Biochemistry of Diabetes and Atherosclerosis [1 ed.] 978-1-4613-4852-8, 978-1-4419-9236-9

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BIOCHEMISTRY OF DIABETES AND ATHEROSCLEROSIS

Biochemistry of Diabetes and Atherosclerosis Edited by

JAMES S.C. GILCHRIST

PARAMJITS.TAPPIA

Division of Stroke Vascular Disease St. Boniface General Hospital Research Center 351 Tache Avenue R2H 2A6, Winnipeg, Manitoba Canada

Institute of Cardiovascular Sciences St. Boniface General Hospital Research Center 351 Tache Avenue R2H 2A6, Winnipeg, Manitoba Canada

THOMAS NETTICADAN Institute of Cardiovascular Sciences St. Boniface General Hospital Research Center R2H 2A6, Winnipeg, Manitoba Canada

Reprinted from Molecular and Cellular Biochemistry, Volume 249 (2003)

Springer Science+Business Media, LLC

Biochemistry of diabetes and atherosclerosis / edited by James S.C. Gilchrist, Paramjit S. Tappia, Thomas Netticadan. p. ; cm. - (Developments in molecular and cellular biochemistry; 42) Includes bibliographical references and index. ISBN 978-1-4613-4852-8 ISBN 978-1-4419-9236-9 (eBook) DOI 10.1007/978-4419-9236-9 1. Atherosclerosis-Molecular aspects. 2. Diabetes-Molecular aspects. 3. Diabetes-Complications-Molecular aspects. I. Gilchrist, James S.C. (James Stuart Charles) II. Tappia, Paramjit S. III. Netticadan, Thomas. IV. Series. [DNLM: 1. Diabetes Mellitus-metabolism. 2. Diabetes Mellitusphysiopathology. 3. Arteriosclerosis-metabolism. 4. Arterio sclerosisphysiopathology. W K 810 B615 2003] RC692.B526 2003 616.4'62-dc21 2003044530

Copyright © 2003 by Springer Science+Business Media New York Originally published by Kluwer Academic Publishers in 2003

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Molecular and Cellular Biochemistry: An International Journal for Chemical Biology in Health and Disease CONTENTS VOLUME 249, Nos. 1 & 2, July 2003 BIOCHEMISTRY OF DIABETES AND ATHEROSCLEROSIS Drs. James S.C. Gilchrist, Paramjit S. Tappia and Thomas Netticadan Preface T. Nagasawa, N. Tabata, Y. Ito, N. Nishizawa , Y. Aiba and D.D. Kitts: Inhibition of glycation reaction in tissue protein incubations by water soluble rutin derivative R.S. Faustino, S. Sobrattee , A.L. Edel and G.N. Pierce : Comparative analysis of the phenolic content of selected Chilean, Canadian and American Merlot red wines H.P. del Valle, E.C. Lascano, LA . Negroni and A.J. Crottogini : Absence of ischemic preconditioning protection in diabetic sheep hearts : Role of sarcolemmal KATP channel dysfunction B. Huisamen : Protein kinase B in the diabetic heart M. Gyongyosi, W. Sperker, C. Csonk a, D. Bonderman, I. Lang, e. Strehblow, C. Adlbrecht , M. Shirazi, U. Windberger, S. Marlovits, M. Gottsauner-Wolf, P. Wexberg , M. Kockx, P. Ferdinandy and D. Glogar: Inhibition of interleukin-Hl convertase is associated with decrease of neointimal hyperplasia after coronary artery stenting in pigs N. Satoh and Y. Kitada: Effects of MCC-135 on Ca" uptake by sarcoplasmic reticulum and myofilament sensitivity to Ca" in isolated ventricular muscles of rats with diabetic cardiomyopathy D.N. Umrani, D.N. Bodiwala and R.K. Goyal: Effect of sarpogrelate on altered STZ-diabetes induced cardiovascular responses to 5hydroxytryptamine in rats M. Strniskova, M. Barancik, J. Neckar and T. Ravingerova: Mitogen-activated protein kinases in the acute diabetic myocardium T. Matsubara , T. Ishibashi , T. Hori, K. Ozaki, T. Mezaki, K. Tsuchida , A. Nasuno , K. Kubota, T. Tanaka, T. Miida, Y. Aizawa and M. Nishio : Association between coronary endothelial dysfunction and local inflammation of atherosclerotic coronary arteries P. Religa, K. Bojakowski, Z. Gaciong, J. Thyberg and U. Hedin : Arteriosclerosis in rat aortic allografts: Dynamics of cell growth, apoptosis and expression of extracellular matrix proteins B. Murali, D.N. Umrani and R.K. Goyal : Effect of chronic treatment with losartan on streptozotocin-induced renal dysfunction R. Chen, S. Xiong, Y. Yang, W. Fu, Y. Wang and J. Ge: The relationship between human cytomegalovirus infection and atherosclerosis development E. Aasum, A.D. Hafstad and T.S. Larsen : Changes in substrate metabolism in isolated mouse hearts following ischemia-reperfusion J.e. Chatham, B. Bouchard and C. Des Rosiers : A comparison between NMR and GCMS 13C-isotopomer analysis in cardiac metabolism K.R. Bidasee, K. Nallani, B. Henry, U.D. Dincer and H.R. Besch Jr: Chronic diabetes alters function and expression of ryanodine receptor calcium-release channels in rat hearts A. Pourmoghaddas and A. Hekmatnia: The relationship between QTc interval and card iac autonomic neuropathy in diabetes mellitus V.Z. Lankin, A.K. Tikhaze, v.v. Kukharchuk, G.G. Konovalova, 0.1 . Pisarenko, A.I. Kaminnyi, K.B. Shumaev and Y.N. Belenkov: Antioxidants decreases the intensification of low density lipoprotein in vivo peroxidation during therapy with statins D. Wilson, H. Massaeli, G.N. Pierce and P. Zahradka: Native and minimally oxidized low density lipoprotein depress smooth muscle matrix metalloproteinase levels D. Wilson, H. Massaeli, J.e. Russell, G.N. Pierce and P. Zahradka: Low matrix metalloproteinase levels precede vascular lesion formation in the JCR :LA-cp rat K. Carvajal, G. Banos and R. Moreno-Sanchez: Impairment of glucose metabolism and energy transfer in the rat heart T. Ravingerova, J. Neckaf and P. Kolar : Ischemic tolerance of rat hearts in acute and chronic phases of experimental diabetes B. Ziegelhoffer-Mihalovicova, I. Waczulfkova, Libusa Sikurova, J. Styk, J. Carsky and A. Ziegelhoffer: Remodelling of the sarcolemma in diabetic rat heart s: The role of membrane fluidity Index to Volume 249

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Molecular and Cellular Biochemistry 249: 1, 2003. © 2003 Kluwer Academic Publishers.

Preface It is believed that there are more than 150 million diabetic patients worldwide and it has been estimated that around 300 million will be afflicted by this condition by 2025 . Much is known about the health risks of obesity, diabetes, hypertension and hyperlipidemia as causal factors in cardiovascular disease, however, despite the fact that diabetes and cardiovascular disease is costly to health care, and that the rising mortality rate is predominantly associated with the increase in cardiovascular complications, there is an essential need to expand our current knowledge in order to develop more effective drugs and nutritional strategies for the treatment of diabetes and its complications. Diabetes is an autoimmune, inflammatory disease affecting many different organ systems and exhibiting both primary and secondary defects. Because diabetes affects a wide range of cellular systems, a multidisciplinaryeffort has been mounted over the past several decades using a wide range of investigative techniques and methodologies in order to identify molecular mechanisms responsible for cellular dysfunction. Because insulin has such a profound influence on protein and energy metabolism, a number of primary defects at various levels of sub-cellular signaling, intracellular calcium handling , protein expression and energy regulation are often a primary consequence of diabetes. Accordingly, with the increased sophistication of biochemical and molecular investigative techniques over the last 30 we have witnessed great progress toward unraveling the details of its etiology.

Much remains to be determined however. A major target of the secondary complications of diabetes is the vascular system . We have known this for a long time but exactly how this occurs is unclear and continues to be a very fertile area of research. The work reported in this focused issue of Molecular and Cellular Biochemistry is a compilation of new multi-disciplinary research efforts that are being driven to broaden our current understanding of diabetes and cardiovascular disease as well as provide the basis for the development of novel therapeutic interventions. Finally, we would like to thank all of our contributors and we hope that we have produced a readable and practical update of present information that will be of interest to health professionals and fellow researchers involved in this very important area. James S.c. Gilchrist Division of Stroke Vascular Disease St. Boniface General Hospital Research Centre Winnipeg, Manitoba, Canada E-mail: ｪｧｩｬ｣ｨｲ｀ｳ｢ Ｎ｣｡［ｪｭ･ｳｾｩｬｨｲｴ｀｢ Paramjit S. Tappia and Thomas Netticadan Institute of Cardiovascular Sciences St. Boniface General Hospital Research Centre Winnipeg, Manitoba, Canada

Molecular and Cellular Biochemistry 249: 3-10,2003. © 2003 Kluwer Academi c Publish ers.

Inhibition of glycation reaction in tissue protein incubations by water soluble rutin derivative Takashi Nagasawa,' Nobuaki Tabata,' Yoshiaki Ito,' Naoyuki Nishizawa, I Youichi Aiba' and David D. Kitts' 'Food and Health Scienc e, Faculty ofAgriculture, Iwate University, Morioka, Iwate ; "Ioyo Sugar Refining Co. Ltd., Tokyo, Japan ; 3Food, Health and Nutrition, Faculty ofAgricultural Sciences, The University ofBritish Columbia, Vancouver, BC, Canada

Abstract In the Maillard reaction, nonenzymatic glycation reaction reversibly produces Amadori rearrangement products which subsequently lead to the formation of irreversible advanced glycation end-product (AGE). These reactions are important in the pathogenesis of complications associated with diabetes. This study examined the antioxidant activity of rutin and related efficacy to inhibit glycation in three distinct tissue protein sources . Rutin and the rutin analogue exhibited significant antioxidant activity in a liposomal model reaction similar to quercetin. Incubation of rat muscle and kidney proteins with 50 mM glucose for 5 days resulted in the generation of N-fructoselysine (FL), a biomarker for initial stage glycation. The addition of G-rutin, a water soluble glucose derivative of rutin, to the incubation medium (0.1 mM) reduced (p < 0.05) FL production. AGE content in both muscle and kidney proteins was also increased (p < 0.05) with the addition of glucose in the incubation mixture , but completely suppressed by the presence of G-rutin. On the contrary, inhibition of FL and AGE formation by G-rutin was found to be comparatively less effective in bovine serum albumin than both muscle and kidney proteins. These results demonstrate that the antioxidant activity of G-rutin corresponds to a strong affinity to suppres s the formation of both initial and advanced stages of Maillard reaction in tissue protein sources. (Mol Cell Biochem 249: 3-10,2003) Key words: rutin, N-fructoselysine, advanced glycation end-products, muscle, kidney

Introduction The non-enzymatic reaction occurring between glucose, or other reducing sugars with amino groups of protein, peptides or certain amino acids results in the Maillard reaction [1]. Initial products of the Maillard reaction are generated from the conversion of unstable Schiff base adducts to form stable Amadori rearrangement products. The rearrangement products undergo further transformation to irreversible, advanced glycation end-products (AGEs) complexes . AGEs are known to accumulate in both plasma proteins and slow turnover tissue protein sources such as collagen and lens of aged and diabetic subjects [2-10]. Moreover, accumulation of tissue AGEs has been linked with diabetic complications that include vascular diseases [II], diabetic neuropathy [12], and

renal failure [13] . Oxidative stress may be an underlying contributor to these diseases, because formation of AGEs can be enhanced by free radicals. Moreover, the formation of AGEs can result in the production of reactive oxygen species (e.g. ROS) that catalyze glycoxidation reactions [11]. It follows therefore, that dietary intervention strategies which may include increased consumption of antioxidant containing fruits and vegetables could potentially suppress the accumulation of AGEs of tissues in diabetic susceptible subjects and thus reduce the risk to the onset of diabetic related chronic diseases . Tissue AGEs accumulation can be controlled by several inhibitors which prevent AGEs formation. Aminoguanidine is one example of a glycation inhibitor which acts by trapping reactive dicarbonyls and reducing the formation of free radi-

Address/or offpr ints :T. Naga sawa, Food and Health Science , Department ofAgro-bioscience, Faculty of Agriculture, Iwate Univer sity, 3-18-8 Ueda , Morioka, Iwate 020-8550, Japan (E-mail: tnaga@iwate-u .ac.jp)

4

cals [3,10,14]. Bioflavonoids have also been shown to reduce formation of glycated hemoglobin [8]. In the latter example, the antioxidant activity of certain bioflavonoid constituents may explain the mechanism for inhibiting glycation reactions by reducing the generation of ROS that otherwise contribute to increased oxidative stress. Rutin (quercetin-3-0-J3rhamnosylglucose), containing in buckwheat and pagoda tree (Sophora japonica L.), is one example of an antioxidative bioflavonoid [15, 16] which can suppress glycation of aminoguanidine substrate [3]; albeit the mechanism of action is unknown. G-rutin (4G-a-D-glucopyranosylrutin, Fig. I) is derived by enzymatic transglycosylation [17] and used as an antioxidant and a colorant for foods. The present study examines the in vitro antioxidant activity and tissue anti-glycation properties of a water soluble rutin glucose derivative, G-rutin . We were particularly concerned with determining the relative efficacy of G-rutin to inhibit AGEs in three body protein sources, namely skeletal muscle proteins, kidney proteins and serum albumin. Skeletal muscle protein is the most abundant protein in the body and susceptible to AGE accumulation because of its characteristic slow turnover rate [4]. In the kidney, an AGE receptor has been reported thereby facilitating AGEs accumulation in kidney tissues and possibly related reduced organ function [12]. Serum albumin is also sensitive to high concentration of glucose in diabetic patients . We evaluated the effect of G-rutin on glycation to these proteins measuring NE-fructoselysine (FL) as an index of early stage of glycation and AGE of proteins by Western blotting.

