrate Metabolism����������������������������������������������������������������������   17 4 Mitochondria: The Batteries of Our Cells��������������������������������������������   37 5 Lipid Metabolism ������������������������������������������������������������������������������������   51 6 Protein Metabolism����������������������������������������������������������������������������������   67 7 Alcohol Metabolism ��������������������������������������������������������������������������������   79 8 Metabolism and Obesity��������������������������������������������������������������������������   83 9 Diabetes and Metabolism������������������������������������������������������������������������   97 10 Metabolism in the Brain��������������������������������������������������������������������������  107 11 Metabolism and Heart Disease ��������������������������������������������������������������  117 12 Metabolism in Exercise����������������������������������������������������������������������������  125 13 Cancer and Metabolism��������������������������������������������������������������������������  135 14 We Are Stardust ��������������������������������������������������������������������������������������  139 Glossary������������������������������������������������������������������������������������������������������������  143 Index������������������������������������������������������������������������������������������������������������������  149

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Chapter 1

What Is Metabolism?

Metabolism often has a bad reputation, blamed for making people fat or unable to eat specific foods. The truth is metabolism is essential to maintain us alive, and so important that it defines what life itself is. What is metabolism? Metabolism, in a dictionary-like definition, is the combination of chemical reactions that occur within a living organism in order to maintain life. As surprising as this may seem to us, chemical reactions are happening within us all the time, and there is nothing scary about this. In fact, chemicals is another word with a bad reputation that definitively does not deserve it. Everything you use, hold, eat and breathe is made of chemicals. Living beings, including humans, are composed of chemical substances of all sorts of types, shapes, sizes, and functions. These molecules make up our cells, tissues, and organs, and at the same time have jobs within our cells, tissues, and organs. Molecules don’t stand still over time, and are instead changing into other molecules, in the continuous process of metabolism. In fact, there are tens of thousands of different types of chemical reactions happening right now within your own body. All living beings, by definition, must be able to change the molecules within themselves and with their environment. Therefore, all livings organisms have metabolism. The difference between us, living humans, and a very dead human-shaped Egyptian mummy is that we have active metabolism and are modifying our molecules constantly. The mummy stopped metabolizing long ago and is, consequently, dead. It shouldn’t come as a surprise that we are constantly modifying our molecules through chemical reactions. After all, most of us eat (or incorporate into our bodies) quite a bit of stuff that does not look, act, or function even remotely like us. Unless our food mysteriously disappears inside us, this must mean we change its molecular structure in some way. You can safely eat corn your whole life long without any risk whatsoever of becoming a huge yellow grain. Instead, you will metabolize the molecules in corn and either use them as an energy source or transform them into human molecules. In fact, we all know that, while there is nothing wrong with eating corn in moderation, eating it in excess will help us accumulate fat molecules, which are

© Springer Nature Switzerland AG 2020 A. Kowaltowski, F. Abdulkader, Where Does All That Food Go?, https://doi.org/10.1007/978-3-030-50968-2_1

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chemically different from the main molecule present in corn (starch). But we’ll have more on that later.

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Our fascination with metabolism is not new. Hippocrates (c. 460 – c. 370 BC), certainly one of the most outstanding physicians in history, recognized the importance of moderate nourishment and exercise in the maintenance of health (at least according to early biographers, who unfortunately only wrote about him centuries later). He could not have been expected to further understand the relationship between diet, exercise and nutrition at that point: This was a time in which it was already revolutionary to consider diseases consequences of natural changes in body function, as opposed to superstitious causes or religious deities. Roughly two millennia later, Santorio Sanctorius (1561–1636  AD) conducted experiments that have crowned him the founder of metabolic studies.1 He started by experimenting on himself, weighing all food and liquid he consumed and all urine and stool he produced, and was baffled to find that part of the weight of what he ingested seemed to disappear. What he eliminated never added up to what he ingested. He then went to extreme measures to understand this phenomenon, developing methods to measure sweat (in an unsuccessful effort to find the missing weight being lost) and creating a moveable platform attached to a scale on which a person could stay for extended time periods while weight changes were followed. In a letter to his contemporary Galileo Galilei (1564–1642), he claims to have used this device on more than 10,000 persons over 25 years! His conclusions were that somehow things never added up and that people lost part of what they ingested in a form of “insensitive perspiration”, which he could not measure. It was too early in scientific history for Sanctorius to do much better than that, despite his extensive studies. Antoine Lavoisier (1743–1794) would later discover that matter can change its form, but not its mass, confirming that the food and liquid ingested could not simply disappear, something Sanctorius understood instinctively in his explanation of “insensitive perspiration”. When John Dalton (1766–1844) proposed the atomic theory, the basic understanding of the chemistry of life could begin. Because Sanctorius worked long before these seminal findings, he could never have imagined that the weight difference he was seeing was due to the loss of atoms by breathing. When we breath out, the air that leaves us has about 100 times more carbon dioxide than the air that we breath in (which in turn has more oxygen). The weight changes he attributed to “insensitive perspiration” were in fact because of carbon atoms within carbon dioxide molecules in exhaled air. When you lose weight, you are, literally, losing part of your former body by breathing carbons out of it. Louis Pasteur (1822–1895) was the scientist that began to elucidate the specific chemical reactions within us that metabolize our food. He did this by studying the 1  Eknoyan G. (1999) Santorio Sanctorius (1561–1636)  – founding father of metabolic balance studies. Am J Nephrol 19:226–233.

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process of wine production2,3 How can studying winemaking help us understand our own metabolism? The answer is simple: the microscopic organism (microorganism) that produces wine (Saccharomyces cerevisiae or, more popularly, Baker’s yeast) metabolizes sugars in pretty much the same way as humans. Pasteur was the first to recognize that it was the living activity of organisms so small you can’t see them by naked eye that was responsible for the chemical transformations that ferment grape juice into wine. Consequently, Pasteur was also the first to study chemical transformations promoted by metabolism. (Of note, Saccharomyces cerevisiae also participates in beer and bread production – a very useful microorganism indeed!) Pasteur demonstrated two important characteristics of sugar metabolism. First, he showed that the amount of sugar metabolized by the yeast was a lot lower when air was present than when it was not. This was the first indication that aerobic metabolism, which happens in the presence of oxygen from the air, was more efficient in generating energy. As a result, less sugar is required to obtain the energy necessary to grow the same amount of yeast. Second, he found that the end-product of fermentation in different microorganisms can be either lactate (such as in yoghurt fermentation) or ethanol and carbon dioxide (such as in wine, beer and bread fermentation). Today we know that cells in our bodies, when deficient in oxygen, ferment sugars to lactate through chemical reactions identical to those in microorganisms that make yoghurt. We also know that fermentation to ethanol and carbon dioxide involves the same chemical steps as fermentation to lactate, except for the very last reaction. All of us living organisms metabolize sugars in a very similar manner. This is no coincidence, but rather the result of evolution, and the fact that we all descend from organisms that metabolized sugars this way. Indeed, we all evolved from an organism that had a pathway of chemical reactions, still in use today, that made efficient use of the energy in sugar molecules, and thus helped living organisms thrive. Over the next decades after Pasteur’s discoveries, many researchers worked on understanding the step by step transformations that ferment sugars. The early 1900s saw a burst in the understanding of metabolic pathways, with the principles of lipid (fat) metabolism described in 1905 and the urea cycle (vital in protein metabolism, as we shall see further on), the first cyclic metabolic pathway (with a circular configuration), described in 1932. The most complex of the central metabolic mechanisms, because it involves not only chemical transformations, but also energy storage as electrochemical gradients, much like batteries, was described in 1961, when Peter Mitchell proposed the “chemiosmotic hypothesis”. This hypothesis explains how the energy released from nutrient oxidation (by the oxygen we breathe) is transformed within mitochondria into the energy necessary for our cells to function. We will discuss this fascinating process later.

 Schwartz M. (2001) The life and works of Louis Pasteur. J Appl Microbiol 91:597–601.  Berche P. (2012) Louis Pasteur, from crystals of life to vaccination. Clin Microbiol Infect 18:1–6.

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Timeline: hallmarks in the understanding of metabolism

After the main pathways were uncovered, smaller but vital metabolic routes were and still are the focus of intense research. In addition, scientists are now focused on understanding what certainly are the most complicated aspects of metabolism: how all these chemical reactions work together in a coordinated manner, how disease states affect metabolism, and how we can fine-tune metabolic processes that go awry.

Chapter 2

How Metabolism Works

The knowledge accumulated about metabolism today is enormous; so large that we build intricate flowcharts to make sense of it. The fascinating complexity can be seen in the illustration of the central metabolic reactions below, which represents only the tip of the iceberg of what we know today.

© Springer Nature Switzerland AG 2020 A. Kowaltowski, F. Abdulkader, Where Does All That Food Go?, https://doi.org/10.1007/978-3-030-50968-2_2

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Metabolic Map showing the main metabolic chemical reactions occurring in living organisms. Prepared by the late Prof. Donald Nicholson (University of Leeds). Copyrights (used with permission) are assigned to the The International Union of Biochemistry & Molecular Biology. The Sigma-Aldrich Corporation has for many years organized and published these Metabolic Pathways Charts. Complete versions are freely available for download at http://www.iubmb-nicholson.org

You may notice by examining this chart that the chemical reactions that are shown are connected, since the product of one chemical reaction is the reactant in the next reaction. This stepwise modification of molecules is how metabolism

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works. The tandem reactions that transform an initial molecule into a final substance form what we call metabolic pathways. Because of the routes formed, illustrations showing the way metabolic reactions are organized are aptly called metabolic maps. Comparisons between the organization of metabolism and the organization of transport systems go beyond the fact that both are illustrated in the form of maps. There are many similarities between the structure of metabolism and that of urban transport systems. For example, in metabolism, some reaction pathways are very commonly used, with many molecules processed through them all the time. They act as “metabolic highways”, processing a large number of molecules. An example of a “metabolic highway” is the glycolytic pathway, the route that processes all sugars and other carbohydrates. On the other hand, some metabolic pathways are not used very often, and process a lower number of molecules, functioning similarly to small local roads. Despite their low throughput, these pathways are critically important for the few molecules they process. For example, a defect in the ability to metabolize the amino acid phenylalanine leads to an inability to process proteins that contain this amino acid (including almost all animal proteins), in a disease called phenylketonuria. Babies are routinely screened for this disease at birth, as avoiding the ingestion of phenylalanine in breast milk avoids the very serious brain damage that happens when these babies accumulate phenylalanine they cannot break down (metabolize) because they lack the pathway to do so. Indeed, the recognition and ability to detect this defect in a metabolic pathway has guaranteed normal brain development in hundreds of thousands of persons worldwide, who would otherwise be severely disabled.

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Another parallel between metabolism and transport systems is that metabolism has both two-way reactions and one-way reactions, in a manner similar to two- or one-­way streets. What this means is that some chemical reactions in metabolism can happen in both directions (substance A can be transformed into substance B, and substance B can be transformed into substance A), while in other reactions molecule B can be produced from molecule A, but molecule A cannot be produced from molecule B  – these are one-way molecular streets, or irreversible metabolic transformations. One-way metabolic pathways are important, because they explain how we process our molecules. For example, proteins can produce both carbohydrates and fats within our bodies. When you eat excess protein, your body will store at least part of that protein as fat. However, humans cannot produce protein from the atoms in stored fat nor from carbohydrate molecules they eat. This happens because fats and carbohydrates lack an atom that is crucial to the structure of proteins – nitrogen. On the other hand, proteins can become either fats or sugar – as long as they get rid of nitrogen. Overall, all this means we can only rebuild proteins from other proteins, and explains why lack of adequate amounts of protein in your diet can lead to serious health problems.

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Another example is that humans can produce fat from carbohydrates, but not carbohydrates from fat, because the carbohydrate-to-fat pathway is a one-way pathway in humans. Interestingly, many microorganisms and plants can do what we cannot: transform fat into carbohydrates, because they have a pathway we don’t have (the glyoxylate pathway). And yes, you read it right: proteins can become fat, but fat cannot become proteins. Carbohydrates can become fat, but fat cannot become carbohydrate. Overall, everything that humans eat in excess can be stored as fat. The sad reality is that we are very well prepared, metabolically, to gain weight by storing fat molecules!

Interconversions between the main groups of nutrients in our metabolism. Carbohydrates can become fat, as can proteins. Because they lack nitrogen, no other nutrients can produce proteins



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At this point in our description of metabolism, we need a quick introduction regarding the mechanisms in which all those metabolic chemical reactions (symbolized by the arrows on the metabolic map we saw before) actually happen. These chemical reactions are coordinated by a group of highly specialized proteins called enzymes, which are responsible for virtually every chemical transformation in our body. Enzymes are large molecules that have complex 3D structures which are both useful and also strikingly beautiful, as you will see in the figure below, showing the hugely enlarged structures of a few enzymes involved in metabolism.

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Enzyme structures. Metabolic enzymes are complex 3-D structures that create environments within them that facilitate (catalyze) chemical reactions. A few examples of metabolic enzyme structures are shown: hexokinase (top left), phosphofructokinase (bottom left), pyruvate dehydrogenase (top right), arginase (bottom right) and ATP synthase (center). Source: Protein Data Bank

Enzymes are more than beautiful structures. Because they are somewhat like molecular sculptures, enzymes create spaces within them that have specific chemical and physical properties. These spaces provide locations in which the chemicals of our cells can fit and sit relatively still for a while, and also promote the enormous acceleration of chemical reactions, known as catalysis. Within the environment of the enzyme, chemical changes can happen much more quickly, because the chemicals are brought together, and the properties of the space within the enzyme help these changes occur. Enzymes act similarly to cellular cheerleaders or matchmakers. They bring molecules together and encourage them to change. In this manner, they make chemical reactions happen much faster. In fact, most enzymes make reactions happen between 10,000 and 1,000,000 times faster! Like matchmakers, enzymes can bring molecules together and thus speed things up considerably. However, and this is a very important point, enzymes cannot make reactions happen if they weren’t chemically feasible in the first place. They are matchmakers and cheerleaders, but not miracle workers. For example, common table sugar (sucrose) can be oxidized by oxygen in the air and become water and carbon dioxide (CO2), which is a gas and simply floats away into the air. However, the reactions that do this are so enormously slow that you can keep sugar in your pantry for years and not notice any discernable decrease in the weight of your sugar because it floated away as a gas. When you eat this sugar, enzymes within your cells vastly accelerate this chemically feasible reaction (through pathways we shall see ahead), and within minutes you are breaking this

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sugar down and breathing out CO2 generated from it. Your enzymes accelerated the chemically possible reactions to break it down. On the other hand, CO2 in the air and water cannot spontaneously produce sugar, and therefore this reaction does not happen on its own, nor in the presence of enzymes. So, overall, each chemical reaction that happens within us is coached into existence by a sculptural molecule called an enzyme, that creates an environment that is just right for that reaction to happen. In this manner, enzymes essentially provide and pave the roads of our metabolic pathways, allowing much faster travels for our molecules.

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Enzymes are effectors not only of the chemical reactions, but also of metabolic regulation. Imagine a city in which traffic was completely free to flow as every vehicle saw fit – it would be chaotic! In the same manner as cities have mechanisms to control traffic, including traffic lights, speed limits and radars, metabolism has systems to control the flow of molecules through its molecular pathways. Within each one of our cells (which are separated from other cells by space-­ marking membranes much like medieval cities are surrounded by walls), metabolism is regulated by the cell’s own energy levels. When we talk about energy levels within a cell, we are basically referring to the amount of a specific molecule called adenosine triphosphate, or ATP, the main molecule used as an energy source for processes that require investments. ATP is a small molecule that has three phosphate groups (the red and yellow structures at the bottom of the ATP molecule shown below). Breaking off the last phosphate group generates adenosine diphosphate (ADP) and a free phosphate molecule, and releases energy that powers the cell for many different energy-consuming processes. This energy, of course, does not flow all of a sudden to the location or molecule that will make use of it, like some kind of spiritual entity or bolt of lightning. The beauty of ATP in metabolism resides in the fact that Life has developed to use the chemical energy that binds phosphate and ADP. What actually happens is that this energy is used by making the phosphate in ATP react with other molecules, transferring this chemical group to them (phosphorylating them). These new molecules with the additional energy received from the phosphate are said to be activated (or phosphorylated) and subsequently can easily shed this phosphate and use the energy it gave them to undergo reactions that were not feasible before. The ADP molecule formed by removing phosphate from ATP is later linked once again to a phosphate, re-forming ATP (an energy-giving chemical the cell can easily use), in metabolic pathways that obtain the energy to do so from our food and stored energy sources, such as fat. In essence, ATP is the energy currency of the cell: metabolic pathways that conserve energy convert this energy to ATP, while pathways that need energy use the energy stored in ATP.

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Spatial representation of an ATP molecule (Sourcefile: ATP exp.qutemol-ball.png)

When cells use up a lot of energy to do something such as grow, divide, contract (as muscle cells do) or deal with information (in the case of neurons in your brain), ATP is broken down and releases energy that fuels these cellular processes. The result is that ATP levels within the cell decrease and ADP levels increase, indicating a shortage of energy the cell can use. These lower levels of ATP don’t last long, because low ATP also changes the cell’s metabolism. One of the features of metabolic pathways that ensures our survival is that they are regulated, and ATP, in addition to being produced by metabolism, is a metabolic regulator. There are certain steps in metabolic routes that release energy (to form ATP) which are inhibited when ATP levels are high. As a result, when ATP levels fall due to energy-consuming activities in the cell, these pathways are uninhibited and activated. The result is that ATP is quickly produced again, and ideal levels are restored. ADP and other molecules that accumulate when energy levels are low also contribute toward metabolic regulation in a cell. When their levels increase, indicating low energy levels, they activate pathways that produce more ATP and restore the cell’s energy levels. The result of metabolic regulation by energy levels is that ATP never stays low for long within a heathy cell. The levels fluctuate up and down within fractions of seconds, as pathways are activated and inhibited by the increase and decrease of easily usable energy, reflected as ATP levels. This maintains the cell’s ability to use energy for necessary functions. It also ensures that energy stored in our food and reserve molecules is not wasted when replenishing ATP levels is not necessary. You may be asking yourself at this point why cells keep stocks of energy-rich molecules such as fats instead of stocking up on ATP, the molecule that will ultimately be produced from the fat stores and used for processes that require energy. The reason is that ATP, while an excellent and easy source of energy, is not a practical way to store this energy. In fact, one molecule of triacylglycerol, the main fat

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molecule we store as an energy source, is capable of generating hundreds of ATP molecules. It can also be stashed away in the cell in compartments that don’t let water in, which both decreases the space and weight needed for storage and the chances that the molecule will react with other molecules in undesirable manners, changing its structure and spoiling it. In fact, we all know that water-excluding lipid molecules such as fats keep well: you can have unrefrigerated oils sitting around for months in your kitchen without seeing them go rancid (changing their structure). So why not use fats as direct energy sources for the cell’s needs? Why transform them into ATP? While fats are excellent storage molecules, releasing the chemical energy in them is not an easy process, requiring instead a complete metabolic pathway with multiple chemical reactions and many different cell components. In contrast, the energy in ATP can be released in one step, by a specialized group of enzymes that are aptly called kinases (from the Greek kinesis, movement). Overall, storing energy as large molecules such as fat, and then transforming them into small molecules that can easily break down, such as ATP, works well in terms of cellular metabolic organization. That is why this form of energy management has been selected and kept throughout different groups of living beings.

A monetary analogy between metabolism and banks. As in banks, metabolism has ‘safes’, where energy is stored. These are glycogen and fats. To get whatever is stored in these ‘safes’, you have to use clerks  – whose metabolic counterparts are glucose and acetyl-CoA (more on this later!) – to get to the energetic currency, ATP. We also have a form of metabolic “special credit”, which involves “paying interest” – proteins. These fuel their energy into metabolism by special clerks, called amino acids

In fact, ATP is (if there is any) the molecule of life as we know it. Every living being on Earth uses it as an energy source. Every living being on Earth synthesizes it and dies when unable to replenish ATP. Single molecules of ATP in our bodies are

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broken down and re-synthesized thousands of times a day. ATP stability even defines the limits of life on Earth: living beings can exist at temperatures over the boiling point of water, but not over the temperatures in which ATP molecules spontaneously degrade. Life requires ATP. Life requires a constant flow of energy, to and from ATP.

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While ATP is both the energy source for cellular processes and an important controller of cellular energy metabolism, multicellular organisms (organisms with many cells) such as ourselves must have a system to control energy metabolism in a coordinated manner among all their tissues, and not just in individual cells. Again, this is similar to traffic control: while in a very small town all you need are a few traffic lights, in a large city you need to have these traffic lights and traffic detection systems coordinated by a central office which can tweak each one of these systems in order to solve problems that affect different neighborhoods.

Cells and tissues as traffic networks. Cells could be thought of as towns (whose boundaries are in red at the left), where the chemical energy flows around via the molecules that take part in the various metabolic pathways (grey streets). This flux is controlled by “traffic lights”, which are the key enzymes in the pathways that sense the levels of regulators, particularly ATP. But cells are inserted in a country (your body, in dark blue to the right), and are thus connected by highways (blood flow), from which they receive supplies (nutrients) and communication (chemical signals) from both federal (brain, in the form of neurotransmitters) and state (glands, as hormones) governments (the federal capital is in red and state capitals are in orange). The “state roads” in green correspond to blood vessels, while “federal roads”, in blue, correspond to the nerves

While ATP controls metabolic processes within each cell, it does not usually leave cells, and stays within the individual cell it was produced in. That means it does not work as an indicator to coordinate metabolism between different cells. Instead, we have specialized molecules that coordinate metabolic processes in our

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whole body, acting on many cells at a time. Metabolism is also controlled by hormones, or chemical messengers that are produced in one part of the body, circulate around (frequently in the blood) and change how many other cells within the body work. There are many different hormones that control metabolism in our bodies, and we will talk about them in detail later. You probably heard of at least one of these hormones before: insulin. Insulin is produced by special cells in our pancreas, a long and flat organ inside our abdomen. It is released into the blood after we eat and indicates to the whole body that it should use the nutrients from the food we ate to remake things that need rebuilding (such as broken down proteins) and stock up on energy reserves for later. It increases the production of muscle proteins, glycogen (a storage form of carbohydrates) and fats. So, if we did not have any insulin, we would not get fat? That is in fact true: people who develop type 1 diabetes, usually children and teenagers, cannot make insulin and lose a lot of body weight. But that is not a good thing at all – they also have dangerously high glucose levels in their blood and will have serious health issues and die if they do not receive the insulin they need. On the other hand, if they are given too much insulin, their blood sugar levels can get dangerously low and also lead to health issues (mainly in the brain) and death. Insulin is, as many other hormones, a “Goldilocks” molecule: neither too much nor too little of it is good. You need it at just the right amounts, which a healthy person is perfectly capable of maintaining, because the cells in the pancreas that release this hormone do so in a regulated manner. Insulin is such a central molecule to control energy metabolism in multicellular organisms that it appeared very early in the evolution of living beings that have many cells (and possibly even earlier, given some lines of evidence in single-cell organisms). Caenorhabditis elegans has insulin,1 despite being a very small flat worm that is about the size of a period on a printed page. It has around 1000 cells (a mere handful compared to our ~37 trillion cells2). Not only does this worm have insulin, but it works in the same way as insulin in humans, helping the worm store energy molecules for later use. Flies also have insulin, as do snails, fish, cows and pigs (from which insulin was isolated early on to treat diabetics). This just comes to show that integrating metabolism in the whole body of an animal is essential, and that is done by hormones such as insulin.

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Having seen the basics of what metabolism is, namely a large collection of chemical reactions in our bodies that transforms molecules, and how it works in a 1  Girard L.R., Fiedler T.J., Harris T.W., Carvalho F., Antoshechkin I., Han M., Sternberg P.W., Stein L.D., Chalfie M. (2007) WormBook: the online review of Caenorhabditis elegans biology. Nucleic Acids Res 35:D472–475. 2  Bianconi E., Piovesan A., Facchin F., Beraudi A., Casadei R., Frabetti F., Vitale L., Pelleri M.C., Tassani S., Piva F., Perez-Amodio S., Strippoli P., Canaider S. (2013) An estimation of the number of cells in the human body. Ann Hum Biol 40:463–471.

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step-wise and regulated manner, we are now ready to see what happens to the molecules we eat as they are transformed by our metabolism. We will follow the journey through our bodies of each of our main nutritious molecule types (carbohydrates, proteins and lipids), as they are metabolized and transformed.

Chapter 3

Carbohydrate Metabolism

Carbohydrates, often nicknamed carbs, are a group of molecules often found in our food and that have somewhat of a bad metabolic reputation in most people’s mind, although they are sometimes also divided into “bad carbs” and “good carbs”, therefore redeeming at least part of them. In reality, carbohydrates consist of a very diverse group of molecules with many different sizes, shapes and functions. They are of utmost importance, as illustrated by the fact that the most abundant biological molecules on Earth are carbohydrates. A good place to start understanding carbohydrate metabolism is by recognizing what carbohydrates are. The name says a lot, derived from carbon and hydrate, which means that the carbon atoms within these molecules are chemically hydrated, or bound to water molecules. This characteristic defines the structure of carbohydrates, in which most carbon atoms reacted with water, so that many oxygen and hydrogen atoms from the water molecule remain in the molecular structure of the carbohydrate. The basic structure of most carbohydrates contains only carbon, oxygen and hydrogen atoms, and in this way is quite different from other biological molecules such as proteins or DNA, which have nitrogen atoms too.

© Springer Nature Switzerland AG 2020 A. Kowaltowski, F. Abdulkader, Where Does All That Food Go?, https://doi.org/10.1007/978-3-030-50968-2_3

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A sucrose (table sugar) molecule, left, and a small part of the structure of a starch molecule, right. Both are common carbohydrates and are often found in our foods: sucrose is abundant in many fruits and gives them their sweet taste. Starches are present in most vegetables and are the main component of dietary staples such as rice, potatoes, and bread. Note the presence of many – OH groups, derived from water, which are typical of carbohydrates

Apart from having many –OH groups linked to carbon atoms, the structure of carbohydrates is very variable. The smallest carbohydrates (with complicated names: dihydroxyacetone and glyceraldehyde) have three carbon atoms, while the largest carbohydrates (storage molecules such as starch in plants and glycogen in animals) typically have at least 500 carbon atoms, and frequently have thousands of them. The carbohydrate we will most talk about is glucose. A glucose molecule only has six carbon atoms, so it is a fairly simple carbohydrate. Six-carbon carbohydrates are indeed considered the building blocks for the majority of all other classes of carbohydrates, including starches. In fact, the structure of starch molecules is so big that only a small portion could be included in the figure, above (this pattern repeats on and on in a similar fashion, where the dotted lines are). Because these structures are so different, carbohydrates also have many different functions in humans and in the living beings we humans eat as food (just about everything you eat is derived from organisms that were once alive – there is no way around this reality!). Carbohydrates can be energy storage molecules for later use by that organism (such as the starch in potatoes or wheat, as well as glycogen molecules in our livers and muscles), attractive energy-packed molecules made by plants to entice animals that help disperse seeds or pollinate (such as sugars in fruit and nectar), the outer structure of plants, bacteria and insects (among others), participants in immune processes (defense systems against invading microorganisms), lubricants in your joints, as well as many other functions. We have metabolic pathways to construct all the carbohydrates that we need for our bodies to function, starting with the raw material to build these molecules, which is usually the carbohydrates that we eat. Carbohydrates in our food fall essentially into one of three categories: sugars (smaller carbohydrate molecules which usually taste sweet), starches (larger carbohydrates which are slower to digest) and fibers (carbohydrate molecules we cannot digest, and therefore are eliminated in our feces). They are all important, and we will start discussing them by the group that isn’t digested, and therefore is not metabolized within our bodies: fibers.

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The three types of carbohydrates. Six-carbon molecules (monosaccharides, top) are the basic components of carbohydrates. Sugars (left) are the simplest type of carbohydrate, with one or just a few basic units. Starches (center) are composed of hundreds to thousands of units, which are connected in tandem and in branches. Although these are huge molecules, we can digest them (we are able to break the bonds between the basic units). Fibers (right) can be either big or small molecules. Their main characteristic is that we cannot digest them, because we lack enzymes to digest the bonds between their basic units



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While we have bones to scaffold our bodies to their typical human shape, plants use carbohydrates, in particular a specific carbohydrate called cellulose, to maintain their structure. Without cellulose plants would be blobs of liquid matter. Cellulose is a polymer  – a large molecule formed by many repeats of a smaller molecule linked together – in which the repeated smaller molecule is glucose. Glucose is fully metabolized by humans. However, cellulose is not, because in cellulose, the glucose molecules are linked together by a particular type of chemical bond called the β glycosidic link. Many microorganisms and animals, including insects, can break this kind of bond, but humans cannot, because we do not have enzymes capable of doing this. This means that these large carbohydrate molecules remain in our intestines and are not broken down to become small enough to be absorbed. But that does not mean these fibrous carbohydrates aren’t important for you. Not being absorbed means that fibers give the intestines more bulk to grip on, increasing

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the movement that pushes the food forward, helping to normalize bowel movements (if not in excess, of course). Fibers also decrease the speed in which we absorb the nutrients in our food, avoiding spikes in blood sugar and stabilizing nutrient levels for longer periods of time after a meal. This helps you feel hungry less often. They additionally help keep a healthy population of bacteria in our gut (yes, your gut is full of microorganisms, and that is normal and healthy). So be sure you get some daily roughage. How much? Enough to get regular bowel movements, neither too dry nor too liquid. Where do you get fibers to keep your gut healthy? Fruits and vegetables contain fibers, at variable degrees. Vertebrate animal foods (such as fish, beef, chicken and pork, the bulk of animal products western humans eat) do not contain fibers because these animals have bones as skeletons, like we do. Insects and crustaceans, on the other hand, do not have skeletons, but are instead surrounded by chitin, a non-­ digestible carbohydrate that constitutes their exoskeletons (the outer hard part of their bodies). Therefore, insects and plant-based foods have fiber, while the larger animals we eat do not. Since few people in the western world eat large quantities of insects or shrimp with their shells, most of our fiber comes from plants. Some vegetable products are processed to decrease their fiber content, as is the case for flour and rice. This processing helps us use the energy contained in these staple foods, since it decreases the amount of non-digestible molecules and enriches the digestible starch content. For this reason, our ancestors began refinement processes. Today, we have perfected this refinement to a point that many humans have diets that only include refined carbohydrates, and therefore are too low in fiber. This can cause constipation and discomfort due to the low speed the food travels through the intestines if no other fiber source is ingested. For most of us with varied diets, however, it is perfectly fine to consume grain products that have been processed to reduce the fiber content in moderation, as long as enough fiber is obtained overall. Once again, balance in fiber ingestion is key.

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Fruits and vegetables also contain varying amounts of starch, a large molecule that is likewise a polymer of glucose molecules. The difference between starch and fiber is that starch contains glucose units linked by α glycosidic links, which humans are capable of breaking into individual glucose molecules and absorbing. These glucose units will later be transformed into other molecules, stored, or broken down to generate energy. Starch is the most prominent molecule in many food staples including bread, pasta (and other wheat products), rice, potatoes, beans and corn. Most cultures use at least one source of starch as a staple in their diets. Indeed, starches are an excellent source of the chemical energy necessary to keep us alive. They have a somewhat bad reputation as inducers of obesity, but the reality is that starches cause obesity only when eaten in excess. Starch is a healthy energy source when used proportionally to the demands of a person. A chemical ‘cousin’ of starches – glycogen – can also be found in some animal cells, where it functions as a source of carbohydrates. We will talk about glycogen in more detail later.

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Finally, sugars are small carbohydrates which generally taste sweet to us, and are therefore something most humans enjoy eating. There is an evolutionary reason for this, as we will soon see. Sugars found in our food can be disaccharides, which means they are composed of two molecules linked together. Sucrose (also known as table sugar, composed of glucose and fructose and present in many fruits and vegetables) and lactose (found in milk and composed of glucose and galactose) are common disaccharides in our food. Other sugars we eat are monosaccharides, or smaller sugars such as glucose, fructose and galactose, that have not been linked together to form larger sugars. All of these disaccharide and monosaccharide sugars taste sweet. Because they are small molecules, sugars are absorbed fast during digestion. Eating these types of carbohydrates as part of their naturally occurring source, such as within fruits, is recommendable, since fruit are also rich in many other nutrients (including starches) and have fiber, therefore decreasing the rate in which these sugars are absorbed. Incidentally, fruits taste sweet because they are rich in sugars. The problem with sugars (and the reason they are often called “bad carbs”) appears when they are eaten in large quantities and in a refined form, such as table sugar. When eating sugar alone, it is absorbed quickly, spiking up the levels of glucose in your blood, and releasing a lot of insulin, the hormone which makes many of our cells accumulate fats as a form of storage molecule for the excess sugar in the blood. This results both in fat accumulation and can also lead to a dip in blood sugar levels soon after you eat because of the high levels of insulin. In other words, refined sugar can cause your blood glucose levels to “crash and burn”, and therefore is not an ideal way to provide energy for your body (although some sugar will not harm a healthy person).