OH OH

HO

OH

0

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R=H-Rutin R=Glucose; G-Rutin OH OH

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Fig . 1. Structures of rutin and G-rutin.

Materials and methods Materials Rutin and quercetin were obtained from Sigma Chemicals Co. (St. Louis, MO , USA). G-rutin was supplied from Toyo Sugar Refining Co. Ltd. (Tokyo, Japan). 2, 2'-Azobis (2amidinopropane) dihydrochloride (AAPH) was purchased from Wako Chemicals USA Inc . (Richmond, CA, USA). Phosphate buffers made from distilled deionized water were eluted through a Chelex-l00 chelating resin column to eliminate the existence of transition metal ions prior to use [18]. Bovine serum albumin (Fr. V) was purchased from Sigma Chemicals Co. Anti- AGE mouse monoclonal antibody (6D 12) was purchased from Trans Genic Inc . (Kumamoto, Japan). Nll-carbobenzoxy-NE-fructoselysine was prepared by the method described by Watanabe et al. [4]. All other chemicals and solvents were analytical grade.

Antioxidant methods (in vitro) Flavonoid protection against AAPH induced liposomal peroxidation was performed according to the method of Hu and Kitts [18]. A 10 mg mL- 1 lecithin stock solution in 10 mM phosphate butter (pH 7.4) was made by ultra sonication of soybean lecithin for 2 h in an ice-water bath using a Bransonic 200 sonicator (Branson cleaning equipment Co., Shelton, CN, USA). The reaction mixture contained 3 mg ml' liposome and 25 ｾｍ of CuCl 2 or 2 mM of AAPH, as well as 100 ｾ of flavonoids in phosphate buffer. Liposomal peroxidation was initiated by incubating the reactants at 37°C . Conjugate diene measurements were taken against a 10 mM phosphate buffer (pH 7.4) . Concentrations of conjugate diene were calculated according to an extinction coefficient a = 295,000 M:' em:' [18]. The interassay variability of this test was less than 4.3%.

Incubation of tissue proteins The animal care protocol for this experiment was approved by the Faculty of Agriculture, Iwate University Animal Research Committee under the Guidelines for Animal Experiments of Iwate University. Gastronemius muscle (200 mg) samples were collected from 5 week old male Wistar strain rats which were anesthetized with diethylether and homog enized with 2 mL of water containing 0.5 mL of 100 w/v % trichloroacetic acid. Tissue extracts were centrifuged at 1,000 g and the pellet was recovered and washed with diethyl ether before drying. Kidneys were collected from the same rat and the acid extracted proteins were processed as described for

5 the skeletal muscle. Incubation of extracted proteins (10 mg mL-1) from skeletal muscle, kidney or bovine serum albumin (BSA) sources were conducted in 2 mL of 0.2 M sodium phosphate buffer (pH 7.4) including 0.01% gentamycin sulfate. Following a short adjustment period, the addition of 2 mL of 100 mM glucose to the incubation mixture followed. G-rutin, rutin or quercetin were dissolved in dimethylsulfoxide at various concentrations and added the protein incubation mixture. Aminoguanidine was dissolved in 0 .2 M sodium phosphate buffer and added the incubation mixture to represent a positive control. Incubation mixtures were held at 37°C for 5 days in a shaking water bath. The reaction was terminated by the addition of 1 mL 10 w/v % trichloroacetic acid and the contents of the incubation mixture were centrifuged (1,000 g) to recover the tissue protein pellet.

Measurement ofNr-fructoselysine (FL) FL, a common marker for identifying the initiation stage of glycation [4], was analyzed by HPLC in the form of furosine, after hydrolysis of proteins or NU-carbobenzoxy-N-fructoselysine (standard) with 7.75 M hydrochloric acid at 110°C for 24 h. The hydrolyzate was evaporated under reduced pressure and the resulting residue was re-dissolved in HPLCgrade water immediately prior to HPLC analysis. HPLC was performed using a 15 mM sodium dodecylsulfate (SDS)0.1 M phosphate buffer (pH 6.0)/acetonitrile = 80120 as a mobile phase and a LiChrospher 100 RPI8(e) column (4 .0 x 250 mm, Merck, Germany). Absorbance detection was set at 280 nm.

Western blotting Tissue protein pellets recovered from the incubation suspension were dissolved in sample buffer for subsequent identification analysis using SDS polyacrylamide gel electrophoresis (SDS-PAGE). Duplicate SDS-PAGE were performed using 10% gels, where one gel was stained with Coomassie Brilliant Blue (CBB) and proteins identified on a sister duplicate gel were transferred to a nitrocellulose membrane. Western blots were blocked with skim milk and incubated sequentially with anti-AGE antibody. AGE protein bands were visualized by chemiluminescence and exposed to Hyperfilm (Amersham, UK) for final identification.

Other assays

Statistics All results are expressed as mean ± S.E.M. Students r-test was used to test for significant difference between treatments with values considered significantly different at p < 0.05 .

Results The relative affinities of rutin, G-rutin and quercetin to protect against peroxyl radical-induced lipid peroxidation is shown in Fig . 2. Lipid peroxides were produced within 10 min after incubation exposure to the thermalysis products of AAPH in the soybean liposome assay. Quercetin, rutin and G-rutin showed similar significant (p < 0 .05) protection against peroxyl radical induced lipid oxidation compared to the control sample not containing antioxidant. Relatively longer lag phases (e .g. 8 min) and corresponding reduced propagation phases of quercetin, G-rutin and rutin resulted in peak concentrations of conjugate diene lipid oxidation products that were 39, 33 and 52 nmol mg" lecithin, respectively after 100 min incubation, compared to 329 nmol mg" conjugate diene at 25 min for the control sample. The incubation of skeletal muscle proteins with 50 mM glucose resulted in a marked increase (p < 0.01) in protein carbonyl content. This effect was reduced (p < 0.05) with the addition of 1 mM aminoguanidine, and equally inhibited in incubation mixtures that contained G-rutin, rutin and quercetin, respectively, at the same concentration (Fig. 3). The inhibition of carbonyl generation in muscle proteins was similar for both 0.5 mM and 1 mM G-rutin, but less (p < 0.01) than control and 0.1 mM G-rutin, respectively. The concentration of FL in muscle protein was increased lO-fold when exposed for 5 days to the glucose containing

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The protein carbonyl content was measured using 2,4-dinitrophenylhydrazine as described previously [19]. Protein concentration in all tissue protein sources was determined by the method of Markwell et al. [20].

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60

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Incubation time (h) Fig. 2. Relative effect of antioxidant inhibition of peroxyl radical-induced liposome peroxidation. All analysis points represent mean of three samples. Que - quercetin; Rut - rutin; G-R - G-rutin .

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(kDa) Fig. 3. Inhibit ion of increase in protein carbonyl content of skeletal muscle by G-rutin in vitro after incubation with 50 mM glucose . Glc - without glucose ; G-R - G-rutin; Rut - rutin; Qur - quercetin ; AG - aminoguanidine. *p < 0.05 vs. without inhibitor.

200 116 97.4 66.2 45

incubation mixture (Fig. 4A). The presence of aminoguanidine in the incubation mixture resulted in a 50% inhibition (p < 0.05) ofFL formation after only 3 days incubation. The comparative response to G-rutin inhibition ofFL was less marked with no change occurring in FL formation until 5 days of incubation. The presence of both G-rutin and aminoguanidine to the incubation mixture completely inhibited AGE formation of muscle proteins that were incubated with glucose for 5 days (Fig. 4B) . The addition of glucose to the incubation mixture containing kidney protein also resulted in an increase (p < 0.01) in kidney protein FL content. The presence of G-rutin to the incubation mixture significantly inhibited (p < 0.01) glucose induced FL formation in kidney proteins when incubated for 5 days (Fig. 5A). In contrast, generation of FL in BSA incubated with glucose was less inhibited by the addition of Grutin (Fig. 5B) . Figures 6 and 7 show the results of Western blotting of kidney proteins and BSA incubated with glucose alone and with glucose and G-rutin. A 116 kDa protein in kidney was identified as a AGE marker protein that was sensitive to the various incubation treatments. Incubation of kidney proteins with glucose resulted in a 15-fold increase in the AGE marker protein, relative to control levels after 5 days of incubation. This response was returned to control levels with the addition of G-rutin (Fig. 6). BSA was also shown the increase in AGE (Fig. 7) as indexed by the presence of the 66.2 kDa protein . The relative response was weaker than that observed for skeletal muscle (Fig. 4) and kidney (Fig. 6); however, notwithstanding this, the presence of G-rutin was found to reduce AGE formation.

-

Incubation time (day)

Glc

G-R AG

1....-

.......

135135135135

+

+ +

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Fig. 4. In vitro inhibition of glycation enhancement of skeletal muscle by G-rutin after incubation with glucose . Panel A, N£-fructoselysine endpoint measure ; Panel B, Western blot analysi s of AGE. Glc - 50 mM glucos e; G-R - O. I mM G-rutin ; AG - I mM aminoguanidine. *p < 0.05 vs. without inhibitor.

Discussion The structure-antioxidant activity relationship of many flavonoids has been shown to depend on both the position and degree of hydroxylation of the compound. For example, the presence of 3-hydroxyl-4-keto group or a 5-hydroxyl-4keto group (when the A ring is hydroxylated at the C5 position) provide antioxidant activity. Catechol group, C 3,C4-di-orthohydroxyl groups on the B-ring also protect against free radical mediated lipid peroxidation by donating hydrogen ions, thus stabilizing active radicals by reducing activity. The structure-activity relationships of flavonoids collectively elicit antioxidant activity both free radical scavenging as well as metal ion chelation [15]. In the former example, chelation of transition metal ions will render them catalytically inactive. Rutin (quercetin-3-rhamnosylglucoside) and quercetin are examples of naturally occurring flavonoids which possess many of the specific structural components that contribute to

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Fig . 5. Inhibition of N'-fructoselysine production in kidney protein (Panel

A) and BSA (Panel B) by G-rutin after incubation with glucose. Glc - 20 mM glucose ; G-R - 0.1 mM G-rutin . *p < 0.05 vs , without G-rutin .

-

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antioxidant properties [15, 16, 21]. The 3-g1ycosylation of quercetin with disaccharide yielding rutin has been reported to reduce the antioxidant activity relative to non-glycosylated flavonoids [21]. G-rutin is a water soluble rutin glucose derivative which also exhibits antioxidant capacity in vivo by inhibiting DNA and protein oxidation [22]. The present study demonstrates similar antioxidant activity for both G-rutin and quercetin which were slightly greater than rutin, in the peroxyl radical induced lipid peroxidation assay. Thus, specific steriochemical differences between rutin and G-rutin were sufficiently different to produce relatively small differences in antioxidant activity compared to the quercetin standard in the liposome model assay . The selection of other

Incubation (days) 135135135

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Fig . 7. Inhibition of AGE production in BSA by G-rutin after incubation

with glucose . Panel A, Western blot of 66 kDa AGE marker protein; Panel B, calculated % of control. Glc - 20 mM glucose ; G-R - 0.1 mM G-rutin .

flavonoids such as myricetin, which has an additional hydroxyl group on the 5' position, or naringenin which has only one hydroxyl group on the B-ring, would have produced a greater range of relative antioxidant activities in the liposome model assay used herein . The hyperglycemic condition and predisposition to oxidative stress are well-documented conditions underlying the diabetic condition [23,24] . An enhanced predisposition to oxidative stress due to marked alterations in tissue antioxidant enzymes has been proposed as a possible underlying cause for the adverse health manifestations of diabetes [25]. Agents with antioxidant or free radical scavenging power may inhibit oxidative reactions associated with glycation [26]. Reduced oxidative stress in the diabetic condition has also been observed in experimental animals and in human subjects following the administration of antioxidants, such as vitamin E and certain polyphenols [27-29] . Antioxidant supplementation with vitamin E has also been shown to reduce lipoprotein oxidation in diabetic rats, thus indicating that ROS are involved in various aspects of tissue damage that accompany diabetes [30]. Furthermore, antioxidants can reduce tissue injury in diabetic subjects by protecting against the generation of superoxide anion that results from the formation of AGEs during reaction of Amadori rearrangement product [31]. Garcinol, a polyisoprenylated benzophenone derivative found in the Garcinia indica fruit, is a recent example of a plant constituent that possesses antioxidant and

8 anti-glycation activities [32]. It could be suggested that a dietary intervention which includes greater consumption of naturally occurring antioxidant rich food sources known to complement the cellular mechanisms involved with removal of ROS in response to AGE formation of diabetic subjects could lead to potential improvement of the oxidative status. In the present study, protein carbonyl content of muscle tissue incubated with glucose was shown to be markedly increased with the formation of AGE products. Events associated with the non-enzymatic reaction of protein with reducing sugar results in the production ofROS with the generation of Amadori reactive products [1]. The positive control standard aminoguanidine used in this study is a known inhibitor of glycation, and acts to interfere with reactive carbonyl groups of AGE [33]. Giardino et al. [14] have also demonstrated that aminoguanidine suppresses AGE production by trapping reactive dicarbonyls and thereby impeding conversion to AGE and related generation of free radicals. Our findings herein, that showed a reduced formation of reactive protein carbonyl content of the different tissue and muscle protein sources tested with exposure to G-rutin, as well as rutin and quercetin, corresponded to the similar in vitro antioxidant activity noted for these three compounds. Thus it is reasonable to suggest that they are involved in the potential reduction of ROS that is related to AGE formation. FL content, is a specific marker for early stage glycation [4] compared to specific fluorescent aggregates which represent advanced glycated end-products (AGEs). In the present study, FL was not reduced in skeletal muscle protein and BSA when incubated in the presence of G-rutin . On the other hand, G-rutin was very effective at reducing FL production in kidney tissue. These observations corresponded to the fact that the positive control, aminoguanidine was affective at inhibiting FL formation in all protein sources. The reason for the variable affinity of G-rutin to reduce FL production in all tissue protein sources, compared to aminoguanidine is not certain at this time. A possible explanation, for the apparent specific tissue protein protection against early stage glycation observed herein with G-rutin and not with aminoguanidine, could be the different chemical reactants that result in FL and subsequently AGE formation. Other workers have specifically shown that the formation of 3-deoxyglucosone derived from Amadori products reacts directly with protein [13] and does not solely involve the generation of free radicals. There is additional evidence to suggest that antioxidant activity may not be the only mechanism required to protect against early stage glycation for all reactants. Rather, mechanisms of action underlying the activity for various glycation inhibitors is very complex and specifically related to variables that include differences in substrate sources of glycation and possibly stage of glycation. In support of this conclusion it is important to note that several AGEs, including Nt-(carboxymethyl)lysine (CML) can

be generated by reacting protein with reducing sugar [34]. In the present experiment, we used anti-AGE mouse monoclonal antibody (6D 12), which specifically reacts with CML and not with FL [35], to assess AGE formation. Results of Western blotting analysis that showed a complete inhibition of AGE formation attributed to G-rutin, clearly demonstrate the strong anti-glycation affinity for this compound. It is therefore of interest that G-rutin also displayed a relatively weaker affinity to reduce FL production, as ascertained with the fluorescent measurements. The apparent different findings based on different end-point measures assessing antiglycation activity may reflect the different sensitivities of the two assays to show inhibitory effect. Alternatively, it can not be discounted that several different mechanisms may exist to protect against glycation reactions and therefore may be specific for different sources of AGE inhibitors. For example, the keto acid, pyruvate, protects against AGE formation by inhibition of Schiff base formation [36], were as a thiazolium compound prevents protein cross -linking [37]. Strong chelation activity of trace metal ions that otherwise can catalyze glycation has also been shown for garcinol, a polyiso-prenylated benzophenone derivative from Garcinia indica and plant pigment used for food preparations [32]. Finally, a synthetic thiazolidine derivative has been reported to inhibit AGE formation through changes in signal transduction [38]. Oddetti et al. [3], have previously shown that rutin reduces collagen-linked fluorescence in streptozotocin-induced diabetic rats; however, the mechanism was not clearly delineated because rutin, like quercetin is also an inhibitor of aldose reductase activity [39]. Other studies have also indicated that flavonoids (e.g. a mixture of diosmin and hesperidin) decrease HbA]C concentration in response to increased antioxidant enzyme activity [8], which further emphasizes the importance of reducing oxidative stress in order to protect against AGE formation . The antioxidant activity of G-rutin shown in the present study in the in vitro phospholipid micelle model system certainly fits with the potential affinity to protect against AGE through its capacity to scavenge free radicals. In conclusion, our findings clearly indicate that G-rutin is a potent glycation inhibitor, especially with kidney proteins that are susceptible to early and advanced glycation reactions . In the present study we have extended the findings of others that have demonstrated certain anti-glycation properties of antioxidants using simple in vitro reducing sugar-protein model systems . Defining antioxidant potential of G-rutin in the micelle system and employing specific tissue proteins sources with relevant measurements of complex AGE formation herein, have provided strong evidence that an antioxi dant property of G-rutin and related flavonoids is related to anti-glycation properties attributed to nonenzymatic reactions between proteins and reducing sugars. The concentration of G-rutin used in this experiment was 1 mM which is about

9 0.08% . This concentration is easy to achieve through foods. Future studies are required to include in vivo experiments to re-establish the anti-glycation properties of G-rutin, if the results are to have realized beneficial effects on the management of diabetic complications.

Acknowledgements The authors wish to acknowledge the financial assistance from Natural Sciences and Engineering Council of Canada (DDK) for conducting this study.

References 1. YaylyanVA,Huyghues-Despointes A: Chemistry of Amadori rearrangement products:Analysis, synthesis, kinetics, reactions, and spectroscopic properties . Crit Rev Food Sci Nutr 34: 321-369, 1994 2. Chellan P, Nagaraj RH: Protein crosslinking by the Maillard reaction : dicarbonyl-derived imidazolium crosslinks in aging and diabetes. Arch Biochem Biophys 368: 98-104, 1999 3. Odetti PR, Borgoglio A, De Pascale A, Rolandi R, Adezati L: Preventation of diabetes-increased aging effect on rat collagen-linked fluorescence by aminoguanidine and rutin . Diabetes 39: 796-801, 1990 4. WatanabeH, Ogasawara M, Suzuki N, Nishizawa N, Ambo K: Glycation of myofibrillar protein in aged rats and mice. Biosci Biotechnol Biochem 56: 1109-1112, 1992 5. Ryle C, Leow CK, Donaghy M: Nonenzymatic glycation of peripheral and central nervous system proteins in experimental diabetes mellitus. Muscle Nerve 20: 577-584, 1997 6. Schleicher ED, Wagner E, Nerlich AG: Increase accumulation of the glycoxidation product Nt-(carboxymethyl)lysine in human tissues in diabetes and aging. 1 Clin Invest 99: 457--468, 1997 7. Frye EB, Degenhardt TP, Thorpe SR, Baynes lW: Role of the Maillard reaction in aging of tissue proteins. 1 Bioi Chern 273: 18714-18719, 1998 8. Manuel y Keenoy B, Vertommen 1, De Leeuw I: The effect of flavonoid treatment on the glycation and antioxidant status in type I diabetic patients . Diab Nutr Metab 12: 256-263, 1999 9. Verzijl N, DeGroot 1, Oldehinkel E, Bank RA, Thorpe SR, Baynes lW, Bayliss MT, Brjlsma lWl, Lafeber FPIO, TeKoppele 1M: Age-related accumulation of Maillard reaction products in human articular cartilage collagen . Biochem 1 350: 381-387,2000 10. Singh R, Barden A, Mori T, Beilin L: Advanced glycation end-products: A review. Diabetologia 44: 129-146,2001 II. Chappey 0, Dosquet C, Wautier M-P, Wautier l-L: Advanced glycation end products, oxidant stress and vascular lesions. Eur 1 Clin Invest 27: 97-108, 1997 12. Sima AAF, Sugimoto K: Experimental diabetic neuropathy: An update. Diabetologia 42: 773-788, 1999 13. Miyata T, Saito A, Kurokawa K, VanYpersele de Strihou C: Advanced glycation and lipoxidation end products: Reactive carbonyl compounds-related uremic toxicity. Nephrol Dial Transplant 16(suppI4): 8-11 ,2001 14. Giardino I, Fard AK, Hatchell DL, Brownlee M: Aminoguanidine inhibits reactive oxygen species formation, lipid peroxidation and oxidant induced apoptosis, Diabetes 47: 1114-1120, 1998

15. Rice-Evance CA, Miller Nl, Bolwell Po. Bramley PM, Pridham JB: The relative antioxidant activities of plant-derived polyphenolic flavonoids . Free Radic Res 22: 375-383, 1995 16. Liao K, Yin M: Individual and combined antioxidant effects of seven phenolic agents in human erythrocyte membrane ghosts and phosphatidylcholine liposome systems : Importance of the partition coefficient . 1 Agric Food Chern 48: 2266-2270, 2000 17. Suzuki Y,Suzuki K: Enzymatic formation of 4G-a-glucopyranosylrutin. Agric Bioi Chern 55: 181-187, 1991 18. Hu C, Kitts DD: Evaluation of antioxidant activity of epigalloocatechin gallate in biphasic model systems in vitro: Mol Cell Biochem 218: 147155,2001 19. Nagasawa T, Hatayama T, Watanabe Y,Tanaka M, Niisato Y,Kitts DD: Free radical-mediated effects on skeletal muscle protein in rats treated with Fe-nitrilotriacetate. Biochem Biophys Res Commun 231: 37--41, 1997 20. Markwell MAK, Hass SM, Biebra LL, Tolbert NE: A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87: 206-210, 1986 21. Foti M, Piatteli, M, Baratta MT, Ruberto Gl : Flavonoids, coumarins and cinnamic acids as antioxidants in a micellar system. Structureactivity relationships. 1 Agric Food Chern 44: 497-501, 1996 22. Funabiki R, Takeshita K, Miura Y, Shibasato M, Nagasawa T: Dietary supplement of G-rutin reduces oxidative damage in the rodent model. 1 Agric Food Chern 47: 1078-1082, 1999 23. KakkarR, ManthaSV, Radhi 1, Prasad K, Kalral: Increased oxidative stress in rat liver and pancreas during progression of streptozotocininduced diabetes . Clin Sci 94: 623-632, 1998 24. WestIC: Radicals and oxidative stress in diabetes. Diabet Med 17: 171180,2000 25. Wohaieb SA, Godin DV: Alterations in free radical tissue -defense mechanisms in streptozocin-induced diabetes in rat. Effects of insulin treatment . Diabetes 36: 1014-1018, 1987 26. Elgawish A, Glomb M, Freedlander M, Monnier VM: Involvement of hydrogen peroxide in collagen cross-linking by high glucose in vitro and in vivo. 1 Bioi Chern 271: 12964-12971 , 1996 27. Lean MEl, Noroozi M, Kelly I, Bums 1, Talwar D, Sattar N, Crozier A: Dietary flavonols protect diabetic human lymphocytes against oxidative damage to DNA. Diabetes 48: 176-181, 1999 28. Sharma A, Kharb S, Chugh SN, Kakkar R, Singh GP: Evaluation of oxidative stress before and after vitamin E supplementation in diabetic patients. Metabolism 49 : 160-162,2000 29. Sanders RA, Rauscher FM, Watkins IB III, Effects of quercetin on antioxidant defense in streptozotocin-induced diabetic rats. 1 Biochem Mol Toxicol15: 143-149,2001 30. Morel DW, Chisolm GM: Antioxidant treatment of diabetic rats inhibits lipoprotein oxidation and cytotoxicity. Lipid Res 30: 1827-1834, 1989 31. Mossine VV, Linetsky M, Glinsky GV, Ortwerth Bl, Feather MS: Superoxide free radical generation by Amadori compounds : The role of acyclic forms and metal ions. Chern Res Toxicol12: 230-236,1999 32. Yamaguchi F, Ariga T, Yoshimura Y, Nakazawa H: Antioxidative and anti-glycation activity of garcinol from Garcinia indica fruit rind. 1 Agric Food Chern 48: 180-185,2000 33. Brownlee M, Vlassara H, Kooney A, Ulrich P, Cerami A: Aminoguanidine prevents diabetes-induced arterial wall protein cross-linking. Science 232: 1629-1632, 1986 34. Ahmed MV, Thorpe SR, Baynes lW: Identification of Nt-carboxymethyllysine as a degradation product of fructose -lysine in glycated protein, 1 Bioi Chern 261: 4889--4894,1986 35. Ikeda K, Higashi T, Sano H, linnouchi Y, Yoshida M, Araki T, Veda S, Horiuchi S: Nt-(Carbox ymethyl)lysine protein adduct is a major immunological epitope in proteins modified with advanced glycation end products of the Maillard reaction. Biochemistry 35: 8075-8083, 1996

10 36. Zhao W, Devamanoharan PS, Varma SD: Fructose-mediated damage to lens a-crystallin: Prevention by pyruvate. Biochim Biophys Acta 1500: 161-168,2000 37. Vasan S, Zhang X, Zhang X, Kapurniotu A, Bernhagen J, Teichberg S, Basgen J, Wagle D, Shih D, Teriecky I, Bucala R, Cerami A, Egan J, Ulrich P: An agent cleaving glucose-derived protein crosslinks in vitro and in vivo. Nature 382: 275-278, 1996 38. Miyata T, Veda Y,Asahi K, Izuhara Y, Inag i B, Saito A, Van Ypersele

de Strihou C, Kurokawa K: Mechanism of the inhibitory effect of OPB -9195 [(±- 2)-isopropylidenehydrazono-4-oxo-thiazolidin-5ylacetanilide] on advanced glycation end product and advanced lipoxidation end product formation. J Am Soc Nephrol 11: 17191725,2000 39. Varma DS, Kinoshita JH : Inhibition of lens aldose reductase by flavonoids : Their possible role in the prevention of diabetic cataracts. BiochemPharmacoI25:2505-2513,1976

Molecular and Cellular Biochemistry 249: 11-19. 2003. © 2003 Kluwer Academic Publishers.