Digestion and absorption of carbohydrates. While digestion of starches starts in the mouth, its bulk digestion and consequent absorption happens slowly in the gut. Meanwhile, sugars, far simpler molecules, can be rapidly digested and absorbed in the mouth and in the initial segment of the intestines

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Unfortunately, despite the common knowledge that sugars are not a healthy food option, many people today eat a large amount of it, because sugar is present in many types of processed foods, including hidden within foods that are savory in taste. There are two reasons for adding sugar into processed food: First, adding sugar increases stability and shelf life, since it decreases the free water in the food that bacteria can use to grow, promoting spoilage. This is of interest to the food companies, that can use added sugar to make the shelf life of the food they produce longer. Second, humans like sweet foods, so adding it to prepared foods makes us like, and consequently buy, these processed products more often. Again, food companies are benefitted by adding sugars to food for this reason. But why do we like sugar so much, if it isn’t all that good for us? The answer, as with almost anything in Biology, is related to evolution. We evolved over hundreds of thousands of years to survive short lives, living maybe 30 or 40 years (if you were lucky to survive childhood), having kids and dying early. Only during the last century did we get average life expectancies over 40 years of age. Eating sweet foods rich in sugars gives you a fast energy source that helped humans for most of our evolutionary history. On the other hand, we only started very recently to develop the consequences of excess calories leading to obesity, such as diabetes and heart disease, which appear much later in life. Basically, evolution of our tastes and food preferences prepared us for a reality in which we no longer live: short lives with limited food availability. So if you like super-sweet foods and have a hard time avoiding them, even though you know they aren’t healthy, it really isn’t your fault. Blame evolution!

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Now that we have seen what carbohydrates are, we can start to understand how sugars and starches, the kinds of carbohydrates we metabolize, are chemically transformed within us. Carbohydrate metabolism actually begins in the first place food goes inside you: your mouth. Your mouth is an amazing place, where food is ground down using your teeth (the hardest part of your body). Enormous forces are generated to macerate food by the muscles that hold your jaws in place. In fact, the masseter muscle, the main one responsible for moving your lower jaw up and down, is the strongest human muscle when compared by weight. While food is mechanically ground up using the strength of this incredible muscle and the hardness of our teeth, it is also being mixed with saliva, giving it a pasty consistency that helps it move down the rest of your digestive tract. Saliva also starts the digestion of our food, and therefore promotes the first step of carbohydrate metabolism. Simple sugars such as glucose and fructose can be absorbed into your blood, leaking through the mucus membranes that cover the whole nine-meter-long journey food makes from your mouth until the end of your intestines. On the other hand, larger sugars such as sucrose, lactose and starches need to be broken down to monosaccharides such as glucose, fructose and galactose to be absorbed. Saliva contains amylase, an enzyme that starts breaking down the carbohydrates in your food into

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smaller pieces, including some sugars such as glucose and maltose (a disaccharide composed of two glucoses, also found in malt, a component of beer). Because these sugars are produced when you chew starchy foods, you get a bit of a sweet taste, which, as we saw before, is a taste humans are evolutionarily selected to like. As a result, eating starches makes you happy, basically from the moment you start chewing them. But these first sugar molecules you taste are just a teaser: the bulk of glucose molecules in starch will be absorbed further on during their journey through your gut, in your intestines. The speed in which sugars are absorbed affects the way your blood sugars change. As we saw, refined table sugar leads to rapid increases in blood sugar and a fast and large response in insulin production, which makes the blood sugar quickly decrease soon afterwards. On the other hand, starches promote a slower increase in blood sugar levels, maintaining these levels steady over time, especially when digested together with fibers, which help keep a slow and stable absorption of these nutritious carbohydrates. Once in the blood, the sugar molecules you absorbed from the food you ate move quickly around the body and will be taken up and metabolized by many different types of cells within you.

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Before we take a look at what happens to carbohydrates after they are absorbed, we’ll take a brief detour to talk about a sugar many adult humans are not able to properly absorb: lactose. Lactose is a disaccharide composed of two simple sugars (galactose and glucose) linked together with by a bond with the fancy name of β-1  →  4 glycosidic bond (don’t worry  – you don’t have to remember that!). An enzyme called lactase, which is secreted by our intestines, can break this bond, releasing the simple sugars that make up lactose. These simple sugars are then absorbed from the intestines into the blood stream. Mammals, including humans, produce lactase as infants, because they drink milk, which contains lactose as a source of energy for the baby. As a result, baby mammals are able to digest and absorb the simple sugars in milk and use them to live and grow. Adult mammals do not drink milk in the wild, but humans started doing this some 7000–9000 years ago. However, not all human adults produce lactase, so many of us are incapable of digesting lactose in the milk we drink. The reason, once again, lies in evolution. The domestication of mammals as a source of milk by humans happened only recently in evolutionary history, and did not involve all parts of the world. As a result, many humans lose the ability to produce lactase as they age and are not expected to nurse anymore. This was of little or no consequence until animal milk began to be consumed by adult humans, so it was a characteristic that was not weeded out by evolution. In fact, about 65% of the human adult population today has some level of lactose intolerance, with levels as high as 95% in specific regions of Asia and Africa. Lactose intolerance means they are not capable of producing enough lactase to properly digest the lactose found in dairy products. This intolerance is not limited to adult humans: adult pet animals such as cats and dogs can also be lactose intolerant, for the same reasons we are.

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The result of a lack of lactase in your gut is that, if the sugar lactose is ingested, it remains in the intestines and is not absorbed. This sugar retention helps the gut to retain water (sugars attract water) and can lead to diarrhea. Worse still, the bacteria in our gut are perfectly happy to digest the leftover lactose, and in fact thrive on it, growing very quickly and making large quantities of gasses inside us, causing bloating and cramps. The result is that soon after ingesting a product containing lactose, a person who does not produce enough lactase to break it down can feel quite sick and uncomfortable. The symptoms vary a lot individually, depending on how much lactase each person still produces, and how much lactose they ate. Often times a lactase-deficient person can tolerate small quantities of lactose such as in yoghurt (in which bacteria digested most of the lactose in the milk before you ate it) or cheese (in which lactose is partially removed during production) but not products that contain more lactose, such as milk itself. Although evolution served us poorly in terms of being able to break down lactose as adults, it gave us the keen intelligence and curiosity that makes humans natural scientists, thus creating a wonderful tool to counteract lactose intolerance. While the avoidance of any milk product can certainly relieve lactose intolerance symptoms, today we have the choice of using lactase made by microorganisms to help us digest this sugar. Lactase is industrially purified from yeasts or bacteria, and then taken in the form of lactase pills or added previously to milk products, making them lactose-­ free. In fact, you may notice lactose-free milk tastes a bit sweeter than regular milk, because the galactose and glucose produced by breaking down lactose are sweeter-­ tasting than lactose itself. The result of the production of lactase is that today anyone, even those who have no lactase of their own, can enjoy dairy products without bloating, by adding back the enzyme they lack. With lactase present, lactose is broken down and absorbed as simple sugars into our blood stream from our intestines.

Lactose, lactase and malabsorption. Left scheme: lactose digestion and absorption by our intestines in the presence of normal lactase activity. Right scheme: undigested lactose due to low lactase activity leads to its metabolism by bacteria, that proliferate and produce large amounts of gasses

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The production of lactase to help digestion in persons lacking this enzyme is just one of many examples we will see in which we, humans, have developed scientific knowledge to produce a useful solution for a metabolic problem. In the case of lactase, this scientific development was useful. In other cases, such as in diabetes (which we will discuss later on in this book), we will see that Science is essential and lifesaving.

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We now return to the journey of carbohydrates through our body. Up to now we saw that, with the exception of fibers, carbs are broken down into smaller sugars and absorbed into our blood, mainly at our intestines (the longer part of our digestive tract and where most nutrients are absorbed). The blood that leaves our intestines is drained into a specific blood vessel called the Portal Vein, which takes this nutrient-thick blood to its first destiny during its journey around our body: your liver. Because of the unique anatomy of the blood flow from the intestines, the liver always starts nutrient handling in our bodies, and has the first pick in the processing of molecules we absorb from food. In fact, the liver has a decisive role in metabolism, processing the molecules that come from the food we eat, modifying them, storing some of them, and sending others off to other organs. The liver is also extremely flexible from a metabolic standpoint. If a metabolic transformation can be done, it most probably can happen in the liver. When glucose molecules from your dietary carbohydrate intake arrive at the liver through the blood, they can be transported across the membranes of liver cells and enter these cells. Glucose could then also leave the liver cell, but instead, inside the liver cell it is transformed into a new molecule, by adding a phosphate from ATP to its sixth carbon. The new molecule formed, glucose-6-phosphate, has two advantages over glucose itself: First, the negative charges around the phosphate group trap this molecule inside the cell, keeping it from going back into the blood. Second, the reaction that transforms glucose uses the energy currency of ATP as the supplier of the phosphate group, which transfers some of the energy in the ATP to the glucose molecule. Glucose-6-phosphate is now a higher-energy molecule which is trapped within the liver cell and cannot leave immediately. This happens because the membrane transport systems that mediate the import/export of glucose to and from the liver cells cannot recognize glucose with this phosphate tag. Once formed, glucose-­6-phosphate will be transformed into some other molecule within that cell, and is usually stashed away to be used when food within that body is scarce. Glucose-6-phosphate is trapped within a cell, but this molecule is not the final molecular destiny for the carbohydrate you ate. Stocking up on either glucose or glucose-6-phosphate is not a practical way for our bodies to save up on excess food to use later for energy, since these molecules take up a lot of space (because they attract water). These molecules are also not as chemically stable as a stock molecule should be, meaning that they can degrade spontaneously or react with other molecules if left waiting around inside the cell, losing the regulated use of their precious chemical energy. In fact, glucose-6-phosphate molecules keep this chemical form

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for mere hundredths of seconds, since they are further modified quickly in the chemical transformation pathways that make up our metabolism.

Glucose in the blood, absorbed from carbohydrates in your food, gets inside liver cells and is transformed into many different molecules. Glucose enters the liver cell and is modified into glucose-6-phosphate, a form unable to leave the cell again. This glucose is now at a crossroads: it can become glycogen, a large storage molecule composed of hundreds or thousands of glucose units. It can become other sugars and antioxidants, through the pentose pathway, or follow the glycolytic pathway and ultimately be broken down to acetyl CoA. Acetyl CoA is another metabolic crossroads molecule, and can be broken down to release energy (in the form of ATP) or joined to many other acetyl CoA molecules to produce fat

Glucose-6-phosphate within a liver cell can follow different paths, because it sits at something of a metabolic crossroads. This single molecule can go three different ways after a carbohydrate-rich meal: to produce other sugars through the pentose pathway, to produce the stock molecule glycogen, or to follow the glycolytic pathway (also known as glycolysis) and ultimately transform glucose-6-phosphate, a 6-carbon molecule into smaller, 2-carbon molecules. The road taken by glucose-­6-­ phosphate at this crossroads is determined by the capacity of each route (or the number of molecules they can take up at any time) and the regulation of each pathway. This regulation occurs through the mechanisms of metabolic regulation we saw before, including the energy state of each cell (as seen by ATP quantities) and

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levels of regulatory circulating hormones such as insulin and glucagon. Let us now take a look at the three metabolic pathways glucose-6-phosphate can take.

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One of the most important metabolic destinies for glucose in the liver is the production of glycogen. Glycogen is a storage molecule, produced to stock up on the energy contained in glucose that you just ate, so that it is available later, when you haven’t got any glucose to absorb in your intestine, such as the time between your meals. This storage is important, since glucose is not only a vital source of energy, but also the only source some cells use, including your neurons, the cells in your brain that participate in thought processes, and your red blood cells, that take oxygen all around your body. So that our brains keep on working even when we haven’t just eaten carbs, our liver stocks up on glucose in the form of glycogen when we just ate, and then releases glucose from glycogen when needed. Glycogen is a large molecule typically formed of thousands of glucose units. To make glycogen, glucose molecules are stuck together side by side through chemical reactions promoted in our cells. The result is a string of glucose molecules that looks similar (at a sub-microscopic level) to beads on a pearl necklace. Separate strands are then linked together at branches every 8–14 glucose units, producing a large molecule with many, many branches poking toward the outside and a solid core of compact glucose “beads” crowded in the inside, somewhat similar to a Koosh ball, but in which each strand of glucose molecules splits at some point. Each one of our liver cells has many of these large molecules deposited inside of them, with glycogen making up about 5% of the liver’s total weight. Although individual biological molecules are so small they are not usually visualized using conventional microscopes, glycogen molecules are so large they can be seen with simple staining techniques, giving a grainy aspect to liver cells – each grain is an individual glycogen molecule containing thousands of glucose molecules linked to each other. Storing glucose in the form of glycogen has many evolutionary advantages that lead animals to stock up on glucose in this manner. (Plants accumulate starch, which is very similar in structure to glycogen, for the same reasons.) The first advantage is that glycogen is not capable of leaving a cell because of its large size, which makes it unable to cross the cell membrane. That means it stays put inside liver cells until it is broken down into individual glucose molecules, which can then leave the liver. This happens when the liver cell is “told” that person has not eaten for some time, mainly by the presence of the hormone glucagon in the blood. The result is that glycogen is formed in the liver after you eat, while lots of glucose molecules are in the blood (stimulated by the hormone insulin). Glycogen holds on to this glucose until you haven’t eaten and blood sugar levels start lowering, promoting the increase in glucagon. Glycogen then releases some of its glucose, keeping blood sugar levels high enough for your cells to function. A second advantage of storing glucose in the form of glycogen molecules is that it occupies a lot less space. While each glucose molecule inside the cell is surrounded by many water molecules, a glucose unit within a glycogen molecule is

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typically not bordered by water, but instead mostly encased by other glucose units from the same glycogen molecule. This exclusion of water makes glycogen a much more compact way to store energy in a cell than glucose. Excluding water molecules from this storage site also has the advantage of making a molecule more stable, and less prone to react with other molecules brought there by the water solution. As we will see later on, glucose is a molecule that can spontaneously react with other molecules in our body. This reactivity of glucose generates many of the problems related to diabetes, a condition in which glucose levels in the blood are high. Storing the glucose molecules in glycogen prevents this reactivity. As a result of these many advantages, our liver has quite a bit of glycogen stocked up within it, and is thus capable of using this glycogen as needed. When you just ate and a lot of glucose is in your blood, this sugar is delivered to the liver, transformed into glucose-6-phosphate within liver cells and can then be incorporated into a glycogen molecule, making it bigger, with more glucose units in it. This will keep the glucose you ate stashed away within the safe and compact environment of the glycogen molecule until enough time has passed for your blood glucose levels to drop once again. When your blood glucose levels drop, glucose molecules are again released from glycogen, and can leave the cell, go into the blood and keep your blood sugar levels stable. The process of growing and shrinking our glycogen molecules in your liver keeps blood sugar levels in their “sweet spot” (pun intended): neither too low, so that cells that need glucose such as neurons and red blood cells can work, nor too high, leading to undesirable reactions between glucose and other molecules, as we shall see when we discuss diabetes, a condition in which carbohydrate metabolism goes haywire. Glycogen is thus an important molecule in the liver and helps maintain glucose levels steady throughout the body. But this handy form of storing glucose is not present only in the liver. Glycogen can also be found in many other cell types, and is abundant in our muscles. Although our muscles are not the first stop for the blood after it leaves our intestines, glucose does reach our muscles after we eat, and is incorporated into glycogen molecules in the same manner as in the liver. In fact, in addition to the liver, our muscles are very important in removing excess sugar from our blood soon after we eat. However, muscle cells do not help maintain glucose levels when we are hungry like our liver cells do. Instead, glycogen in our muscle cells store glucose molecules to be used by those muscle cells alone. This happens because muscle cells are not able to convert glycogen back into glucose, but only into glucose-6-phosphate, which cannot leave the cells. As a result, glycogen in the muscle fuels the muscle’s energy needs, while glycogen in the liver fuels the whole body’s energy needs. In other words, the muscle is greedy about its own glycogen, while the liver is altruistic.

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We now understand one of the possible destinies for glucose after it enters the cell and gets trapped in it as glucose-6-phosphate: becoming part of the large glucose

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storage system which is glycogen. Let us explore another possible destiny for the “crossroads” molecule glucose-6-phosphate, which is the pentose pathway. The pentose pathway is often the bane of undergraduate students when studying the basics of metabolism. The reason this metabolic pathway creates havoc in students´ mind is that it does not have one specific molecule as its final “molecular destination”, unlike most other metabolic pathways they study. Instead, the pentose pathway is somewhat of a metabolic network of interconnected small trails in which molecules can change into many other molecules and arrive at different final molecular destinies, for a myriad of unique metabolic functions. For the purposes of this book (which will not examine metabolism at a college level), it suffices to say that the pentose pathway helps provide electrons necessary to produce fats, helps remove free radicals (more on that later) and also generates different sizes of sugars. These are many and very different functions indeed! The production of different-sized sugars includes producing pentoses (5-carbon sugars) necessary for DNA and RNA synthesis, that give the pentose pathway its name. It is, in sum, a flexible pathway, with many uses in metabolism, and another possibility for the metabolism of glucose 6 phosphate.

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In addition to becoming glycogen or following one of many molecular destinies in the pentose pathway, glucose-6-phosphate has another pathway it can follow: the glycolytic pathway. This is the last pathway we will see for this molecule, but by no means the least. In fact, it is more of a metabolic highway than a pathway, capable of transforming a large quantity of molecules, and thus responsible for a very significant part of glucose metabolism in the liver and, indeed, in most of our cells. The glycolytic pathway starts out with our already familiar glucose 6 phosphate, the 6-carbon sugar that has a phosphate group which keeps it from leaving the liver cell it was produced in. This molecule is broken down into two 3-carbon molecules, generating some energy-containing ATP in the process. In the liver, the three carbon molecules produced by glycolysis are almost all converted to a two-carbon molecule known as acetyl coenzyme A, or acetyl CoA for short, by removing one carbon atom in the form of carbon dioxide, or CO2 (more on that later…). Acetyl CoA is another important “crossroads” molecule in metabolism, and therefore we will name it here. The main destinies for acetyl CoA in the liver are two: producing fat or being completely degraded to CO2 and water (yes, we need to drink water, but we also generate water within our metabolic reactions, about 300 mL of it per day, or a bit more than a cup). The “metabolic decision” regarding which path will be taken is again the result of metabolic regulation. When the ATP levels within the cell rise and fat production-stimulating hormones such as insulin are present (right after you ate), fat production will be the most prominent fate for the carbohydrate you ate (you knew that already…). On the other hand, when energy levels in the cell go down, as indicated by decreasing ATP levels, the main fate for the carbohydrate you ate and that ended up as acetyl CoA is to yield energy in the form of ATP. In this process, carbs are completely broken

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down into CO2 and water. Let us now take a look at both these metabolic options for the acetyl CoA molecules we have now generated by metabolizing the carbohydrate we ate.

Acetyl-CoA fates. Fed (left) and fasting (right) states



∗∗∗

We will start with the destiny that we all know carbohydrates we eat can have, as we often see it happen in real life: the production of fat. Indeed, humans and most other animals are superbly well prepared to store excess calories ingested as fats or lipids, the more technical term for this class of molecules. Lipids are a chemically diverse group of molecules that includes some names you may recognize from your lab exams, such as cholesterol, fatty acids and triglycerides. The unifying characteristic of all these molecules that classifies them as lipids is that they are poorly soluble in water, and tend to stick together, separating from water solutions that make up the cell. This separation from water makes fats excellent storage molecules, in a manner even more efficient than that we discussed for the carbohydrate glycogen. Separating lipids from water decreases the amount of space and weight needed to store calories (because it eliminates water) and also separates these molecules from chemical reactions that happen in water solutions in the cell, making lipids very stable storage molecules. An example that shows how lipids are stable is that foods that contain high quantities of lipids such as oils, butter, margarine or lard have long shelf lives – they can be kept for a long time in your pantry or fridge without spoilage. For this same reason, processed foods often have a lot of fat – it helps make them last longer.

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Space use is optimized for energy storage in fats as compared to starches, their animal counterpart glycogen, or proteins. In the figure above, we have roughly the same amount of biochemical energy stored in potatoes (starch) and in beef (protein) and olive oil (fat). Oil weighs much less and occupies much less space. This is partly because starch, despite its compact structure, still attracts a lot of water because of its highly hydrophilic glucose units

Storing excess food energy as lipids also has the advantage of packing a lot of calories in very little weight. Because lipids are richer in electrons per carbon atom, they are capable of generating a lot more energy (in the form of our “energy currency” molecule ATP) than carbohydrates or proteins. The reason electrons are so important for energy funneling into ATP will become clearer in the next chapter, but for now it suffices to say that the same amount of lipid, in weight, has roughly twice the calories compared to carbohydrates or proteins. That means that eating a diet rich in lipids will result in ingesting more calories than the same amount of food with mostly carbohydrates or proteins. It also means that storing lipids can stash away about double the amount of energy in the same amount of weight, when compared to storing energy in the form of glycogen. Given that lipids are a stable and efficient way to store energy for later use, it should come as no surprise that we are evolutionarily programmed to store any excess food as fat. This characteristic kept us and other animals alive during many millennia of hunger and uncertain food sources. But in modern days in which food can be easily obtained in large quantities, this biological property has contributed to a continuous and unhealthy expansion of our waistlines. Indeed, healthy humans are made up of somewhere between 10 and 25% lipids, depending on their gender (men have less fat), age, athleticism, and other characteristics. Obese humans frequently have 50% of their weight composed of fat, more than double the healthy amount. Most of the lipid accumulated as a storage molecule is in the form of triacylglycerol or triglycerides, large molecules that contain three long carbon chains (called acyl chains) and that can generate a lot of energy when broken down. These molecules are also composed of a glycerol molecule that holds onto the three long carbon chains, that in humans are usually around 16 carbons long, each. The result is a

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molecule that resembles somewhat the shape of a three-legged spider (at a molecular level, of course).

A triglyceride molecule. Each one of these molecules contains three long carbon chains linked by a glycerol molecule (in red)

Triglycerides are formed in our bodies from many different molecules, including carbohydrates. The reactions that form lipids are signaled by many different stimuli, but most importantly by the excess of energy circulating in our bodies, as happens after a meal rich in carbohydrates, which increase the levels of insulin in our blood. The pathway followed is that the carbohydrate is absorbed as simple sugars and follows the glycolytic pathway we just saw, where each glucose molecule generates two acetyl CoA molecules and two CO2 molecules, which we eliminate by breathing them out. The acetyl CoA molecules can generate triglycerides by being chemically stitched together, making a chain of carbon molecules that grows by two carbons every time an acetyl CoA is incorporated. After 8 acetyl CoAs are stitched together, you have a long and thin 16-carbon molecule called palmitic acid, which is part of a class of molecules called fatty acids (more on that later). Three chains of these fatty acids, all around 16 carbons long (but ranging from a bit shorter to a bit longer than that) are stitched together to form the three-legged spider-shaped molecule we call a triglyceride. After individual triglyceride molecules are formed, thousands of these molecules then get together, attracted to each other by their poor interactions with water, forming little drops of fat inside our cells, much like oil forms drops on the surface of a pot of water. These lipid droplets wait around inside our cells, mainly in cells that are specially adapted to make them, until there is a metabolic signal to break these molecules down and use them as energy. This signal is typically the presence of the hormone glucagon and the parallel decrease in insulin levels, both stimulated by low levels of blood sugars, which appear long after a meal (we will see these hormones in a lot more detail later when discussing the metabolism of obesity and diabetes).

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Acetyl-CoA molecules are stitched together to form fats. Every acetyl-CoA molecule has 2 carbon atoms. Joining them in tandem eight times yields a long-chain molecule called palmitic acid, which is a fatty acid. Three fatty acids (mostly palmitic acid) make a triglyceride molecule

Triglycerides are formed and stored in many parts of our bodies, including our livers. In fact, after a rich meal, our livers become very fatty and soft, with a consistency like butter. This characteristic is observed in foie gras, which consists of the triglyceride-rich liver of very well-fed duck or goose. Indeed, much like we are evolutionarily selected to like energy-rich sweet foods, we are also evolutionarily selected to enjoy energy-rich fatty foods, which is another reason (besides the long shelf life) that companies add a lot of triglycerides to processed foods. But the liver’s ability to accumulate lipid droplets would seem almost amateur when compared to the most specialized cells that both produce and store triglycerides: our fat cells, more technically known as adipose cells. These cells, which are spread around our whole bodies (including our bellies, thighs and all those other places that tend to grow bigger every time you overeat), are highly efficient in removing glucose from the blood, sending it down the glycolytic pathway, producing acetyl CoA and then stitching together these two carbon molecules until triglycerides are produced. In fact, triglycerides accumulate in such large quantities in these cells that lipid droplets formed by them can take up more than 90% of the cell. These fat deposits are our energy packs, stocking up the excess we ate for later use. You may think at this point that adipose cells are bad news, and the reason you must watch not to eat too much in order not to need ever-wider clothes. But adipose cells are not just an energy savings account for our bodies. They are also very important in metabolic control, helping to maintain our blood glucose levels by removing the excess after a meal. In fact, scientists have found that lab animals altered so they don’t have adipose cells, and therefore with no body fat deposits, are

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diabetic, because they cannot control their blood glucose levels!1 The simple fact is that fat accumulation in our bodies is good, in moderation.

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Having seen how glucose (and all other carbohydrates) can become fat, we can now see the last possible route for these molecules in metabolism: complete oxidation to CO2 and water, producing a large amount of ATP. Completely breaking down glucose molecules involves taking the glycolytic pathway and then producing acetyl CoA and some CO2, as we saw up to now. Each of these acetyl CoA molecules are then broken down themselves to two CO2 molecules, within a pathway often called the Krebs Cycle, or more formally the Citrate Acid Cycle or (in an attempt to use the most complicated name possible) the Tricarboxylic Acid Cycle. This elegant metabolic pathway often sits in the middle of metabolic maps, given its central importance in metabolism. It breaks down not only glucose, but also proteins and fats, since, as we shall see in the next chapters, all these molecules follow metabolic pathways that transform them into acetyl CoA, and then into CO2 within the Krebs Cycle. As the name suggests, this metabolic pathway was first uncovered by Hans Krebs (1900–1981), who earned the Nobel Prize in 1953 for understanding how this pathway works. As the name also suggests, this metabolic pathway is cyclic, which gives it the distinct circular aspect when drawn in metabolic maps. At the time in which Dr. Krebs worked, deciphering how metabolism transformed molecules was an exercise in knowing what molecules were in a specific tissue, knowing some of the chemical reactions that happened between these molecules through experimental evidence, and then putting the sequence of these reactions together, including all these molecules and reactions, like a metabolic puzzle. They also had to keep in mind that all changes proposed along the metabolic pathway had to be chemically feasible in order to happen. Hans Krebs excelled at predicting these step-by-step transformations and contributed toward the understanding of many metabolic pathways at the time. He was particularly good at understanding that metabolic pathways could be cyclic, having participated in the description not only of the Krebs Cycle, but also of the Urea Cycle (which we will see in protein metabolism) and of the Glyoxylate Cycle (which humans do not have). What exactly is a cyclic metabolic pathway? A cyclic metabolic pathway is a sequence of metabolic transformations in which one molecule acts both as an entry molecule, combining itself with another molecule, and as an exiting molecule a few steps ahead. Because the same molecule is formed and used, it cycles around the pathway, and these forms of metabolic transformations are well represented by sequences of chemical reactions in circles within metabolic maps. In the specific 1  Moitra J., Mason M.M., Olive M., Krylov D., Gavrilova O., Marcus-Samuels B., Feigenbaum L., Lee E., Aoyama T., Eckhaus M., Reitman M.L., Vinson C. (1998) Life without white fat: a transgenic mouse. Genes Dev 12:3168–81. Shimomura I., Hammer R.E., Richardson J.A., Ikemoto S., Bashmakov Y., Goldstein J.L., Brown M.S. (1998) Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: model for congenital generalized lipodystrophy. Genes Dev 12:3182–94.

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case of the Krebs Cycle, the molecule that both enters and exits is called oxaloacetate (no need to remember that name) and has four carbon atoms. The cycle begins when oxaloacetate, with four carbons, combines with acetyl CoA, with two carbons, producing a six-carbon molecule known as citrate (which lends the cycle one of its technical names). This citrate molecule then undergoes a series of reactions we don’t need to see in detail, but that involve two steps in which one carbon is removed from the molecule, generating two CO2 molecules. When six-carbon citrate has completed these chemical transformations and lost two carbons, it regenerates four-carbon oxaloacetate, and this newly formed molecule can again start the cycle by combining with a new acetyl CoA. As you can imagine, deciphering the chemical steps in a cyclic pathway can be trickier than understanding a linear pathway, in which molecules do not participate in more than one part of the pathway, so Dr. Krebs’ ability to imagine this more complicated metabolic construction is most definitely a breakthrough in understanding metabolism.

The Krebs Cycle as an example of a cyclic pathway. In a cyclic pathway, the entering molecule (in this case, acetyl-CoA) is taken up by another molecule that is steadily churned around in a series of transformations in which the atoms of the entering molecule are removed in the form of simpler molecules (in this case, CO2). The energy that held the atoms of the entering molecule together is thus transferred to other molecules (in this case, ATP and NADH) in this process, the initial molecule is recycled and can take another entering molecule into the cyclic pathway

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In addition to producing CO2 and completely breaking down glucose molecules, the Krebs cycle includes a step in which ATP is generated. The two ATPs produced from two acetyl CoAs here (remember that one glucose yields two acetyl-CoAs during glycolysis) add to two ATPs formed in the glycolytic pathway to provide energy for your cells. But this total of four ATP molecules per glucose molecule is far less than glucose produces for us, which is somewhat variable, but in the range of over two dozen ATP molecules per glucose. The rest or our ATP is generated in a process called Oxidative Phosphorylation, orchestrated by our mitochondria, and is such a fascinating and central process that it deserves a chapter of its own.

∗∗∗

Before we move on to understand where and how most of our energy in the form of ATP is made, let us do a brief recap on what happens to the carbohydrates we eat. As we saw, carbohydrates are broken down to their simple sugars in our digestive system, and then absorbed by our blood and distributed to our cells. Within our cells, glucose, the most abundant carbohydrate-derived simple sugar, is converted to glucose 6 phosphate, which is imprisoned within the cell. This “crossroads” molecule can then either become glycogen (a form of storage that animals use to have glucose at hand when in need), follow the pentose pathway and become other sugars or follow the glycolytic pathway and break down into two smaller three-carbon molecules, which are then broken down to two-carbon acetyl CoA and CO2. Acetyl CoA is also a crossroads molecule, capable of generating triglycerides (also known as fat) or entering the Krebs cycle to be completely broken down to CO2. We have followed our carbohydrates on quite a whirlwind of a journey, but the best has yet to come, because we are now ready to understand how these carbohydrates, and all other energy sources we eat, produce the bulk of our energy-packed ATP molecules, within our mitochondria.

Chapter 4

Mitochondria: The Batteries of Our Cells

We just saw how carbohydrates are metabolized, either generating storage molecules or breaking down into the single carbon molecule CO2, which we then expel from our bodies by breathing out. We also briefly mentioned that we break down carbohydrates because this allows us to use the energy generated in this process to produce the energy-packed molecule our cells use to fuel all their functions: ATP. We now need to understand how breaking down carbohydrates to CO2 generates most of the ATP carbohydrates produce. This process happens within mitochondria, a part (also called organelle) of our cells that is responsible for many metabolic pathways (including the Krebs cycle we just saw). Mitochondria also generate the largest part of our ATP, through a process called oxidative phosphorylation. They are our main energy power source; in essence, the batteries of our cells. The name “mitochondria” is the plural for mitochondrion and derived from a Greek description of the shape of these parts of our cells, meaning something like “thread granules”. Mitochondria are tiny parts of our cells, somewhere around 1 micrometer long, which means you would need to line one million of them side by side to add up to 1 m in length. They are separated from the rest of the cells by membranes, structures similar to the skin around an egg yolk. These membranes are very important to generate energy, as we will see a bit further on. Mitochondria vary a lot in size and form, and can be shaped like anything from small spheres to long spaghetti-like strands. They are also dynamic, changing their size and shape all the time. Most of all, they are important. They are essential for all complex life forms (such as us humans), and occupy somewhere around 25% of our cells’ inner space. They have a lot of real estate within cells because they have a lot to do.

© Springer Nature Switzerland AG 2020 A. Kowaltowski, F. Abdulkader, Where Does All That Food Go?, https://doi.org/10.1007/978-3-030-50968-2_4

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cristae

outer membrane

matrix

intermembrane space

inner membrane

Mitochondrial structure. Mitochondria are very versatile in shape and can change their form and size a lot, but a characteristic feature is the presence of two membranes, of which the inner one is filled with folds called cristae. This substantially increases the area of this membrane, and thus provides lots of sites for ATP production, as we will see later

Mitochondrial functions within the cell are quite varied, and we will name and explore just a few of the jobs coordinated by these multipurpose cellular components. They participate in the metabolism of carbohydrates, proteins, and fats. They generate and remove free radicals (we will talk about them later). They control cell differentiation and cell death, including cell suicide. They control immunity, or the ability to fight infection. They control body weight and heat production. Most of all, they transfer most of the energy in the molecules we eat into the user-friendly energy form of ATP. In this last function, mitochondria are indeed the batteries of our cells, not only because they provide energy, but also because they do so in a manner very similar to the way batteries generate energy.