Comparative analysis of the phenolic content of selected Chilean, Canadian and American Merlot red wines R.S. Faustino, S. Sobrattee, A.L. Edel and G.N. Pierce National Centre for Agri-Food Research in Medicine, and the Division of Stroke and Vascular Disease, St. Boniface General Hospital Research Centre, Department of Physiology, University ofManitoba, Winnipeg, Manitoba, Canada

Abstract Flavonoids are a group of naturally occurring antioxidant compounds found in wine that are thought to have therapeutic importance in cardiovascular disease [1, 2]. The flavonoid content of red wines can differ as a function of the variety of wine examined [3-7] . Since there is a paucity of data on the content of these antioxidants in Merlot wine, we used high performance liquid chromatography to identify and compare catechin , epicatechin, rutin, transresveratrol and quercetin levels in selected Merlot wines from Canada, Chile and the United States. Additionally, antioxidant content was correlated with the price of the wine. Catechin content was the most abundant when compared to the other four phenolic compounds. The concentrations of each compound in the Merlot wines also varied as a function of the country of origin . Catechin and transresveratrol occurred in significantly lower concentrations in Merlots from the United States. The lowest levels of rutin were observed in Canadian Merlots. Quercetin occurred at significantly higher levels in Chilean Merlots. Wine prices were inversely correlated with catechin concentration. Merlot wine represents a source of antioxidants that may have an impact on cardiovascular disease. (Mol Cell Biochem 249: 11-19,2003) Key words: flavonoids, antioxidants, heart disease, atherosclerosis, catechin, resveratrol, quercetin

Introduction Elevated cholesterol and elevated low density lipoprotein (LDL) levels are associated with coronary heart disease and atherosclerosis. Recently, the participation of an oxidized form of LDL has been implicated in the generation of an atherosclerotic plaque [8, 9]. It has been hypothesized that the prevention of LDL oxidation by antioxidants may be a useful therapeutic strategy [10-12]. Thus, the identification and characterization of antioxidants is potentially important information in the study of cardiovascular disease. A number of foods and beverages have been identified that contain antioxidants that may possess health related benefits when ingested. The consumption of wine has been reported to induce cardiovascular benefits [2]. Referred to as the 'French paradox' , there is a low incidence of coronary heart disease (CHD) within the French population despite the prevalence

of many risk factors for CHD [13]. It was suggested that a possible explanation for this paradox may be due to a relatively high wine consumption in France that may confer protective effects with regard to cardiovascular disease. In vitro studies have demonstrated antioxidant properties within wine that may be responsible for this protective cardiovascular effect [7, 14]. Catechin, epicatechin, rutin, quercetin are four phenolic substances belonging to a broad class of molecules collectively referred to as flavonoids. Flavonoids are phenol compounds that possess potent antioxidant capacity within red wines [15, 16]. Transresveratrol is a polyphenolic phytoalexin that has also been reported to possess a variety of biological activities [3,17-21], including protection against oxidation [22-24]. Not all wines, however, possess similar levels of antioxidants. Red wines, for example, contain far greater quantities of antioxidants than white wines . The concentrations of the various

Address for offprints: GN . Pierce , Division of Stroke and Vascular Disease, St. Boniface General Hospital Research Centre , 351 Tache Avenue , Winnipeg , Manitoba, Canada. R2H 2A6 (E-mail : [email protected])

12 phenolic species also differ depending upon where the wine is produced, how it is produced and the conditions under which the cultivars are grown. The flavonoid content can also vary according to the type of red wine examined. For example, the catechin content of a Pinot Noir variety is much greater than that of a Cabemet Sauvignon [25]. Merlot wine is a variety of red wine that has recently gained popularity in North America. The purpose of the present study was to investigate the levels of catechin, epicatechin, rutin, quercetin and transresveratrol in Merlot wine . In view of our knowledge that different climates can influence phenolic content, we also assessed and compared the levels of these different compounds in selected Merlot wines of Chilean, Canadian and American origin to determine if Merlot wine produced in widely varying environmental regions of the world would contain very different phenolic concentrations.

Materials and methods Catechin, epicatechin, rutin, transresveratrol and quercetin were purchased from Sigma-Aldrich and were used as standards for peak identification and quantification. A Waters Model 501 coupled to a Rheodyne 7725i injector was used for solvent delivery. A Waters 484 Tunable absorbance detector was used for solvent detection, together with Waters Baseline 810 Software to determine concentration. A Prodigy reversed phase column, 4.6 x 150 mm, 511particle size from Waters/ Millipore was used for the stationary phase with a flow rate of 0.5 mL/min. The solvents used for the separation were : Solution A = 5% acetic acid, 15% methanol, 80% ddl-l.O; Solvent B =5% acetic acid, 20% methanol, 75% Hp; Solvent C = 5% acetic acid , 45% methanol, 50% Hp Standards were injected at time zero and solution A was run for 0-5 min at a wavelength of 280 nm, followed by solution B for 5-20 min . The wavelength was changed to 306 nm and solution C was run for 20-45 min followed by solution A again for 45 min to 1 h.

Several brands of Chilean, Canadian and American Merlots were used for this study (Table 1). These were chosen in a random fashion from stock solutions commercially available in Canada .After establishing standard curves for each concentration of the phenolic species, the injection syringe was rinsed out twice with the sample to be injected and a total volume of 25 III of wine was injected into the injection port. Individual standards were examined at arbitrary points throughout the course of the experiment to verify the validity of the original standard curve.

Statistical analysis The concentrations of catechin, epicatechin, rutin, transresveratrol and quercetin were measured and analyzed using one way ANOVA. Values were reported as mean concentration ± S.D. Phenolic content was plotted against price and the degree of correlation was determined by calculating their Pearson Product Moment Correlation coefficient.

Results Typical retention times of the catechin, epicatechin, rutin, transresveratrol and quercetin standards from a typical HPLC analysis are shown in Fig. 1a. Concentration curves were determined for each of the five standards used. Figure 1b shows the concentration curve of the catechin standard as a representative example. It displays a linear relationship for area under the peak and the concentration of catechin. Correlation coefficients for catechin, epicatechin, rutin, transresveratrol and quercetin were 0.9962, 0.9945, 0.9920 , 0.9938 and 0.9977, respectively (data not shown). Once all of the concentration curves were determined for the five standards, samples of the different wines were examined. A representative chromatogram of the flavonol content of a Merlot wine is shown in Fig. 2. A 25 III aliquot of

Table 1. Wines used in this study

Chilean

Canadian

American

Concha y Toro (1997) Walnut Crest (1998) Sunrise Chilean (1997 ) Santa Rita (1997) Santa Isabella* La Playa Merlot (1995) Casa La Postolle (1996) Vina Tarapaca (1996) Santa Monic a (1996) Valdivisio (1995)

Mission Hill Merlot (1996) Peller Estates * Jack son Triggs Merlot (1996) Sumac Ridge Merlot (1995) Bighorn Vineyards * Calona Vineyards (1996) Konzelm ann Estates (1996) Inniskillin Merlot (1996)

Erne st and Julio Gallo (1998) Corbett Canyon (1997) Sutter Home (1996) St. Francis Estate Reserve (1994) Mystic Cliffs (1996) Montere y Vineyard (1995) Rutherford Hill (1996)

Various wines from Chile, Canada and the United States were analyzed for catechin, epicatechin, rutin, transre sveratrol and quercetin contents. Listed are names and vintage s. *denotes wines which were comprised of several different cultivars and did not have a specific vintage.

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I Representative HPLC chromatograms showing separation of wine phenolics. (a) Illu strated is the elution profile of : B - ca techin, C - epic atechin, D - rutin, E - tran sresveratrol and F - quercetin standards attained by HPLC analysis. A - represent s injection point. (b) Standard curve obtained for different concentrations of catechin injected into the system, R = 0.99615 .

Mystic Cliffs Merlot was injected and the catechin, epicatechin, rutin, transresveratrol and quercetin components of the wine (labelled B-F, respectively) can be identified as well as a variety of additional unidentified peaks , The phenolic content of Merlot wine was analyzed, Catechins account for the majority of the phenolics in Chilean and Canadian wines, but epicatechins comprise the majority of the phenolics analyzed in American wines (Figs 3a-3c), Quercetin was the third most abundant antioxidant in all three groups, with rutin and transresveratrol accounting for less than 5% of the phenolic content in all wines examined, The overall flavonoid composition of the Merlot wines from all three countries is detailed in Fig, 3d, The relative abundance

F 2. Chromatogram illustrating separation of wine phenoli cs in Merlot wine, Analysis of red wine by HPLC analysis displ ays other comp ounds in addition to the phenolics selected for this study (indicated by arrows). Ainjection point , B - catechin, C - epicatechin , D - rutin, E - transre sveratrol and F - quercetin.

of the phenolic species in this study is catechin> epicatechin > quercetin> rutin> transresveratrol. The absolute concentrations of the individual phenolic compounds were compared among the Chilean, Canadian and American Merlots used in this experiment. Figure 4 illustrates the differences among the Merlots analyzed from the three countries of origin . Catechin was present in comparable levels in both Chilean and Canadian Merlots but it was not as high in the American wines (Fig. 4a). There was no statistical difference in the concentration of epicatechin found in the three groups (Fig, 4b) , Chilean Merlots possessed more rutin than Merlots from the United States or Canada (Fig. 4c). Transresveratrol content was detected in minute amounts in all of the wines examined (Fig, 4d). American wines, however,possessed a significantly lower amount of transresveratrol than Merlots from the other two countries. Quercetin composition was similar for both Canadian and American Merlots but was significantly higher in Chilean wines (Fig. 4e). It was of interest to determine if the price of the wine was correlated in any way with the flavonoid concentration. The correlation between catechin concentration and the price of the wines from the three different countries was examined (Fig. Sa), Although Chilean and Canadian Merlots exhibited a negative correlation between catechin concentration and wine price, only the Chilean red wines demonstrated any significance (R =-0.7, P < 0.05). No relationship was observed for American Merlots. Overall, a statistically significant negative correlation was found for catechin content and wine price (Fig.5b). Epicatechin content in the Merlots was examined as a function of the cost of the wine (Fig, 6). No significant association of price and epicatechin content was found for Chilean ,

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as a percentage of total composition in the wines . Total composition (100%) was the total amount (in mg) of all five compounds investigated in this study. (a) Phenolic distribution in Chilean Merlots . (b) Phenolic distribution in Canadian red wines . (c) Phenolic distribution in American wines. (d) OveraIl phenolic distribution in all wines studied.

Canadian and American wine (Fig. 6a, p > 0.05 for all countries). Overall, there was no significant correlation between epicatechin concentration and pricing (Fig. 6b). There was a significant negative correlation between rutin concentration and the price of Chilean Merlots (Fig . 7a). Wines from the other two countries exhibited no relationship between wine price and rutin content. Overall, rutin concentration demonstrated no significant correlation to pricing, when examined in all wines studied (Fig. 7b). Although Merlots from all three countries displayed a positive correlation between the concentration of transresveratrol and price of the wine (Fig . 8a), this was not statistically significant (p > 0.05) within countries or when all wines were examined (Fig. 8b). Merlot wines from Chile, Canada and the United States showed no significant correlation between quercetin concen tration and wine pricing (Fig. 9a) . Overall, no statistically

significant correlation of quercetin concentration to wine price was exhibited over all the wines examined (Fig. 9b).

Discussion This is the first report of phenolic content in Merlot wines from Canada, Chile and the United States. While the distribution of the phenolics follows the trend seen in most red wines, the relative amounts of each of the five species show slight variations from the amounts observed in earlier studies [25,26]. The red wines in this study show higher amounts of epicatechin than that reported in a variety of French and Italian red wines in an earlier study [25,27] . We also detected low amounts of rutin in all three Merlots, similar to the concentrations found by Goldberg et al. [26], although they detected rutin in only two of the eighteen wines used in their

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study. The remaining phenolics (catechin, transresveratrol and quercetin) had similar concentrations to that observed by Goldberg et al. [26]. Earlier studies using HPLC analysis have demonstrated that this distribution is typical of red wines [2831]. Therefore, with the exception of the elevated epicatechin and rutin content, the Medot wines exhibited similar phenolic levels as found in other red wines .

The phenolic content of non-alcoholic beverages such as grape juices is very different. Grape juice contains a lower amount of the monomeric catechins and epicatechins than are present in wine [32] . Resveratrol is found in high concentrations in the skins and seeds of grapes and is produced in response to environmental stresses [17, 33, 34] . However, a recent report by Gilly et al. demonstrated that resveratrol in

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grape juice is rapidly degraded by an endogenous tyrosinase [17]. Therefore , a direct comparison between grape ju ice and wine would not reflect initial, absolute resveratrol concentrations in either beverage. The content of catechin, epicatechin, rutin, transresveratro1 and quercetin in Merlot red wines from the United State s, Chile and Canada was also analyzed for comparative purposes. American Merlots appeared to possess the lowest concentration of both catechin and transresveratrol in comparison to wines from the other two countries where as both rutin and quercetin were found in the highest amounts in Merlots from

Chile. Canadian wines appe ared to be in between the two countries in most comparisons. The overall distribution of phenolic content in all red wines studied identified catechins and epicatechins as the most common phenolics, with the remaining three accounting for a small percentage of the compound s studied (1-5 %). Since each phenolic species differ s with regard to its own antioxidant potency, these differences in phenolic compo sition may result in differences in the antioxidant capacity of the wine s. Quercetin is the most potent antio xidant of the five pheno lic species examined [35] . We would hypothe size, therefore , that the ability of Chilean wine

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Fig. 8. Correlations of transresveratrol content to wine price . (a) Trans resveratrollevels from Chile, Canada and America were plotted against price (p > 0.05 in all cases). (b) A slight positive association is shown for transresveratrol and wine price (R = 0.1, P > 0.05) .

to function in an antioxidant capacity may be greater than the wines from the other two countries. However, this remains to be directly tested. If antioxidant content provides significant health-related benefits, its relative content may provide added value. The phenolic content of red wine influences its flavour , stability, appearance and overall aesthetic quality [36]. It was of interest, therefore, to determine the potential correlation of phenolic content and pricing of the wines. Overall, only catechin exhibited a statistically significant association with price (R =-0.5, p < 0.05, Fig. 5b). Surpris ingly, this was a

negative correlation. Within specific countries, only the association between catechin content and price in Chilean Merlots was determined to be statistically significant. We may conclude that similar antioxidant content may be obtained from Merlot wine across a very wide price range. If one is interested in purchasing a Merlot wine high in antioxidant content , it would appear to be erroneous to use the price of the wine as an indirect indicator of antioxidant content (at least within the price range used in this study) . Our data have demonstrated the presence of differences in the phenolic content of wines from three different countries.

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These data and their conclusions are limited by several factors. The environment in which the grapes are grown may affect the final flavonoid content. Temperature, humidity [37, 38] and soil nutrients are a few examples of variables that can influence phenolic composition of wines. Additionally, the method of harvesting and processing and even the container in which the wine is fermented affects flavonoid content [36]. Finally, the age of the wine is another determinant of phenolic concentration in red wine [25]. Together, these variables can affect the quantity of phenolic constituents within the Merlots. We have tried to control the age of the wine as a

3.

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6.

7.

Riemersma RA, Rice-Evans CA, Tyrell RM, Clifford MN, Lean MEl : Tea flavonoids and cardiovascular health. Q J Med 94: 277-282, 2001 Rimm EB, Katan MB, Ascherio A, Stampfer MJ, Willett WC : Relation between intake of flavono ids and risk for coronary heart disease in male health Professionals. Ann Intern Med 125: 384-389,1996 Kopp P: Resveratrol , a phytoestrogen found in red wine . A possible expl anation for the conundrum of the 'French paradox' ? Eur J Endocrinol 138: 619-620, 1998 Nigdikar SV, Williams NR, Griffin BA, Howard AN : Con sumption of red wine polyphenols reduces the susceptibility of low-density lipoproteins to oxidation in vivo. Am J Clin Nutr 68: 258-265, 1998 Iijim a K, Yoshizumi M, Hashimoto M, Kim S, Eto M, Ako J, Liang YQ, Sudoh N, Hosoda K, Nakah ara K, Toba K, Ouchi Y: Red wine polyphenols inhibit proliferation of vascular smooth mus cle cell s and downregulate expre ssion of cyclin A gene . Circulat ion 101: 805-811, 2000 Abu-Amsha R, Croft KD, Puddey IB, Proudfoot JM, Beilin LJ: Phenolic content of various beverages determines the extent of inhibition of human serum and low-density lipoprotein oxidation in vitro: Identification and mechani sm of action of some cinnamic acid derivatives from red wine. Clin Sci 91: 449-458,1996 Kerry NL, Abbey M: Red wine and fractionated phenolic compounds prepared from red wine inhibit low density lipoprotein oxidation in vitro. Atherosclerosis 135: 93-102,1997

19 8. Steinberg D: A critical look at the evidence for the oxidation of LDL in atherogenesis. Atherosclerosis 131(suppl) : S5-S7, 1997 9. Berliner lA, Navab M, Fogelman AM, Frank JS, Derner LL, Edward s PA, Watson AD, Lusis Al : Atherosclerosis: Basic mechanisms . Oxidation, inflammation and genetics . Circulation 91: 2488-2496,1995 10. Giugliano D: Dietary antioxidants for cardiovascular prevention . Nutr Metab Cardiovasc Dis 10: 38-44, 2000 II. Frei B: Molecular and biological mechanisms of antioxidant action. FASEB 1 13: 963-964, 1999 12. Chopra M, Thurnham DI: Antioxidants and lipoprotein metabolism. Proc Nutr Soc 58: 663-671 ,1999 13. Renaud S, Lorgeril MD: Wine, alcohol, platelets , and the French paradox for coronary heart disease. Lancet 339: 1523-1526, 1992 14. Teissedre PL, Frankel EN, Waterhouse AL, Peleg H, German JB: Inhibition of in vitro human LDL oxidation by phenolic antioxidants from grapes and wines . J Sci Food Agric 70: 55-61, 1996 15. Miyagi Y, Miwa K, Inoue H: Inhibition of human low-density lipoprotein oxidation by flavonoids in red wine and grape juice. Am J Cardiol 80: 1627-1631, 1997 16. deWhalley CV, Rankin SM, Hoult lRS, lessup W, Leake DS: Flavonoids inhibit the oxidative modification of low densit y lipoproteins by macrophage s. Biochem Pharmacol39: 1743-1750, 1990 17. Gilly R, Mara D, Oded S, Zohar K: Resveratrol and a novel tyrosinase in carignan grape juice. 1 Agric Food Chern 49 : 1479-1485, 2001 18. Pace-Asciak CR, Hahn S, Diamandi s EP, Soleas G, Goldberg DM: The red wine phenolics trans-resveratrol and quercetin block human platelet aggregation and eicosanoid synthesis: Implications for protection against coronary heart disease . Clin Chim Acta 207-219,1995 19. Pendurthi UR, Williams IT, Rao LVM: Resveratrol, a polyphenolic compound found in wine, inhibits tissue factor expres sion in vascular cells. Vase Bioi 19: 419-426,1999 20. Rotondo S, Rajtar G, Manarin i S, Celardo A, Rotilio D, Gaetano GD, Evangelista V, Cerletti C: Effect of trans-resveratrol, a natural polyphenolic compound, on human polymorphonuclear leukocyte function. Br J Pharmacol123 : 1691-1699, 1998 21. El-Mowafy AM, White RE: Resver atrol inhibit s MAPK activity and nuclear transloc ation in coronary artery smooth muscle : Reversal of endothelin-I stimulatory effects . FEBS Lett 451 : 63-67, 1999 22. Stojanovic S, Sprinz H, Brede 0 : Efficiency and mechanism of the antioxidant action of trans-resveratrol and its analogues in the radical liposome oxidation . Arch Biochem Biophys 391: 79-89, 2001 23. Wu 1, Wang Z, Hsieh T, Bruder 1, Zou 1, Huang Y: Mechanism of

24.

25.

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27.

28. 29. 30. 31.

32.

33.

34. 35.

36.

37.

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cardioprotection by resveratrol, a phenolic antioxidan t present in red wine. Int J Mol Med 8: 3-17, 2001 Fremont L, Belguendouz L, Delpal S: Antioxidant activity of resveratrol and alcohol -free wine polyphenols related to LDL oxidation and polyunsaturated fatty acids. Life Sci 64: 2511-2521, 1999 Landrault N, Poucheret P, Ravel P, Gasc F, Cros G, Teissedre P-L: Antioxidant capacities and phenolics levels of French wines from different varieties and vintages. J Agric Food Chern 49: 3341-3348, 2001 Goldberg DM, Tsang E, Karumanchiri A, Diamandis EP, Soleas G, Ng E: Method to assay the concentrations of phenolic constituents of biological interest in wines . Anal Chern 68: 1688-1694, 1996 Ghiselli A, Nardini M, Baldi A, Scaccin i C: Antioxid ant activity of different phenolic fractions separated from an Italian red wine. J Agric Food Chern 46: 361-367, 1998 Lamuela-Ravent6s RM, Waterhouse AL: A direct HPLC separation of wine phenolics . Am 1 Enol Vitic 45 : 1-5, 1994 Roggero I-P,Archier P, Coen S: Wine phenolics analysis via direct injection: Enhancement of the method. J Liq Chromatogr 14: 533-538 ,1991 Roggero 1-P, Coen S, Archier P: Wine phenolics: Optimization of HPLC analysis. 1 Liq Chromatogr 13: 2593-2603, 1990 Salagony-Auguste M-H, Bertrand A: Wine phenolics-analysis oflow molecular weight components by high performance liquid chromatography. J Sci Food Agric 35: 1241-1247,1984 Arts lCW, Putte Bvd, Hollman PCH: Catechin contents offoods commonly consumed in the Netherlands. 2. Tea, wine, fruit juices, and chocolate milk. 1 Agric Food Chern 48 : 1752-1757,2000 Jeandet P, Bessis R, Sbaghi M, Meunier P, Trollat P: Resveratrol content of wines of different ages: Relationship with fungal disease pressure in the vineyard . Am J Enol Vitic 46 : 1-4, 1995 Siemann EH, Creasy LL: Concentration of the phytoalexin resveratrol in wine. Am 1 Enol Vitic 43: 49-52,1992 Makris DP, Rossiter IT : Comparison of quercetin and a non-orthohydroxy flavonol as antioxidants by competing in vitro oxidation reactions . 1 Agric Food Chern 49: 2001 Auw 1M, Blanco V, O'Keefe SF, Sims CA: Effect of processing on the phenolics and color of Cabernet Sauvignon , Chambourcin, and Noble wines and juices. Am 1 Enol Vitic 47 : 279-286, 1996 Martinez-Ortega MV, Carcia-Parrilla MC, Troncoso AM: Resveratrol content in wines and musts from the south of Spain. Nahrung44: 253256,2000 Del Alamo M, Bernal Jl., Gomez-Cordoves C: Behaviour of mono saccharides, phenolic compound s, and color of red wines aged in used oak barrels and in the bottle . J Agric Food Chern 48: 4613-4618, 2000

Molecular and Cellular Biochemistry 249: 21-30, 2003. © 2003 Kluwer Academic Publishers .

Absence of ischemic preconditioning protection in diabetic sheep hearts: Role of sarcolemmal KATP

channel dysfunction Hector F. del Valle, Elena C. Lascano, Jorge A. Negroni and Alberto J. Crottogini Department of Physiology, Pharmacology and Biochemistry, Favaloro University, Buenos Aires, Argentina

Abstract Sarcolemmal ATP-sensitive potassium (KATP) channels have been mentioned to participate in preconditioning protection. Since these channels are altered in diabetes, it would be possible that preconditioning does not develop in diabetic (D) hearts. The purpose of this study was to assess whether early (EP) and late (LP) ischemic preconditioning protect diabetic hearts against stunning in a conscious diabetic sheep model and whether diabetes might have altered KATP channel functioning . Sheep received alloxan monohydrate (1 g) and were ascribed to three experimental groups: control (DC, 12 min of ischemia (I) followed by 2 h of reperfusion (R)), early preconditioning (DEP, six 5 min 1-5 min R periods were performed before the 12 min I) and late preconditioning (DLP, same as DEP except that the preconditioning stimulus was performed 24 h before the 12 min I). Regional mechanics during reperfusion was evaluated as the percent recovery of wall thickening fraction (%WTH) expressed as percentage of basal values (100%) and KATP behaviour was indirectly assessed by monophasic action potential duration (MAPD) and sensitivity to glibenclamide blockade (0.1 and 0.4 mg/Kg) . The results were compared to those obtained in normal (N) sheep . EP and LP protected against stunning in normal sheep (%WTH: NC = 63 ± 3.7, NLP = 80 ± 5** , NEP = 78 ± 3*, *p < 0.05 and **p < 0.01 against NC) whereas contrary results occurred in diabetic ones , where DLP (%WTH =60 ± 4) afforded a similar recovery to DC (%WTH = 54 ± 5) and DEP surprisingly worsened instead of improving mechanical function (%WTH =38 ± 6, p < 0.01 against DC) . KATP channel behaviour appeared altered in diabetic hearts as shown by MAPD during ischemia in normal sheep (153 ± 9 msec) compared to diabetic ones (128 ± 11 msec, p < 0.05) and by the sensitivity to glibenclamide (while 0.4 mg/Kg blocked action potential shortening in normal and diabetic animals, 0.1 mg/Kg completely blocked KATP in diabetic but not in normal hearts, p < 0.05). A sarcolemmal KATP channel dysfunction might afford a primary approach to explain the absence of ischemic preconditioning protection against stunning in diabetic sheep. (Mol Cell Biochem249: 21-30,2003)

Key words : diabetic heart, ischemic preconditioning, myocardial stunning, KATP channel, ischemia and reperfusion, sheep

Introduction In 1986 Murry et al . described an important endogenous cardioprotective mechanism against infarction which received the name of ischemic preconditioning [1). The phenomenon is considered as the most important mechanism of cardioprotection described up to the present. The first works pointed out protection against infarction [1-3J but later, preconditioning development was described against arrhythmias

[4, 5J and both systolic [6, 7J and diastolic [8J stunning. Ischemic preconditioning has two well recognized phases : early preconditioning (which appears immediately after the stimulus and disappears within 3 h) and delayed or late preconditioning (which appears 12-24 h after the stimulus and remains for at least 48-72 h) [9J. Even though the phenomenon has been studied in a great variety of experimental models, preconditioning has almost always been described in 'normal' hearts and there is rela-

Addressfor offp rints : H.P. del Valle, Favaloro Univer sity, Solis 453, Buenos Aires (078), Argent ina (E-mail: [email protected])

22 tively little experience in pathologic hearts (e.g. diabetic, hypertrophic). Although many authors found classic or early ischemic preconditioning protection aga inst infarction in diabetic [10-13] or hypertrophied [14, 15] hearts, the afforded cardioprotection has been controversial and notoriously, there are no reports in which early and late preconditioning protection against stunning or infarction has been studied in a large pathologic animal model with co-incident cardiovascular pathology such as diabetes. Diabetes mellitus is a disorder of carbohydrate, lipid and protein metabolism that affects many organs. In addition to contractile abnormalities, this disease causes disturbances in the function of cardiac subcellular organelles, including the sarcolemma, sarcoplasmic reticulum and mitochondria [16] . This pathology is also associated [16] with several abnormalities in energy metabolism, depressed Na+-Ca2+ and Na+-H+ exchange activities, decreased sarcoplasmic reticular Ca 2+ and Na+-K+ pump activities, and elevated antioxidant defenses. All or many of the mentioned alterations might explain the reported differences in response to ischemic injury in diabetic vs. normal hearts [17,18]. Diabetes also alters the functioning of vascular and myocardial ATP-dependent potassium channels (KATP channels) [19-21] and in addition, channel density appears to be diminished in diabetic hearts [20,22]. Many authors have identified the KATP channel as a major contributor to preconditioning protection against infarction and stunning [23]; thus , it is probable that the cardioprotection afforded by preconditioning would be reduced in diabetic hearts . Since the development of large conscious animal models with co-incident cardiovascular pathology has been recommended for the study of ischemic preconditioning [24] and because many authors have mentioned preconditioning as a ' healthy heart phenomenon' [11,25] ; our objective was to evaluate whether early and late preconditioning against stunning could be obtained in diabetic conscious sheep, and whether KATP channels might playa role in the response to ischemia-reperfusion events in diabetic hearts.

Materials and methods Animal treatment Male castrated Hampshire Down sheep aged 6 to 8 months, weighing 27-30 Kg were used and treated according to the 'Guide for the Care and Use Laboratory Animals' , published by the US National Institute of Health (NIH publication No . 85-23, revised 1996).