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Batteries work by promoting chemical oxidation and reduction (redox) reactions within them that generate a positive and negative side in each battery. This difference in charge between the positive and negative sides is the source of energy for the equipment they power. Mitochondria are quite similar. They also promote redox reactions that generate a positive and negative side as an energy source to generate ATP and power our cells. To understand how they do this, we should first understand what redox reactions are. Put in very simple terms, redox reactions are reactions in which molecules transfer electrons to other molecules. Electrons are the small and mobile parts of atoms and molecules that have negative charges. Because electrons decrease the

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total charge of a molecule due to their negative nature, their addition to a molecule is known as reduction of that molecule. On the other hand, the molecule that loses electrons is said to be oxidized, a term used because oxygen is very good at oxidizing things (rust is an example). The important part here, however, is not all these names for types of chemical reactions, but the fact that transferring electrons involves a flow of energy. When electrons go from a substance that has less affinity for electrons to a substance that has more affinity, energy is released. In the case of rust, the energy is released as heat. In batteries, this energy is used to power electronics. In our bodies, the energy released from redox reactions is used to power our cells.

Oxidation and reduction as sources of electron flow in a battery. In this cartoon, electrons flow (electrical current) from the negative pole (substance B) to the positive pole (substance A) through the circuit of the fan, transferring energy from the flowing electrons (e) to the fan engine, making it spin. Substance B, which is providing electrons, is said to be oxidized during this process, while substance A, which receives the electrons, is reduced



∗∗∗

While glucose and other molecules are broken down to CO2, they are also oxidized, which means they lose electrons. Electrons are removed from glucose as it passes through the glycolytic pathway, generates acetyl CoA and, mostly, during the Krebs cycle. These electrons are collected by two electron-carrying molecules known by their rather complicated biochemical names of nicotinamide adenine dinucleotide and flavin adenine dinucleotide, or by their acronyms NAD and FAD. NAD and FAD act as electron pickup and delivery systems, taking electrons from the partially broken-down glucose molecules within all chemical pathways we saw, and bringing them to the mitochondrial membrane, where these electrons will participate in the main reactions that generate ATP. In fact, NAD and FAD are

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electron carriers not only for glucose metabolism but also for the metabolism of proteins and lipids, as we shall see in future chapters. NAD and FAD are chemicals formed inside our bodies using nicotinamide (vitamin B3) and riboflavin (vitamin B2) as chemical building blocks. Vitamins are molecules that are necessary for us in some capacity within our bodies, but that we are not capable of producing on our own and must therefore get through our diets. But you don’t have to run to the pharmacy to get your vitamins in pills now that you know that. Most people with healthy, varied, diets have no lack of the vitamins necessary to generate NAD and FAD. In fact, with the exception of specific needs that can be identified by medical professionals, vitamin supplements bring no health benefits, and in some cases may actually be detrimental to your health.

Oxidation and reduction as sources of electron flow in the mitochondrial inner membrane. In this cartoon, electrons flow (electrical current) from the electron carriers NAD and FAD to oxygen molecules through the proteins of the transport chain in the membrane, transferring energy from the flowing electrons to these proteins, making them pump protons (H+) from the inside of the mitochondrion to the intermembrane space, creating an electrochemical gradient that stores energy for ATP production. NAD and FAD are thus oxidized and recycled to take up more electrons in the Krebs cycle, and oxygen is reduced to water



∗∗∗

In 1960, most of the central metabolic pathways had been uncovered reaction by reaction by painstakingly quantifying chemical products in cells, following individual chemical transformations and proposing theoretical sequences of reactions that were chemically possible within what was known to exist in cells, then testing these theories to verify if they were true. The metabolism of glucose as we saw it up to now was fully understood, as was the basic metabolism of proteins and lipids.

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There was just one very important point that was not clear yet, which was how the electrons collected by NAD and FAD during the metabolism of these molecules generated large amounts of ATP within mitochondria. NAD and FAD in their reduced electron-rich forms were known to interact with mitochondrial membranes, in which their electrons were transferred through a series of components in the membrane to the final destination of these electrons in our metabolism: oxygen. Indeed, we need about 500 l per day of oxygen, which we breathe in from the air that comes into our lungs, so that our mitochondria can add electrons to this gas. Mitochondria are constantly reducing oxygen we inhale into water molecules we eliminate through our urine, sweat or breath. Yes, you actually produce water within your mitochondria! It is not as much as you need, which is why you also have to drink water, but it is not an insignificant amount (about 250–300 milliliters a day). Mitochondria are in fact very clean “cell batteries”, since the product of the redox reactions within them is ecologically-friendly water. By 1960, the fact that NAD and FAD brought electrons to mitochondrial membranes and transferred these electrons to oxygen, generating water, was known. These reactions were also known to release energy, which was, somehow, coupled to the production of ATP, also within the mitochondrial membrane. By “coupled”, we mean that the processes of reducing oxygen to water and producing ATP were linked somehow. Scientists could see in their experiments that there was no ATP production without oxygen reduction. Oxygen reduction was also strongly inhibited if ATP production was halted. The fact that these two processes were coupled meant that they were connected by some kind of metabolic phenomenon, and everyone in the field was looking for what promoted the coupling between these two processes. All ATP production described until then, and all metabolically coupled processes, were connected by molecules. For this reason, researchers at the time were seeking a high-energy molecule that linked NAD and FAD oxidation by oxygen to the production of ATP in mitochondria. Because so many high energy compounds in metabolism have phosphate links (including ATP itself), the elusive compound was nicknamed ~P (read as “squiggle P”), referring to some unknown chemical entity with a phosphate. Everyone was looking for ~P because this was the last central obscure step in energy metabolism, and describing it would make the understanding of the degradation of all the molecules we eat complete. But no one could find ~P. If this molecule was so central in the generation of energy for our cells, why couldn’t it be detected somehow? Then a brilliant and somewhat eccentric scientist called Peter Mitchell came up with a very radical idea: what if the link between NAD and FAD oxidation in mitochondria and the generation of ATP was not a molecule, but instead an electrical gradient (or a difference in positive and negative charges) across the mitochondrial membrane? What if ~P could not be found because everyone was looking for a chemical, instead of looking for other ways to store energy such as an electrochemical gradient?

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He wrote about this very radical idea in a paper in 1961, published in the prestigious scientific journal Nature.1 Unfortunately, his idea was not immediately well received. The scientific community found his paper a difficult read (even in hindsight, with today’s knowledge, it still is not easy to understand). The idea was also too different to be accepted immediately within the mindset of that age. To make matters worse, the paper was written by Peter Mitchell alone without any co-authors to support him, and was purely theoretical, without any experiments to prove his somewhat avant-garde theory (for the times, at least). It did not help that Mitchell was a bit of a character too. He had very vast interests, ranging from the origin of life to metabolism to architecture to how people could communicate more effectively. Soon after publishing that very provocative paper on how mitochondria generate ATP, he left the University of Edinburgh, and pursued most of the rest of his scientific interests in a restored mansion in Cornwall called Glynn House, a private laboratory funded by a charitable foundation managed by Dr. Mitchell and his longtime colleague Jennifer Moyle. Glynn House was pretty much his own private laboratory, since it was supported by an endowment made possible by Peter Mitchell himself and his brother. All these characteristics of Dr. Mitchell, plus his rather exotic and completely theoretical theory, made the Chemi-Osmotic Hypothesis (as he called it) very hard to swallow. The idea that the oxidation of NAD and FAD in mitochondria were coupled to ATP synthesis through an electrical gradient across a membrane was only accepted years later, as attempts to experimentally disprove the theory largely failed, slowly opening the minds of the scientific community toward the hypothesis. Mitchell was also helped immensely by plant scientist André Jagendorf, who like many others had initially dismissed Mitchell’s idea. Prof. Jangendorf admits his skepticism openly when describing himself first listening to a talk by Mitchell2: “His words went into one of my ears and out the other, leaving me feeling annoyed they had allowed such a ridiculous and incomprehensible speaker in.” Later, after a personal visit, Dr. Jangendorf was intrigued enough to give the controversial theory a try and conducted a crucial experiment showing that within plant chloroplasts, ATP synthesis was promoted by a gradient across the chloroplast membranes. Soon after, Mitchell and Moyle followed suit and experimentally demonstrated that generating an electrical gradient across the mitochondrial membrane was enough to synthesize ATP, even in the absence of reduced NAD and FAD, or oxygen. The Chemi-Osmotic Theory was well proven by then, and its acceptance by the scientific community was very well established in 1978, when Mitchell was named a Nobel Laureate in Chemistry for this discovery. What we know today is that, as electrons brought to the mitochondrial membrane by NAD and FAD pass through a series of electron transporters (known as the mitochondrial electron transport chain), the redox reactions that occur change the 1  Mitchell P. (1961) Coupling of phosphorylation to electron and hydrogen transfer by a chemiosmotic type of mechanism Nature 191:1448. 2  Jagendorf A.T. (1998) Chance, luck and photosynthesis research: An inside story. Photosynth Res 57:215–29.

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structure of the components of the transport chain, and allow these components to transport protons (which have positive charges, H+) from inside mitochondria to the outside. This makes the mitochondrial membrane negative on the inside and positive on the outside, and generates a form of energy very similar to that in a battery, because now the positive protons on the outside are attracted to the negative inside. Because membranes are not environments protons can go through easily, these protons must go back inside mitochondria by passing through a specialized protein, ATP synthase, which (as the name implies), produces ATP using the energy released by the proton coming back into the negative inside of the mitochondrion.

Coupling of proton pumping through the inner mitochondrial membrane to ATP synthesis. The transport of protons from the inside to the outside of mitochondria powered by the oxidation of NAD and FAD and reduction of oxygen promotes a strong separation of electrical charges across this membrane, since protons are positively charged. The protons pumped out are strongly attracted to the negative charge in the mitochondrial core. They can solve this is by going through a spectacular molecular machine within the same membrane, ATP synthase, so that ATP synthesis then associates (in technical terms, ‘couples’) their flow back into the mitochondrion to the production of ATP

Overall, what this means is that the chemical energy in your food is released by oxidation of this food (using oxygen you breathe) and produces an electrical gradient across membranes in mitochondria (negative inside), another form of energy. This energy is then converted back into chemical energy by producing the phosphate chemical bond in ATP. The energy in ATP is used by the whole cell in a myriad of processes including movement, thinking and building new cell materials. Our cellular batteries (mitochondria) power our lives.

∗∗∗

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At first glance, the idea that oxidation of molecules in our food is coupled to the production of ATP through an electrical gradient across the mitochondrial membrane may seem like an extremely lucky guess by Dr. Mitchell, particularly since he postulated that this happened without any experimental evidence to prove it. But in reality, he creatively based his hypothesis on a good number of already known facts that no one else had managed to piece together, and which he discussed at length in his original publications. If he was “guessing”, it was highly educated guesswork, to say the least, which we scientists prefer to call “hypothesizing”. One piece of evidence scientists already had at that point was that the mitochondrial membrane had to be intact to allow for coupled oxidation of NAD and FAD with ATP production. With broken mitochondrial membranes, oxidation happened (measured typically by following oxygen consumption over time) but no ATP was produced, and instead the energy from these reactions was released as heat. The fact that the membrane had to be intact to produce ATP helped Dr. Mitchell shape the idea that the membrane was needed to separate something between the outside and inside of mitochondria. Indeed, the reactions that lead to the production of mitochondrial ATP all happen inside mitochondrial membranes. This is rather unusual. All other metabolic pathways we saw before happen in water-based cellular environments, such as that within mitochondria (Krebs cycle) or the inner part of the cell (the cytosol, where glycolysis happens). Mitochondrial ATP, on the other hand, is produced inside the membrane, which is composed of lipid molecules that do not mix with water and which comprise a very small amount of the cell’s volume. This again is evidence that these membranes must be important for the production of ATP. So why are the membranes important? If the ATP-synthesis function is lost when the membranes are still there, but broken and leaky (full of holes, such as produced when frozen or treated with detergents), it must be because the membrane is separating something that is really crucial for ATP production between the inside and outside of mitochondria. But what would this ‘something’ that membranes were separating be? Dr. Mitchell must have pondered... A clue as to what membranes separated, which is discussed in the original hypothesis paper, comes from the effect of a molecule named 2,4 dinitrophenol, which has an interesting history that involves everything from explosives to weight loss to the creation of the modern form of operation of the U.S.  Food and Drug Administration (FDA).

∗∗∗

2,4 Dinitrophenol is a molecule that when purified and dry is a yellow powder which is quite explosive (about 80% of the explosive strength of TNT). It also has a number of industrial uses, including the production of dyes, pesticides, and wood preservatives, and can be purchased in large quantities from chemical companies at relatively inexpensive prices. In the early 1930s (decades before Dr. Mitchell’s groundbreaking hypothesis), a group of researchers at Stanford, noticing that workers in factories that used 2,4

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dinitrophenol were unusually slim, tested the metabolic effects of this chemical both on laboratory rats and human test subjects. This was before most rules for lab testing were in implemented. So, as shocking as giving people random chemicals to see what they did may seem now, the researchers of the time were not doing anything illegal, although they were rather daring.3 They found that rats and humans had the same result when treated with low doses of 2,4 dinitrophenol: their body temperatures increased, and their “respiratory quotient” decreased. The “respiratory quotient” is a metabolic measurement of how much of the energy from food is converted to useful energy in a body. If the respiratory quotient decreases, less energy from food is being used for work, and more is released as heat, which explains the increased body temperatures. These results explained why factory workers exposed to 2,4 dinitrophenol were unusually slim – they were unable to use the energy in their food fully, and lost part of it as heat, therefore storing less of this energy as fat. This also gave the researchers an idea that this chemical could be used as a weight loss drug, although at the time they had no clue how it decreased the use of energy in food and increased heat production. Within a year, the group reported excellent weight loss results using 2,4 dinitrophenol in over 100 people. They initially also reported not to have found any side effects. Word of an inexpensive and easy way to lose weight spread quickly around the U.S., and within a year the same Stanford group estimated that 100,000 persons might be using the compound, often prepared locally from the industrial powder and used as self-medication, without strict dosage control or the supervision their group had over the study volunteers.4 The original proponents were now worried about this widespread use of 2,4 dinitrophenol with so little knowledge of its effects, and expressed this officially. Their worries were well founded, since all sorts of undesirable effects were described in the following years, from the development of skin lesions and cataracts in young users to deaths caused by overheating in persons taking higher than recommended doses, in an attempt to lose weight faster. In 1938, the situation with 2,4 dinitrophenol and its use as a weight loss inducer lead to a major change in the manner in which the FDA worked. Up until then, the agency could only post warnings about dangerous foods or drugs, but not control their use. They also had no jurisdiction over cosmetics, as weight loss pills were considered at the time. From 1938 on, the FDA declared all use (even medically supervised) of 2,4 dinitrophenol illegal and subject to prosecution, and its widespread application quickly disappeared. Unfortunately however, it has not completely vanished from the black market even today, and sporadic cases of 3  Cutting WC, Mehrtens HG, Tainter ML, (1933) Actions and uses of dinitrophenol promising metabolic applications. JAMA 101:193–5. Tainter ML, Stockton AB, Cutting WC, (1933) Use of dinitrophenol in obesity and related conditions a progress report JAMA 101:1472–5. 4  Parascandola J (1974) Dinitrophenol and bioenergetics: an historical perspective Mol Cell Biochem 5:1–2.

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intoxication, mainly by persons looking for a quick weight loss fix, still appear in hospitals. The problem is that, while highly toxic, it does in fact promote weight loss quickly, and also can be purchased as a chemical at a low price, prompting dangerous self-medication.

∗∗∗

But how did 2,4 dinitrophenol so effectively promote weight loss, sometimes even to the point of causing death? Dr. Mitchell postulated that this was related to the fact that this molecule can pass inside membranes both when carrying an extra proton (protonated) and also when not carrying this extra proton, due to its chemical characteristics. In doing so, it provided a pathway for the protons outside mitochondria to return inside. This pathway was not the ATP synthase, and therefore did not produce ATP, and instead dissipated the energy in the proton gradient as heat. Essentially, 2,4 dinitrophenol was promoting a short circuit in the battery mechanism of mitochondria, wasting the energy produced by breaking down food, and thus leaving less excess available to produce fat. This short circuit mechanism is known as uncoupling of mitochondria, since in its presence, the oxidation of NAD and FAD is no longer coupled to the synthesis of ATP.

2,4-Dinitrophenol and the action of uncouplers of proton transport in ATP production. As a highly soluble molecule in the inner mitochondrial membrane that can bind protons, 2,4-dinitrophenol creates a proton flow shunt back to the mitochondrial core, deviating from their usual path through ATP synthases, thus removing the energy that fuels ATP synthase to produce ATP

Today, many other uncoupling molecules are known, all with the same property of being able to carry protons across membranes, thus allowing them to enter mitochondria while bypassing ATP production. A few natural uncoupling pathways have

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also been described in mitochondria, which we will talk about later, when we discuss the metabolism of obesity.

∗∗∗

We now understand that mitochondria are indeed the batteries of our cell, and that they have a positive and negative side which fuel the production of ATP, our main source of chemical energy. But our journey into the understanding of how mitochondria work will only be complete when we see the protein that makes ATP in mitochondria and uses the electrical gradient to do so. ATP synthase, the enzyme that does this, is arguably the most beautiful protein in Biology. You can see the mushroom-like structure of ATP synthase in the figure bellow, a large blow-up of the structure a single molecule that all of us have millions of within our mitochondria. The structure here is from the bacteria E. coli, but that really does not matter much because ATP synthase has pretty much the same structure in all living organisms, from bacteria to man. In fact, ATP synthase is present in all classes of life forms on Earth today, and is thus predicted to already exist in the last universal common ancestor (nicknamed LUCA) of all life forms that inhabit the world today. It is a molecule central to life.

Molecular structure of ATP synthase. The portion of the protein shown on the bottom is inserted in the mitochondrial membrane and is where protons pass through it, entering mitochondria. The mushroom-cap shaped portion on the top is inside mitochondria and the place where ATP is formed. The bottom and middle (“stalk”) portions rotate. Prepared using Protein Database (pdb. org) deposit 5T4O (E. coli)

So what is so special about this protein besides the fact that it generates almost all the chemical energy we use? Why are scientists so fascinated by this particular protein’s structure? Because it turns, round and round, spinning at about 130 revolutions every second! The bottom part of the protein (which sits inside the membrane

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in mitochondria) and the “stalk” portion of this mushroom-shaped molecule turn continuously in response to protons passing through it, like an axle in a car. It is this circular motion that changes the structural properties and makes the top part of the “mushroom cap” synthesize ATP. ATP synthase is basically a tiny molecular sized motor. It is somewhere in the range of 45 nanometers tall (a nanometer is one billionth of a meter) and 20 nanometers wide. That means you would have to align 50,000 of these molecules side by side to reach a structure as wide as a meager millimeter. That is a very small motor indeed. And yet it turns, continuously, and does so using the electrical energy from the mitochondrial battery system. This property of the protein is so unique that many scientists are working on how to explore this wonder of Nature in order to make very small (nano) machines. The way ATP synthase works was uncovered by Paul D.  Boyer and John E. Walker, who were awarded the 1997 Nobel Prize in Chemistry for their achievement. Dr. Boyer’s work, which began in the 1950s, was carefully investigating the properties of ATP synthase as an enzyme, describing both how it actually makes ATP (which is a bit advanced for the purpose of this book) and also describing the surprising idea that it had three different portions in its structure that physically rotated in an alternated fashion (each one had a specific property at each specific point of the rotation). Even without techniques to build a three-dimensional model of the protein, Dr. Boyer’s careful study of how the enzyme worked gave him a clear idea that its function involved a protein that turned. He went as far as calling ATP synthase a “molecular machine”,5 and was lucky enough to live and work in science long enough to have his work confirmed by structural analysis of the protein. Dr. Boyer died in 2018, 2 months shy of his 100th birthday, after elucidating not only how ATP synthase works, but also about 20 other enzymes. Dr. Walker, who began his work on ATP synthase in the 1980s, determined the structure of the enzyme, which consists of understanding the organization in space of the atoms it is composed of. This is a scientific challenge given the large size of this protein (relative to other proteins) and the fact that part of it is inside a membrane, which makes structural determination harder. When he did resolve the structure, the reason for the behavior of the enzyme Dr. Boyer had described became immediately apparent. The fact that it indeed rotated was beautifully illustrated by its shape, in which the central stalk changes position within the “mushroom cap” portion of the protein. These structures helped understand the way the protein worked even further, at an atomic level. More than 20  years after becoming a Nobel laureate, Dr. Walker remains the emeritus director of the Mitochondrial Biology Unit in Cambridge and an active scientist, still studying structural aspects of the systems that produce ATP. His research unit is decorated by a large three-dimensional model of the ATP synthase

 https://www.nobelprize.org/prizes/chemistry/1997/press-release/

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made out of LEGO blocks, to a scale of 50 million times larger than the actual protein.6 How cool is that? ∗∗∗



We now know what mitochondria do. They energize cells, acting as an intracellular battery. They do this by using electrons collected by the transporting molecules NAD and FAD from carbohydrates (and other molecules, as we shall see ahead) and reacting these electrons with the oxygen we breathe, generating water. In promoting these redox reactions, mitochondria generate an electrical gradient across their membranes, negative inside and positive outside, which fuels the production of ATP. ATP is produced by ATP synthase, a rotating enzyme that allows protons, attracted by the negative charge of the interior of mitochondria, to enter through its stalk. This generates the energy to make the enzyme turn around and leads to the chemical reaction that produces ATP. How much ATP do these incredible cell parts with battery properties and rotating molecular motor proteins produce? About the same as you weigh, every single day. How is that even possible? It only is possible because every ATP molecule is broken down to ADP and phosphate and then re-synthesized many, many times during a day, and the addition of all that is produced is your weight, but these molecules obviously don’t all exist at the same time within you.

ATP daily balance. Although the amount of ATP we use to keep us alive every day is huge, the actual pool of ATP we have inside us at any given time is small, but constantly recycled at very high rates. This can only be sustained by the extreme activity and efficiency of our mitochondria

The fact that we make this much ATP every day illustrates how important this molecule is as an energy source for all the things that keep us alive. Indeed, ATP is not produced only from carbohydrates, but also from other types of molecules. We will now see how lipids (fats) are metabolized, producing very large quantities of ATP.

 http://www.maxlilley.com/portfolio/lego-exhibition-design/

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Chapter 5

Lipid Metabolism

In July 2010, an article published in the scientific periodical Science garnered attention not only among scientists, but all around the world.1 Lead by Craig Venter and entitled “Creation of a bacterial cell controlled by a chemically synthesized genome”, the article was presented in non-scientific outlets as the description of the first synthetic life form, a lab-made bacterium capable of living and reproducing. Readers were fascinated by the future possibilities of not only manipulating Life but in fact creating it, while critics condemned the work as potentially dangerous. Religious objections were also voiced rather emphatically, even claiming Venter and his group were “playing God”.2 The limits between an endeavor that can be done and one that should be done are debatable and ever-changing, and Craig Venter is certainly a person capable of pushing boundaries in Science. Before creating this bacterium, he proposed to sequence the human genome through a different technique than the NIH-run Human Genome Project, secured private funding to do it and published the complete sequence in parallel with the NIH group lead by Francis Collins, in 2001. He also led projects to sequence the DNA of different organisms that are found in oceans (basically setting out to sequence oceans) and set individual human genome sequencing in motion (starting with his own) for purposes of personalized medicine, among many other boundary-pushing activities. When creating the first lab-made bacterial genome, the group lead by Venter was not fazed by possible controversy, and even marked their genome with a genetic “watermark”, supposedly aimed at setting it apart from the original organism it was constructed to mimic. This watermark contained, in a coded genetic alphabet, the 1  Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang RY, Algire MA, Benders GA, Montague MG, Ma L, Moodie MM, Merryman C, Vashee S, Krishnakumar R, Assad-Garcia N, AndrewsPfannkoch C, Denisova EA, Young L, Qi ZQ, Segall-Shapiro TH, Calvey CH, Parmar PP, Hutchison CA 3rd, Smith HO, Venter JC. (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science. 329:52–6. 2  https://www.theguardian.com/science/2010/may/20/craig-venter-synthetic-life-form

© Springer Nature Switzerland AG 2020 A. Kowaltowski, F. Abdulkader, Where Does All That Food Go?, https://doi.org/10.1007/978-3-030-50968-2_5

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names of all 46 contributing authors of the work and a few quotations. Intriguingly, it also had a secret web address. When accessed, the webpage contained an e-mail, so that readers who broke the bacterial code could send a message to the authors and prove they had, indeed, broken the code. The secrets within this genome only contributed to the hype around the achievement. Although it was a monumental feat described as the first synthetic life form in many news outlets, Venter’s group never claimed to have created life itself, but described it instead as the “creation of a bacterial cell controlled by a chemically synthesized genome”. In other words, a genome had been synthesized chemically, and controlled a bacterial cell. However, the cell itself was not lab-made. Instead, what the group did was produce a very large lab-made DNA molecule which contained all the information this bacterium needed to function (as well as the “watermark” information). They then placed this DNA within a pre-existing bacterial cell. Once within the cell, the lab-made DNA was able to control the cell’s function and cell division, showing that the synthetic DNA molecule had, indeed, all information necessary to maintain and propagate life. It just could not start a living being on its own. But why did they need a ready-made cell to put the lab-made DNA into, in order to make it capable of generating new cells? Although DNA is somewhat of the blueprint for life and contains the information necessary to produce all proteins (and therefore the controlling molecules in metabolism), there are a few functions DNA cannot do alone. One of them, and a vital one for life, is the production of membranes. Membranes are lipid structures (more on that later) that separate one cell from another. They exist in every life form, from bacteria to man, and are essential to separate cells and organisms from one another. Interestingly, although we know how the lipid molecules in membranes are made, scientists today have yet to find out how to build a bacterial membrane (or the membrane of any living organism) without starting with a ready-made membrane. We only know how existing membranes are expanded and replicated. Lipids, it so happens, are still less understood today in terms of structure, function and organization than many other biological molecules, but that does not make them less important, nor less fascinating.

∗∗∗

Lipids, or fats, are often seen as the villains of metabolism, molecules nobody wants to have in their bodies. In reality, these are indispensable molecules, that not only stock up on energy for later use (as we saw before) but also participate in many functions in the cell. Indeed, if it weren’t for lipids, cells could not exist. The term lipid refers to a variety of biological molecules that share the unifying characteristic of being poorly soluble in water. Because they don’t interact well with water, they tend to separate from the majority of the cell, which is composed of molecules dissolved in water. As we saw earlier, this allows our cells to stock up on a large amount of energy in a very efficient way in the form of triglycerides, which are the three-legged spider-shaped molecules we store and call fat. But there is

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another very important component of our cells that is a lipid: membrane lipids, mostly composed of molecules called phospholipids. Phospholipids have structures similar to triglycerides, except that instead of three long carbon chains (the spider leg-shaped part), one of the carbon chains is substituted by a structure containing phosphate (the same phosphate group we saw earlier as part of ATP – Nature tends to repeat itself in structures, even when the function of these structures is quite different). Phosphate is a part of phospholipids that has a negative charge, which therefore interacts well with water. As a result, the overall effect of a phospholipid is a molecule in which a large portion is not water soluble (the two long carbon chains), but one smaller part does interact well with water (the phosphate part known as the polar head). As a result, if you put phospholipids into water, they tend to aggregate in a manner in which their heads interact with water on two sides of two opposite-facing layers of phospholipids, and the carbon chains interact with each other in the space between the polar heads, in a way that does not let water pass. In other words, these molecules line up next to each other and create a barrier between two water-based portions of the cell. This barrier is a biological membrane, and is an essential component of all cells. We already saw how membranes are important in mitochondria, separating a positive and a negative side, and allowing them to function similarly to batteries to produce energy. But membranes do a lot more than that. They separate many different parts of the cell, and ultimately characterize what is the cell and what is its surrounding environment. There is a membrane around the nucleus of the cell, the portion that stores our DNA, with the genetic information on how to produce our proteins. You can think of this membrane as the “skin” that separates the yolk from the egg white. An egg is a very, very large cell, which allows you to see its structure without a microscope, and the yolk is its nucleus. It is separated from the rest of the cell (the egg white) by a membrane.

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Phospholipids and their role in the structure of membranes. Phospholipids are fairly cylindrical molecules in which most of the structure doesn’t interact with water, so water molecules tend to push them away from them. However, at one of its tips, phospholipids have a tiny bit that has phosphate, and that water is attracted to. So phospholipids are shoved around to have their waterinteracting parts facing water spontaneously, while the large bits water doesn’t like are compacted in a double layer of phospholipid molecules. That is why the interior of the membranes is very impermeable to water, ions and other substances that are soluble in water

Cells are also separated from each other by membranes. You may have noticed that eggs have a second membrane right inside the eggshell. This is the cell membrane or plasma membrane, and delineates the area of each individual cell. The separation between cells allows each cell to have a different set of proteins, distinctive metabolism and therefore specific functions. It also protects the content within each cell by creating a thin water-free barrier around it. Phospholipids don’t only protect individual cells, but also our whole bodies. The visible part of our skin, which covers and protects our bodies, is composed of dead cells and has a high content of lipids, including many phospholipids, which came from the membranes of skin cells before they died. This is what allows you to shower or get caught in a rainstorm without dissolving – the lipids keep the outside water away from the water environments that form your cells in the rest of your body. Because all cells have membranes, and almost everything we eat is (or was) alive, any kind of food we eat has some amount of lipids. Obviously, some kinds of foods have more lipids, such as those which come from cells which accumulate a lot of triglycerides. Examples of high lipid foods include animal fat or plant seeds.

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Foods also have variable content of another lipid that we hear a lot about: cholesterol, which is also an important component of membranes.

∗∗∗

Cholesterol is yet another molecule that has a bad reputation but does not deserve it. In reality, it is a lipid all animals have, and it has an essential role in the maintenance of the structure of cell membranes, where it is present alongside phospholipids and maintains the membrane stable, but not rigid, exactly as a membrane should be. Because it is a membrane component, most of the cholesterol present in eggs is within that membrane that separates the white from the yolk, which is the reason eliminating the yolk reduces the cholesterol content in eggs, and egg white consumption became something of a dietary health fad (although, as we shall see, dietary cholesterol does not affect your cholesterol levels very much). Additionally, cholesterol is also important as a precursor molecule to produce some hormones (including sexual hormones estrogen, progesterone and testosterone) as well as vitamin D. The importance of cholesterol is illustrated by the fact that an average human synthesizes about one gram of cholesterol every day, which is a lot more than a person with a reasonable diet eats. Additionally, if you eat a meal high in cholesterol, the production of this molecule by your body is slowed down to make up for the extra cholesterol you ate, keeping the overall amount within you constant. In other words, although cholesterol in foods is demonized as unhealthy, it has a much smaller role in changing your body’s cholesterol levels than the amount you produce, unless of course you have a diet that is very cholesterol-rich. Foods that contain cholesterol are exclusively those that are of animal origin, since cholesterol is a lipid that is only produced in significant quantities by animals. No plant-based food has cholesterol, because plants have a different molecule, phytosterol, which has the same role in plant membranes, but is not used by humans. So if you see a bottle of vegetable oil or a tub of margarine with a text proudly declaring it contains no cholesterol, you certainly know it is a true statement, but can question the company’s bragging rights, given these products never contain cholesterol. In fact, plant phytosterols (which are abundant in fatty plant products such as avocado, nuts and seeds) can compete with cholesterol you eat and result in decreased absorption of cholesterol. As a result, these foods may help reduce cholesterol levels in people with high levels of this lipid in their blood. When you get a blood test to measure cholesterol levels, you usually have the results split between different types of packages of cholesterol, including HDL cholesterol and LDL cholesterol, also known informally as “good” and “bad” cholesterol, respectively. The truth is that the cholesterol molecule is the same in all cases, but the HDL and LDL forms refer to the way this cholesterol molecule is circulating in the blood. Cholesterol does not mix well with water (that is why it is a lipid), so it circulates in the blood associated with many other lipid molecules and with specific proteins that keep these lipids together. These lipid-protein structures are known as lipoproteins.

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HDL is an abbreviation for high density lipoprotein, and refers to a protein-rich lipoprotein that indicates that the body does not have excess cholesterol produced or absorbed from digestion. That is why this form of cholesterol in the blood is known as “good cholesterol”. The presence of high levels of HDL is, in fact, associated with good heart health. You can improve your HDL levels by a number of lifestyle changes including diet and exercise, although there is a strong genetic factor involved, too. On the other hand, LDL is an abbreviation for low density lipoprotein, and is a lipid transport form that takes cholesterol from the liver to the tissues. High LDL levels in the blood indicate that the cholesterol being produced by the liver is not in balance with what is being used in the rest of the body. That is why this form of cholesterol is known as “bad cholesterol”. In fact, high LDL levels are associated with poor heart and circulatory health. On the other hand, LDL is important as a form of cholesterol transportation for its numerous functions in the body. As a result, the “bad cholesterol” label given to this lipoprotein is rather unfair  – you need LDL, just not too much of it.