Diabetic conscious sheep model Five weeks before instrumentation diabetes was induced with alloxan monohydrate infused at a total dose of 1 g (25 ± 4 mgt

Kg). The drug was dissolved in 10 ml sterile saline and administered over 1 min via a jugular vein to sheep that had been fasted for the previous 24 h, as performed in dogs [26]. To ensure diabetes maintenance, venous blood samples were taken in the fasted state on 2 consecutive days before alloxan injection and twice a week after drug infusion (Fig. 1). Glu cose, triglycerides, total cholesterol, HDL, LDL creatinine, total proteins and albumin were automatically determined by a Hitachi 912 Automatic Analyzer (Boehringer, Mannheim Systems) while glycated hemoglobin was determined in a IMX analyzer (Abbot Laboratories, Argentina) and ketonuria, urinary pH , urinary proteins and glycosuria by using a Multistix 10 SG (Bayer, Argentina) (Tables 1 and 2). To perform the glucose tolerance test, glucose (l g/Kg) was slowly infused through a venous catheter in the relaxed, conscious sheep (Fig. 1). The procedure and the obtained results were similar to that mentioned in dogs [26] and sheep [27] . To make comparisons between the diabetic and normal metabolic state, 6 non-diabetic animals were used.

Surgical procedure As described [8,28], after sedation with acepromazine maleate (0.3 mg/Kg), anesthesia was induced with thiopental sodium (20 mg/Kg). Following intubation and connection to mechanical ventilation (Neumovent 910, Cordoba, Argentina), anesthesia was maintained with 3% enflurane carried in oxygen and fentanyl citrate 0.1 mg. A thoracotomy was performed at the fifth intercostal space and after pericardiotomy a pres sure microtransducer (Konisberg P7, Pasadena, CA, USA) was inserted in the left ventricular cavity. Tygon fluid-filled catheters were inserted in the mammary internal vein (for drug infusion) and in the left ventricle (for Konigsberg calibration). The left anterior descending coronary artery was dissected just distal to the second diagonal branch, and a pneumatic cuff occluder was positioned around it. To obtain left ventricular wall thickness (WTH), a pair of piezoelectric crystals (5 Mhz) was placed within the zone to be rendered ischemic. All wires and catheters were tunneled subcutaneously to emerge between the scapulae and the thoracotomy was closed without pericardial closure.

Experimental protocol One week after surgery, the animals were studied standing in a cage. The fluid filled ventricular catheter was connected to a pressure transducer (DT-XX, Viggo-Spectramed, Oxnard, CA, USA) previously calibrated using a transducer calibration system (Xcaliber, Viggo-Spectramed). The zero pressure point was set approximately at the level of the right atrium, and the signal generated by the Konigsberg transducer was

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Time (days) F g 1. Biochem ical profil e of the diabetic sheep model. Panel A shows glycemic levels in normal and diabetic anima ls. Blood glucose levels increased after alloxan injec tion and were mai ntai ned over 4 weeks. Oral glucose tolerance curves did not differ notoriou sly between norma l and diabetic sheep (panel B). Noteworthily, although the alloxan dose (I g) resulted in diabete s develo pment , it did not cause a toxic effect nor a decompensated state as panel C shows. After a period of instability, biochemical and weig ht measurements remain ed stab le during 5 weeks. ' p < 0.000 1 diabetes vs. norm al sheep (r-test) , Data are mean ± S.E. Seric crea tinine is expressed in flg/IO ml for bett er representation .

adjusted to match that of a Statham transducer. The ultrasonic pair of crystals was connected to a sonomicrometer (Triton, San Diego, CA, USA) and calibrated in mm using the internal calibration . At each acquisition time all signals were digitized at 4 msec interval during 15 sec using a personal computer equipped with an NO converter (National Instruments Lab-PC , Austin, TX, USA) and software developed in our laboratory.

To assess difference s in the re sponse to ischemia-reperfu sion events between healthy and pathologic hearts, experiments were performed in non-d iabetic (normal) and diabetic sheep. The animals were randomly ascribed to six experimental groups: (1) normal control ischemia (NC, n = 9): after 20 min of basal recording s sheep underwent 12 min of regional ischemia followed by 120 min of reperfusion; (2) diabetic control ischemia (DC, n = 7): same as in (1) ; (3) nor-

Table 1. Blood biochem ical profile in norm al and diabetic shee p

Normal Diabetic

Glycemia

Glycosilate

(mg/dl)

HGb (%)

59 ± 4.5 190±61

3.3 ± 0.3 4.4 ± 0.3'

Ketonemia Albumina (g/dl) +/-

- t-

3.72 ± 0.03 3.60 ± 0.11

Total plasma Total cholesterol HDL cholesterol proteins (gldl) (mg/dl) (mg/dl)

LDL cholesterol Triglycerides Seric creatinine

7.06 ±0.1 6.90 ± 0.3

26 ± 1.5 45.4 ± 51

47.5 ± 2 90 ± 101

23 ± 2.8 48.6 ± 61

HGb - hemoglobin. Student' s t-test: *p < 0.05, I p < 0.0 1 norm al vs. diabetic group . Data are mean ± S.E.

(mg/dl)

(mg/dl) 5.2 ± I 48.3 ± 71

(mg/dl) 1.2 ± 0.3 1.3 ± 0.5

24 Table 2. Body weight and urinary biochemical profile in normal and dia-

betic sheep Body weight Glycosuria (Kg) (mg/dl) Normal Diabetic

35 ± 3.1 25.9 ± 0.9*

Urinary pH Urinary proteins Ketonuria (mg/dl)

Negative 7.6 ± 0.5 1090 ± 2291 6.7 ± 0.3

Negative +/++

Negative 90 ± 20 1

Student's r-test: *p < 0.05 and I p < 0.01 normal vs. diabetic group . Data are mean±S.E.

mal early preconditioning (NEP, n = 8): six 5 min ischemia; 5 min reperfusion periods were performed 45 min before the 12 min ischemia; (4) diabetic early preconditioning (DEP, n =6): same as in (3); (5) normal late preconditioning (NLP, n =7): the same as the early preconditioning protocol except that the preconditioning stimulus was performed 24 h before the 12 min ischemia; and (6) diabetic late preconditioning (DLP, n = 6): same as in (5). The signals of 15-25 consecutive steady beats were recorded at each acquisition time: basal (after stabilization of left ventricular pressure and dimensions), preischemia (immediately before ischemia), ischemia (at 12 min of the ischemic period) and reperfusion every 10 min during the first hour and every 20 min during the second hour. Measurements ofleft ventricular regional (percent wall thickening fraction) and global function (end systolic pressure [Pes], end diastolic pressure [Pd], the maximum [PI max] and minimum [PI min] values of the time derivative of left ventricular pressure [PI] and heart rate [HR]) were performed. A 12 min ischemic period was used because this short-term regional ischemia induced considerable deterioration of myocardial function without myocyte death and afforded complete recovery of function [28]. To study KATP channel functioning in normal and diabetic sheep hearts, open chest protocols were performed. KATP channels were indirectly studied by measuring action potential duration and by assessing their response to the blocking effect of two different doses of glibenclamide (0.1 and 0.4 mg/Kg). Monophasic action potentials [28] were measured by placing a Ag/AgCl suction bipolar electrode on the epicardium within the zone to be rendered ischemic. Control recordings were taken in 5 normal and 5 diabetic sheep during basal, preischemia, ischemia (at 2 and 12 min occlusion), and at 2 min of reperfusion. Of the remaining 20 sheep, 5 normal and 5 diabetic were treated with glibenclamide 0.4 mgt Kg, and 5 normal and 5 diabetic with glibenclamide 0.1 mgt Kg. The drug was infused 30 min before ischemia [28] and all experimental recordings were acquired as in control.

Data analysis End diastole was defined to occur at the onset of the rapid upstroke of the digitally obtained P' max while end-systole

was defined as the time point where P' min reached 10% of its minimal value and end ejection was established to occur at P' min. Percent (%) regional wall thickening (WTH) was calculated as: %WTH

= 100. (WTHe -

WTHd)/WTHd

where WTHe is maximum ejective wall thickness between end-systole and end-ejection, and WTHd is end-diastolic wall thickness. At each acquisition time, Pes , Pd, HR, P' max, P' min and %WTH were calculated from each recorded beat and the average of processed beats was the value assigned to the corresponding acquisition time. The value assigned to reperfusion for global hemodynamic variables was the mean integral value (trapezoidal rule) of the first , second, third and fourth half hours, whereas %WTH was measured at 10, 20, 30, 40, 50, 60, 80, 100 and 120 min of reperfusion and referred to its basal value considered as 100% . Monophasic action potential duration (MAPD) was determined at a repolarization of 90% (MAPD90) of maximal plateau amplitude [28].

Statistical analysis Values were expressed as Mean ± S.E . To compare global hemodynamics throughout the protocol, the protection afforded by early and late preconditioning, and MAPD between diabetic and normal control groups, an ANOVA one way for repeated measures test was employed. When statistical differences resulted in p < 0.05, a post hoc analysis using a Scheffe test was performed. Student's 't ' -test was used to compare glycemic levels, biochemical profile and the effect of control ischemia and early preconditioning protocol on %WTH between normal and diabetic sheep.

Results Characterization of diabetic state in sheep Table 1 shows the biochemical profile of blood proteins, lipids and glucose while Table 2 shows changes in urine and body weight in diabetic sheep. Diabetes altered all parameters and this results are completely in accordance with those reported by other authors [27, 29] regarding the characterization of diabetes in ruminants and specially in sheep. It is important to mention that diabetes in our model did not result in an unbalanced state as shown by global hemodynamics (Table 3), regional function (Table 4) and the stabilization in glycemia, cholesterol, creatinine and body weight after the third week (Fig. 1). The slight proteinuria seen in Table 4

25 might be considered within the normal range in sheep [27] and not necessarily as cause of diabetes or alloxan-induced renal toxicity (as shown by the maintenance of seric creatinine levels in Table 1 and Fig . 1).

among experimental groups . These results might be explained on the basis of the small ischemic area in all groups (less than 20% of the total left ventricular mass , data not shown).

Regional contractile behaviour during ischemia and reperfusion

Hemodynamic data Hemodynamic data are shown in Table 3. Note that although a significant rise in Pd was observed during ischemia, it returned immediately to its preischemic value during reperfusion. HR, P' min, P' max and Pes remained unchanged throughout the experiment and there were no differences

The data in Table 4 shows that %WTH at the start of the protocol (basal condition) was similar in all groups. This result and those shown in Table 3 seem to indicate that cardiovascular function was not affected after 4 weeks of diabetes and supports the assumption that our model was a model of compensated diabetes.

Table 3. Hemod ynamic values of left ventricular global function throughout the protocol in diabetic and normal sheep