Cholesterol and its different “transport parcels” in the blood. Cholesterol is a highly insoluble molecule in water that participates in the structure of membranes, as well as acting as the precursor of many hormones and bile salts. It is transported to and from cells in the blood by complex aggregates of lipids and proteins, called lipoproteins. The lipoprotein that provides cholesterol to cells is called LDL, while the one that removes it is HDL

Some individuals have very high levels of circulating LDL cholesterol because they produce cholesterol in their livers, are able to package it as LDL and send it

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into the bloodstream normally, but lack the protein necessary to pick up this cholesterol in the non-liver cells. All cells are able to produce cholesterol, but many rely on the liver’s hefty production to generate a good amount of the cholesterol they need. The lack of ability in peripheral cells to pick up liver-produced cholesterol results in two problems: one is that every cell must now produce all of its own cholesterol, which can stress these cells.3 The second problem is that LDL cholesterol is produced but not used, and therefore LDL remains high in the blood. This condition is associated with damage to your arteries and poor heart health. Thankfully, effective drugs exist for persons with this form of familial hypercholesterolemia (a fancy name for genetic predisposition to high LDL cholesterol in the blood). They act by inhibiting the production of cholesterol in the liver, preventing the accumulation of LDL. Because most cholesterol is produced in our bodies and not ingested, these drugs are often much more effective than restricting cholesterol in food.

∗∗∗

We’ve seen what triglycerides, phospholipids, and cholesterol are. We now need to understand a final characteristic of lipids before we see what happens when we eat them. I’m sure you have heard of saturated, unsaturated and trans fats. Let’s understand what these terms mean. Saturated, unsaturated and trans are terms that refer to the structure of the long carbon chain that many lipids, including triglycerides and phospholipids, have. These long strings of carbon molecules linked to each other are called fatty acids, because they come from fat, and have an acidic characteristic when separated from the rest of the molecules they come from. Fatty acids are not all made equal. Some have only single chemical bonds between the carbons, and are loaded with the maximum number of electrons and hydrogens that a carbon chain can hold. These are known as saturated fatty acids. Saturated fatty acid chains in triglycerides are very flexible in space, because single chemical bonds between carbons can bend around easily. This allows these chains to pack very close together, and makes the lipid more dense when at room temperature. Lipids that are rich in saturated fats are solid (unless heated), and include animal fat (lard) and butter. Some fatty acids have one or more double bonds between carbon atoms, which means they are unsaturated (a term that alludes to the fact these molecules could hold a few more electrons if they were saturated). Double bonds between carbons generate a kink in the long chain of the fatty acid structure, which does not move around in space like a single bond. The result is that unsaturated fats cannot pack together as closely, and therefore are liquid at room temperature. Examples include vegetable oils. Some unsaturated fatty acids have double bonds in places we cannot build within our bodies, but are still important for the structures of our membranes, such as 3  Oliveira HC, Cosso RG, Alberici LC, Maciel EN, Salerno AG, Dorighello GG, Velho JA, de Faria EC, Vercesi AE. (2005) Oxidative stress in atherosclerosis-prone mouse is due to low antioxidant capacity of mitochondria. FASEB J. 19:278–80.

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omega fatty acids. These lipids must be eaten so we can use them, and are present in many foods including fatty fish, nuts and seeds. Trans fats are also technically unsaturated fats, since they have double bonds between carbon atoms. However, these double bonds are not in the cis configuration that is most common in Nature. Instead they are in a trans form. Cis and trans are names given for structurally different positions in which a double bond can be in a molecule. Very few trans fats are produced naturally, but industrial processing (hydrogenation) of vegetable oils (such as in the production of margarine) makes high quantifies of trans fats. The trans configuration is a special one in our diet because this double bond does not make a kink in the carbon chain, but instead keeps it straight, in one place. That means trans fats can pack well together and is the reason why margarine is more solid than the vegetable oil it is made from. On the other hand, trans fats are rare in Nature, so we have a very limited ability to metabolize this kind of chemical bond. That means that once you eat these fats, they tend to stick around longer in your body than other kinds of fats.

Fatty acids and their role in the structure of different lipids. Depending on the way the carbon atoms are linked together in the fatty acid chain, these are classified as saturated or unsaturated. Among the unsaturated, most common types in Nature have cis bonds, which bend the carbon chain and make them loosely packed, and thus liquid. Artificially produced trans-unsaturated fatty acids do not have these bends, and are solid. Fatty acids are integral parts of lipids we have seen before, such as triglycerides and phospholipids

In fact, ingestion of high quantities of trans fats is associated with increased LDL cholesterol levels, and poor heart health. No level of trans fat ingestion is currently

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considered healthy, and processed food containing high levels of hydrogenated oils are best avoided. To a much lesser extent, saturated fats can increase LDL and compromise heart health when in excess (but are fine in moderation), while unsaturated fatty acids are generally associated with good heart health (but also pack a lot more calories than proteins or carbohydrates, so watch out to avoid them in excess).

∗∗∗

We saw earlier that eating carbohydrates makes you happy because we have taste receptors for sugars derived from them, and evolved to perceive their taste as delicious. A similar effect happens with fats. In addition to enjoying eating foods with fats for their smell and texture, humans have also evolved to perceive fats as a taste (although the exact mechanism for this taste perception is still being studied4), and enjoy fatty tastes. This is of course important, since fats are a vital part of our diets – lack of fat in your diet can make you deficient in vitamins that are not soluble in water (vitamins A, D, E and K) and other essential nutrients, including omega-­ unsaturated fatty acids. The problem with fats, as with carbohydrates, is not the act of eating these nutrients, but instead the act of eating them in excess. Again, the reason we enjoy eating fats (and therefore sometimes eat too much of them) is evolutionarily determined. While eating too much fat is undeniably unhealthy for long term living such as we aspire today, short-term fat ingestion has the immediate advantage of being an important source of energy. Indeed, fats have about double the amount of energy (calories) in one gram compared to a gram of carbohydrates or protein. This means that when our ancestors had access to fatty foods, they had a large energetic advantage, and therefore eating these foods helped them survive. The result is that today, when many of us have a surplus of food, our taste buds and brain conspire against us and often stimulate us to eat more fat than we should. While we enjoy eating fats, the very act of eating them also changes the way we respond to them. In the short run, fatty foods change the speed in which food passes through your stomach and intestines, slowing it down. The presence of fat in your stomach and intestines also releases special hormones from your stomach and intestines that circulate in the blood and control hunger within the brain. This gives you a greater feeling of being satiated, and decreases the amount of food you eat in the immediate future. In the long run, on the other hand, eating fatty foods makes you respond differently to the amount of food you eat. Scientists are still working to understand the mechanisms behind this, but it seems that eating a lot of fat in the long term can make your satiety response less accurate. This means that you will no longer feel full when you should, and will therefore be hungry more often. More hunger often means eating more, and more food usually results in more fat accumulation in your body. 4  Liu D, Archer N, Duesing K, Hannan G, Keast R. (2016) Mechanism of fat taste perception: Association with diet and obesity. Prog Lipid Res. 63:41–9.

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The taste for fatty foods is not only determined by what you eat, but also genetically, including by the type of taste receptors you have. In fact, taste preferences seem to run at least in part in families, as indicated by many studies, including those following food preferences of twins5 – identical twins have the same genetic information and tend to have more similar food preferences than fraternal twins, who share the same upbringing but do not have identical DNA. Unfortunately, there are associations between the genetic predisposition to enjoy fatty foods and the development of obesity. Once again, this is a trait that was determined by evolution, but which started to work against us recently, when food availability increased worldwide.

∗∗∗

Lipases, the enzymes that break down lipids, are present in your saliva. So, as with carbohydrates, lipids are digested starting in your mouth. However, most of our lipases are produced by the pancreas and are added to what you ate in the small intestine, meaning that most lipid digestion happens after your food went through your mouth, esophagus and stomach, finally reaching your small intestines. Although lipases are enzymes that break down lipids, which do not mix well with water, these enzymes are water-soluble. This means that lipases can only work on the surface of fat globules in our food. To help out with this, your small intestine also collects bile salts, which are produced by the liver, using cholesterol as a precursor, and kept in your gall bladder until they are released into the intestines. These salts act like detergents, breaking down fat globules to smaller droplets, and helping them have larger surfaces interacting with water, so the lipases can break them down better and faster. That is why a person who has had gall bladder problems such as stones and inflammation is encouraged not to eat large amounts of fats after having the gall bladder surgically removed. Because bile salts cannot be stored without a gall bladder in the amounts necessary to help break down fats, lipids cannot be digested in large quantities. Fats don’t have to be broken down much to be absorbed in our intestines. Most of the lipids we eat are triglycerides, and these have to be broken down into three pieces: two fatty acid chains on their own (free fatty acids), and one fatty acid chain linked to the glycerol that used to keep all three of them together. Although these are still large molecules, around 10–20 carbons each, they can be absorbed because they are largely insoluble in water, and therefore more soluble in the membrane lipids of our intestines. Similarly, phospholipids we eat only have to be broken down into their fatty acids, while cholesterol can be absorbed without being broken down at all.

5  Reed DR, Bachmanov AA, Beauchamp GK, Tordoff MG, Price RA. Heritable variation in food preferences and their contribution to obesity. (1997) Behav Genet 27:373–87.

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Lipases, bile salts and the digestion of fats. Lipases are the enzymes that break up fats into fatty acids that can then be absorbed by the intestine. Bile salts act as detergents, that largely increase the surface area of lipid droplets and thus the access of the water-soluble lipases to their lipid substrate

The intestinal cells that absorb fats from our food pack these fats up in a form of lipoprotein called chylomicron. These lipoproteins are the same form of lipid transport package we saw when we discussed cholesterol transport in the blood, except larger. Chylomicrons are collected by lymph vessels (an alternative form of transport to blood vessels) and then by larger blood vessels. They circulate around the body, and the fats within them can be collected by all sorts of different cells. Within the final cell destination, the fats can be used as a source of energy immediately, or, most frequently, stored for later use as triglycerides. Much of the storage of lipids from our food, in fact, happens within our adipocytes, or fat storage cells. From this description, you may notice that there aren’t a whole lot of metabolic transformations between the lipids you eat and the fat you accumulate in your cells. Basically, the general structure of the fat molecules is maintained. When you have less metabolic transformations, you have fewer chemical reactions in which energy is released as heat, which means you lose less of the energy in the food you ate and use more of it. This means you can use more of the energy from lipids. Add to that the fact that lipids have more calories per gram than carbohydrates or proteins, and it is easy to understand why overeating fats is an effective way to get fat. But the fact you ate fats is not enough to determine how much fat you will accumulate and how much you will break down and use. Hormones such as insulin (the hormone released after you eat, in particular carbohydrates), glucagon (the hormone

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circulating when you haven’t eaten for some time) and adrenalin (the hormone released when you are scared or exercising) also affect how much of these lipids you accumulate or break down. We will talk a lot more about that later, but for now it suffices to say that when insulin is around, you will store lipids. So eating excess fats together with sugars, which release a lot of insulin, such as in delicacies like ice cream or donuts, is a very, very effective way to get fat (but you already knew that, right?). We now know how lipids in our food become fat in our body. Next, we will see how fat is broken down to generate very large amounts of energy for us in the form of ATP.

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Fat droplets in our cells are broken down when specific enzymes that break down lipids are stimulated by hormones. Fittingly, the enzyme within our cells that breaks down these lipids is called hormone-sensitive lipase. The hormones that activate this enzyme are glucagon (the hunger hormone) and adrenalin (the “be alert and act fast” hormone). By breaking down lipids, these hormones ensure that our cells have energy to survive when we haven’t eaten recently or need to be alert and act fast. Lipid droplets that are broken down by hormone-sensitive lipase are all over our body, but in most quantity in two tissues: the liver and adipose (fat) cells. We saw earlier that these were the two main places that produced triglycerides (fat) from carbohydrates. Not coincidentally, these are also the main places triglycerides are stored, and will be used from when you are hungry or frightened. Hormone-sensitive lipase breaks down lipids into a glycerol molecule and three free fatty acids. Glycerol is used for energy, but is quite small compared to the rest of a triglyceride, and therefore much less important. The fatty acids can be broken down to produce energy within the same cell as they were produced in, or the fatty acid can travel in the blood until a cell absorbs it and uses it as an energy source, as is most frequent for fat stored in the adipose tissue. In fact, fatty acids appear in your blood and can be detected in routine blood tests, and their levels increase when you are hungry or scared. Once within the cell that is using a fatty acid as a source of energy, the fatty acid will enter mitochondria, where it will be oxidized to generate ATP. This happens in steps, some of which we already saw, because fatty acid oxidation involves some steps that are the same as carbohydrate oxidation. The first step in fatty acid oxidation to generate energy is called beta oxidation, and involves breaking down the long linear chain of carbons that the fatty acid is composed of into smaller two-carbon molecules. Fatty acids are composed of a long string of carbons, usually between 12 and 20 carbon atoms in humans. Beta oxidation breaks these carbon chains down, taking off two carbons at a time, and adding a coenzyme A molecule to these two carbons. This makes a molecule we already met: acetyl CoA. This means fatty acid metabolism produces the exact same two-­ carbon product as carbohydrate metabolism. In fact, the acetyl CoA produced from fatty acids then follows the exact same path as the acetyl CoA from carbohydrates:

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it enters the Krebs cycle, where electrons are removed from it by electron-­ transporting molecules NAD and FAD. The electrons are then transferred to oxygen within the mitochondrial membrane, and generate ATP through oxidative phosphorylation. All this occurs through the same familiar pathways we saw before.

Fatty acid oxidation and its yield in ATP production compared to glucose. Oxidation of a typical 16-carbon fatty acid molecule, palmitic acid, leads to the formation of 8 two-carbon acetylCoA molecules that can be further metabolized to produce ATP. Meanwhile, oxidation of the 6-carbon glucose molecule yields only 2 acetyl-CoA, as 2 carbons are lost in the form of CO2

The specific pathway for fatty acids is therefore beta oxidation, or the breakdown of the fatty acid into acetyl CoA. Beta oxidation adds an extra level of energy production for fatty acids, because each removal of two carbons from a fatty acid also involves oxidation, with electrons collected by NAD and FAD. This means that fatty acid metabolism generates a lot of electrons to feed into the mitochondrial membrane, and therefore a lot of energy to produce ATP. Fatty acids, and lipids in general, are highly energetic. A typical fatty acid can generate somewhere around four times the amount of ATP that a glucose molecule produces. This is possible because lipids are very rich in electrons, and therefore can reduce more NAD and FAD molecules than carbohydrates. Overall, triglycerides are broken down to fatty acids by hormone-stimulated lipases, which can circulate in the blood to an energy-needy cell. Within this cell the fatty acid is broken down into multiple acetyl CoA molecules, generating reduced NAD and FAD. Acetyl CoAs are also broken down to carbon dioxide in the Krebs

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cycle, generating more reduced NAD and FAD. The reduced transporters feed electrons into the mitochondrial membrane, where ATP is generated in large quantities, given the large number of electrons available. That is how, in a nutshell, you burn off fats. While most tissues go through this process to completely break down fatty acids, the liver does something slightly different, which once again demonstrates its altruistic function. The liver generates ketones, another important source of energy for our cells, and the molecules we will focus on next.

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Ketogenic diets are all the rage at the moment. They promise to cure every and all ailment you can imagine, as well as make you slimmer, stronger, more intelligent and live longer. All sorts of special supplements are sold claiming to make your body do better on this diet, or induce a ketogenic state on their own. Do they work? If so, how do they work? And what are ketones, anyway? As with most, if not all fad diets, we are sorry to state that the hype does not live up to the facts for ketogenic diets. The diet consists of various forms of strict carbohydrate restriction and high fat consumption, conditions which, as we will see shortly, increase your production of ketones. The fad around this dietary intervention seems to have begun from the real scientific finding that a specific group of patients who have seizures which do not respond well to medication can see some benefits when on a ketogenic diet. The fad may also have grown from the fact that diets poor in carbohydrates tend to lead to weight loss (as we will see later in more detail). However, an important point this trend ignores is that not every form of weight loss is desirable, nor healthy. It also overlooks the fact that a dietary intervention that has positive effects for a specific disease is not automatically a dietary intervention that is good for anything and everybody. So what exactly are ketones, and why does eating a lot of fat and no carbohydrates make us produce more of them? Ketones are produced in the liver when it breaks down fatty acids to acetyl CoA through beta oxidation. Instead of further breaking down the acetyl CoA produced, the liver, in its altruistic function of providing energy for other organs, joins two acetyl CoAs together, producing four-­ carbon molecules (acetoacetate and β-hydroxybutyrate), known collectively as ketones. Ketones are then released from the liver into the blood, and can easily go to many other organs, since they are water-soluble (while fatty acids are not). In other organs, they are re-converted to acetyl CoA, enter the Krebs cycle and generate ATP for that cell. Basically, ketones are a liver export product that can be used as a handy source of energy by other organs. Skeletal muscle and heart are organs that use a lot of ketones donated by the liver, although they are useful for many other parts of our body, including the brain. The presence of ketones in the blood is normal under fasting conditions (when we are burning fat) and is part of the physiological regulation of energy sources within us.

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Ketone production in the liver and its use by other organs. Ketones are produced from fatty acids in the liver, whose incomplete break-down there generates acetyl-CoA molecules, which are then condensed into ketones

By eating a diet very poor in carbohydrates, a person restricts their production of insulin (more on that later), while keeping glucagon (the hunger hormone) levels high. If you add a very high amount of fat to this same situation, you are stimulating a lot of fat metabolism, both by making fat available and by promoting the hormonal condition that stimulates this form of metabolism. The result is that by eating almost no carbs and a lot of fat, you stimulate your liver to produce a lot of ketones. That is how ketogenic diets work. But the fact that ketones are normal, natural, metabolites (products of metabolism) does not mean that they are all good all the time, nor that keeping their levels high through a diet is desirable. In fact, some very undesirable conditions involve increased ketone levels. Diabetics can go into ketoacidosis, which is a life-­ threatening medical condition promoted by their inability to produce insulin (more on that later). Ketoacidosis, or the excess of ketones above levels found in normal times between meals, is a medical emergency that can also occur in anorexia, some inherited metabolic diseases and even in healthy people on ketogenic diets. In 2004,6 a previously healthy 40-year-old New  York woman sought medical attention because she had severe shortness of breath, nausea and vomiting. When 6  Chen TY, Smith W, Rosenstock JL, Lessnau KD. (2006) A life-threatening complication of Atkins diet. Lancet. 367:958.

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examined, she was found to have dangerously acid blood and very high ketone levels, but she had no history or sign of being diabetic or having a metabolic disease. When questioned, she reported she had been diligently following a diet (the Atkins diet) with no carbohydrates and very high fat content, including monitoring the presence of ketones in her urine, which had increased largely over the month she had dieted. This woman was the first reported case in the medical literature of ketoacidosis as a life-threatening medical emergency promoted by diet alone, without a metabolic disease to promote it. Luckily, after her acute ketoacidosis was stabilized and she was advised to follow a more balanced diet, her condition normalized completely. However, other case reports of ketoacidosis as a medical emergency in people following ketogenic diets have appeared after this, showing that this is a possible complication, albeit a rare one. Even if a person does not end up in the emergency room on a ketogenic diet because of very abnormal ketone levels, this form of diet still may have less severe complications. The ketones we produce as forms of energy are 4-carbon molecules, but they can spontaneously lose one carbon and become a 3-carbon molecule called acetone. Acetone is a chemical found in many places, including nail polish remover, and which can be quite toxic when in high quantities. It is the reason for the foul smell in the breath of an uncontrolled diabetic or a person who hasn’t eaten for a long time. It is reactive, which means it can react with and change molecules in our body in undesirable ways. You can also have tissue damage due to changes in the blood acidity in ketogenic diets. Finally, the lack of insulin and low blood sugar can lead to confusion (“brain fog”) and loss of muscle mass. You will certainly lose weight by cutting carbohydrates completely from your diet and eating large amounts of fat, but is it worth all these complications? We think not. We will talk a lot more about dietary interventions and how they affect your body when we discuss the metabolism in obesity. But first, we have to see how our final group of molecules found in food are metabolized: proteins.

Chapter 6

Protein Metabolism

Protein supplements were a 12.4-billion-dollar industry in 2016, and are projected to grow more than 6% a year until at least 2025,1 given their perceived health effects. Composed of different proteins or amino acids (the building blocks of proteins), these supplements promise to promote muscle growth and repair, prevent joint pain, satisfy food cravings, help lose weight, generate perfect skin and all sorts of other health benefits. But do they work? In order to understand what supplements can do and cannot do, we need to understand what proteins are and how we metabolize them. ∗∗∗



Proteins are all over the place within living beings. All the enzymes we saw up to now transforming molecules within us are proteins. Many hormones are proteins too, as are the molecules that participate in the transport of metabolites, in immunity, in movement, in thought development, and basically every other thing that a living being does. The reason proteins participate in so many different functions is related to the fact that they can have many different shapes and, as a result, very different biochemical properties and purposes. Proteins are produced based on information in our DNA, and are composed of sequences of amino acids linked together, side by side, forming a long strand. Amino acids are, therefore, the building blocks of proteins. This strand of amino acids then folds itself in space and acquires all sorts of different shapes that proteins have. There are 20 different amino acids in our proteins. They all have both an acid and an amino group (hence their name), and differ by their side chains, the third part of an amino acid, which has variable chemical characteristics and sizes. The changes in characteristics of the side chains are what makes a protein have its particular shape and function. The combination of 20 different possibilities of side chains for  https://www.grandviewresearch.com/industry-analysis/protein-supplements-market

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© Springer Nature Switzerland AG 2020 A. Kowaltowski, F. Abdulkader, Where Does All That Food Go?, https://doi.org/10.1007/978-3-030-50968-2_6

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each of the amino acids in a protein chain (and there are hundreds or thousands of them) leads to an enormous number of potential chemical characteristics, and therefore, a very wide variety of biological functions. On the other hand, the amino and acid groups that all amino acids have are all pretty much the same, and are what is used to chemically stitch them together.

Amino acids, peptides and proteins. There are 20 amino acids, and the only thing that makes them different from each other is the composition of the side chain, which are represented in the picture by –R.  When connected through the amino group of one amino acid linked to the acid group of the other, amino acids form chains called peptides if short, and proteins, if long. What makes one protein different from another is their amino acid sequence, that ultimately determines their shape and, thus, their function

Many of the molecules we have seen up to now have acid characteristics (including fatty acids). Although the term “acid” is often associated with dangerous substances in everyday life, having these biological acids within us is not something to be afraid of, first because biological acids are weak, unlike strong acids that can destroy your tissues, and also because our bodies have a lot of mechanisms to keep your cellular acidity balanced. Therefore, acid groups within these molecules are normal chemical properties of our molecules, not new to us, nor different in terms of metabolism. Meanwhile, the amino group is different, because it has an atom that other molecules we have seen before do not usually have: nitrogen. While our bodies can change molecules around by rearranging atoms, it cannot change atoms into other

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atoms by promoting atomic fission (such as that which generates energy in nuclear reactors) or fusion (which powers the sun). Carbohydrates and lipids typically do not contain nitrogen, but proteins do, because this atom is part of the amino group structure. This means that, when proteins are metabolized and transformed into lipids or carbohydrates (humans can do both these transformations, as we shall see), the nitrogen atoms of the proteins have to be removed and somehow eliminated from our bodies. This also means that proteins within us cannot be produced from lipids or carbohydrates, because they lack nitrogen atoms.

Possible interconversions between the three groups of energetic nutrients within human metabolism. Proteins, amino acids, carbs, and fats can be used to provide energy for ATP synthesis, but they are not freely interconvertible. Carbs can become fats, and amino acids can become either carbs or fats, but the reverse processes are not possible



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Proteins also begin their metabolism in your mouth: saliva has proteases, enzymes specialized in breaking down proteins. This means that some amino acids are released from proteins when we start chewing on protein-containing food (which is basically any food, albeit in varying quantities). Your tongue has a type of receptor that detects one of the more common amino acids, called glutamate, and produces a taste known as umami, which is quite delicious to us. Once again, this means that eating proteins makes us happy as soon as we start to chew. And once again, this is a trait that was selected by evolution to guarantee that we eat proteins, which helps us survive.

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The fact that the amino acid glutamate is perceived by us as a delicious taste is used as a mechanism to enhance flavors through the addition of MSG to foods. MSG, a popular cooking additive, is an abbreviation of monosodium glutamate, and is a salt form of glutamate that you can add directly to food. When this added glutamate reaches your tongue, it activates the umami receptors and results in an agreeable taste perception. As a result, it is used to give extra flavor. MSG was first proposed for use as a food additive in 1908 by Japanese biochemist Kikunae Ikeda, who searched for components in a popular seaweed that were the reason it gave such agreeable flavor to foods. Ikeda found that the seaweed was rich in glutamate, and that the purified amino acid also promoted a nice taste perception when eaten. He then patented the use of MSG as a food supplement. The use of MSG grew over decades, given that it actually does give food a flavor humans enjoy. As its use grew, MSG also caused controversy, and was anecdotally associated with a series of undesirable symptoms including headache, flushing and palpitations soon after its ingestion, known popularly as the Chinese Restaurant Syndrome (because MSG is commonly used in oriental restaurants). Despite many such descriptions, strong scientific evidence linking these immediate physical effects to MSG itself is still lacking.2 MSG was shown in the 1970s3 to promote obesity in laboratory animals when injected into them soon after they were born, due to its effects on the brain. However, this happens only with injected MSG in newborns, and not with MSG ingested with food or in adults. As a result, the additive is classified as generally safe by most reputable medical groups and the FDA. MSG is not the only food additive that uses our taste perception of amino acids. Aspartame is a molecule composed of two amino acids linked together (aspartate and phenylalanine). Although it is made from amino acids, it produces a sweet taste for us humans, similar to sugars. Sweet tastes resulting from amino acids are another adaptation of evolution to make us enjoy and therefore eat proteins, which helps us survive. The fact that aspartame tastes sweet without being a sugar has been used commercially since 1965, when it was first introduced as a sweetener or sugar substitute. Although the chemical link between the amino acids in aspartame is not stable enough to allow it to be cooked or baked like table sugar (later studies developed new sweeteners that are heat-resistant), this molecule is still useful in uncooked foods, mainly for persons such as diabetics who should not ingest sugar. Because aspartame is much sweeter than ordinary sugar (about 200 times), it also requires much less quantity to be perceived by us, which means you get less calories from aspartame for the same amount of flavor. This has long been used as a manner to decrease the number of calories in sweet foods for people trying to lose weight. Interestingly, however, although aspartame consumption at the normal levels found in foods is considered safe by most reputable health-monitoring agencies 2  Obayashi Y, Nagamura Y. (2016) Does monosodium glutamate really cause headache?: a systematic review of human studies. J Headache Pain. 17:54. 3  Bunyan J, Murrell EA, Shah PP. (1976) The induction of obesity in rodents by means of monosodium glutamate. Br J Nutr. 35:25–39.

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based on many scientific studies,4 many other reports have shown that consuming foods with this sugar substitute does not help prevent obesity nor diabetes. In other words, the use of aspartame is not harmful in itself, but it also may not help prevent diseases, which is the reason many people consume it. Unfortunately, similar results are seen with other sugar-substitute sweeteners. The reasons why substituting calorie-rich sugar for calorie-poor sweeteners is ineffective in terms of health benefits are not yet clear to researchers (but we are working on it!). Most of these results come from studies that follow human habits, and it may just be that people who choose to use sweeteners do so because they are prone to obesity in the first place. Sweeteners may also give people the impression they can eat foods without guilt, but in reality they just substitute sugar, while the foods that contain them can be caloric in many other manners. Indeed, products with the “diet” label do not contain sugar and are adequate for sugar-free diets, but may actually contain more calories from fats or proteins than non-“diet” products. Finally, sweeteners may also change the bacteria in our gut and modify the way our brain perceives our energy needs and translates them into hunger, both characteristics linked to diabetes and obesity. Overall, proteins are broken down in our mouth and give us agreeable taste sensations. This is a useful evolutionary trait to make us eat enough protein, which we need, but also is a characteristic used by food additives to make eating more pleasurable. However, although some break-down of proteins happens in the mouth, resulting in an enjoyable taste, most protein digestion happens in the stomach and intestines.

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In your stomach, an enzyme called pepsin breaks the long strands of amino acids in proteins to smaller strands (called peptides). Then, in the intestines, a group of different proteases (enzymes that digest proteins) complete the job and break proteins down completely to their individual amino acids. These amino acids (all 20 different types we saw make up a protein) are absorbed by your intestines, reaching your blood and circulating around your body until they are further metabolized. Knowing that proteins must be broken down into amino acids to be absorbed explains many properties of proteins as medications and within nutrition. This is the reason why medical drugs that are proteins, such as life-saving insulin (a necessity for type 1 diabetics), cannot be taken orally. Ingested insulin would be broken down to its amino acids, which are exactly the same amino acids found in any other protein, and therefore would not be insulin anymore, but instead the building blocks that produce proteins. That is why insulin and other protein drugs such as gamma-­ globulin or some vaccines are typically injected, not ingested. It is consequently understandable why drugs that are proteins don’t work when you eat them, because they are broken down to their building blocks, or amino 4  Lohner S, Toews I, Meerpohl JJ. (2017) Health outcomes of non-nutritive sweeteners: analysis of the research landscape. Nutr J. 16:55.

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acids. The same is true for any protein ingested, including some highly popular dietary supplements found at your local pharmacy. While drugs are highly regulated (for example by the FDA in the USA) and must prove they are effective in treating a condition, supplements are typically sold without having to prove that they work. Astoundingly, in some countries supplements do not even need proof that they are not harmful to be marketed! This makes the supplement market particularly prone to selling hyped, ineffective, and sometimes dangerous products. A popular supplement which promises more than it can deliver is collagen, which is a protein. While collagen is an extremely important protein within us (it is our most abundant protein), and maintains the structure of our skin and joints, eating collagen in the form of a supplement will not result in these same collagen molecules reaching your skin or joints. Instead, the collagen protein you ate will be broken down to amino acids, which circulate in your blood, and can then be used to build any protein within you (which may or may not be collagen) or can be used to build lipids or carbohydrates (as we shall soon see). So unless you are deficient in protein ingestion (and if you have a balanced diet, you are not), collagen supplements will do nothing for you, even though they are sold as the solution for wrinkles and achy joints. Even if you believe you are deficient in the amino acids in collagen after all that we just stated here, and really want to take a collagen supplement, you can do yourself a favor and eat gelatin instead, since it is made from skin and joints and is basically collagen, and therefore is a much cheaper way to get the same amino acids. Finally, collagen supplements are also sometimes sold as “hydrolyzed collagen”, which basically means that the collagen in them is pre-digested to its amino acids. This is of no use at all, because amino acids can become many different molecules, not only collagen. In fact, if you need the protein to be hydrolyzed to be able to use it, you have serious digestive issues that cannot be fixed with a supplement. Although as a supplement it is rather useless, collagen actually has an interesting structure and history. When explorers navigated the World in the the eighteenth century, a Royal Navy surgeon named James Lind decided to study the effects of nutrition on scurvy, in a process that is described by many as the first reported controlled clinical trial to treat a disease. Scurvy is an illness described since antiquity (including by Hippocrates) and involves loss of integrity of the skin and other surfaces, including the mucous membranes within the mouth and nose. Symptoms include thin and brittle skin, poor wound healing, swollen gums and nose bleeds. Scurvy was frequently observed among sailors, who typically had poor diets while at sea, and was known since the seventeenth century to be improved by citrus in the diet. Lind believed the acid in citrus was somehow responsible for the improvement of scurvy, and separated sailors with scurvy into groups that received different acids, including vinegar and even vitriol, which is diluted sulfuric acid. He found that only the group that got citrus fruit (oranges and lemons) as the acid source improved from scurvy, and concluded it was not caused by lack of acid in general, but instead by the lack of a citrus fruit component. It took some time after his findings, but by the late 1700s lemon juice began to be widely used during long-­ term voyages to prevent scurvy, with very positive results.

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Today we know what causes scurvy and why it can be prevented or treated with citrus juice. Citrus fruits are rich in vitamin c, which is necessary to produce collagen. Collagen is a protein produced from the same amino acids as any other protein, but after it is assembled, some of these amino acids are modified by enzymes that require vitamin c to work. If vitamin c is absent, collagen cannot be completely produced, and the lack of adequate collagen leads to thin and brittle skin, seen in scurvy. In other words, if you want to make sure you are producing collagen well, making sure you eat fruits that are rich in vitamin c is much more effective than taking collagen supplements. There is no need to take vitamin c supplements, which contain too much of it, and which will just be eliminated in your urine.

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Amino acids from proteins you eat will circulate in your blood after meals, and can follow many different metabolic pathways, resulting in the production of different molecules. Many of these amino acids will be incorporated in proteins. Our body continuously produces proteins for the different functions they have, replenishing proteins that are old and dysfunctional, or creating new functional proteins for new roles within our cells. Building proteins requires amino acids as building blocks, and these amino acids frequently come directly from our diet and are taken up from the blood by our cells. In fact, building proteins in our body requires eating proteins, since amino acids cannot be produced from other types of molecules we eat. However, the reality that we need to have amino acids as materials to build proteins does not mean that increasing the levels of amino acids in our diets will necessarily increase protein production. Delivering bricks at a construction site is necessary to build a brick wall, but not sufficient for building it. Likewise, eating amino acids is necessary to build protein, but not enough. The production of every individual type of protein is regulated in different manners, and although protein production in general is boosted by some metabolic hormones like insulin and thyroid hormones, the production of each individual protein needs other specific cell signals to happen. For example, the proteins that make up our muscles are long fibrous structures that slide alongside each other to contract or extend a muscle. That is what makes you move. These proteins need amino acids to be made, just as every other protein, but unfortunately you will not beef up your muscles just by eating a beef steak, taking a whey protein supplement or ingesting any other source of abundant protein. Muscle growth requires signals within the muscle, such as those induced by exercise, for example. If you don’t have the signals but eat a whole lot of protein, the amino acids in the protein you ate will not go into your muscle. They can be incorporated into other proteins and, when in excess, stored as other molecules. What other molecules? If you guessed that the excess protein you eat typically ends up being stored as fat, you got it right. It is a sad but true metabolic fact that our bodies are very good at storing anything we eat too much of as fat. This knowledge of course should make you question what all those protein supplements, that are sold supposedly to make your muscles grow, are good for. The

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truth is that whey protein supplements are a smart sales idea, that uses leftover protein from dairy industries that was previously used for animal feed. They are not necessarily bad for you (although there is some still inconclusive evidence they can increase kidney disease), but if you have a typical western diet, you probably have more than enough protein from your food, and adding a protein supplement powder will not make you build any more muscle than you already would build from your normal exercise. The exact same amino acids that are in these supplements, in fact, can be found in foods with high protein content, which are both more affordable and, most importantly, much more tasty to eat.

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In addition to being incorporated into proteins, we mentioned that amino acids from your food can become other molecules, including both the carbohydrate glucose and fats, mainly triglycerides. This often happens after amino acids are absorbed into your blood from your intestines. As with any other molecule absorbed from food, the amino acids from your intestines go to the blood and pass first through the liver before going to the rest of the body. In the liver, these amino acids can stay in the liver itself – this organ produces many proteins for its own function as well as proteins that circulate in the blood. But the presence of amino acids in the liver also activates a metabolic pathway that initiates the transformation of amino acids into other molecules. As we saw earlier, proteins and amino acids contain nitrogen, an atom lipids and carbohydrates don’t have. This means that in order to transform amino acids into fats or carbohydrates, the nitrogen atom must be removed. The removal of nitrogen from amino acids happens in the liver in a metabolic pathway called the urea cycle, which, as the name implies, produces urea. Urea is the form in which the nitrogen atom of these amino acids is removed through our urine, and the reason urine has its distinct color and smell. Interestingly, the urea cycle is regulated by the presence of amino acids in the liver. The higher the amino acid levels in the blood, the higher the amino acids in the liver, and consequently the higher the activity of the urea cycle. Thus, the more protein you eat, and the more that is left over after proteins are made, the more nitrogen you eliminate from these amino acids, transforming them from building blocks for proteins to building blocks for fats or carbohydrates. This is the mechanism in which eating excess protein makes you accumulate fat, and not buff up your muscles. Amino acids in your blood, apart from making proteins, can be transformed into either fat or carbohydrates, specifically the carbohydrate glucose. The “metabolic decision” regarding which of these destinies will be predominant depends on the presence of hormones. In the presence of insulin, the hormone that is released when your blood sugars are high, the excess amino acids you ate and that had their nitrogen removed will generate fats, which will be stored for later use. Since people usually eat meals that have both proteins and carbohydrates, excess protein in your food very often becomes fat.

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When insulin levels are low and glucagon, the hunger hormone is high, amino acids that lost their nitrogen atoms can become the carbohydrate glucose, through a process called gluconeogenesis (or the new formation of glucose from molecules that are not carbohydrates). This metabolic process occurs in the liver and is vital to keep blood sugar levels stable when we haven’t eaten carbohydrates (remember we have cells, including in our brain, which only use glucose as an energy source). Gluconeogenesis occurs both when you eat a meal with protein in it, but no carbohydrates, and when you did not eat at all. When you are hungry and have no amino acids from your food in the blood, gluconeogenesis needs a molecule to start out with to make glucose, and the molecules used are amino acids from proteins within us. Since we do not have storage protein molecules, when we are hungry, proteins from our muscle are broken down, and their amino acids go to the liver, where the nitrogen atom is removed to form urea and the rest of the amino acid is transformed into glucose, keeping your blood sugars stable.

Proteins, amino acids, and their metabolic fates within the body. Proteins within your cells are constantly rebuilt and degraded into amino acids. These can leave cells reach the liver, where they can undergo different transformations, depending on the prevailing signals

At this point you are probably worried about losing muscle tissue with this process every time you get a bit hungry, but it isn’t really something to worry about, just a normal process. These same muscle proteins will be rebuilt the next time you eat, in a process stimulated in part by insulin released as a result of the carbohydrates in your next meal. Just like growing and shrinkage of fat molecules or glycogen, the use of muscle for gluconeogenesis is normal and part of the cycles your body goes through daily.

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In summary, we saw that proteins we eat become amino acids in our digestive system and can then be metabolized in our bodies to build new proteins, build fats or glucose, depending on how much protein you ate and the levels of your hormones. That is an overview of protein metabolism, in a nutshell. We will now look at how a specific amino acid called phenylalanine is metabolized in order to understand why some people cannot eat this amino acid.

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You may have noticed that many foods contain a warning on it: “Phenylketonurics: contains phenylalanine“and may have questioned if you should be afraid of food that has such an ominous warning full of complicated words. You shouldn’t. Phenylalanine is the name of one of the 20 different amino acids we have seen that are the building blocks for our proteins. All of us have quite a bit of this amino acid within us, and almost all of us can eat phenylalanine without any worry whatsoever. In fact, we do so every time we eat animal products and some vegetables which are rich in this specific amino acid. However, about 1 in every 12,000 persons is born with a metabolic defect called phenylketonuria (or PKU, for short). These persons lack an enzyme called phenylalanine hydroxylase (yes, scientists love difficult names…), which makes them unable to metabolize, or break down, the amino acid phenylalanine. For every one of the 20 amino acids we have, there is a group of enzymes which form a metabolic pathway to break down that specific amino acid. We aren’t going to talk about these pathways, because they are way beyond the scope of this book, but we now understand enough about metabolism to realize that if you don’t have an enzyme necessary to break down phenylalanine, and you eat more phenylalanine than you can incorporate into your proteins, this amino acid will accumulate, because the pathway for its metabolism is not there. Phenylalanine accumulation leads to devastating consequences: it stunts brain development, leading to severe cognitive limitations, seizures, tremors, stunted growth, vomiting, and a musty odor which is a consequence of the accumulation of this amino acid. It also leads to skin and hair discoloration because melanin, which gives us skin pigmentation, is derived in part from phenylalanine. But, thanks to Science, we don’t have almost anyone around with these awful symptoms of PKU anymore, although about 1 in 12,000 people worldwide are born with it. That is because we know how to diagnose and treat this disease. PKU was first understood in 1934 by Norwegian Ivar Asbjørn Følling, who noticed that some children with developmental delays had very high phenylalanine and molecules derived from it in their blood and urine. By the early 1950s doctors recognized that a diet very low in phenylalanine could help these children, but they could only provide better management for their symptoms with this diet at the time the children were diagnosed, when they already had significant brain damage. The problem then was that phenylalanine is present in human breast milk, which is the most complete and healthy food for most newborns, but leads to brain damage in babies with PKU. A way to identify these children before they had symptoms was

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necessary. An American medical doctor, Robert Guthrie, invented such a method, using small blood samples collected on filter paper from a needle prick to inexpensively test for high phenylalanine in newborns. This not only allowed for the identification of newborns with PKU, but also introduced the practice of newborn screening for diseases, which has grown over the years to encompass many other disorders. Newborn screening for PKU is now required almost worldwide, and in many countries, babies are not released from maternity wards before getting its results. That is a safeguard to protect children against severe brain damage. Newborns identified with PKU are fed a special formula that has very low levels of phenylalanine, and are later introduced to a diet poor in this amino acid, excluding all foods that contain the warning label regarding the presence of phenylalanine, as well as most animal protein. With this early dietary intervention, they can lead normal lives. The global use of newborn screening tests for PKU is a wonderful example of how Science can cause a strong impact on lives.

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Gout is another disease that can be controlled in part by a diet with lower amounts of animal protein, and that is much more common than PKU. It involves sudden and intense pain attacks in the joints, with swelling and redness. It often is most intense in joints that are colder, such as those in hands and, especially, the feet. With time, it can lead to chronic pain, arthritis and stiffness. The inflammation in gout is associated with the accumulation of uric acid in the joints, which is another molecule we produce that has nitrogen atoms in it and that we normally eliminate in our urine (hence the name). Gout is caused by many genetic and lifestyle factors, including by the person’s diet. Additionally, once symptoms begin, they can often be controlled at least in part by a diet with low meat content. So what does gout and uric acid formation have to do with proteins in meat? The answer is nothing – the proteins are not the problem. Coincidentally, meat also has a high content of purines, the molecules that generate uric acid when broken down. Purines are a large group of molecules which include our energy currency molecule ATP (and there is a lot of ATP in meat) and our “recipe book” molecule DNA. That is why reducing the amount of ATP-rich meat and other purine-rich foods (including some vegetables) reduces bouts of gout.

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Having seen that diets high in animal protein sources can increase chances of developing gout (in conjunction with other genetic and lifestyle factors), and that eating a lot of protein does not increase your muscles (but instead generally leads to fat accumulation), you may ask yourself if you should forego animal protein altogether. Indeed, vegan and less restrictive vegetarian diets have often been portrayed as healthy options.

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While a number of studies have shown that the typical North American vegan or vegetarian is healthier than the typical North American fast-food omnivore, these are certainly not the most equitable comparisons to determine the role of animal products in health. Indeed, when more similar lifestyles and food variety are compared, there seem to be no overt health differences between vegetarians and non-­ vegetarians, while strict vegan diets may compromise some health aspects, due to lack of certain vitamins and possible low protein content and amino acid variety. Overall, while there are solid arguments to be made for vegan diets based on ethical and ecological principles, these justifications do not seem to extend to health aspects. As we shall see in future chapters, most healthy options are those that include a variety of nutrients, from a wide variety of sources. These diets should include protein (because protein is our main source of nitrogen, and can only be produced from proteins in our diet), with all the amino acids we need (some vegetable protein sources do not have all amino acids), but do not require excess protein. As with most dietary advice, moderation and variety is key.

Chapter 7

Alcohol Metabolism

Alcoholic beverages have long been appreciated by humans. Jugs from the late stone ages with traces of fermentation products in them have been found, evidence of purposeful production of alcoholic beverages. However, even the 12,000 years that separate us from this age are short in terms of evolution. In other words, we started producing beverages with high content of ethanol (the main alcohol in these drinks) much later than we became evolutionarily equipped to metabolize alcohols. All animals have enzymatic pathways to metabolize alcohol, since all animals will eat something slightly fermented or rotten once in a while, containing alcohols. This shows that metabolic pathways to metabolize alcohol are important to survive across a wide range of organisms. The reason for the existence of metabolic pathways that involve alcohol is not only that alcohol can be found spontaneously in foods that animals eat, and therefore must be amenable to being metabolized or eliminated from these organisms, but also because alcohol is an excellent source of energy. Using this chemical energy gives the animals that ingest alcohol an evolutionary advantage.

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Humans started to use fermented foods early on not only because they enjoyed the effects of ingesting alcohol, but mostly because fermented foods have longer shelf lives than fresh foods. Fermentation is a process in which microorganisms metabolize the nutrients in a food, generating products such as alcohol (in wine, beer and bread) or lactate (in yoghurt), as well as smaller quantities of other molecules that give these products their unique tastes. We then eat both the microorganisms that grew in this food and the nutrients that are left over, as well as those they produced, using all of these molecules as fuel for our bodies. The microorganisms in fermented foods do not cause disease and are healthy for us. They also grow fast and release molecules that inhibit the growth of other microorganisms, including those that promote food spoilage and cause food poisoning. The result is that fermented foods tend to last longer than non-fermented foods. This © Springer Nature Switzerland AG 2020 A. Kowaltowski, F. Abdulkader, Where Does All That Food Go?, https://doi.org/10.1007/978-3-030-50968-2_7

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was vital for human survival during most of our history, in which refrigeration, canning and other modern conveniences were not an option to conserve food. In fact, during most of our recorded history, drinking fermented liquids such as ale or wine was often much safer than drinking water, since purification methods for water were not yet developed. Alcoholic beverage drinking was, consequently, standard practice for most urban dwellers, including children. Not only do alcoholic beverages have less pathogens than untreated water, but also fermented foods last better than the fresh foods they were produced from: yoghurt and cheese last longer than milk, pickles last longer than fresh cucumbers, etc. But why do humans like alcohol so much, to the point we risk getting drunk? Well, before getting drunk most people go through a state in which they feel good, happy and relaxed, as if their inner brakes were lifted off. This is indeed true, as seen when we look at our brain circuitry. Alcohol acts by promoting a general switch-off of our neurons, although not all neurons are responsive in the same way to alcohol. The neurons that participate in our inhibitory circuits, such as those that make us shy or restrained, are among the first to be switched off by alcohol ingestion. As a result, the neurons that are involved in the circuits that make us bold and happy are released from being constantly told off by their refraining inhibitory counterparts. However, if alcohol continues to be ingested, eventually even these merry neurons will be switched off – and unconsciousness may be an unpleasant result.

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We now understand why we have long enjoyed alcoholic beverages. The next step is to see how alcohol is metabolized. Alcohol present in beverages (ethanol) is small enough and soluble enough to be absorbed as ethanol throughout the digestive system, and thus is directly present in the blood soon after it is ingested. Like many other nutrients, it is metabolized mainly in the liver, although other organs can also participate. Ethanol in the liver is metabolized by two different enzymes, alcohol dehydrogenase and acetaldehyde dehydrogenase (yes, scientists love long, difficult, names). Both these enzymes remove electrons from ethanol, which are transported by NAD (the electron-transporting molecule we saw previously) to the mitochondrial membrane, generating energy in the form of ATP. In addition to producing energy in these two steps of its metabolism, ethanol transformation by these enzymes results in the production of acetyl CoA, the same energy-rich molecule that is the result of carbohydrate, lipid and amino acid metabolism. Acetyl CoA produced from ethanol is identical to all other acetyl CoAs we saw before, and will then enter the Krebs cycle and generate more energy by the same mechanisms as other nutrients. The result is that ethanol is a small, two-­carbon molecule, but it has a lot of chemical energy in it, and therefore is highly caloric. Indeed, if energy levels in the cell and person are high, the acetyl CoAs produced from alcohol will, by metabolic regulation, be diverted to produce fat, storing that energy for later. As a result, alcoholic beverages are a significant source of excess calories in the diets of many people, who often don’t suspect that they are drinking

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in so many calories in the form of alcohol. Just to exemplify, a typical can of beer has slightly over 150 calories (the same as two slices of toast), while a single glass of wine can have around 200 calories (the same as a cup of cooked white rice).

Alcohol metabolism and its relationship with ATP and fat production. Alcohol metabolism is mediated by oxidizing enzymes called dehydrogenases that remove electrons from alcohol, reducing NAD. The reduced NAD can now be a source of electrons in mitochondria, leading to ATP production. In addition, the end product of this pathway is acetate, that can be converted into acetyl-CoA and produce fatty acids

In addition to being a good source of calories, ethanol metabolism can generate some unwanted effects depending on how much you ingest and how well equipped you are to metabolize this molecule. Everyone knows that ingesting large amounts of alcohol can acutely make you feel drunk. These symptoms are largely caused by the presence of acetaldehyde in your body, which is the product of alcohol dehydrogenase, and is formed before the second enzyme in alcohol metabolism, acetaldehyde dehydrogenase, acts. Acetaldehyde not only makes you feel drunk when it is produced in excessive alcohol ingestion, but also is a reactive molecule that can change other biological molecules, and is therefore involved in tissue damage such as liver cirrhosis seen in chronic alcoholics. Interestingly, although we are able to metabolize alcohol, evolution only equipped us with the enzymatic capacity to metabolize low levels of alcohol, found in spontaneously fermented foods, and not necessarily the large concentrations of alcohol found in purposefully fermented beverages such as those that our late stone age ancestors started to develop a taste for. That is the reason we can feel sick when we drink too much of it. Indeed, alcohol ingestion can become a medical emergency, mainly when the person has not eaten carbohydrates, since it can inhibit

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gluconeogenesis, or the production of glucose from proteins (which we saw in the last chapter), and can lead to low blood sugar levels. The ability to metabolize alcohol varies widely within the population, but is markedly deficient in persons of Asian origin. This happens because a large part of the Asian population has a much less effective form of acetaldehyde dehydrogenase. As a result, acetaldehyde is produced normally in a person of Asian descent (by alcohol dehydrogenase), but is accumulated abnormally, because the next enzymatic step in alcohol metabolism, acetaldehyde dehydrogenase, is deficient. As we saw before, acetaldehyde is the molecule that causes most of the symptoms of being drunk. Many Asian persons consequently present flushed cheeks, palpitations and cognitive changes when they ingest even very small amounts of alcohol. They can then either choose not to drink alcohol to avoid these unpleasant sensations or continue drinking and have more symptoms than most people have, while drinking less alcohol.

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We have now seen how all our most important sources of calories are metabolized, generating either energy in the short run or molecules that are stored away within us to generate energy when needed. Next, we will see what determines how much we store for future use, understanding the relationship between metabolism and obesity.

Chapter 8

Metabolism and Obesity

There is no denying that people around the World are getting fatter. According to the World Health Organization,1 global obesity rates have tripled since 1975. The world has close to two billion overweight individuals today, of which more than 650 million are medically obese. Hunger was an important limiting factor for lifespan during most of our evolutionary history, but tables have turned, and today most people live in countries in which being overweight kills more than being undernourished. Because obesity is ever-growing and affects more and more children – more than 380 million children are overweight today – some countries such as the USA are expected to have a decrease in lifespan over the next decades unless the current trend for increased body weight can be stopped. Obesity is, thus, a very serious health issue. We have discussed why we tend to become obese earlier. Evolution prepared us for a world with frequent famine and in which we had to work hard to find food. On the other hand, very recently food became more readily available for most of us, but we are still metabolically and mentally “programmed” by evolution to like food, to eat too much of it, to seek high calorie foods and to store any excess very efficiently in the form of fat. The result is the rampant obesity epidemic we see around us today. Although evolution is to blame for expanding waistlines, its health consequences cannot be ignored in the name of an evolutionary explanation. Obesity increases your chances of developing heart diseases, stroke, many types of cancer, type II diabetes, dementia (including from Alzheimer’s disease), asthma, mental health issues, gout, gallbladder stones, sleep apnea, arthritis and many other serious and debilitating health conditions. Maintaining a healthy body weight can prevent these diseases and increase life and health spans significantly, so it is definitively worth doing your best to overcome the overeating temptations evolution makes you have, in a quest to prevent obesity.

1  https://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight 27th, 2019

© Springer Nature Switzerland AG 2020 A. Kowaltowski, F. Abdulkader, Where Does All That Food Go?, https://doi.org/10.1007/978-3-030-50968-2_8

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Undeniably, preventing obesity depends on dietary intake versus use of calories, such as from physical activity. We saw that anything you eat in excess, above the energy needs for your daily activity, can, and most usually will, become fat. However, fat production and use for energy is regulated not only by how much you eat, but also by the hormones that regulate energy metabolism. These hormones are, in turn, controlled by the type of food you eat. We will now start to understand how hormones regulate our overall metabolism by seeing why we tend to lose weight when following a popular weight-loss group of diets, those that are low in carbohydrates.

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A large number of diets intended to lead to weight loss suggest limiting carbohydrate intake to a large extent. This includes Atkins, Dukan, South Beach, Paleo(lithic), Low Carb, Keto(genic), and many other popular weight loss regimens. The reason so many diets for weight loss suggest cutting carbs is that, without a doubt, this helps people lose weight, through mechanisms we will soon see. However, we will also see that weight loss alone is not necessarily a sign of increased health. We will discuss here how low carbohydrate diets fit into the wellbeing scenario. We already spoke a lot about evolution here, and how we evolved to live short lives and reproduce quickly under conditions in which finding food was hard, thus preparing our bodies to stock up on energy whenever available. Consequently, you may have already questioned why a diet such as the “paleo” diet justifies its supposed beneficial effects. The argument made by the proponents of these diets is that we did not evolve to eat the farmed food we have available today. It is a fact we did not evolve for that, but we did not evolve to live past 40 years of age either, nor to read books, use machines, have modern health care or spend much of our childhoods getting an education. Thus, this argument made by the paleo diet followers does not hold weight, simply because evolution did not prepare us for the life we want to live today. We also spoke a bit about ketones, and how they are important sources of energy in metabolism. These molecules are produced by the liver from fats, but, when in excess, can be quite toxic, as in diabetes. So you already know a bit about “keto” diets. We now need to better understand how diets that restrict carbohydrates work to reduce weight, regulating metabolism in the body as a whole. Weight loss is an undeniable fact associated with low carb diets in general, although we will discuss a little later why this weight loss is not necessarily always accompanied by a gain in health or lifespan. Limiting carbohydrate intake prevents weight gain and may promote weight loss because it decreases the amount of insulin in the blood. Insulin is a hormone produced by specialized cells called beta cells in your pancreas. These cells synthesize the insulin peptide (a small protein) and keep it within them until your blood glucose levels rise. This typically happens when you eat carbohydrates, either as

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simple sugars (which raise these levels quickly) or as starches (which do so more slowly, and in a more sustained manner). When your blood glucose gets higher, a series of metabolic reactions are triggered within beta cells resulting in the release of insulin from these cells into the blood. As a result, in healthy people, high blood sugars lead to higher insulin levels in the blood.

Insulin secretion and the effects of eating the same amount of energy in the form of sugar or starches. Insulin is a peptide hormone that is secreted by beta-cells in response to rises in blood glucose concentration. Beta-cells are found in special structures present in the pancreas, the islets of Langerhans. Other cell types are present in these islets and secrete other hormones, such as alfacells that secrete glucagon in response to reductions in blood glucose. Sugars are a form of carb that is quickly absorbed, so the concentration of glucose in the blood spikes, leading to massive insulin secretion. If the same amount of energy is ingested as starch, the rise in blood glucose is lower and steadier, so the stimulus for insulin release is more gradual

Insulin in the blood circulates and binds to insulin receptor proteins on almost all our cells, significantly changing almost every aspect of metabolism within them. The sites within our body in which insulin action is most important for our metabolism are the liver, muscle and fat (adipose) tissue. In the liver, insulin stimulates protein production, increases glucose absorption into the cells and production of both glycogen (the carbohydrate storage molecule we have that is similar to starch from plants) and lipids (which can stay in the liver or be exported to other organs). Both glycogen and lipids are produced from the glucose that was absorbed by the liver, so glucose has the double effect of acting as a source of molecules to make

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glycogen and lipids and a stimulant of the release of insulin, which activates glycogen and lipid production. In muscles, insulin also leads to more protein production, more glucose absorption and more glycogen buildup. In the adipose (fat) tissue, insulin leads to more glucose uptake and triglyceride production from that glucose. Overall, insulin results in an increase of your body’s mass, both because proteins are produced and because glycogen and fat are generated as energy-storage molecules. It also results in a decrease in the levels of glucose in the blood, since this molecule is taken up by all these different organs and transformed within them. As a result, insulin is very important to prevent the unwanted effects of chronically elevated blood sugar, which we will see more about when we discuss metabolism in diabetes.

Insulin actions in the liver, muscle and adipose tissue. Insulin is a stock-up signal for these organs, and “tells” them to take up glucose and amino acids, as well as make large storage molecules such as glycogen (liver and muscle), fats (liver and adipose tissue), and proteins (liver and muscle)

So what do low carbohydrate diets do? By limiting the presence of glucose in the blood derived from carbohydrates you eat, these diets limit the secretion of insulin into the blood by beta cells. The result is that less glycogen, protein, and fat are

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produced because there is less insulin to stimulate these pathways. Your body grows less because of less insulin. Low insulin makes the effects of another metabolically-active hormone important: glucagon, which is basically a counterpart to insulin’s effects. Glucagon is also a peptide (small protein) and is also produced in the pancreas, but in a group of cells known as alfa cells. Glucagon is released when blood sugar levels are low, and circulates throughout the body binding to glucagon receptor proteins in many different cells, thus changing metabolism in most organs. Overall, glucagon leads to loss of body weight because it promotes degradation of glycogen, proteins and lipids. That explains why a diet that reduces insulin levels and stimulates the effects of glucagon, such as restricting carbohydrates, leads to weight loss. Glucagon is not the only hormone that stimulates shrinkage of glycogen and triglyceride energy storage molecules. Adrenaline (also called epinephrine) is a hormone secreted by your adrenal glands when you are stressed by something and also when you exercise. Similarly to glucagon, it leads to large degradation of triglycerides and glycogen, making lipids and glucose available as energy sources. That is the reason why exercise and stress lead to weight loss. Although it is similar in results, adrenaline acts faster and for less time than glucagon, and is very effective in muscles, preparing them so you have energy available for a fight or flight situation.

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If being obese increases the incidence of so many illnesses, and restricting carbohydrates is proven to decrease body weight by the mechanisms we just saw, it should follow that low carb diets are a great way to age well, right? The truth, unfortunately, is not so simple. The problem with the idea that lowering carbohydrate intake can prevent obesity and thus obesity-related diseases is that the link between obesity and age-related diseases is a correlation, and correlations are not necessarily an indication of cause. In other words, we know that people with higher body weights develop these diseases more often than thinner people. But the reason they develop these diseases is not necessarily just because they are overweight. Correlations can merely indicate that things tend to go together because they may have indirect links. Instead, the relationship between obesity and age-related diseases could be related to other characteristics that are more often found in obese persons, such as specific genetic characteristics, place of residence, diet, lifestyle, etc. Adding to this complication, each age-related disease may be mediated by a different aspect associated directly or indirectly with obesity. Overall, this means that losing weight will not necessarily prevent age-related diseases, even though these age-related diseases are correlated with obesity. A second point to consider when attempting to gain health through a dietary intervention is that not all forms of weight loss are equal, and therefore not all forms of weight loss can prevent health issues associated with obesity. Weight loss in low carb diets involves not only loss of fat, but also of muscle, as insulin is necessary to build muscle, and will not be released in the absence of carbohydrates. Unfortunately, muscle loss in many studies is associated with poor health in aging.

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That may be the reason why a large meta-analysis2 (a study of scientific studies, involving more people and therefore more trustworthy) has shown that very low carbohydrate intake in humans is associated with a higher mortality rate. Indeed, this study of more than 15,000 adults with relatively normal total food intake (neither very low nor high, therefore reflecting most people) showed that eating both too much or too little carbs had a poor outcome in terms of increasing your risk to die. The “sweet spot” for carbohydrate intake was around 50–55% of total caloric intake, and presented the lowest risk. Overall, this suggests that, although low carb eating is very effective for weight loss, it is not necessarily the best diet to maintain in order to age well. While the study conclusively shows that 50–55% carbs are associated with lowest mortality risk, once again, it does not indicate causes for this. In fact, we do not know why this risk is lowered at this level of carbohydrates. It may be just an association, it may be because of muscle mass loss, or it may be because of excess ketones in low carb diets. On the other hand, higher carb diets, which are also associated with increased risk, may produce this risk because of diet-induced obesity, or high insulin levels, or any other of a myriad of factors we as scientists don’t fully understand yet (but we are working on it!).

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While we are on the subject of carbohydrates, it is worth remembering that, as we saw earlier, not all carbohydrates are equal in terms of their metabolic effects. While starches release glucose slowly into your blood, elevating blood sugar levels gradually, simple sugars such as sucrose (table sugar) are absorbed and elevate blood sugars much faster. Consumption of these simple sugars is not associated with a healthy outcome in scientific studies. Thus, although we are evolutionarily selected to love sweet foods, health-wise these are best avoided, and used for occasional consumption only. Avoiding simple sugars does not involve only steering away from sweets, but also keeping an eye out for hidden sugars in savory foods. Many processed savory foods contain large amounts of sugar in them, both because this makes us like their taste and because this helps them keep longer. To avoid them, it helps to avoid highly processed foods. Read food labels and keep an eye out for sucrose, glucose, fructose and other simple sugars; the earlier on the list an ingredient appears, the higher the quantity in that food. If you look through processed food labels, sugar content is often high within them.

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If limiting carbohydrates is an effective way to lose weight, but not necessarily gain health, maybe the way to go is to reduce fat consumption? Once again, there 2  Seidelmann SB, Claggett B, Cheng S, Henglin M, Shah A, Steffen LM, Folsom AR, Rimm EB, Willett WC, Solomon SD. (2018) Dietary carbohydrate intake and mortality: a prospective cohort study and meta-analysis. Lancet Publ Health 3:E419–E428.

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are metabolic advantages in limiting fat intake, but going “low fat” is also not the answer to a universally healthier life. As we saw earlier, fats are very caloric. A gram of fat has about double the number of calories compared to carbohydrates or protein. As a result, it is easier to gain excess calories eating fats versus other nutrients. Fats also are minimally transformed within us when eaten, and can be deposited as basically the same triglyceride molecules as you ate. This “metabolic economy”, or a smaller amount of chemical transformations, means less of the energy content of these molecules are lost within our cells when converting fat in food to fat deposits within us. As a result, eating fat is in fact a very effective way to gain fat. So is going on a low fat diet the solution? The answer again is not so simple. One problem with low fat diets is that lipids are actually important from a nutritional standpoint. There are a number of vitamins (A, D, E and K) and other essential nutrients such as omega fatty acids that we have to eat because our cells can’t make them. These nutrients can be found only in fats and oils. In other words, cutting out lipids is not good for you because you’ll lack important nutrients. Another problem with “low fat” diets is the kind of foods eaten. Many processed products are sold as “low fat” or “light” foods, supposedly to aid weight loss. People then eat them believing to be contributing toward weight loss, but the truth is that these labels do not indicate that the foods can help prevent obesity. Indeed, “low fat” just indicates there is less fat than a similar full-fat product, although calories from fat or other components may be quite high in both. “Light” indicates that there is a 30% reduction in some component of the product relative to the original, which may or may not decrease total calories, but certainly does not prevent the food from having a considerable caloric value. In fact, processed foods in general have high fat contents, which gives them an agreeable flavor and enhances their shelf lives. As a result, reducing this fat content in 30% may be a reduction from a very, very high content to a still very high fat and calorie content, all while gaining a seemingly health-conscious “light” sticker… What is the wrap-up on fats in diets versus health results? As with carbs, neither too much nor too little is a good idea. Once again, moderation is key.

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We have now collectively seen that the ingestion of both too little or too many carbs, lipids or proteins is unhealthy. So what is the ideal diet for healthy aging? Overall, the best scientific consensus is that diets that include all three groups in moderation are best. Furthermore, diets that include a large variety of foods, mostly in unprocessed form (therefore avoiding the high fat and sugar contents of processed foods), present the best clinical outcomes. Eating quality food in moderation will ensure good availability of micronutrients (vitamins, minerals and such) as well as moderate quantities of calorie sources necessary to keep you alive and well, but not gain weight. This kind of eating, which is naturally typical of people from the Mediterranean regions (which have rich agriculture, and therefore traditionally varied diets),

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includes a solid (but not excessive) basis of carbohydrates, with many grains, vegetables and fruits, as well as varied, moderate, amounts of protein sources and sensible amounts of fats. It is both a delicious and nutritious way to eat, and as such, a more sustainable long-term dietary regimen. A good health-maintenance lifestyle should also include exercise, and once again, moderation in exercise also works. Even small increments in daily movement, like taking the stairs, playing with a pet outdoors or walking more, have strong health benefits. These forms of physical activity are also much more economic, attainable and sustainable than exaggerated fitness schemes often marketed by commercial organizations. Finally, a question that often arises is how much weight loss is associated with improved health. Fitness and health industries constantly bombard us with pictures of rail-thin bodies as models of ideal health, particularly for women; Science does not support this. Many models have overly low body weights, and lower body weight is not always the same as better health. Of course, weight is influenced by height, and the fact that we can vary quite a bit in height makes it harder to compare us. A simple measurement that overcomes the effect of height is the body mass index (BMI), which divides your weight by your height squared, in kilograms and meters. Although it isn’t a perfect solution (it can be influenced by different levels of muscle or bone mass, for example), it is the simplest way to compare body weights over large populations and understand health associations. As a result, many studies have employed it. - different heights - different weights

same BMI

- different heights - same weight

different BMIs

Body mass index (BMI) as a tool to estimate obesity in humans. In the left cartoon, the two persons have different heights and weights, but their BMI is similar. Conversely, in the right cartoon the two persons have the same weight, but their heights are different, so their BMI is different

In a Science Advisory from the American Heart Association (publications from large associations of professionals are always better, because they represent a consensus from that group of specialists), both underweight and overweight persons are found to have higher disease and mortality risk. This means that the rail-thinness of models, that often reflects a BMI below the normal number of 18.5 Kg/m2, is less healthy than a normal body weight, with a BMI between 18.5 and 24.9 Kg/m2. On the other hand, BMIs of 25 and over, in the overweight and obese range, are also not

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healthy. So once again, moderation in weight loss is ideal, in order to avoid being overweight, but also not reaching underweight levels.