Basal

Preischemia

Ischemia 12 min

Reperfusion 30 min

Reperfusion 60 min

Reperfusion 90 min

Reperfusion 120 min

Norm al

NC NEP NLP

102.1 ± 3.8 103 ± 4.6 101 ± 4.4

104 ± 3.4 100 ± 5 102 ± 5.3

109 ± 5.7 103 ± 7.2 105.7 ± 4.9

101.4 ± 3.4 102.6 ± 5.8 103.2 ± 4.6

103 ± 4.6 100.7 ± 3.6 98.6 ± 4.3

100±3.4 100.5 ± 3.9 101.5 ± 4.4

101 ± 5.3 100.4 ± 4.6 103.4 ± 5.2

Diabetes

DC DEP DLP

99.8 ± 3.4 103 ± 2.5 98 .9 ± 1.7

99 ±4 100 ± 3.5 100 ± 2.4

99 ±4.3 103 ± 5.3 101 ± 4.6

98.7 ± 4.3 100 ± 2.8 99.8 ± 3.1

100 ± 3.2 102.1 ± 3.3 98 ± 3.1

99 .8 ± 4.3 102 ±4.6 100.9 ±4.2

100.9 ± 2.4 102.9 ± 4.2 99.2 ± 4.9

Normal

NC NEP NLP

10 ± 2.5 9 ± 3.1 11 ± 0.6

9±2 11±2 10.8 ± 1

15±3.tt 16 ± 3.41 15.4 ± 1.61

11.2 ± 2.3 10 ± 2.4 11 ± 1.4

10.2 ± 3.2 9.1 ± 2.1 9 ±2.3

1O± 2.1 9 ± 3.2 10.6 ± 2.2

1O±2 9 ± 1.9 11.2 ± 2.3

Diabetes

DC DEP DLP

10 ± 0.7 9± 1 12 ± 2.3

11 ± 2 9±1 11 ± 3

15.4 ± 2.31 15.6 ± 2.61 16 ± 2 1

10.2 ± 1.1 11.4 ± 2.4 10.7 ± 2.3

10 ± 2.3 9.8 ± 1.4 11 ± 3

11 ± 3.2 10 ± 2.2 12 ± 3.6

9.3 ± 3.1 9±2 11.7 ± 4.2

Normal

NC NEP NLP

93 ± 5.2 88 ± 4.7 90±4

89 ± 6.5 86 ± 4.6 87.4 ± 5.2

90 ±4 0.9 ± 3.2 89 ± 3.6

91.3 ± 4.2 84 ± 3.4 87.5 ± 6.9

89 ±4 86.2 ± 3.6 90 .3 ± 4.5

88.5 ± 3.4 90 ± 5.3 88.8 ± 4.4

92.3 ± 5.3 87.6 ± 4.3 87.6 ± 5.3

Diabetes

DC DEP DLP

97 ± 4.4 86 ± 4.6 94 ± 5.4

99 ± 4.6 82 ± 3.4 84.4 ± 2.3

90 ± 4.4 89 ±4 100 ± 4.6

93.8 ± 5.2 95.1 ± 5.8 93.7 ± 5.4

94 ± 4.3 92 ± 3.4 97 ± 3.2

96.2 ± 3.6 89.1 ± 3.3 94.4 ± 4.6

96.2 ± 4.6 88.6 ± 4.3 90.8 ± 2.7

Norm al

NC NEP NLP

2700 ± 119 2350 ± 111 2259 ± 122

2769 ± 190 2380 ± 132 2242 ± 130

2670 ± 115 2389 ± 130 2235 ± 120

2698 ± 129 2437 ± 115 2312 ± 143

2612 ± 156 2472 ± 146 2230 ± 143

2768 ± 138 2480 ± 200 2352 ± 167

2692 ± 213 2859 ± 232 2239 ± 209

Diabet es

DC DEP DLP

2600± 121 2590 ± 144 2300 ± 135

2549 ± 156 2690 ± 120 2292 ± 163

2498 ± 160 2720 ± 148 2380 ± 145

2602 ± 157 2650 ± 158 2373 ± 165

2590 ± 165 2641 ± 176 2235 ± 129

2600 ± 159 2600 ± 146 2319 ± 169

2600 ± 210 2486 ± 143 2258 ± 126

Norm al

NC NEP NLP

-2264 ± 190 -2155 ± 148 -2110 ± 111

-2261 ± 132 -2135 ± 161 - 2140 ± 105

-2108 ± 170 -2208 ± 166 -2198 ± 115

-2000 ± 140 -2211 ± 141 -1968 ± 105

-2160 ± 124 - 2102.3 ± 124 -1861 ± 126

-2272 ± 108 -2002 ± 154 -1808 ± 138

-2165 ± 123 - 205 1 ± 172 -1728 ± 122

Diabetes

DC DEP DLP

-1983 ± 158 -1997 ± 197 -1882 ± 185

-1993 ± 155 - 1900 ± 190 -1920 ± 194

- 1995 ± 186 -1894 ± 200 -1900 ± 182

-1840 ± 152 -1853 ± 104 -1792 ± 76

- 1830± 174 -1718 ± 186 -1801 ± 103

-1982 ± 159 -1698 ± 102 -1900 ± 100

-1832 ± 155 -1808 ± 180 -1896 ± 190

Pes

Pd

HR

P' max

P'min

NC - normal control; NEP - norm al early preconditioning; NLP - normal late preconditioning; DC - diabetic control sheep ; DEP - diabetic early preconditioning ; DLP - diabetic late preconditioning; Pes (mmHg); Pd (mmHg); HR (beat/min); P' max (mmHg/sec) ; P' min(mmHg/sec) . I p < 0.01 against basal and prei schemic values (one way ANOVA for repe ated measures followed by Scheffe test) . All data are mean ± S.E. There were no differences between group s for any of the con sidered parameters throughout the entire protocol.

26 Table 4. Comparison of %WTH (real values) between normal and diabetic sheep groups before starting the protocols

Normal sheep NEP

NC

34.3 ±4.4

35.2 ±4.9

Diabetic sheep DEP

NLP

DC

33.6 ± 3.6

34.4 ± 4.2

DLP

33 ± 3.2

35.4 ± 4.6

%WTH - percent wall thickening fraction; NC - normal control ; NEP - normal early preconditioning; NLP - normal late preconditioning; CD - control diabetic sheep ; DEP - diabetic early preconditioning; DLP - diabetic late preconditioning. There were no differences among groups (one way ANOYA for repeated measures) . Data are mean ± S.E.

Figures 2 and 3 show mechanical recovery during reperfusion in normal and diabetic sheep when control ischemia and preconditioning protocols were performed. Diabetic hearts exhibited less improvement in functional recovery during control reperfusion when compared to normal sheep (Fig. 2). Interestingly, Fig. 3 shows that although ischemic preconditioning protected against stunning in normal sheep (Fig. 3A) its protection did not develop in diabetic ones (Fig. 3B). Noteworthily, while late ischemic preconditioning afforded a functional recovery similar to that obtained in diabetic sheep subjected to control ischemia, early preconditioning worsened instead of improving myocardial recovery. The latter appears to be in part explained by the existence of a 'cumulative ischemic damage ' caused by the brief preconditioning periods in diabetic hearts, in contrast to the action of these brief ischemialreperfusion intervals as a trigger stimulus to elicit preconditioning protection in normal hearts (Fig. 4). The mechanical behaviour occurred during reperfusion in the absence of global functional changes (Table 3) which indicates that the ischemic area was small enough to be well compensated by the rest of the ventricular mass and that regional measurements were not influenced by changes in global hemodynamic parameters.

Sarcolemmal KATP channel functioning in normal and diabetic sheep hearts As stated, KATP channel functioning was indirectly assessed by measuring MAPD and the response to glibenclamide. Fig-

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Fig. 2. Recovery of wall thickening fract ion during reperfusion after a

12 min control ischemia in normal (NC) and diabetic (DC) sheep . The figure shows that diabete s impaired functional recovery from stunning. *p < 0.05 DC vs, NC (r-test). Data are mean ± S.E.

o

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40

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80

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Fig. 3_ Recovery of wall thickening fraction during reperfusion for control (NC and DC), early (NEP and DEP) and late (NLP and DLP) preconditioning protocols in normal (panel A) and diabetic (panel B) sheep . Noteworthily, early preconditioning was shown to impair regional functional recovery from stunning in diabetic animals. One way ANOYA for repeated measures followed by Scheffe : *p < 0.05 and'p < 0.01 NC and DC against LP and EP. Data are mean ± S.E.

27

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Fig . 4. Early ischemic preconditioning periods in normal (NEP) and diabetic (DEP) sheep . The figure shows the progressive decay in wall thickening fraction during brief ischemia and reperfusion episodes. There appear s to be a 'cumulative deleterious effect' in diabetic animals . While healthy animal s quickly recovered their function at 15, 10 and 5 min before the prolonged ischemia, diabetic hearts were not able to improve their motility . T-test: *p < 0.05 and ' p < 0.01 NEP vs. DEP (r-test), Data are mean ± S.E.

ure 5 shows action potential recordings and the response to glibenclamide blockade in normal and diabetic hearts. MAPD appeared to be longer in diabetic sheep heart (D-MAPD 90 =351 ± 9 msec, p < 0.05) when compared to the normal control (N-MAPD 90 =280 ± 7 msec) before ischemia and during reperfusion (D-MAPD 90 =360 ± 6 msec vs. N-MAPD 90 = 277 ± 10 msec, p < 0.05) (Fig. 5 left panel). During ischemia, when action potential duration diminishes as a consequence of KATP channel activation, MAPD in diabetic hearts (D-MAPD 90 = 128 ± 11 msec) was shorter than in normal ones (N-MAPD 90 =153 ± 9 msec , p < 0.05). The response to glibenclamide differed notoriously between normal and diabetic sheep; while 0.4 mg/Kg completely blocked action potential shortening during ischemia in both groups (Fig. 5A), 0.1 mg/Kg had a 100% blocking effect on action potential shortening in diabetic animals but it did not completely block action potential shortening in normal sheep (Fig . 5B) . This last result and the previous one regarding MAPD differences in both sheep groups during ischemia led us to ascribe them to an altered KATPchannel functioning in diabetic hearts.

Discussion The present work is the first to study the effects of early and late ischemic preconditioning in a diabetic conscious animal model. The main findings regarding ischemia/reperfusion events in the diabetic heart were : (a) diabetes resulted in less functional recovery from stunning after a reversible ischemia (Fig. 2); (b) early and late preconditioning did not protect the

heart against stunning (Fig. 3); (c) early preconditioning stimuli had a 'cumulative deleterious effect' (Fig . 4) accounting for a lower mechanical recovery during reperfusion (Fig. 3); and (d) sarcolemmal KATP channel dysfunction in the diabetic heart might provide an explanation to the mentioned results as shown by the differences in MAPD and in the sensitivity to glibenclamide blockade with respect to normal hearts (Fig . 5). Our findings reinforce for the first time in a in vivo large mammal model the altered behaviour in KATP channel functioning reported in in vitro experiments in diabetic rat [19, 20,30] and mouse [31] hearts . Regarding the lack of mechan ical recovery from stunning and the absence of early preconditioning protection against stunning during diabetes, our results are completely in accordance with those that have shown that diabetic hearts are more sensitive to ischemic injury (stunning) [17] and those that did not find preconditioning protection against infarction [11,13] and arrhythmias [32]. Nevertheless, some studies have mentioned that diabetic hearts are less sensitive to ischemia [18] whereas others have found early preconditioning protection against infarction [10, 33] and endothelial dysfunction [12]. The conflicting data would be explained by differences in the experimental model, the species subject to study and the type and duration of the diabetic state.

Animal model We decided to employ sheep in our study because: (a) diabetes develops in sheep [29], (b) sheep is a docile animal that allows to perform experiments in the conscious state, and (c) this model has been previously employed to study preconditioning [8] and stunning [28] . It is important to mention that alloxan , at the employed dose, did not result in altered kidney function and that the small urinary protein content could not be ascribed to a toxic drug effect since the employed dose was three times lower than the minimal one shown to exert renal toxicity in sheep [27]. One limitation of our model would be the type and the duration of the induced diabetes; although type I diabetes (or insulin dependent) would result in a similar clinical and/or pathological manifestations as type II diabetes, it might have particular events in the development of the pathology that turns it different from type II. However, preconditioning protection has been proved to develop in both type II [10] (including human cardiomyocytes [34]) and in type I diabetic hearts [11-13, 33]. Regarding the maintenance of diabetes in sheep, it was similar to the one reported in rats [33] and dogs [13]. Nevertheless, the duration of diabetes seems not to be determinant in the experimental findings [10, 11]. After 3 weeks of diabetes induction the metabolic profile remained stable (Fig. 1) and was compatible with that previously

28 Action potential duration

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Fig. 5. Left panel shows monophasic action potential duration (MAPD) during preischemia, ischemia (at 12 min) and reperfusion (at 2 min) in normal and diabetic hearts . Note prominent differences in action potential duration in diabetic vs normal sheep . Panels A and B show change s in MAPD during ischemia and reperfusion (percent change from its basal values considered as 100%) in normal and diabetic hearts before and after glibenclamide infusion . The figures show MAPD during preisch emia (0 min) , ischemia (at 2 and 12 min) and reperfusion (2 min) . Glibenclamide at high doses (0.4 mglKg) blocked action potential shortening in both normal and diabetic animals (panel A) while glibenclamide at low doses (0.1 mg/Kg) completely blocked action potential shortening (measured at 90% of repolarization) during ischemia in diabetic but not in normal hearts (panel B). This might be explained on the basis of a differential KATP channel functioning during ischemia in both groups . One way ANOV A for repeated measure s followed by Scheffe: *p < 0.05 vs. non-glibenclamide treated normal group and vs. diabetic with glibenclamide 0.1 mg/Kg ; 'p < 0.01 vs. non-glibenclamide treated normal and diabetic groups. Data are mean ± S.E.

found in sheep [27,29] and dogs [35]; moreover, all animals showed mild symptoms of established diabetes (polyuria, polyphagia, polydipsia).

Lack of recovery from stunning and absence of preconditioning protection in diabetes: An explanation based on KATP channel dysfunction Sarcolemmal KATP channels are important structures present in many tissues and are of particular interest in the cardiovascular system where they have been suggested to playa cardioprotective role during ischemic episodes [23], their activation increasing the outward potassium current and reducing action potential duration (APD) [36,37]. It has been speculated that their cardioprotection would be attained

through this action potential shortening, decreasing the time of Ca 2+ influx through Ca 2+ voltage dependent channels and avoiding the deleterious effects of Ca2+ overload [36, 37]. This mechanism appears to be implicated in sarcolemmal KATP channel protection against stunning and arrhythmias [28, 36, 37]. More recently, these channels have been mentioned to be involved in preconditioning protection [23, 38], and specifically they appear to participate in its anti-stunning effect [38]. Our results show that action potential duration differs between diabetic and normal sheep hearts (Fig. 5) in control conditions (before ischemia). This finding is in coincidence with previous reports which have described a sustained action potential lengthening in vitro [19,20,31]. These differences appear to be explained on the bases of K+ and/or Ca 2+ altered currents [19, 20, 39]. Both during ischemia and early reperfusion (when activated KATP channels shorten action

29 potential protecting the heart against ischemia-reperfusion injury) [37] the electrical activity in diabetic hearts exhibited a different behaviour when compared to normal ones. Although the exact mechanism for these latter observations is not fully known, it is probable that sarcolemmal KATP channels are altered in diabetes. An altered sensitivity to variations in ATP levels [39] and changes in the physical structure [19] of the channel due to changes in the transcription or expression of channels proteins [39], might have lately determined a considerable alteration of the outward K+ current [20] affording a plausible explanation to our results . Whatever the involved mechanism, action potential lengthening (specially at the start of reperfusion) [37] may result in Ca 2+ overload (an increase in APD diminishes Ca 2+ extrusion via the electrogenic Na+ICa 2+ exchanger). Since unpublished data from our laboratory have shown that action potential lengthening due to KATP channel blockade cause Ca 2+ overload in sheep [Lascano et al., in press], it could be assumed that KATP channel dysfunction in diabetes caused an inadequate Ca 2+ handling during ischemia and reperfusion which finally determined the lack of functional recovery from stunning, the 'cumulative ischemic damage' during triggering episodes and the absence of early and late preconditioning protection in diabetic sheep. The attribution of mechanical results obtained in diabetic sheep to KATP channel altered behaviour appears to be reinforced by the different vasodilatory response due to KATP channel dysfunction in diabetic patients [21]. Moreover, the reduction in the outward K+current together with the decrease in KATPchannel density [20, 22] described in hearts from experimental diabetic animals might afford an explanation to the prolongation of the QT interval in diabetic patients [30]. In addition, the different response to glibenclamide blockade also reflected a differential behaviour of sarcolemmal KATPchannels in diabetic hearts (it could be speculated that diabetes has altered the configuration of the sulfonylurea receptor) [39]. Since sulfonylureas have been shown to have deleterious cardiovascular actions (mainly due to KATP channel blockade) [28,37,40], the grater sensitivity to glibenclamide blockade seen in our model might be a plausible explanation to the high mortality due to cardiovascular events observed in diabetic patients treated with these compounds [40]. In summary, KATP channel dysfunction in diabetic hearts could afford a physiopathologic approach to the development of diabetic cardiomyopathy and could establish a rational explanation to the high cardiovascular risk observed in these patients .