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Insulin doesn’t only regulate the production and break-down of glycogen, proteins and fats in our body. This hormone also acts within our brains, adjusting another aspect of weight control: your hunger. Deep inside your brain is a small region called hypothalamus, which is responsible for many important functions, including regulating the relationship between the brain and hormones, sleep, thirst and hunger. All of these are very basic and evolutionarily early functions of the brain. As a result, these functions are present in all animals with brains. When insulin reaches this region in the brain, it activates local hypothalamic insulin receptors that result in a reduction of signals in the brain that make you hungry. Therefore, whenever you eat and your blood sugar levels increase, insulin is released into the blood, and reaches your brain, inducing a slow-­ down in further eating, because it decreases your appetite. That is the reason why you aren’t usually hungry soon after eating. It is also the reason why eating starches, that keep your blood sugar levels high for longer times because they are absorbed slowly, keeps hunger at bay for longer than eating sugar, that causes a fast and short-­ lived peak in insulin levels. But insulin is not the only hormone that regulates hunger. In 1949, an interesting group of mice appeared spontaneously in The Jackson Laboratory, a biomedical institution that is one of the largest and best sources of mouse models to study how our bodies work (as well as how bodies do not work properly when diseases happen). While many of their mouse models, particularly today, are created in a controlled manner using genetic engineering tools, the odd mice that appeared there, dubbed the Ob mice, developed spontaneously. Scientists maintaining the facility noticed that some mice were noticeably fatter than their counterparts, weighing as much as three times more than normal. These animals had good reason to be this big: they were insatiable, eating much more than their relatives. Hence, they were named Ob mice, short for obesity. The spontaneous appearance of such mutant mice in laboratories dedicated toward maintaining animal colonies is not a complete fluke. In reality, laboratory animals are often inbred, or continuously crossed between close relatives, in order to decrease their genetic diversity as individuals, and thus give scientists a more even group of animals to study (which makes finding the effects of specific alterations easier). However, as with humans, if you cross too many times within the same family, you run the risk of propagating rare gene mutations and having more genetic diseases. That is why interbreeding within families is discouraged in most cultures, and illegal in most countries. In the case of the niche world of laboratory animals, however, this can bring about some really interesting findings. Ob mice appeared at a time when there was little to help understand what caused this extreme obesity in these animals, except for the observation that it was genetically passed through generations in a recessive manner. This suggested this extreme

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mouse obesity was caused by one faulty gene which had to be acquired in the mutated form from both parents in order to produce obesity in the offspring. In the 1980s, however, the understanding of the molecular structure and organization of DNA allowed geneticist Jeffrey Friedman from the Rockefeller University to ask what was the gene that was faulty in Ob mice.3 He found that this gene contained the information to build a protein that was unknown until then, which they named leptin. The name came from the Greek leptos, meaning slim. Animals lacking leptin could not keep slim. Leptin is a hormone which is produced in our adipose tissue. When you eat too much and gain fat, leptin is released in higher quantities in the blood by the same adipose cells that stock up on fat in the form of triglycerides. It circulates in the blood until it reaches the hypothalamus, where it acts similarly to insulin, decreasing appetite. The difference between insulin and leptin in terms of hunger suppression is that leptin remains in circulation for days at a time when you gain fat tissue, unlike insulin that goes up and down as blood sugar levels increase and decrease throughout the day. The result is that leptin can help control long term weight gain. It is a major reason why you are less hungry for a few days after a particularly heavy eating period. Leptin not only decreases your hunger, but also, through a signaling loop initiated in the brain, activates fat burning processes in your adipose cells that involve decreasing ATP production and increasing heat release. This pathway was discovered when scientists noticed that Ob mice, despite their substantial girth, were cold-­ sensitive, and therefore produced less heat than the average mouse. Indeed, leptin was found to act in two different ways to decrease body weight, limiting energy intake by preventing hunger and increasing energy expenditure by promoting loss of chemical energy from fat degradation in the form of heat. Overall, these effects maintain healthier body weights in normal animals. Leptin does not exist only in mice – humans have it too, and it works in much the same manner. This of course could make you wonder why obese people today aren’t all taking leptin in order to curb obesity, both by decreasing hunger and by increasing loss of calories as heat. Indeed, giving leptin to the Ob mouse completely reverses its hunger and weight gain. Leptin was also found to be highly effective as a treatment for a few human families which have the same mutation as the mice, leading to loss of any leptin in their blood (mostly due to intermarriage in highly isolated communities). But, unfortunately, leptin does not work for the vast majority of obese humans. Instead, most obese people don’t have a lack of leptin in their blood, and have instead quite a bit of it – more than normal. What happens is that leptin is produced as it should be by the enlarged adipose tissue in obese people, but it does not work correctly when it reaches the brain. Most obese humans have become what is known as leptin-resistant: they have leptin, but their brain does not adequately respond to

3  Friedman JM, Halaas JL. (1998) Leptin and the regulation of body weight in mammals. Nature. 395:763–70.

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it, so their appetite is not suppressed, and they continue to feel hungry even when overweight. We don’t yet fully understand why people become leptin resistant (but we are working on it!). However, knowing this clearly shows why giving leptin to curb obesity does not work. Once we understand fully why the brain does not respond well to this hormone, we may be able to create more effective treatments for hunger experienced in obesity.

Secretion and actions of leptin in healthy and obese subjects. In the upper panel, a normal situation is shown, in which fat uptake and insulin signaling promote adipose tissue growth and consequently leptin release. Leptin acts on the hypothalamus to decrease appetite and promote fat burning. The lower panel presents the situation in obesity in which, despite increased fat stores and leptin blood levels, the hypothalamus is less sensitive to leptin, and the actions it triggers there are not completed

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Since the discovery of leptin, other hormones that control hunger have also been described. We now know that molecules that are released from your stomach, gut and muscle are also involved in hunger regulation. We are also gaining significant knowledge regarding the genetic determinants behind obesity (which is highly tied to your genetic background, even when conditions such as your environment and childhood diet are factored out). As we gain more knowledge we will, hopefully, be able to treat this important medical condition better.

∗∗∗

Obesity is unfortunately not only a condition limiting human life and health spans. It is also growingly affecting the animals closest to us: our pets. Pet cats and dogs are now increasingly obese, and have very similar consequences to their health because of this, with decreased life expectancy and increased age-related diseases associated with obesity. The reason for pet obesity is pretty much the same as with most humans: excess food versus low energy use, promoted by an evolutionarily-selected tendency to overeat. But pets also have a specific characteristic within evolution, which is selective breeding by humans. Humans have long bread their pets in very controlled manners, aiming to keep and enhance certain aspects such as appearance, intelligence, and ability to respond to human commands. This has led to specific changes in the promotion of obesity in labrador retrievers, a dog breed which is particularly prone to being overweight. A group of researchers searched the genome of labradors for clues as to why they gain weight so easily.4 They found that many labradors had a defect in the gene that produces a protein called pro-opiomelanocortin (also known as POMC), which is important to control hunger. Many labradors do not have the functional gene, and therefore have lost an important mediator of hunger control in their brains. This means that when they look at us humans with their loving puppy eyes, they most certainly love us, but are also chronically hungry and are begging for food. Therefore, they love us in part because we are a source of food for them... Even when well fed, dogs with this defect feel hungry, and will thus overeat if allowed access to food. Even more interestingly, the faulty gene was found to be much more common in the retrievers selected to become assistance dogs, relative to labradors that are household pets. This means that the lack of adequate hunger control somehow correlates with the ability to learn and become service dogs. Indeed, the process of training to become a service dog is often based on food rewards. It makes sense that an individual dog that is particularly hungry due to a mutation in POMC would be more motivated to respond to food treats. As a result, the seemingly defective

4  Raffan E, Dennis RJ, O’Donovan CJ, Becker JM, Scott RA, Smith SP, Withers DJ, Wood CJ, Conci E, Clements DN, Summers KM, German AJ, Mellersh CS, Arendt ML, Iyemere VP, Withers E, Söder J, Wernersson S, Andersson G, Lindblad-Toh K, Yeo GS, O’Rahilly S. (2016) A deletion in the canine POMC gene is associated with weight and appetite in obesity-prone labrador retriever dogs. Cell Metab. 23(5):893–900.

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animals have an advantage in their behavior – the ability to learn to act as service animals based on food rewards. This trait was unknowingly selected over generations by us humans, and is the reason why this breed is particularly prone to obesity today. Overall, the finding that a specific dog breed has evolved to respond well to a human need is unsurprising. Dogs are the most long-lasting human pets, and have, during millennia, evolved many traits that differ from the wolves they descend from. These traits led to better understanding and interaction with us humans, and include many more taste receptors for carbohydrates (which wolves rarely eat and do not enjoy, but humans have an abundance of), as well as a keen perception of human wants and needs, and how to manipulate us humans in their favor. The next time a labrador retriever looks at you lovingly to beg for food, remember that they certainly love us, but this love is fueled by a selected metabolic error that can easily make them unhealthy by overeating.

∗∗∗

Obesity in our pets and working dogs is undesirable, although it was in part selected by human interaction with these animals. Curiously enough, we have not only developed hungry and obesity-prone dogs, but also changed the metabolism of other animals we breed. An example related to obesity are pigs, another animal kept by humans for thousands of years, which have also evolved to serve our needs better. Wild pigs do not have a tendency to become obese, while domesticated pigs most certainly do. Indeed, obesity in pigs is probably the main reason we rear them. This tendency toward obesity is the result of thousands of years of pig selection by humans, who crossed the larger animals until the resulting offspring were more satisfying to human needs in terms of growth and fat content. Scientists have recently discovered a reason for pig obesity, based on modern understanding of mechanisms affecting obesity in animals: it is related to the lack of functional uncoupling proteins.5 Uncoupling proteins are proteins in mitochondria which decrease ATP production by allowing part of the energy of our food to be converted into heat instead of ATP. They act quite similarly to 2,4 dinitrophenol, the toxic substance that was used as a diet pill in the early 1900s, decreasing the efficiency of mitochondrial “battery” function in a manner similar to a short circuit. Because domestic pigs don’t have adequate uncoupling proteins, they conserve most of the energy of their food very well, and therefore gain a lot more weight and fat tissue than their wild counterparts. On the other hand, conserving energy well and not generating heat has its downside for domestic pigs. Young piglets can lose heat very easily in cold weather, but not generate it, since their uncoupling proteins are not functional. This makes

5  Berg F, Gustafson U, Andersson L.t(2006) The uncoupling protein 1 gene (UCP1) is disrupted in the pig lineage: a genetic explanation for poor thermoregulation in piglets. PLoS Genet. 2(8):e129.

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piglets very cold-sensitive, a characteristic that pig breeders must keep in mind in order not to have newborn piglet deaths.

∗∗∗

Uncoupling proteins, as pathways that determine the amount of “short circuiting” in our mitochondria, are also involved in the regulation of human body weight. Just as the lack of uncoupling proteins in pigs leads to obesity, the high activity of these proteins in humans has been linked to an ability to resist weight gain even with calorie-rich diets.6 Certain humans simply present more activity of these proteins, and therefore can eat a lot and remain lean. The activity of these same proteins has also been related to the ability to keep warm in cold weather. Activating uncoupling proteins in humans seems to be a wonderful way to prevent obesity, allowing for loss of energy as heat instead of packing up excess food as fat. It is in fact an active area of research in metabolism, with the aim of treating obesity. Unfortunately, these proteins have proven quite difficult to activate directly with medications, which has moved scientists to try to understand how we can regulate their activity within our bodies in different ways. Hopefully these studies will not only help treat obesity but also further expand our understanding of this condition and how best to avoid its undesirable health effects.

6  Nedergaard J, Cannon B. (2010) The changed metabolic world with human brown adipose tissue: therapeutic visions. Cell Metab. 11(4):268–72.

Chapter 9

Diabetes and Metabolism

On January 23, 1922, 14-year-old Leonard Thompson was treated with an experimental injection at the University of Toronto that would quickly change the lives of diabetic children worldwide. In reality, it was a last-ditch attempt to do something for a critically ill child. He had already gotten a similar experimental injection a few days earlier, with no improvement. Both injections had a mix of substances isolated from a specific part of animal pancreases, albeit isolated using slightly different techniques. To the utter joy of all involved, the second injection worked, and within the next few days the young patient improved dramatically. Until 1921, type I diabetes, or the form of diabetes that Leonard Thompson had and that often affects children and adolescents, was a death sentence. About 24 in 10,000 children who were previously healthy would develop weight loss, increased hunger and thirst as well as an odor characteristic of the large production of ketones. They would then gradually worsen until they became comatose and finally died. There was no available treatment capable of helping in any significant manner. Indeed, the only known manner to decrease the production of ketones leading to coma and death was to fast these children, but fasting a child who already had significant weight loss was not really of any help at all. Type I diabetes occurs when pancreatic beta cells die, and the body becomes unable to produce and secrete insulin. The body then responds to the lack of insulin with all the metabolic processes that occur during normal periods of low insulin, such as fasting periods between meals. Although blood glucose levels are high, in type I diabetes cells do not absorb this glucose due to lack of insulin. Instead, glucagon-type signaling promotes muscle protein break-down, releasing amino acids which are used to produce more glucose in the liver. This elevates glucose levels in the blood to even higher levels. Wasting muscle tissue in order to elevate already high blood sugar levels may seem to be an inadequate metabolic response, and it certainly is an undesirable one. However, our individual cells are not equipped with intelligence. Instead, they respond to the lack of insulin with the metabolic response that always occurs in the absence of this hormone: protein degradation in muscles and glucose production in © Springer Nature Switzerland AG 2020 A. Kowaltowski, F. Abdulkader, Where Does All That Food Go?, https://doi.org/10.1007/978-3-030-50968-2_9

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the liver. In reality, the only phenomenon capable of conferring apparent rationality to cellular responses is evolution, and evolution does not prepare us for a disease that affects less than 1% of the population. In addition to promoting the loss of muscle and increases in blood glucose levels, type I diabetes also leads to break-down of fat in all tissues that store it, increasing the levels of fatty acids in the blood. Part of these fatty acids are used as fuel in many organs, including the liver. In the liver, the breakdown of fatty acids produces ketones, similarly to conditions in which low carbohydrates are ingested, which we saw before. The difference is that in type I diabetes the production of ketones is much higher, because of the chronic and complete lack of insulin.

Metabolic disturbances in type 1 diabetes. In the absence of insulin, our body works as if there were no glucose available, although its levels are high, leading to a life-threatening wasting condition

Unlike type II diabetes, which we will soon discuss, type I is not linked to obesity, and is instead related to the immune system which attacks and destroys beta

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cells within clusters of cells known as islets in the pancreas. The reasons for this destruction are not yet fully understood, but seem to include both genetic and environmental factors. Until 1921, this destruction of beta cells took the lives of all young patients affected. After 1921, scientific knowledge had advanced to the point where we were capable of supplementing insulin, the protein that was lacking in these patients.

∗∗∗

The story of how insulin, type I diabetes and scientific endeavor are interrelated is an inspirational story of how Science works, and how pursuing curiosity drives improvements in our lives, which in turn allow us to pursue new scientific advances. In order to reach the point where diabetes could be treated with insulin, scientists needed to know that this disease was caused by a molecule that came from beta cells in pancreatic islets (the name “insulin” was given to the molecule because it came from these islets). Pancreatic islets were first described in 1869 by German anatomist Paul Langerhans, and are thus often called islets of Langerhans. He saw that groups of cells which looked different from the rest of the pancreas dotted its surface, like tiny islands. He not only described the pancreatic islets, but also the different types of cells in them, including beta cells, although at the time the function of these different cell types was not known. The idea that the pancreas was involved in the pathology of diabetes came from experiments conducted in 1889 by Joseph von Mering and Oskar Minkowski. These scientists were trying to understand the function of the pancreas using a technique that may sound cruel by today’s standards, but was actually how the function of most organs and tissues in our body was discovered: by cutting it out and seeing what happened to an animal. At a time in which sterile surgical procedures were still under development, doing this was no small feat. Using dogs, the scientists carefully removed the pancreas and managed to keep the animals alive after the surgery, recording their progress over the next weeks. They found that animals without a pancreas developed symptoms that were exactly the same as diabetic children: hunger, thirst, weight loss, high glucose in the blood and urine. Like diabetic children, the animals also progressed into a coma and died. From these experiments, scientists in the late 1800s knew that the pancreas contained the factor that, when absent, promoted diabetes. While cutting out an organ seems today like a crude experiment to study its function, in reality removing a biological component to discover what it does is a technique still in use today. We don’t do it with whole organs now, but we do eliminate specific genes, proteins and functions of molecules, then observe what the consequences are. Indeed, scientists have developed thousands of so-called “knockout animals” in which there is an absent gene, and the roles of this gene can be studied. These animals are an invaluable tool in the understanding of diseases as diverse as diabetes, Parkinson’s, Alzheimer’s, obesity, cancers, genetic childhood disorders

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and more. They are also instrumental for the development of new therapies in these diseases. After the pancreas was identified as the source of a factor that, when absent, caused diabetes, further studies early in the twentieth century pinpointed the pancreatic islets as the part of the pancreas that produced this factor. Eliminating the islets but preserving the rest of the pancreas caused diabetes. Conversely, eliminating the rest of the pancreas (which produces enzymes that aid digestion) but preserving the islets did not cause diabetes. From these findings, to separate this factor from animal islets and use it to treat a diabetic human patient became the dream of many researchers. In 1920, Canadian surgeon Frederick Banting, then just recently returned from war, had such an idea, and approached well-established University of Toronto researcher John Macleod with a proposal to try to isolate pancreatic islet factors to treat diabetes.1 Macleod, by most accounts, was not overly optimistic about the feasibility of this idea or Banting’s abilities, but still assigned Banting a position, and provided a laboratory research assistant and dogs to conduct his studies. Charles Best, at that point just hired as a research assistant in Macleod’s lab, reportedly won a coin toss and was assigned as Banting’s assistant. This was a very lucky moment for Banting, for Best had excellent laboratory technique, which was certainly instrumental in their studies, which began in 1921. Many different techniques were tried to isolate an extract from islets that could rescue dogs that had developed diabetes after having the pancreas removed. The process was extremely tricky because the rest of the pancreas has digestive enzymes, including enzymes that break down proteins, and these would degrade insulin in the islet extract. After many failed attempts, they developed the idea of ligating the pancreas first, a surgery that impeded the elimination of digestive enzymes, and made that part of the pancreas die, while still preserving the islets. After a few days, in a second surgery, they were able to remove the islets and make an extract. Incredibly, this extract was able to keep a diabetic dog alive, as well as decrease its blood glucose levels. These exciting results were presented in a conference in December 1921, and met with a mix of hope but also substantial criticism, possibly because of Banting’s nervous and unconvincing public speaking abilities. The truth was that even the Toronto group knew that their technique still needed work, because although they had proved this form of treatment was possible, the surgical procedures involved were so complex they had trouble making functional extracts consistently. Around that time James Collip joined the group, and helped both with the production of large quantities of extract necessary to start human treatment attempts,

 https://www.sciencehistory.org/historical-profile/frederick-banting-charles-best-james-collipand-john-macleod (accessed April 7th, 2019)

1

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and to overcome technical difficulties. They found that including the use of alcohol and strict cooling contributed to isolate functional insulin. These conditions were later identified as more important in the final outcome than the pancreatic ligation surgery itself. The group then jumped headfirst into human tests, treating Leonard Thompson in early 1922, followed by other children, with very positive results. Soon the Toronto group was known for having the ability to treat type I diabetes not only by the medical community, but also by families of sick children, who would travel far in an attempt to save these children from a disease that until then meant certain death. Many of the patients treated by the group early on went on to live for decades using insulin, and followed the development of better extracts and treatment techniques throughout their lives. Banting and Macleod received the Nobel prize in 1923 for their discovery, and shared it with Best and Collip. By then, insulin was already produced in commercial quantities at the University of Toronto and by Eli Lilly, now using pancreases from pigs and cows obtained from slaughterhouses. Physicians were also trained by the University of Toronto on how to administer insulin to treat diabetics. The seriousness of the disease, added to the effectiveness of the treatment, led to one of the fastest and most effective laboratory bench-to-bedside transfers in modern medicine. ∗∗∗



But the relationship between insulin, diabetes and scientific development did not stop in the early 1920s. Instead, understanding insulin became an interest for many scientists, and this led to new knowledge, which in turn once again helped treat diabetes, as well as many other conditions. After 1923, many people researched the properties of the molecule that cured this disease,2 in studies that helped understand its size and characteristics. This also helped develop tools to study properties of other proteins. In the 1950s, Frederick Sanger and colleagues made a major breakthrough in Biology related to insulin: they painstakingly determined, one by one, the sequence of amino acids that composed this small protein, thus determining the primary structure of insulin. Understanding the structure of a molecule is an essential step in comprehending how this molecule works, since structure determines function. Indeed, Sanger was awarded the Nobel prize in 1959 for determining the first sequence of amino acids in a protein. Knowing the structure of insulin allowed scientists to produce it by chemically stitching amino acids together in the right sequence, instead of isolating it from animals. Once again, insulin was the first protein in which this scientific  Ward CW, Lawrence MC (2011) Landmarks in insulin research. Front Endocrinol (Lausanne). 2:76.

2

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breakthrough was achieved. This synthetic insulin was shown to be as effective as the natural molecule, proving that the structure indeed determined its function. However, the process of making it this way was very difficult and costly, not amenable to large-scale commercial production needed to treat patients, so the use of animal insulin continued for some time still. In 1969, the way the amino acids in insulin arranged themselves in three-­ dimensional space was determined by another Nobel laureate, Dorothy Crowfoot Hodgkin, who used the pattern of how X-rays were diffracted by insulin to generate a 3D model of its structure. Again, this was the first time the structure of a protein was uncovered. X-rays had been used in the 1950s to determine the structure of DNA, a huge achievement at the time, but DNA has much less complexity in its structure than proteins, and the determination of the structure even of a small protein such as insulin was, again, a major breakthrough. In the early 1970s, scientists had advanced to the point in which they understood a good deal about how DNA carries genetic information, and how this information determines sequences of amino acids and allows us to make proteins. They also knew that the pathways to make proteins from the information in DNA were essentially the same in all life forms, from bacteria to humans. Knowing this, scientists could start to dream of stitching DNA parts together in microorganisms to make them produce proteins that were of interest to us. Once again, the first protein that came to mind was insulin.3 Until then, insulin was still produced from animal pancreases, mostly from pigs, in a process that required one metric ton of pancreases to produce around 500 g of insulin. There was a justified concern that this production, in face of the growing need for insulin worldwide, was not sustainable. Animal insulin is also slightly different from human (pigs have one different amino acid; beef, three) and can carry contaminants from the animals it is isolated from. A better source of insulin was clearly necessary to meet the World’s production needs. Efforts were then made during the 1970s to insert the genetic information that produces human insulin into harmless genetically-modified (recombinant) bacteria, transforming these bacteria into efficient animal-free insulin-producing machines. This incredible feat, which involved not only very new techniques but also a lot of ethical discussion related to the limits of what should or should not be done in terms of recombinant DNA technology, was achieved in the late 70s. In the early 80s, human insulin produced in bacteria was already on the market, and is practically the only insulin used today.

 Johnson IS. (1983) Human insulin from recombinant DNA technology. Science. 219:632–7.

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Breakthroughs in biomedical sciences associated with insulin research. Insulin was not only very important as a subject of research on its own, but also led to crucial developments in Science. This is recognized by the many Nobel prizes granted to insulin researchers

Insulin production through recombinant DNA techniques jump-started a whole line of applications that use genetically-modified organisms. This includes proteins to treat blood clotting diseases, to boost blood cell production, to modulate the immune system, promote growth and many others. Applications for genetically-engineered organisms are not limited to the medical field. They are also useful in industrial processes such as those that require the production of specific enzymes. Increasingly today, we are developing improved foods through this technology, that have more nutrients, grow faster, use less resources or require less pesticides. Genetically-modified foods therefore help our environment while feeding our growing population. Just as people were first afraid of the technology for insulin production, but later embraced it, many people today are weary of genetically-modified foods, despite the fact that they are much more closely studied, and therefore more thoroughly understood, than “normal” foods.

∗∗∗

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While type I diabetes is a rare disease, type II diabetes is an unfortunately common disease today, and a strong determinant of quality of life as we age. Roughly 90–95% of diabetics in the world today have type II diabetes, and in some countries, 25% of the population over 65 has this disease. Both type I and type II diabetes involve increases in blood glucose levels, but the diseases are quite different in the population they affect and the metabolic changes that generate them. Type I diabetes is most typically diagnosed in children and teenagers, while type II usually develops later in life. Type I is not associated with obesity or dietary history. Type II is very strongly associated with obesity, to the point in which the disease can be prevented and even reversed by weight loss, in some cases. This is not to say that type II diabetes does not have a genetic component and should be “blamed” only on the person’s dietary choices. In fact, type II diabetes is more strongly dependent on genetic factors than type I. Just as discussed previously for obesity, our tendency to overeat and like particularly caloric foods is very largely determined by our genes, which consequently also determine our tendency to develop type II diabetes. Type II is also different from type I at a molecular level. While type I diabetics are unable to produce insulin, type II diabetics often even have high insulin levels in their blood. Normally, high insulin results in lowering of blood glucose because, as we saw before, insulin helps our cells absorb this sugar and produce other molecules (including glycogen and fats) from it. But in type II diabetics, the problem lies primarily not in the production and secretion of insulin in the pancreas, but in how the other cells in the body perceive and respond to insulin. Type II diabetics have defects in insulin receptor proteins (that bind insulin in different cells) and also in other proteins that allow a cell to respond to insulin with the changes in metabolism it is expected to produce. The result is that insulin is present in the blood, but cells do not respond to it, and do not absorb blood glucose as they should. Glucose consequently remains in the blood, and can thus slowly lead to damage in nerves and the circulatory system. Unlike type I diabetes, in which symptoms are serious from the onset and the disease cannot go undiagnosed for long, type II diabetes can be a silent disease, in which symptoms are not apparent and can go on for years without a diagnosis. This happens because the loss of responses to insulin is usually partial, and therefore massive ketone production leading to ketoacidosis is rare. The loss of insulin signaling can also be tissue-specific, affecting some organs, but not others. Typically the liver does not respond well to insulin in type II diabetics, and therefore breaks down proteins and produces glucose, worsening the high glucose levels in the blood. On the other hand, the adipose tissue, where we produce and store fat, can often respond well to insulin early on. So type II diabetics, unlike type I, often can gain weight in the form of fat, all while having high blood sugar levels and a liver that breaks down proteins to produce more circulating glucose.

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Blood glucose and insulin levels during a typical day in healthy, obese and type 2 diabetes patients. Glucose concentrations in the blood fluctuate during the day as a result of meals as well as insulin and glucagon secretion. Before overt type 2 diabetes, when blood glucose is sustainably high, obese individuals present a normal blood glucose pattern, albeit at the expense of increased insulin release after each meal. This characterizes insulin resistance. In type 2 diabetes, insulin is present, but unable to reduce blood glucose to normal levels in between meals

All these undesirable metabolic occurrences are, unfortunately, happening more often because type II diabetes is a disease associated with longer lifespans, easy access to high-calorie food and low exercise, all of which have increased over the last few decades. Treatment strategies have evolved over the years, and include not only insulin (which can work when in higher quantities in type II diabetics), but also drugs that decrease glucose absorption, promote insulin secretion in the pancreas, and decrease glucose production in the liver. Treatment can also include food-intake limiting procedures such as bariatric surgery. Plans with how to deal with the disease are highly specific for each patient, since the presentation of type II diabetes is very variable. Also central in the control of type II diabetes is a limitation of food intake and increase in exercise. Unfortunately, this is the part which most patients have trouble adhering to, given their genetic tendency to overeat. In this sense, new scientific findings are also helping control food intake and cravings. Once again, the understanding of the mechanisms of the disease is central toward the control of its effects.

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In this sense, type II diabetes still presents new challenges in its comprehension, since it involves complex changes in molecules within insulin signaling pathways in many different organs, and which do not present themselves in the same manner in every person. As always in Science, we already know a great deal, and can do a great deal because of that, but there is still a lot more to understand.

Chapter 10

Metabolism in the Brain

In the early 1980s, a group of seven patients with puzzling symptoms appeared in northern California1,2 All were relatively young (ranging from age 26 to 42) and all had very quickly developed a severe inability to move most, if not all, of their body. Apart from that, they had very little in common. Some were initially thought to have a psychiatric disorder, such as catatonic schizophrenia, which also affects the ability to move. However, upon examination, their limbs had the type of rigidity that is characteristic of Parkinson’s disease, and not present in schizophrenia. What did not fit well within the diagnosis of Parkinson’s was the young age of these patients and the fact that the rigidity had come upon them very quickly. A typical Parkinson’s disease patient is older and has gradual onset of symptoms. The confirmation that they had Parkinson’s disease came with finding that they exhibited other characteristics of the disease (including tremor, cognitive changes and facial skin alterations). They also responded very well to L-dopa, the drug that helps supplement the neurotransmitter that is low in this disease, correcting the motor problems patients have. (Neurotransmitters are chemicals that mediate communication between your brain cells.) In fact, their response to L-dopa was exactly the same as in long-term Parkinson’s disease patients, including the appearance of a serious side effect, dyskinesia, a different neurological disorder characterized by excessive involuntary movement, which can complicate treatment of Parkinson’s rigidity. But why was there a sudden cluster of seven people with early and quick onset of Parkinson’s disease in northern California? The atypical age and progression of the disease, as well as the fact that all patients lived in the same region, suggested an environmental cause.

 Langston JW. (2017) The MPTP story. J Parkinsons Dis. 7:S11–S19.  Langston JW, Ballard P, Tetrud JW, Irwin I. (1983) Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis. Science. 219:979–80. 1 2

© Springer Nature Switzerland AG 2020 A. Kowaltowski, F. Abdulkader, Where Does All That Food Go?, https://doi.org/10.1007/978-3-030-50968-2_10

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After extensive investigation, the medical teams involved determined that the only common characteristic of these patients was that they had used a new recreational drug described as “synthetic heroin”, which had recently appeared in local underground markets. The medical team was then able to obtain samples of this drug through police raids and “friendly dealers”, and determined its chemical composition. “Synthetic heroin” contained a substance called 1-methyl-4-phenyl-4-­­ propionoxy-piperidine (yes, biochemists love long names, but we nicknamed it MPPP, to keep it simpler…). MPPP is an opioid analgesic which was the intended molecule for “synthetic heroin” preparations. However, the samples also contained large amounts of another chemical, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (nicknamed MPTP), a side product of the synthesis of MPPP, which was not intended to be there. MPPP was a fairly new abuse substance in the 1980s, created a few years before in Maryland by a chemistry graduate student named Barry Kidston. He used his professional knowledge to develop, in an adapted lab in his home, new opioid molecules. His aim was to produce novel legal drugs that could generate the same “high” as opiates, that were already regulated by authorities. Kidston reasoned, in a rather tongue-in-cheek manner, that making new and not yet prohibited molecules circumvented illegality… In 1976 he developed a synthesis protocol to make MPPP, and used this home-­ made purified chemical himself for several months without any undesired side effects (at least not undesired to him). He then got lazy and attempted to simplify the synthesis of MPPP through a series of shortcuts. Soon after he used this shortcutter “sloppy batch” (as he described it), he exhibited signs of Parkinson’s disease, and sought medical attention. When he described his story and symptoms, doctors suspected his neurons were damaged by the drugs he took, but were not able to link his Parkinsonism to a specific drug of the many ones he had used while “experimenting” (very unscientifically) on himself. Kidston died a few years later from his continued drug abuse, and his brain was examined by a medical team. It showed signs typical of Parkinson’s disease: the destruction of cells in a particular area of the brain, the substantia nigra (which gets its name from its dark color). Both his clinical story and the autopsy results were published by the doctors who originally attended him.3 Publications such as these are important for doctors to later link to new findings and, possibly, solve medical mysteries. In fact, the doctors in California read this publication, and it helped them understand their seven-patient cluster appearance of Parkinson’s disease. They added to the understanding of the Kidston case by identifying MPTP as a major component of the drug that caused the symptoms. Their patients had not experimented with many homemade new chemicals like Kidston had, and only used MPPP preparations containing MPTP. This suggests that MPTP, found in poorly prepared MPPP, 3  Davis GC, Williams AC, Markey SP, Ebert MH, Caine ED, Reichert CM, Kopin IJ. (1979) Chronic Parkinsonism secondary to intravenous injection of meperidine analogues. Psychiatry Res. 1(3):249–54.

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was the molecule that caused Parkinsonism. The California group, with this new knowledge, also published their results, so the scientific community would learn from their findings.34 At this point, most people would think that the world learned that poorly prepared “synthetic heroin” can cause severe brain damage, further supporting the notion that illegal drugs should, in fact, remain illegal. However, scientists are by nature creative, and immediately saw a potentially useful spin-off for the link between MPTP and Parkinson’s disease. Only one day after the publication of the paper describing the California patients, MPTP, which was retailed as a laboratory chemical by a major company that supplies research facilities, completely sold out. The reason was that animal researchers all over the world wanted to test if this chemical could be used to generate models of Parkinson’s disease in lab rats and mice, and thus use them to better understand the disease. At the time, there was no way to induce the disease in animals in order to study it. Indeed, MPTP worked as an inducer of Parkinson’s in animals, and is used to date to study the disease. MPTP also promotes the changes found in Parkinson’s disease in cultured brain cells, another model used by scientists to understand the disease and develop new ways to prevent and treat it.

∗∗∗

Mouse models using MPTP to promote Parkinson’s disease are not perfect (as with any other research model), but have very greatly advanced our knowledge regarding how this disease happens, including finding links between energy metabolism and its neurological symptoms. How does MPTP promote Parkinson’s disease? The answer lies in changes that it promotes in mitochondria within the brain. After MPTP was described as capable of reproducing Parkinson’s disease symptoms in lab animals, a natural insecticide and fish toxin called rotenone was found to have similar effects.4 Rotenone was already well known by researchers who study metabolism, because it has a well-established effect on mitochondria. Rotenone inhibits the removal of electrons from NAD, one of the molecules we saw that shuttle electrons to the mitochondrial membrane after they are removed from molecules broken down by the many metabolic pathways we followed. Within the mitochondrial membrane, these electrons reduce oxygen we breathe, producing water and the energy currency of the cell, the energy-rich ATP molecule. When rotenone is present, electrons cannot be removed and build up in NAD. The result is that the process of oxidative phosphorylation in mitochondria is stagnated, and our cells don’t produce energy in the form of ATP in mitochondria. This leaves the cells with very little energy to survive, and is the reason high doses of rotenone kill just about any animal. 4  Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT. (2000) Chronic systemic pesticide exposure reproduces features of Parkinson’s disease. Nat Neurosci. 3(12):1301–6.

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Low doses of rotenone, however, particularly when used directly in the brain, did not kill animals, but lead to a loss of function in a particular area of the brain, the substantia nigra, which causes symptoms of Parkinson’s disease. This was a clue that Parkinson’s disease was related to a failure in energy metabolism in the substantia nigra, and particularly involved a decrease in the ability to remove electrons from NAD. A second clue in the puzzle related to the understanding of how Parkinson’s disease occurs was finding out that MPTP can generate molecules within our cells that act very similarly to rotenone, also inhibiting NAD oxidation. In other words, two separate treatments that lead to Parkinson’s disease affect the same metabolic process within mitochondria. This suggests that this mitochondrial process is a core event in the disease. Many researchers then looked into mitochondrial function in patients that developed the disease without being exposed to MPTP or any other mitochondrial toxin. They found that spontaneous Parkinson’s disease patients did in fact have mitochondrial alterations,5 and that some genes associated with inherited forms of Parkinson’s disease were genes that produced mitochondrial proteins responsible for NAD oxidation. Today, we know that the substantia nigra is an area of the brain particularly sensitive to the lack of perfectly functioning electron removal from NAD. When there is even a small inhibition of this process, the neurons (brain cells) in this area die, and the result is the movement disorder seen in Parkinson’s disease. Giving patients the neurotransmitter molecule that these neurons usually produce can help control the movement disorder, but unfortunately does not help recover brain cells, since these typically do not proliferate after we are adults. New surgical procedures have been developed to help more advanced patients, including surgeries that “turn off” areas of the brain that produce too much movement and electrical stimulations in brain areas that are lacking activity (you may be surprised by this, but our brain cells actually work due to electrical activity on their membranes – thinking is electricity!). Immune, genetic and cell therapy options are also quickly being developed, and will hopefully help even more in the near future.

∗∗∗

Parkinson’s is not the only brain disease which can be promoted by inhibiting processes in mitochondria. Huntington’s disease, a devastating and thankfully rare disorder is also associated with mitochondrial defects. This disease is inherited, caused by a problem in a single gene, and children of an affected parent have a 50% chance of developing it later in life. Symptoms typically begin in adulthood, with uncontrollable jerky movements, changes in mood and mental abilities. Scientists have found that the brains of patients with Huntington’s disease, and particularly a part of the brain called the striatum (which causes these movement 5  Keane PC, Kurzawa M, Blain PG, Morris CM. (2011) Mitochondrial dysfunction in Parkinson’s disease. Parkinsons Dis. 2011:716871.

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alterations), have a defect in their mitochondria. These defects involve abnormal use of electrons derived from FAD, the other electron transporter we saw that, together with NAD, helps gather electrons in the mitochondrial membrane, to ultimately generate energy. Not only that, but, additionally, animals treated with 3-­nitropropionic acid, a chemical inhibitor of FAD-linked reactions in mitochondria, have the same movement disorder as Huntington’s disease patients, and also show damage to neurons in their striatum.6 Basically, although the exact metabolic point inhibited is different and the brain area affected is also distinct, two different obstacles in mitochondrial energy-­ producing functions strongly affect the function of cells in two different areas of the brain. This is not surprising since the brain, while corresponding to only about 2% of our weight (an average brain weighs around 1.4  kg) uses around 20% of our energy when we are not exercising.7 It is a demanding tissue in terms of our energy – thinking, coordinating movement and all other brain functions are high activity tasks for our cells, and their energy consumption reflects that. As a result, even small decreases in ideal energy production by mitochondria lead to large effects on the brain, such as in Parkinson’s and Huntington’s disease. Since every area of the brain has slightly different cells and metabolic characteristics, specific defects in mitochondrial function affect some areas of the brain more than others.

∗∗∗

In addition to Parkinson’s and Huntington’s disease, which develop in adulthood, a large and heterogeneous group of childhood diseases that affect the brain and involve defects in mitochondrial function exists. These are collectively known as mitochondrial diseases,8 and many of them involve defects in mitochondrial DNA. The information to build mitochondria lies in approximately 3000 different genes. Almost all of these are stored in the nucleus, as with all other genes our cells hold within them. However, genes necessary to build 13 different mitochondrial proteins are stored inside mitochondria themselves. Indeed, mitochondria are the only part of our cells outside the nucleus that have their own DNA, therefore storing part of their own genetic material. This mitochondrial characteristic has led scientists to conclude that this organelle descended long ago from independent living beings, related to today’s bacteria. Although only a small part of the information necessary to build mitochondria is in their DNA, this information is related to the construction of proteins which are essential for mitochondria to make ATP. And mitochondrial DNA is not only essential; it is also vulnerable. It has protection and repair mechanisms to keep it healthy,

6  Saulle E, Gubellini P, Picconi B, Centonze D, Tropepi D, Pisani A, Morari M, Marti M, Rossi L, Papa M, Bernardi G, Calabresi P. (2004) Neuronal vulnerability following inhibition of mitochondrial complex II: a possible ionic mechanism for Huntington’s disease. Mol Cell Neurosci. 25(1):9–20. 7  https://www.scientificamerican.com/article/thinking-hard-calories/ 8  https://www.umdf.org/what-is-mitochondrial-disease/

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but these are not as effective as those that do the maintenance of DNA in the nucleus. In addition, mitochondrial DNA is in a particularly dangerous place within the cell, since mitochondria generate a lot of free radicals as side products of metabolic reactions that happen in them (we will see more about that later). Free radicals can easily damage DNA.  So DNA in mitochondria gets more damage due to these free radicals, and has a harder time recovering from this damage, as it does not have protection and repair mechanisms as effective as nuclear DNA. The result is that mitochondrial DNA can sometimes accumulate damage. When large enough parts are destroyed, or many copies are damaged (we typically have hundreds of copies of mitochondrial DNA in each cell), mitochondria cannot be produced adequately, and diseases occur. Mitochondrial DNA diseases are inherited from our mothers (from whom we get all our mitochondrial DNA), and vary a lot in their symptoms, since the damage to this DNA and quantity of damaged mitochondrial DNA molecules is different in each patient. Typically, however, these diseases affect the organs that most use energy, including the brain and muscles. Indeed, mitochondrial DNA-related diseases are a significant cause of developmental delays in children, affecting about 1 in 10,000 individuals. Because the defect is located exclusively in mitochondrial DNA, replacing mitochondria before development of the baby can lead to a perfectly healthy child, even if the mother is affected by mitochondrial DNA mutations. This can be done in a lab during in vitro fertilization, using mitochondria from a healthy donor. This procedure has been named mitochondrial replacement therapy, but is most commonly known as “three parent baby” generation, since the baby will have the DNA of its mother and father, as well as a tiny amount of DNA in its mitochondria from a healthy donor that does not have the mitochondrial DNA defects the mother has. Only a very small part of the genetic material comes from the mitochondrial donor, so no effects of this “third parent” will be seen in the child’s personal traits, but this tiny piece of DNA is essential for a healthy baby with fully functioning mitochondria. Mitochondrial replacement therapy has been recently approved as an ethical practice for assisted reproduction clinics in the UK. As a result, mothers with mitochondrial DNA mutations now have a choice, and can have healthy babies while maintaining their family’s genetic characteristics.

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Another disease of the brain which must be mentioned here due to its links to metabolism is Alzheimer’s disease. Approximately 10% of adults aged 65 or over in developed countries develop this disease. The number of people affected is increasing rapidly, as more people reach older ages. The increase in incidence of Alzheimer’s disease is not only related to increased lifespans, but also to our expanding waistlines. For reasons that scientists are still working to understand, obesity strongly increases the chances of developing Alzheimer’s disease, while interventions such as dietary intake moderation and

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exercise prevent the onset and even decrease symptom progression of the disease. Since obesity is increasing in much of the developed world, this disease has become a major cause of death and disability. Once again, we hope that future studies uncovering the mechanisms in which Alzheimer’s disease occurs, and how these relate to obesity, may help us develop new and better strategies to prevent and treat this disease. For now, the knowledge that preventing obesity can help keep yet one more devastating disease at bay will hopefully stimulate more people to watch their food consumption and practice some form of exercise.

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A final disease of the brain that involves metabolism and that we will discuss here is stroke, the result of poor blood flow into the brain. Strokes can happen in young people, and are then often because of problems in the vessels that lead to the brain. However, the most at-risk population are older people, in particular those who have diseases, such as diabetes and changes in cholesterol and other lipids in their blood, because these conditions lead to damage to blood vessels. Damaged blood vessels can have blockages that result in a lack of blood reaching the brain, which causes the stroke. Neurons, the type of cell in the brain responsible for thought processes, are particularly susceptible to a lack of blood flow. This happens because of their metabolic characteristics. Neurons have fewer metabolic pathways than most cells, and therefore are not able to make energy in the form of ATP from fats or amino acids. Instead, they rely almost exclusively on glucose and glucose-derived lactate, which is produced by neighboring cells in the brain, to supply their significant energy needs (thinking is energy-consuming, as we saw before). In addition to their strict “dietary” needs, neurons have very little glycogen as a glucose storage molecule. This means that, if a blood vessel is not functioning properly and glucose-rich blood does not reach the brain, neurons quickly suffer from a lack of energy, which in itself causes cell death because all energy consuming systems in the cell cease to function. In addition to dying because of lack of nutrient-rich blood, neurons can also be destroyed by a secondary process that happens in stroke, called excitotoxicity (yes, yet another complicated name invented by scientists…). Excitotoxicity gets its name from the fact that neurons get “excited” (activated) to a toxic level when stroke or some other brain disease happen. Neurons submitted to a lack of energy sources not only die, but can also lose their inner content, which includes neurotransmitter molecules. Neurotransmitters are the chemicals that promote communication between neurons. In a healthy brain, neurons release these chemicals to communicate with neighboring neurons. This chemical conversation affects the way neurons fire electricity, and these electrical signals, on the other hand, determine the release of their own chemicals to communicate with yet other neurons. Both this chemical conversation and electrical processing are at the basis of our thought processes. In a brain undergoing stroke, neurons release too much of these neurotransmitters, and the result is that the nearby neurons, which may have survived the lack of

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blood flow because they were a little farther from the most damaged area, are now over-activated. This leads to many futile energy-consuming processes within these cells that may well kill these neurons in the periphery of the stroke area too.

Mechanism of excitotoxicity promoted by stroke. Neurons are highly metabolically active cells, that consume huge amounts of glucose, lactate and oxygen to sustain mitochondrial ATP production. ATP is invested to control the emission of electrical and chemical signals that promote our thought processes. Absence of nutrients and oxygen in stroke leads to death of the affected neurons, which then can’t retain the pre-made chemical signals they produce inside them. The uncontrolled release of these chemicals overexcites otherwise healthy neighboring neurons, that may die from this as well

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In other words, the perfect storm for neurons to die happens when blood does not get to the brain: Neurons use a lot of energy, but store very little energy-rich molecules. They have selective “taste” for energy-rich molecules, using only glucose and lactate, which is made from glucose in the brain itself, but not amino acids or fats. And then, to add insult to injury, neurons with low energy levels can release their neurotransmitters at toxic levels for neighboring neurons, killing cells which could have otherwise survived the lack of blood. How can this catastrophic situation be avoided? First, as with other consequences of obesity, prevention is the best way to go. Moderating food intake and exercising decreases the chances of getting clogged arteries, and thus the chances of developing a stroke. Keeping a healthy body weight also changes brain metabolism, making neurons more resistant to excitotoxicity and preventing neuronal loss because of the toxic release of neurotransmitter chemicals from the cells that die. Another important action in stroke is to get medical attention quickly. In the case of stroke because of obstructions of blood flow (which causes most strokes in older people), removing this blockage and getting the blood back into the brain quickly is the best way to limit damage to the neurons. This can be done with medications and/ or surgical procedures. Finally, better medical strategies have been developed during recovery periods to limit damage and recover the areas that were damaged. All of these require medical attention, that should be sought immediately when a stroke is suspected.

Chapter 11

Metabolism and Heart Disease

Your brain isn’t the only organ that can suffer from the lack of oxygen and nutrients caused by a blocked blood vessel. Hearts are also susceptible to damage caused by lack of oxygen and nutrients during heart attacks, technically known as cardiac ischemia. Ischemia means that an inadequate amount of blood reaches a part of the body, while cardiac is the technical term for tissues within the heart. The heart is a hard-working organ, continuously contracting and relaxing so that blood enters and leaves the inner chambers, to then circulate around, taking nutrients and oxygen to every corner of your body. All this work requires energy for the muscles of the heart themselves, and this energy comes from nutrients delivered to the heart by a set of blood vessels known as the coronary system. Heart cells, called cardiomyocytes, are muscle cells that constantly contract and relax, and, by doing that, promote blood circulation. To make this movement possible, hearts are well adapted to produce ATP as chemical energy from just about any kind of nutrient. They are able to use carbohydrates, fats and ketone bodies to generate energy, as long as these nutrients arrive to the cells, through the blood. On the other hand, heart cells have very little nutrients stocked up within them. Unlike the liver or muscle cells that make us move (skeletal muscles), they do not have much stored triglycerides or glycogen, and therefore need steady blood flow to bring in these nutrients. Another reason heart cells need a steady flow of blood is that it brings oxygen, which, as we saw before, is necessary to make ATP in mitochondria. Oxygen cannot be efficiently stored within cells, and must be brought constantly from our lungs through our blood to all our cells. When a heart attack happens, neither oxygen nor nutrients, such as glucose, fatty acids, or ketones, reach heart cells. The heart can continue to work for a short amount of time under these conditions by producing a little ATP without oxygen, generating lactate, as we saw earlier for fermentation. But that does not last long, because the cells don’t have any glucose reaching them to make lactate from. Soon after, the cells cannot make enough ATP within them not only to continue contracting (and acting as a heart cell should), but even to keep alive. They start to die,

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creating a part of the heart with scar tissue and no ability to contract. Having a blockage in the blood vessels that feed your heart cells is clearly not good news. Cardiologists and surgeons, knowing this, have created methods to unblock heart blood vessels, including medications that dissolve blood clots, as well as physical unclogging methods such as catheters and surgery. This ensures that blood can go back to the heart and saves cells that were about to die because of the lack of oxygen and nutrients. But re-establishing blood flow also has a dark side – unfortunately it leads to a large production of so-called free radicals in the heart, and this causes damage, which can also kill heart cells. Because of that, many different strategies are being created to prevent free radical-induced damage to heart muscles caused by re-­ establishing blood flow to the heart during heart attack.

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Free radicals are a diverse group of molecules that have a bad reputation, blamed for causing just about any disease you could imagine. Is this true? And if it is, why don’t we know how to stop these radical molecules in their tracks, and therefore control all diseases? First it helps to understand what free radicals are. By definition, these are especially reactive atoms or groups of atoms (molecules) that have unpaired electrons. All our atoms are composed of a central part, the nucleus, that has protons (which are positively charged) and neutrons (which have no charge), as well as smaller and mobile electrons, with negative charges, moving around the nucleus. Usually these electrons circulate at different energy levels in pairs, since the presence of two electrons in an energy level around the nucleus helps keep the atom or molecule stable. However, a few atoms and molecules known as free radicals have “solitary” unpaired electrons in an energy level (and sometimes even in more than one energy level, making them double or bi-radicals). This makes these molecules less stable and more prone to react with other molecules. Hence their “radical” reputation.

Free radicals as chemical substances lacking electrons. Most stable chemicals have electrons in pairs in their structure. Loss or addition of one electron from this pair makes the substance highly reactive, as it tends to ‘steal’ electrons from other molecules and fulfill the empty space in the electron pair. The usual consequence is that the molecule that had its electron stolen becomes a free radical itself, starting a chain reaction

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While free radicals are reactive, they are also a totally normal part of life. In fact, they are necessary for life. For example, the oxygen you breathe is a free radical (it is actually a bi-radical, with two unpaired electrons). Despite this, we all use this radical molecule to make most of our chemical energy and would not be alive without it. Oxygen is not the only free radical that is an important part of our lives. Nitric oxide is another free radical found in living organisms with many biological roles. It can act as a neurotransmitter (relaying information between brain cells), in protection against infections, and is a mediator of blood vessel relaxation, which controls blood pressure. Blood vessel relaxation is also necessary for a key step in our reproduction: erection of the penis, which perhaps not surprisingly turns out to be a radical-dependent process. When blood vessels in the penis relax, blood flows in and is trapped there, leading to the expansion that characterizes an erection. The relaxation of the blood vessels that promotes this is mediated by the radical nitric oxide. Indeed, sildenafil, sold under the brand-name Viagra (among others), works its magic by decreasing nitric oxide degradation. Without this particular radical-mediated biological activity, we would not reproduce, and would therefore cease to exist. Not only are free radicals useful for life, they are also a very natural consequence of it. As we saw before, most of our energy in the form of ATP is produced in mitochondria, by taking electrons from nutrients and transferring them to oxygen. This takes a relatively stable free radical (oxygen) and produces much less reactive non-­radical water molecules from it, all while releasing the energy necessary to produce ATP. However, a small amount of the oxygen our mitochondria use does not undergo this full process of conversion to water. Either because of minor metabolic imperfections or due to purposeful production, some of our oxygen gets reduced by a single electron, generating a free radical known as the superoxide anion. Superoxide radicals are produced continuously within us, and have many important functions, including a role as signals within cells, activating specific responses. One important response to superoxide radicals is the production of defense mechanisms against free radicals. Superoxide radicals are also an important part of our resistance against the many different organisms that try to invade us every day. As such, they play central roles in our immune system, destroying pathogens inside of us. Superoxide radicals are definitively important, although too much of these molecules is not a good thing: since superoxide anions are actually relatively reactive radical molecules, that can change the structure and function of our proteins, lipids and even DNA, when in large quantities.

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Oxygen as a source of free radicals. When properly used in the mitochondrial inner membrane, a single oxygen molecule reacts with 8 electrons and 4 protons to produce water, which is very stable and not a free radical. If oxygen, for whatever reason, receives an incomplete number of electrons before it is released from the mitochondrial membrane, other highly reactive free radicals are formed, such as the superoxide anion

Because superoxide radical production is a normal part of life, but too much of these radicals is not healthy for our molecules, our bodies developed effective strategies to remove any excess. This includes an enzyme that transforms these radicals into hydrogen peroxide (the same peroxide you buy at the pharmacy to clean wounds is produced inside you, all the time!). Hydrogen peroxide is not a free radical, but is still a reactive molecule. After peroxide is produced, an enzyme system to remove this peroxide, which is linked to the pentose pathway we saw earlier, safely eliminates this reactive molecule. The exact method in which the removal happens is not as important for us here as is understanding that we are well equipped to deal with free radicals in everyday life: we have very good tools in our cells to keep any excess at bay. Interestingly, the ability to remove free radicals within our cells is regulated, often by free radicals themselves. This happens because increased levels of free radicals activate the production of the systems that remove free radicals. The result is that when we produce too many free radicals, our removal systems are beefed up to eliminate them. Once again, we are well prepared to deal with these reactive molecules! The fact that our normal systems to remove free radicals are modulated by the molecules they exclude explains in part why taking antioxidants (molecules that react with and get rid of some free radicals) is not effective against most human diseases. By taking antioxidants you are removing some radicals, but you are also removing the increase in protection that your body normally generates in response

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to free radicals. Therefore, while you are improving removal by adding antioxidants in, you are simultaneously decreasing the antioxidants that were already present in your cells.

The relationship between free radicals and cellular antioxidant systems. Antioxidants are a class of chemicals that can react with free radicals and stop their chain reactions. They do so both directly or by being used by special enzymatic systems. In both cases, the availability of antioxidants and the enzymes that used them is controlled by the steady level of free radicals that are produced in the cells

Antioxidants are also not super-molecules capable of removing all the different types of free radicals that exist within us, and they cannot reach all parts of our cells in which free radicals occur. Overall, in most diseases, and especially in chronic, long-lasting diseases, although free radicals may be involved in the damage associated with the disease, taking an antioxidant does not help, as shown by many scientific studies. An exception for the lack of efficacy of antioxidants in most disease states are acute situations in which the production of free radicals is sudden and can be anticipated. One such situation is returning blood flow to a heart in which a blood vessel was blocked, generating a heart attack. When blood and oxygen return to the heart, this produces a fast and large burst in free radical production, faster than our defense systems can respond. Under these conditions, antioxidants can help, and are in fact used in clinical situations of heart attack to prevent further tissue damage.

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Because free radicals have been around for the billions of years in which life has evolved, we have not only prepared our cells to deal with them, but also harnessed

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their properties for useful purposes. An example of how our bodies have harnessed the signaling effects of free radicals in order to protect us against free radicals is a natural mechanism that can help protect hearts against the damage of a heart attack, known as ischemic preconditioning. As we saw, much of the damage that occurs in a heart attack is because of free radicals generated when the blood flow comes back. In the 1980s, an unexpected finding in an animal research study changed our understanding of this disease. Researchers were artificially promoting heart attacks in lab animals, and were trying to figure out how best to avoid the damage that happened when blood flow was re-­ established. They found that animals that were submitted to a large simulated heart attack had, as expected, large damage to their hearts. On the other hand, if the animals were submitted to a few short episodes of lack of blood reaching the heart (which were too short to cause damage by themselves) before this simulated heart attack, a subsequent longer period without blood promoted much less destruction. This process of heart protection, called ischemic preconditioning, was a natural (endogenous) mechanism of defense against at least part of the damage seen in a heart attack.1 Basically, a heart that went through short periods of lack of oxygen was much better prepared for a heart attack and showed less damage. Indeed, later studies demonstrated that preconditioning also happens in human hearts. While people who have a single large heart attack tend to have substantial damage to the tissue, persons who have smaller episodes before that (such as those who have a partially clogged blood vessel) are at least partially protected from a large heart attack. More studies showed that the effects of ischemic preconditioning were dependent on the presence of free radicals during the short ischemic periods. If these short periods with lack of blood flow happened in the presence of antioxidants, the damage to the heart was once again large, indicating that preconditioning had lost its protective power. In other words, antioxidants had an undesirable effect during preconditioning. This is surprising since, as we saw before, antioxidants can protect against damage when used during a large and damaging heart attack. These experiments show that, while free radicals in large quantities promote damage to ischemic hearts, in smaller quantities (or produced for short amounts of time), they prepare hearts to cope with a large ischemic event. This proves that free radicals are signaling, protective, molecules under preconditioning conditions. We know today that these short periods of lower amounts of free radicals in small heart attacks promote an increase in the heart’s defense systems against these same free radicals, which protects the heart against longer periods without blood. Once again, this explains why taking antioxidants is not the solution to all health problems related to free radicals. In a heart attack, they can help protect during the acute ischemic event. However, they actually make future heart attacks worse if used in a person who has some loss of blood flow to the heart and is developing

1  Murry CE, Jennings RB, Reimer KA. (1986) Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 74(5):1124–36.

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preconditioning mechanisms because of that. Overall, antioxidants are only medically recommended as supplements under very specific conditions.

∗∗∗

Another disease state of the heart that is quite common, especially among seniors, is cardiac hypertrophy, or the abnormal enlargement of the heart. Enlargement of the heart can happen for a number of reasons, including high blood pressure (which makes heart pumping against this high pressure harder), after a heart attack (the remaining healthy tissue has to compensate for the part of the heart that does not work, and does so by enlarging) and problems with heart valves, among many other causes. An enlargement of heart muscles is a normal response to a higher need to pump, and can be a healthy response in many situations (such as with exercise), but can also be excessive and limit heart function, because the growth limits the amount of blood that can enter the heart. The abnormal growth of the heart that leads to cardiac hypertrophy involves changes in free radical production.2 Here, free radicals mostly act as “villain” molecules. Free radicals produced in excess by mitochondria and other sources within heart cells are one of the causes for the excessive growth (hypertrophy) of heart cells, as well as their inability to properly contract (an essential part of the heart’s function). Free radicals also induce the growth of fibrous tissue in the heart, which is not composed of muscle cells, and therefore interferes with contraction of the organ and how it pumps blood. In laboratory settings, some of these undesirable effects of free radicals can be decreased by antioxidants. However, in clinical trials, they mostly have no results. Once again, this is because antioxidants can’t really remove all different kinds of free radicals from all different parts of the cell in long-­ term diseases. New and more effective strategies are being tested to overcome this difficulty. Interestingly, cardiac hypertrophy also involves changes in heart metabolism. The heart is omnivorous in terms of its choice of fuels: it can use just about any molecule to generate energy, but prefers to use fatty acids and ketones, and therefore mostly obtains energy from fats and derived molecules. In hypertrophy, one of the first changes in the heart is a shift toward increased use of carbohydrates such as glucose.3 The exact reasons for this shift are still being studied. It is not an effect of dietary patterns of the person, so changing the content of fat or carbohydrates in your diet will not change heart hypertrophy development directly, despite the metabolic change that the heart undergoes. Although unrelated to diet, the change in metabolism of failing hearts can be used by scientists to understand why hearts grow out of control under these situations, and potentially thwart this excessive growth by preventing this metabolic shift. Researchers are actively studying these possibilities. 2  Seddon M, Looi YH, Shah AM. (2007) Oxidative stress and redox signalling in cardiac hypertrophy and heart failure. Heart. 2007 93(8):903–7. 3  Tran DH, Wang ZV. (2019) Glucose metabolism in cardiac hypertrophy and heart failure. Journal of the American Heart Association 8:e012673.

Chapter 12

Metabolism in Exercise

While heart muscles contract and relax constantly throughout our lives, muscles linked to our bones (skeletal muscles) contract and relax in response to stimuli from our nerves, and are the reason we are able to move our bodies. Skeletal muscles contract and relax due to the presence of motor proteins called actin and myosin. These are long filamentous proteins that slide past each other as muscles contract and relax. Chemical energy, in the form of breaking down ATP, causes actin and myosin to change their shape and slide. This happens in billions of motor proteins within us all the time, and, as a result, skeletal muscle contraction and relaxation accounts for a significant use of our chemical energy.

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Molecular machines behind muscle contraction. Myosin proteins lock on and then jerk over actin filaments in the presence of calcium ions. This movement is powered by ATP use by myosin and leads to shortening of the muscle cells, causing overall contraction of the muscle

As we saw earlier, the use of ATP in a cell decreases the levels of this molecule and activates pathways that replenish it. For this reason, promoting skeletal muscle contraction (such as when exercising) is an effective way to decrease our chemical energy stores (i.e. lose weight). Indeed, skeletal muscles, which amount to about 40% of our body weight, consume around 20–30% of our ATP when we are at rest, and can consume over 90% of our chemical energy when we exercise. Roughly speaking, there are two different modes of energy use in the skeletal muscle during exercise: energy use in the absence of sufficient oxygen to use mitochondrial ATP production (such as occurs during short and intense exercise) and energy use in the presence of oxygen, involving mitochondrial ATP production (such as in longer, more constant, exercise). You have probably heard about these modes by the terms scientists use to refer to them: anaerobic (literally “without air”) and aerobic (“with air”) exercises. These terms can be misleading though, as people tend to think the ways muscles use energy are mutually exclusive, while the truth is it is much more a continuum between these two extremes.

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During short and intense exercise, such as a 100 m sprint, muscles often contract so significantly that blood flow to that muscle is compromised. This means that the

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cells in the muscle do not get enough oxygen to use their mitochondria as a source of ATP. As we saw earlier, in order to make ATP in mitochondria, oxygen must be present to receive electrons derived from NAD and FAD, that collected these electrons from storage molecules while they were broken down. As a result, oxygen is an essential part of the reactions that constitute the most significant source of ATP in our cells: mitochondria. Under these conditions, only two other sources of ATP are present: Creatine phosphate and the glycolytic pathway. Creatine phosphate is a small molecule synthesized from amino acids in our cells which carries a phosphate group with an energy content similar to ATP. When ATP levels decrease, and ADP increases, creatine phosphate donates its phosphate to ADP, producing ATP and creatine. This creatine molecule can later (when the demand for ATP is lower) receive a phosphate group from an ATP and regenerate creatine phosphate stores. Most muscle cells have about three to five times as much creatine phosphate as they have ATP. Despite this substantial quantity, since ATP is very rapidly used up during muscle contraction, creatine phosphate stores can only maintain muscles working for the first 5–10 s of contraction (but these are very vital seconds). After that, other sources of ATP must come into action. Because of this action of creatine phosphate, creatine (minus the phosphate) is a popular supplement sold to supposedly boost performance in exercise. In reality, we eat creatine in our diets any time we eat animal proteins (because animals have creatine in their muscles, just as we do), as well as synthesize it ourselves. As a result, supplementing this molecule may be of little use for most people. In fact, clinical studies have produced mixed results for the effects of creatine in exercise, with some finding no effect, and some finding small effects on the very initial phases of muscle work, when creatine phosphate is an important source of energy. The second source of energy during short, high impact, exercise is glycolysis, or the break-down of glucose through the glycolytic pathway we saw earlier. In the absence of oxygen, glycolysis can happen leading to the formation of lactate. While glycolytic lactate production generates ATP even in the absence of oxygen, it is a lot less than mitochondria can produce from the complete metabolism of glucose to CO2 and water. Thus, ATP levels in the muscle will drop, and the resulting decrease in ATP in muscle cells will activate glycolysis. This makes the glycolytic pathway much faster, in order to maintain ATP production rates sufficient. When the rates of glycolysis accelerate, muscle cells need more glucose (or glucose 6 phosphate) to use as a precursor to produce ATP and lactate. Cells in general do not store glucose, and skeletal muscle cells are no exception. Instead, the skeletal muscle stores large quantities of glycogen, the large molecule composed of thousands of glucose molecules we saw earlier, which is a more efficient and stable way to stock up on glucose molecules. Indeed, muscle cells, because of their abundance (making up about 40% of an average person’s weight) play a very large role in removing glucose from the blood after a meal, producing glycogen from it.

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ATP sources during short-term, high impact, exercise. Creatine phosphate, a molecule that acts as a cell reservoir of high-energy phosphate bonds, rapidly transfers this phosphate to ADP and sustains ATP levels, despite increased ATP consumption. While creatine phosphate is being used up, increased incomplete oxidation of glucose to lactate in glycolysis provides fast, but inefficient, ATP production

When muscles are exercised, in addition to the effects of adrenalin (a hormone we release in our blood under stress, exercise and other situations that involve preparation for movement and alertness), two different stimuli within cells make glycogen break down. The first is the decrease of ATP and glucose 6 phosphate, which activates the pathways that degrade glycogen. The second is calcium. Calcium is an important element within our bodies. It is essential to form bone tissues, and also participates in a myriad of different functions within our cells and organs. Regulating metabolism is one of the many functions of calcium. Another function of calcium is to signal muscle contraction. Whenever our nerves send signals to our muscles indicating that they should contract, this neuronal electrical signal increases calcium levels within muscle cells. The increases in calcium change the configuration of actin and myosin, the proteins in muscles that slide alongside each other, promoting muscle contraction. But the effects of calcium are not limited to promoting contraction. Calcium also regulates glycogen metabolism, promoting its degradation into glucose 6 phosphate. Overall, this means that any time you send a message to your muscle, ordering it to contract, the same messenger (calcium) that makes the muscle contract also accelerates the degradation of the molecule that can produce ATP, necessary to replenish the energy used to contract that muscle. This very elegant system in which

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the same signal (calcium) promotes muscle contraction, using ATP, and the metabolic pathway that regenerates ATP, keeps our muscles moving efficiently.

Calcium signaling in muscle cells. Calcium is important not only to trigger actin-myosin interaction and muscle contraction, but also to shift metabolism to enhance ATP production

Consequently, when you run a 100 m sprint, your muscles contract in response to electrical stimuli from your nerves, which increase calcium in your cells. The increased calcium results both in sliding actions of actin and myosin, promoting muscle contraction, and increased rates of glycogen break-down. This allows the glycolytic pathway to function quickly, generating lactate and ATP, in the absence of oxygen. We have muscles that are highly specialized in this kind of fast action. For this specific mode of contraction and metabolic activity, muscles are poor in mitochondria, which can’t be used without oxygen anyways, and rich in glycogen. Mitochondria tend to have a reddish color, due to the presence of iron and copper in their proteins; mitochondria give muscles a red color. On the other hand, glycogen is a white molecule, and tends to give muscles a whiter color. Muscles specialized in short, intense, contraction are therefore lighter in color due to fewer mitochondria and more glycogen, while muscles that promote longer and more constant contraction are red, due to abundant mitochondria. Short and intense sprints typically end abruptly, and after the exercise period, the person who sprinted will spend a few minutes breathing quickly, until they catch their breath. This period of time is characterized by a return of blood flow to the muscle. This blood not only provides oxygen and allows the muscle to rebuild its

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ATP, creatine phosphate and glycogen stores over time, but also picks up the lactate that was produced in the muscle during the sprint. During this period of intense energy demand in the muscle cell, although ATP production is largely increased, it cannot make up for the overwhelming increase in ATP consumption. The ATP that is used is converted to ADP and phosphate, while also producing protons, which make the cell acid (something most textbooks forget to mention1). As a result, when ATP production does not meet its demand, particularly when it can’t be fueled by nutrient and oxygen delivery through the blood, muscle cells acidify. In parallel, as we showed before, glycogen stores in the cell are being used to form some ATP through degradation to lactate. As a result, acid and lactate appear together, although they are not produced together. Interestingly, the appearance of lactate at the same time as cell acidification is a defense mechanism. The plasma membrane of muscle cells has a protein that transports protons out together with lactate, so the presence of lactate helps remove acid from a cell. Intracellular acidification is a factor that interferes with the interaction of myosin with actin, and is one of the explanations behind muscle fatigue induced by acute exercise.

ATP-ADP cycling in the exercised muscle leading to acid production. If ATP recycling does not equal ATP converted to ADP and phosphate, the protons released in the second process promote an acidification of the cell, which is compensated in part by the export of protons with lactate through the plasma membrane. The simultaneous appearance of lactate and acid in the blood has been erroneously interpreted as net production of lactic acid, while the truth is that the availability of lactate is a defense against intracellular acidification and muscle fatigue

The lactate from your exercised muscles then circulates until it reaches your liver. In the liver, under the stimulus of the adrenalin that you produced from the run, two lactate molecules are converted back to a glucose molecule, through the process of gluconeogenesis. As we saw, making glucose is a costly process, which decreases the ATP levels in liver cells. This results in more mitochondrial activity in these cells, more use of oxygen, and more burning of fats to generate energy in your body after a sprint. So even though your muscles don’t directly use fats in a 100 m  Robergs RA, Ghiasvand F, Parker D. (2004) Biochemistry of exercise-induced metabolic acidosis. Am J Physiol Regul Integr Comp Physiol. 287(3):R502–16. 1

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sprint, you do lose lipids while recovering from short exercise periods, because they are broken down in the adipose tissue and liver. The use of mitochondrial ATP production (consuming oxygen) and the acid produced from all these metabolic changes are the reason you need a few minutes of faster breathing after a short and intense period of exercise.

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Longer exercise periods, with constant and less intense activity, are metabolically different from short exercise periods, because blood flow to the muscle is sufficient to fuel mitochondrial ATP synthesis, as well as bring in fuels for your muscles from all over your body. Under these conditions, skeletal muscle cells use just about any kind of nutrient they can to generate ATP. As with short and intense exercise, longer exercise periods also involve a signal from the brain to the adrenal glands (structures above our kidneys) to secrete adrenalin into the blood. Adrenalin is a hormone that signals our body for alertness and preparedness. It is important in evolution for dangerous situations, such as fleeing from predators, as well as situations that require focus and high muscular activity, such as hunting. Adrenalin thus activates many processes known as “flight or fight” responses. This includes increasing the number of heart beats each minute, as well as regulating blood pressure so that muscles get more blood. Metabolically, adrenalin also acts in many tissues, altogether promoting changes that favor the production of nutrients for muscles and brain. In the liver, adrenalin stimulates gluconeogenesis, or the production of glucose, which increases glucose levels in the blood and provides fuel for the brain to think, as well as for muscles to contract. Also in the liver, triglycerides are broken down to fatty acids, which provide energy for the liver to function, as well as acting as sources of ketones. Ketones are important nutrients for muscle function during exercise, and also fuel the increased heart contractions necessary to maintain blood flow during exercise. In the adipose (fat) tissue, adrenalin also stimulates triglyceride degradation to fatty acids. These fatty acids are used by the liver and muscle as fuel. Finally, in the muscles themselves, adrenalin stimulates degradation pathways that generate ATP, including the break-down of glycogen into glucose 6 phosphate. The difference between long and steady versus short and intense exercise is that the glucose 6 phosphate produced during longer exercise in the muscle is not transformed into lactate predominantly, but fully oxidized to generate the most ATP possible, because oxygen is present, and mitochondria can generate ATP with high efficiency. This way also there is low net acid production in less intense exercise.

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Adrenalin signaling during long-term moderate impact (aerobic) exercise. Adrenalin is released by the adrenal glands and promotes net nutrient output from the liver and adipose tissue, which sustains increased muscular activity

Overall, adrenalin prepares all tissues for “fight or flight”, and thus allows all tissues to prepare for exercise within their own typical functions. The liver and adipose tissue will produce nutrients for other organs. The brain will receive adequate glucose to function, and heart and skeletal muscles will have a range of different fuels to generate the ATP necessary to contract and relax muscles. Overall, during this form of exercise, the body is expending many different types of storage molecules, breaking them down and eliminating their carbons as CO2 in each breath. As a result, weight is lost.

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While the immediate effect of exercise is to lose weight overall, constant exercise is well established to promote gain of muscle mass, or at least to prevent loss of muscle mass as we age. This is extremely important, since losing muscle mass is a strong predictor of unhealthy aging. The more muscle is lost, and the earlier, the

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shorter lifespan a person tends to have. Additionally, low muscle mass in aging is associated with more diseases and less independence. Exercise prevents muscle loss and promotes muscle gain by promoting the production of more muscle proteins (actin and myosin, as we saw before). Interestingly, we don’t really know the mechanisms in which exercise promotes this increase in muscle protein production (but we are working on it!). Many hypotheses and possible pathways are being studied, including changes in protein synthesis and degradation, changes in muscle stem cells and the effects of small local lesions promoted by exercise. These studies are conducted in hope of finding new mechanisms that lead to muscle growth, as well as learning how to control these mechanisms. This could uncover new ways to prevent muscle loss and guarantee healthy aging. Until these ideas reach clinical use, what you can do to keep your muscles healthy is exercise. Any kind of increased body movement helps, and significant benefit is seen even with moderate activities. So move around more to keep yourself healthy, in whatever manner that keeps your interest.

Chapter 13

Cancer and Metabolism

While we want to preserve our muscles during aging, cancer cells growing within us are certainly something we would rather do without. The term “cancer” actually does not refer to a single disease, but instead to over 100 different diseases, with the common characteristic of having cells with abnormal growth, dividing quickly, sometimes generating masses (tumors), and capable of invading tissues and organs as well as spreading around the whole body. Because cancer is not a single disease, it does not have a single cause. However, cancers are promoted by genetic mutations of cells, from inherited, environmental, and lifestyle factors. This means that lifestyle conditions that promote more mutations in cells can increase the odds of developing many different types of cancers. One such condition (promoted both by genetic and environmental situations) is obesity. Indeed, the American Cancer Society guidelines to prevent cancer1 coincide with the American Heart Association and the American Diabetes Association guidelines to prevent metabolic diseases and promote overall public health. These include maintaining healthy weight through dietary moderation and exercise, as well as not smoking. The effects of smoking on cancer are quite straightforward: cigarettes contain many different components that promote genetic mutations in cells (including by generating more unwanted free radicals). On the other hand, how obesity and cancer are related is more complex, and involves also changes in inflammation, altered free radical formation and many other modifications in our bodies that accompany obesity and produce cancer-inducing mutations. Many of these links between obesity and cancer are still being studied and not yet fully understood.

1  Kushi LH, Doyle C, McCullough M, Rock CL, Demark-Wahnefried W, Bandera EV, Gapstur S, Patel AV, Andrews K, Gansler T; American Cancer Society 2010 Nutrition and Physical Activity Guidelines Advisory Committee. American Cancer Society Guidelines on nutrition and physical activity for cancer prevention: reducing the risk of cancer with healthy food choices and physical activity. CA Cancer J Clin. 2012 62(1):30–67.

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Interestingly, cancer cells themselves are not homogeneous. Within a tumor, different cells with different mutations co-exist with each other. Within this environment, a study in evolution happens, in which the cancer cells best suited to survive will be those that reproduce and thrive (albeit in an unwanted type of “thriving” for the individual who has these cells). Adaptations of cancer cells to survive involve growing more, which of course involves metabolism, because cells can only grow if they are capable of using more energy to make the proteins and lipids that they are composed of. Indeed, many metabolic adaptations happen within tumor cells that can determine if they survive or not. Often these involve changes in protein and amino acid metabolism, which is essential in cell growth. All these changes are very specific to each type of cancer (and there are more than 100!), so we won’t go into the details here. There is one metabolic change in cancer cells that is more general and often observed, therefore worth noting: a switch from respiratory metabolism that generates ATP in mitochondria, to fermentation of glucose, generating lactate. This shift in metabolism is known as the Warburg effect, because it was described by 1931 Nobel laureate Otto Warburg. Fermenting glucose to lactate may seem like a disadvantage to a tumor cell, since it generates much less energy. However, many cancers grow quickly, and form masses that are not fully oxygenated by blood vessels, since these do not grow as fast as the tumor. The result is that the cells within them must operate under conditions without or with very low oxygen. As a result, cells that have mutations that favor fermentation over oxidation of fuels can have an edge, despite the overall lower energy production per glucose molecule. They do this through the activation of a pathway that senses low oxygen, described by 2019 in Physiology or Medicine Nobel Prize laureates William G.  Kaelin Jr., Sir Peter J. Ratcliffe and Gregg L. Semenza. Indeed, the lower energy generated by fermentation is simply compensated in these cells by use of high quantities of glucose.

The Warburg effect is a change in metabolism in cancer cells that allows them to thrive in the absence of enough oxygen to sustain ATP production. Cancer cells replicate in an ­uncontrolled fashion that often can’t be sustained by oxygen delivery from blood vessels. Only cells that undergo a switch from mitochondrial to glycolytic ATP production can survive such environments. This change in metabolism is known as the Warburg effect

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While this metabolic adaptation that makes cancer cells thrive is certainly not desirable for us, scientists have harnessed this characteristic to search for cancer cells in our bodies. Since tumor cells that produce energy in the absence of oxygen can only use glucose as an energy source, and use high quantities of glucose (because the energy obtained per molecule is low), glucose use can be a good way to find these cells. Clinical exams to find cancer cells spread around the body (metastatic cells) today involve injecting radioactive glucose (at low, non-dangerous, levels) into the veins of the person and looking for the places where this glucose is broken down by tracing where the radioactivity collects within the body. These places are either tissues that use a lot of glucose like the brain, or tumor masses. Once located, the masses can be treated in a manner which is appropriate for the type of cancer and location. In this case, a metabolic property is useful in diagnosis, and not only in prevention or treatment of a disease.

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As we saw, different cancers are very unlike each other, and therefore only share a few metabolic similarities in the metabolism of the cancer cell itself. On the other hand, cancer as a whole has many effects on the body which are similar in many types of cancer. Because it consists of a fast-growing tissue, cancers take a toll on energy balance. Many nutrients that were formally absorbed by healthy tissues are instead hoarded by cancer cells, and essential nutrients may be lacking for healthy tissues. Furthermore, many cancers induce the release of hormones that promote degradation of lipids in fat cells and proteins in muscles. Cancer cells that do so gain an advantage, since this gives them more molecules to build their cells with. Therefore, cancer cells that promote the release of these hormones have a higher chance to aggressively thrive. One such hormone released in persons with cancer is called tumor necrosis factor alfa, a name given to it because it was first described in the presence of tumors, despite the fact that this is a hormone present in healthy people with functions under normal conditions. Tumor necrosis factor alfa promotes, among many effects, the breakdown of proteins in the muscle and release of their amino acids. These amino acids may then be used by tumor cells to produce their own proteins and grow, giving the cancer cells a growth advantage. Meanwhile, the person’s muscle mass decreases, in a process known as cachexia, which commonly accompanies cancer. Cachexia is not good news – it both helps cancer cells by providing amino acids and decreases the muscle mass in the person’s body. There are today treatments both to halt or slow tumor growth and prevent or reverse cancer-associated cachexia. These treatments are varied, and range from surgical interventions, drugs to slow cancer cell growth or block the effects of muscle-wasting hormones, as well as appropriate nutrition and even exercise, which may help avoid cachexia in some cases.

Chapter 14

We Are Stardust

We have now seen the basics of how metabolism, and our bodies, work, both in health and in a few common diseases. These processes, happening within humans, are a fascinating part of the metabolism that happens on our planet. To maintain their body in a working state, an average person will ingest almost three kilograms of food and beverages a day. That adds up to about a metric ton a year. If around half of that is beverages, all of us are eating an approximate average of 500  kilos of food a year, a rather astounding amount, considering none of us gains anything close to that amount of weight in a year. Metabolism is actually very precise in maintaining our bodies – weight gain or loss is a very small fraction of our overall metabolic activity. Instead, most metabolic activity is focused on maintaining essential functions of rebuilding molecules that broke down and providing energy from the controlled metabolism of energy-containing nutrients. The 500 kilos of food we eat a year are almost all either products of plants, or derived from animals that eat plants. This means that our sustenance depends on plant life on Earth. Plants are amazing metabolic organisms capable of growing using sunlight and CO2 in the air to build their own molecules. They do this using the process of photosynthesis in their chloroplasts, and a complex CO2 fixation process known as the Calvin cycle. Chloroplasts act similarly to mitochondria, generating ATP for plants using an electrical gradient across their membranes as the energy source for this production. However, instead of using the energy from food to power these plant batteries, the generation of the positive and negative sides of the membrane is powered by the Sun, which can change the energy contained in plant chlorophyll molecules. The energy from the Sun thus fuels ATP synthesis in plants. The energy from the Sun also fuels a reaction in chloroplasts which is essential for us animals: splitting water, removing electrons from it and generating oxygen, which we need to breathe in so that our mitochondria can work. With ATP generated by the power of the Sun, and water split so that its electrons are removed, plants can proceed to generate sugars and starch from CO2 in the air. The carbon atoms from CO2 are added to smaller molecules, making them bigger © Springer Nature Switzerland AG 2020 A. Kowaltowski, F. Abdulkader, Where Does All That Food Go?, https://doi.org/10.1007/978-3-030-50968-2_14

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and jump-starting the process that ultimately leads to the production of large carbohydrates in plants such as starch molecules, which we eat regularly. The enzyme that catalyzes this fascinating process of producing carbohydrates from the air (literally) is called rubisco, and is the most abundant enzyme on Earth. We need it to be abundant, because without it, life as we know it could not exist.

Energy flux in plants and microorganisms. Plants derive the energy they invest in building the molecules they are composed of from the Sun. An important factor is the availability of nitrogen atoms linked to carbon-based molecules. These are made possible by microorganisms that take molecular nitrogen in the air and incorporate it into organic compounds

But plants also need proteins, including enzymes that allow them to have metabolic processes such as the ones we just saw. Proteins require nitrogen atoms, which also happen to be quite abundant in the air. However, plants are not able to get nitrogen gas in the air to make proteins from it. For that, they need help from microorganisms. The soil in which plants grow contains bacteria and related microorganisms which are capable of transforming nitrogen gas into ammonia and other related compounds that have nitrogen. This form of nitrogen is then used by plants to generate proteins, which are then incorporated into animals such as us when we eat these plants, or eat animals that ate these plants. Without nitrogen-fixing microorganisms, we would also not be alive. And these tiny organisms work very hard for us: nitrogen fixation is a complex metabolic process that requires copious amounts of ATP, or chemical energy, that they must generate. As a result, we depend on nitrogen and CO2 gases in our atmosphere and microorganisms in the soil generating ammonia, as well as water, so that plants can produce both our food and oxygen. The nitrogen, CO2 and water are trapped in the atmosphere and surface of Earth because of the gravitational force of our planet,

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which attracts and traps larger molecules such as CO2 and nitrogen gas (which are much heavier than hydrogen gas, found pretty much everywhere in space). Large atoms such as carbon, nitrogen and oxygen atoms, on the other hand, are produced from atomic fusion reactions that happen deep in space, within different kinds of stars. These fusion reactions start with small hydrogen atoms. When stars die, because the hydrogen is used up, the larger atoms produced are released. Massive stars, which produce the large atoms that make up our bodies, die in the form of supernovae, releasing energy and these atoms. The atoms will then be attracted by planetary gravitational pulls, and, if an atom is by chance attracted by planet Earth, it can be incorporated into living beings by the processes we just saw. We are all, quite literally, made of stardust. We are formed from atoms made within stars that died long ago and far away. We are formed by atoms picked up by Earth’s gravity, incorporated into microscopic and macroscopic life today. But we are far more than just a concoction of molecules made from stardust atoms. We are a complex and dynamic, ever-changing, collection of molecules. We are a set of molecules that break down and are rebuilt with a different set of atoms, constantly. We are a collection of molecules that are degraded and released into the air or soil as smaller molecules. We are a constant flow of chemical and electrical energy that keeps all these transformations flowing. We are made today of atoms shared by other beings from the beginning of life on Earth until just seconds ago. Life is a constant exchange and transformation of matter, and this process is called metabolism.

Glossary

1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) a side product of the synthesis of opioids which caused Parkinson’s disease 1-methyl-4-phenyl-4-propionoxy-piperidine (MPPP)  a synthetic opioid analgesic 2,4 dinitrophenol  a molecule that was proposed as a weight loss treatment in the 1930s, then banned by the FDA when many complications from its use were recorded 3-nitropropionic acid  a chemical inhibitor of FAD-linked reactions in mitochondria acetaldehyde  a product of alcohol metabolism that makes you feel drunk acetyl CoA  a molecule that is produced within cells when breaking down carbohydrates, lipids or proteins, and that can generate energy or act as a precursor to produce fat adenosine diphosphate (ADP) an ATP molecule that lost a phosphate group, while releasing energy adenosine triphosphate (ATP)  the main molecule used as an energy source for processes that require energy investments; the energy currency of the cell adipose tissue  the part of our body specialized in the production and storage of fat adrenal glands  structures above our kidneys that produce and secrete adrenalin adrenalin  a hormone secreted by your adrenal glands when you are stressed or exercise aerobic  in the presence of air amino acids  the building blocks of proteins amylase  an enzyme that breaks down carbohydrates into smaller sugars anaerobic  in the absence of air antioxidants  molecules that react with and remove free radicals aspartame  a sweetener composed of two amino acids (aspartate and phenylalanine) linked together ATP synthase  the enzyme that generates most of our ATP in mitochondria beta oxidation  the metabolic pathway that breaks down fatty acids into acetyl CoA molecules © Springer Nature Switzerland AG 2020 A. Kowaltowski, F. Abdulkader, Where Does All That Food Go?, https://doi.org/10.1007/978-3-030-50968-2

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biological membrane  a barrier between two water-based portions of the cell, produced by phospholipids organized in a double layer body mass index (BMI)  the division of weight by height squared, in kilograms and meters, used to evaluate body weight in proportion to height cachexia  loss of muscle mass Calvin cycle  a metabolic pathway present in plants that incorporates CO2 from the air into carbohydrate molecules carbohydrates biological molecules in which most carbon atoms reacted with water, so that many oxygen and hydrogen atoms from the water molecules remain in the molecular structure carbon dioxide (CO2)  a gas we eliminate by breathing, produced from the breakdown of larger biological molecules cardiac hypertrophy  abnormal enlargement of the heart cardiac ischemia  heart attack catalysis  acceleration of chemical reactions cellulose  a large carbohydrate plants use to maintain their structure, which we cannot digest, therefore constituting a fiber chitin a non-digestible carbohydrate (fiber) that constitutes the exoskeletons of insects and crustaceans cholesterol  a lipid molecule present in the membranes of animals chylomicron  a structure in which fats from our foods are transported throughout our body collagen  an abundant protein that maintains the structure of our skin and joints creatine phosphate  a small molecule synthesized from amino acids which carries a phosphate group with an energy content similar to ATP enzymes  complex molecules with 3-D structures that create environments within them that facilitate (catalyze) chemical reactions excitotoxicity  a process in which neurons die because of excessive neurotransmitter molecules fatty acids  a straight chain of carbon atoms with an acid carboxyl group at the end; components of most lipids fibers  carbohydrate molecules we cannot digest, and therefore are eliminated in our feces flavin adenine dinucleotide (FAD)  a molecule that picks up and delivers electrons in many metabolic processes free radicals  molecules that have unpaired electrons, which typically makes them more reactive glucagon  a hormone produced in the pancreas, that regulates blood sugar levels, among other roles gluconeogenesis  the metabolic pathway that produces glucose from molecules that are not carbohydrates glucose  a six-carbon carbohydrate which is a building block for many larger carbohydrates such as glycogen and starch glucose-6-phosphate  a glucose molecule in which a phosphate group was added, making it unable to leave the cell and activating it to be further metabolized

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glycogen  a large carbohydrate molecule produced by animals as an energy storage molecule; present in large quantities in the liver and muscles glycolytic pathway (glycolysis)  the metabolic path that breaks down most of our carbohydrates high density lipoprotein (HDL)  informally known as “good cholesterol”; a form of lipid in the blood that, when high, indicates that the body does not have excess cholesterol hormones  chemical messengers that are produced in one part of the body, circulate around (frequently in the blood) and change how many other cells within the body work hypothalamus a region of the brain which regulates sleep, thirst and hunger, among other functions insulin  a hormone produced in the pancreas that controls blood sugar levels, among other functions ischemia  lack of adequate blood flow to a tissue ischemic preconditioning  a natural mechanism that can help protect hearts against the damage of a heart attack ketoacidosis  a medical condition resulting from excess ketone production ketones  molecules produced by the liver by joining two acetyl CoA molecules, and exported to other organs as an energy source kinases  enzymes that transfer phosphate bonds Krebs cycle, citrate acid cycle or tricarboxylic acid cycle  a cyclic metabolic pathway that is the endpoint of breaking down carbohydrates, lipids and proteins lactase  an enzyme that breaks down lactose, and which is insufficient in persons with lactose intolerance lactose  a sugar found in milk and composed of glucose and galactose leptin a hormone which is produced in our adipose (fat) tissue and that regulates hunger lipids  also known as fats, a group of biological molecules that does not dissolve in water, and function as energy stores and separations between cells (membranes) lipoproteins  structures containing lipids and proteins that transport lipids, including cholesterol, in the blood low density lipoprotein (LDL)  informally known as “bad cholesterol” is a form of lipid in the blood that, when high, indicates that the body has excess cholesterol metabolic regulation  processes that modulate the speed and capacity of specific metabolic pathways, controlling their output metabolic regulator  a molecule capable of modulating a specific metabolic process mitochondria  organelles, or parts of our cells, in which many metabolic processes occur, responsible for generating most of our ATP monosodium glutamate (MSG)  a cooking additive which gives food a delicious taste because glutamate is perceived as such by our tongues multicellular organisms  living beings with many cells neurotransmitters  chemicals that promote communication between brain cells nicotinamide adenine dinucleotide (NAD)  a molecule that picks up and delivers electrons in many metabolic processes

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Glossary

nitric oxide  a free radical that participates in many biological processes, including erection omega fatty acids fatty acids with double bonds in places humans cannot make them oxidation  a chemical process in which a molecule loses electrons oxidative phosphorylation the metabolic process in which electron transfer to oxygen, producing water, is coupled to the production of ATP molecules, with high efficiency pancreas  a long and flat organ inside our abdomen that produces insulin and glucagon, as well digestive enzymes pentose pathway  a metabolic pathway with many functions, including generating sugars with different numbers of carbon atoms, including 5-carbon sugars (pentoses) peptides  small strands of amino acids phenylalanine  an amino acid present in most animal proteins phenylketonuria (PKU)  a disease in which the enzyme necessary to break down phenylalanine is absent phospholipids  a type of lipid that is the main component of biological membranes portal vein  the blood vessel that transports blood from your intestines to your liver pro-opiomelanocortin (POMC)  a protein that controls hunger in our brains reduction  a chemical process in which a molecule gains electrons respiratory quotient  a metabolic measurement of how much of the energy from food is converted to useful energy rotenone  a toxin that inhibits the removal of electrons from NAD in mitochondria rubisco  the enzyme that starts the production of carbohydrates from CO2 in plants saturated fatty acids  fatty acids with no double bonds between their carbon atoms starch  large carbohydrate molecules produced by plants as energy storage molecules; the main component of dietary staples such as rice, potatoes, and bread striatum  a part of the brain involved in movement control substantia nigra the part of the brain in which cells are destroyed in Parkinson’s disease sucrose  table sugar sugars  smaller carbohydrate molecules which usually taste sweet superoxide anion  a free radical produced by our mitochondria which has many roles, including regulating defenses against free radicals trans fats  lipids that have double bonds between carbons in a trans configuration, which is rarely produced in living beings, and usually generated by industrial processes such as hydrogenation of oils triglycerides  the main kind of fat molecule we store, formed by three long carbon chains linked to a glycerol molecule tumor necrosis factor alfa  a hormone that promotes the breakdown of proteins in the muscle and release of their amino acids uncoupling proteins  proteins in mitochondria which decrease ATP production by allowing part of the energy in our food to be converted into heat instead of ATP

Glossary

147

unsaturated fatty acids  fatty acids with one or more double bonds between their carbon atoms urea cycle a metabolic pathway in the liver that removes nitrogen from amino acids, forming urea, which is eliminated in the urine vitamins  molecules that are necessary for us, but we are not capable of producing on our own Warburg effect  a shift from oxidative to fermentative metabolism often seen in cancer cells

Index

A Acetaldehyde, 81, 82, 143 Acetaldehyde dehydrogenase, 80–82 Adenosine triphosphate (ATP), 10–13, 25, 26, 29, 31, 34–44, 46–49, 53, 62–64, 69, 77, 80, 81, 92, 95, 109, 111, 113, 114, 117, 119, 125–132, 136, 139, 140, 143–146 Adipose tissue, 62, 86, 92, 93, 96, 104, 131, 132, 143 Adipose tissue metabolism, 62, 86, 92, 93, 104, 131, 132 Adrenalin, 62, 128, 130–132, 143 Aerobic exercise, 126, 132 Alcohol dehydrogenase, 80–82 Alzheimer’s disease, 83, 112 Amino acids, 7, 12, 67–78, 80, 86, 97, 101, 102, 113, 115, 127, 136, 137, 143, 144, 146, 147 Anaerobic exercise, 126 Antioxidants, 26, 57, 120–123, 143 Aspartame, 70, 71, 143 ATP synthases, 9, 43, 46–49, 143

Carbohydrates, 7, 8, 14, 15, 17–38, 49, 59–66, 69, 72, 74, 75, 80, 81, 84–90, 95, 98, 117, 123, 140, 143–146 Cardiac hypertrophy, 123, 144 Cellulose, 19, 144 Chemi-osmotic hypothesis, 42 Chloroplasts, 42, 139 Cholesterol, 30, 55–58, 60, 61, 113, 144, 145 Collagen, 72, 73, 144 Creatine phosphate, 127, 128, 130, 144 D Diabetes type I, 97–99, 101, 104 type II, 104, 105 Diets, v, 2, 7, 20, 31, 40, 55, 56, 58, 59, 64–66, 71–74, 76–78, 80, 84, 86–89, 94–96, 123, 127 Digestion and absorption, 21, 24

B Beta oxidation of fatty acids, 62–64 Bile salts, 56, 60, 61 Body mass index (BMI), 90, 144

E Electron transport chain, 42 Enzymes, 8–10, 12, 13, 19, 22–25, 47–49, 60–62, 67, 69, 71, 73, 76, 80, 81, 100, 103, 120, 121, 140, 143–146 Ethanol, 3, 79–81 Excitotoxicity, 113–115, 144

C Cachexia, 137, 144 Calcium signaling, 129 Calvin cycle, 139, 144 Cancers, 83, 99, 135–137, 147

F Fasting state, 30 Fatigue, 130 Fat synthesis, 69 Fatty acids

© Springer Nature Switzerland AG 2020 A. Kowaltowski, F. Abdulkader, Where Does All That Food Go?, https://doi.org/10.1007/978-3-030-50968-2

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Index

150 Fatty acids (cont.) saturated, 57–59, 146 trans, 57, 58 unsaturated, 57–59, 147 Fed state, 30 Fibers, 18–21, 23, 25, 144 Flavin adenine dinucleotide (FAD), 39–44, 46, 49, 63, 64, 111, 127, 144 Free radicals, 29, 38, 112, 118–123, 135, 143, 144, 146 Fructose, 21, 22, 88 G Galactose, 21–24, 145 Glucagon, 27, 32, 61, 62, 65, 75, 85, 87, 105, 144, 146 Gluconeogenesis, 75, 82, 130, 131, 144 Glucoses, 12, 14, 18–29, 31–34, 36, 39, 40, 63, 74–76, 82, 84–88, 97–100, 104, 105, 113–115, 117, 123, 127, 128, 130–132, 136, 137, 144, 145 Glycogen, 12, 14, 18, 20, 26–31, 36, 75, 85–87, 91, 104, 113, 117, 127–131, 144, 145 Glycolysis (glycolytic pathway), 7, 26, 29, 32–34, 36, 39, 44, 127–129, 145 Gout, 77, 83 H High density lipoprotein (HDL), 55, 56, 145 Hormones, 13, 14, 21, 27, 29, 32, 55, 56, 59, 61, 62, 65, 67, 73–76, 84, 85, 87, 91–94, 97, 128, 131, 137, 143–146 Huntington’s disease, 110 Hypothalamus, 91–93, 145 I Insulin, 14, 21, 23, 27, 29, 32, 61, 62, 65, 66, 71, 73–75, 84–88, 91–93, 97–106, 145, 146 Insulin resistance, 34, 105 Ischemia, 117, 144, 145 K Ketogenic diets, 64–66 Ketones, 64–66, 84, 88, 97, 98, 104, 117, 123, 131, 145 Krebs cycle, 34–37, 39, 40, 44, 63–64, 80, 145

L Lactase, 23–25, 145 Lactose, 21–24, 145 Lactose malabsorption, 24 Leptin, 92–94, 145 Lipases, 60, 61, 63 Lipids, 3, 12, 15, 30–33, 40, 44, 49, 51–66, 69, 72, 74, 80, 85–87, 89, 113, 119, 131, 136, 137, 143–146 Lipoproteins, 55, 56, 61, 145 Liver metabolism, 18, 25–29, 33, 56, 57, 60, 62, 64, 65, 74, 75, 80, 81, 84–86, 97, 104, 105, 117, 130–132 Low density lipoprotein (LDL), 55–59, 145 M Metabolism, v, vi, 1–15, 17–36, 38, 40–42, 47, 51–115, 117–123, 125–133, 135–137, 139, 141, 143, 147 Mitochondrial DNA, 111, 112 Mitochondrion, 37, 40, 43 Monosodium glutamate (MSG), 70, 145 Motor proteins actin, 125 myosin, 125 Muscle contraction, 125–129 Muscle metabolism, 11, 14, 18, 22, 28, 64, 66, 67, 73–75, 77, 85–88, 90, 94, 97, 112, 117, 118, 123, 125–133, 135, 137 N Nicotinamide adenine dinucleotide (NAD), 39–44, 46, 49, 63, 64, 80, 81, 109–111, 127, 145, 146 Nitric oxide, 119, 146 Nitrogen fixation, 140 O Obesity, 20, 22, 32, 45, 47, 59, 60, 66, 70, 71, 82–96, 98, 99, 104, 112, 113, 115, 135 Oxidation, 3, 34, 38–44, 46, 62–64, 110, 128, 136, 143, 146 Oxidative phosphorylation, 36, 37, 63, 109, 146 P Pancreatic beta-cells, 97 Parkinson’s disease, 108

Index Pentose pathway, 26, 29, 36, 120, 146 Phenylalanine, 7, 70, 76, 77, 143, 146 Phenylketonuria, 7, 76, 146 Plant metabolism, 8, 18–20, 27, 42, 54, 55, 85, 139, 140 Preconditioning, 122, 123, 145 Proteins, 3, 7–9, 12, 14, 15, 17, 31, 34, 38, 40, 43, 47–49, 52–57, 59, 61, 66–78, 82, 84–87, 89–92, 94–97, 99–104, 110, 111, 119, 125–130, 133, 136, 137, 140, 143–146 Purine metabolism, 77 R Redox reactions, 38, 39, 41, 42, 49 Reductions, 38–41, 43, 85, 89, 91, 146 Rubisco, 140, 146 S Scurvy, 72, 73 Starches, 2, 18–23, 27, 31, 85, 88, 91, 139, 140, 144, 146 Strokes, 83, 113–115

151 Substantia nigra, 108, 110, 146 Sucrose, 9, 18, 21, 22, 88, 146 Sugars, 3, 7, 9, 10, 14, 18–29, 32, 36, 59, 62, 66, 70, 71, 74, 75, 82, 85–89, 91, 92, 97, 104, 139, 143–146 Superoxide, 119, 120, 146 T Triglycerides, 30–33, 36, 52–54, 57, 58, 60–63, 74, 86, 87, 89, 92, 117, 131, 146 U Uncouplers, 46 Uncoupling proteins, 95, 96, 146 Urea cycle, 3, 34, 74, 147 V Vitamin c, 73 W Warburg effect, 136, 147