Conclusions Since KATP channels have been mentioned as anti-stunning structures and the final effectors of almost all early and late

preconditioning pathways, diabetes-induced altered KATP channel behaviour appears to explain the lower functional recovery from stunning and the absence of preconditioning protection in conscious sheep. Although this work provides a first approach of the diabetic heart response to ischemiareperfusion damage, the exact level at which KATP channels are altered , as well as diabetes-induced dysfunction of other preconditioning pathways has to be further studied . The effect of chronic oral hypoglycemic treatment with sulfonylureas on preconditioning responses in the diabetic heart remain unresolved . Whether the lack of preconditioning and/or the high sensitivity of KATP channel to glibenclamide blockade demonstrated in laboratory animals explain the reported high mortality due to cardiovascular events in diabetic patients remains to be established.

Acknowledgements We thank Julio Martinez and Fabian Gauna for surgical and technical help. Animal care provided by veterinarians Marfa I. Besans6n, Pedro Iguafn and Marta Tealdo and veterinary assistants Juan Mansilla, Juan Ocampo and Osvaldo Sosa is gratefully acknowledged.

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Molecular and Cellular Biochemistry 249: 31-38 , 2003. © 2003 Kluwer Academic Publishers .

Protein kinase B in the diabetic heart Barbara Huisamen Department ofMedical Physiology and Biochemistry, Faculty of Medicine, University of Stellenbosch, MRC Programme for Diabetes and Heart Research, Tygerberg, Republic of South Africa

Abstract This paper summarizes data from different studies all aimed at elucidating regulation of protein kinase B in the diabetic heart. Two rat models of type 2 diabetes mellitus ((i) elicited via neonatal streptozotocin injection (Stz) and (ii) Zucker fa/fa rats), were used as well as different experimental models viz isolated, Langendorff perfused hearts as well as adult ventricular myocytes. Glucose uptake was elicited by a variety of stimuli and the activation of PKB measured in tandem . Basal glucose uptake was impaired in both diabetes models while basal phosphorylation ofPKB differed, showing lower levels in the Stz model but higher levels in the Zucker rats. Neither 100 nM insulin nor 10-8 M isoproterenol could stimulate PKB phosphorylation to the same extent in the diabetic myocardium as in controls, regardless of the method used, but a combination of these stimuli resulted in an additive response. Concurrent glucose uptake however, was not additive . Wortmannin abolished both insulin and isoproterenol stimulation of glucose uptake as well as PKB phosphorylation. In contrast to the above-mentioned results, the protein tyrosine phosphatase inhibitor vanadate, alone or in combination with insulin , elicited PKB phosphorylation to the same extent in diabetic cardiomyocytes as in controls. Despite this, glucose uptake stimulated by vanadate or insulin in combination with vanadate was attenuated. The combination of insulin and vanadate may however be beneficial to the diabetic heart as it resulted in improved glucose transport. Results from the different studies can be summarized as follows: (i) dysregulation of PKB is evident in the diabetic myocardium, (ii) PKB activation is not always directly correlated with glucose uptake and (iii) insulin resistance is associated with multiple alterations in signal transduction, both above and below PKB activation. (Mol Cell Biochem 249: 31-38, 2003) Key words: type 2 diabetes mellitus, PKB , myocardial glucose transport

Introduction Prominent features of the type 2 diabetic myocardium are insulin resistance and attenuated glucose uptake. Protein kinase B (PKB), also called Akt, is a mediator of the metabolic effects of insulin and it was suggested that activation of this kinase might be involved in the stimulation of glucose transport by insulin. For example, stable over-expression of wildtype PKBa or constitutively active mutants ofPKBa increased glucose transport and translocation of the insulin sensitive glucose transporter, glut 4, to levels similar to or greater than those achieved with insulin in rat adipocytes [1], 3T3-Ll adipocytes [2, 3], and L6 muscle cells [4]. In insulin resistance, dysregulation ofPKB activation may result in abnormalities in signalling thereby causing or augmenting the curtailed glucose transport response. However, different signalling

pathways leading to activation ofPKB and enhanced glucose uptake do exist, e.g. /3-adrenergic stimulation elicits glucose transport in skeletal muscle [5], heart muscle [6] as well as adipocytes [7]. Insulin mediates its effects via activation of Pl-S-kinase [8] while the cascade of events turned on by beta adrenergic stimulation, is currently controversial [6,9-11]. The protein tyrosine phosphatase (PTP) inhibitor vanadate is a known insulin mimetic agent with beneficial effects in the diabetic myocardium [12]. The mechanism whereby vanadate exerts its effects is not clearly understood either [13]. Our studies therefore focussed on evaluating the activation of PKB in the diabetic heart in conjunction with glucose uptake using either isolated perfused hearts or adult ventricular myocytes. Two different rat models of type 2 diabetes with insulin, isoproterenol (/3-adrenergic stimulant) and vanadate as stimuli of the glucose uptake pathway(s) were included.

Address fo r offprints: B. Huisamen, Department of Medical Physiology and Biochemistry, POBox 19063, Tygerberg 7505, Republic of South Africa (E-mail: [email protected])

32 We aimed to determine whether (i) insulin resistance is accompanied by dysregulation of PKB, (ii) PKB activation correlates with glucose uptake and (iii) alternative signalling pathway s are utilized to activate PKB.

At the end of the perfusion period, hearts were snap-frozen and stored in liquid nitrogen for further analyses .

Determination ofglucose uptake by perfused hearts

Materials and methods Two rat models of type 2 diabetes were compared, namely a lean model induced in Wistar rats via intraperitoneal injection of streptozotocin (90 mg/kg) on day 4 after birth (Stz) [14] and Zucker obese (ja/fa) rats. Age-matched, sham injected Wistars were used as controls. Animals were fed ad libitum before experimentation (20 weeks) and anaesthetized by intraperitoneal injection of pentobarbitone sodium (0.1 mg/ g). The South African Medical Research Council 's guide for the use of laboratory animals was followed at all times and the project was approved by the ethics committee ofthe University of Stellenbosch.

Blood glucose and serum insulin determinations

The blood glucose was determined at sacrifice using an Accutrend glucose meter (Boehringer Mannheim) while serum insulin was determined with a coat-a-tube commercial kit from Diagnostic Products Corporation (LA).

Perfusion technique

After removal, hearts were arrested in ice-cold Krebs Henseleit (KH) medium (in mM: NaCl 119; NaHC0 3 25; KCl 4.75; KHl04 1.2;MgS04.7Hp 0.6; NllzS04 0.6; CaCI2.2Hp 1.25; Glucose 5) and immediately (within 30 sec) mounted onto the aortic cannula of a Langendorff perfusion apparatus . All traces of blood were washed out before perfusion in a recirculating volume of 20 ml KH plus 2.5 ug/ml adenosine deaminase. The perfusion pressure was kept con stant at 80 mmHg with 95% 0/5% CO 2 as gas phase. The following perfusion protocols were followed : (i) (ii)

Basal : 25 min perfusion without any additives p-stimulation: 15 min stabilization + 10 min 10-8 M isoproterenol (iii) Insulin stimulation: 25 min perfusion with 100 nM insulin (iv) Insulin + p-stimulation: 15 min stabilization + 10 min 10-8 M isoproterenol with 100 nM insulin added at time O. (v) Wortmannin (100 nM) was added at time 0 or 15 min before the end of the protocol for basal values.

Glucose uptake was measured spectrophotometrically on aliquots of perfusate using a hexokinase assay and a Cobas Fara auto-analyzer.

Isolation of cardiomyocytes

Cardiomyocytes were prepared by collagenase perfusion, essentially as previously reported [15]. After isolation, the supernatant was carefully aspirated and the loose cell pellet resuspended in medium A containing (in mM): KCl 6; Na2HP04 1; NaH2P04 0.2; MgS041.4 ; NaC1128; HEPES 10; glucose 5.5; pyruvate 2; CaCl 2 1.25 plus 2% BSA (fraction V, fatty acid free) pH 7.4. The cells were allowed to recover from the trauma of isolation for 1 h before experimentation. After recovery, the viability of the isolated cardiomyocytes routinely exceeded 80% as estimated by the trypan blue exclusion method.

Determination of2-Deoxy-D-glucose uptake by myocytes

2-deoxy-D-glucose uptake was measured essentially as described previously [15]. In brief, cardiomyocytes were suspended in oxygenated medium A minus substrates (final volume 750 ｾｌＩ Ｎ The cells were pre-incubated for 15 min with or without phloretin (400 ｾｍＩ for measurement of noncarrier mediated glucose uptake or with wortmannin (100 nM). Each experimental series was incubated with or without insulin or vanadate under the same conditions for 30 min after which glucose uptake was initiated by addition of 2-deoxyD-[3H]glucose (1.5 ｾｃｩＯｭｌ ［ final 2-deoxy-D-glucose concentration 1.8 ｾＩＮ Glucose uptake was stopped after 30 min The cells by addition of phloretin (final concentration 400 ｾＩＮ were spun down and the pellet washed twice with HEPES buffer and dissolved in 0.5 N NaOH. The protein concentration [16] and radioactivity of these samples were determined.

Preparation of extracts for immunoblotting

After stimulation , cells were quickly centrifuged and washed with ice-cold HEPES buffer without albumin . These cells or frozen material from perfused hearts were lysed in buffer containing (in mM) : HEPES 25, p-glycerophosphate 50 , EGTA 1, p-nitrophenyl phosphate 10, Na3V041 , MgCI22 .5,

33 PMSF 1,DTI 1, 1% Triton X-I 00 and 10 ug/ml, each aprotinin and leupeptin, pH 7.4. The lysates were microfuged (14,000 rpm) for 15 min and the supernatants diluted with Laemmli sample buffer. An aliquot of the supernatant was used for protein determination [17].

Basal glucose uptake As shown in Fig . 1, basal glucose uptake of the perfused hearts was significantly impaired in both diabetes models . In the Stz group, this became significant after 25 min (p < 0.05 vs. control). In the Zucker group significant lower uptake was measured even at 15 min (p < 0.005 vs. control).

Immunoblotting Samples were boiled for 5 min and 20 ug protein separated on 10% SDS-PAGE followed by electrotransfer to Immobilon P membranes. Membranes were blocked for 1 h in Trisbuffered saline, pH 7.6, containing 0.1 % Tween-20 and 5% non-fat dry milk followed by exposure to Phospho-Akt or Akt primary antibody and horseradi sh peroxidase linked secondary antibody. Bands were visualized by the ECL method and intensity quantified by laser scanning densitometry (UNSCAN-IT - Silk scientific corporation).

Basal phosphorylation ofPKB

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Figure 2A depicts the basal phosphorylation state (Ser"73) of PKB as determined in perfused hearts. In the Stz hearts, a lower level of phosphorylation was measured (60.70 ± 7.96 average pixels measured, n =8) while hyperphosphorylation was found in the Zucker hearts (171.11 ± 29.49, n = 8) vs. a control value of 101.56 ± 8.35 (n =7). A representative blot is shown in the insert. Probing lysates prepared from control and Zucker hearts with a phosphorylation independent antibody against PKB confirmed equal expression of the protein (Fig. 2B) .

Results Glucose uptake elicited by insulin and beta-adrenergic stimulation

Biometric data The biometric data pertaining to the 2 diabetes models versus control values are summarized in Table 1. The mean blood glucose of rats from both diabetes models was significantly higher than that of the control group. Serum insulin was not affected in the lean (Stz) group, while that of the Zucker fa! fa group was significantly elevated. Heart weights from both diabetic groups were significantly higher than controls, indicating possible development of hypertrophy.

Myocardial glucose uptake over a 25 min perfusion period is depicted in Table. 2. As also shown in Fig. 1, glucose uptake over this period in the absence of exogenous stimuli was significantly depressed in both diabetes models - from 23.21 ± 2.66 umol/gwwt to 16.07 ± 1.61 in the Stz and 8.39 ± 1.90 in the Zucker group . Insulin stimulated glucose uptake by 50, 84 and 209% above basal values in control, Stz and Zucker hearts respectively at the end of the 25 min period. Although ｾＭｳｴｩｭｵｬ｡ ｯｮ stimulated uptake to 27.25 ± 1.91 umol/gwwt in controls, 19.45 ± 3.69 in Stz and 14.44 ± 3.42 in Zuckers

Table 1. Biometric data of 20 week old rats

Control

Stz

Zucker

Blood glucose (mmol/L)

7.42 ± 0.30 (n = 50)

10.87 ± 0.24* (n = 50)

9.14±0.22* (n =45)

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18.60 ± 1.70 (n = 13)

20.50 ± 1.90 (n =6)

95.20 ± 7.30* (n =6)

BASAL GLUCOSE UPTAKE 30

25

Body weight (g)

279.62 ± 10.35 (n = 50)

Heart weight (g)

0.91 ± 0.02 (n = 50)

290.85 ± 11.27 (n = 50)

618.30 ± 9.4* (n = 50)

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1.44 ± 0.05* (n =50)

Biometric data collected from the control, lean (Stz) and obese diabetic (Zucker) rats. The number of experimental animals is given in brackets . Values represent mean ± S.E.M. Blood glucose and serum insulin were determined as described in 'Materials and methods' .*p < 0.001 vs. control, 'p < 0.01 vs. control.

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34 A:

lation found in the control group (Fig. 3A with representative blots given in Fig . 3B). Beta-stimulation e.g. elevated PKB phosphorylation 500% above basal levels in control hearts with the corresponding values 110% in Stz and 67 % in Zucker hearts . Insulin on the other hand, was able to stimulate a 1000% increase in activity in both control and Stz hearts with a corresponding value of only 148% in Zucker hearts. A combination of insulin and ｾＭｳｴｩｭｵｬ｡ｯｮ was additive in the diabetes groups but not in the control group.

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Fig. 6. Confirmation of relative levels of RyR2 protein. Western blot analyses were used to confirm relative levels of RyR2 in 100 ug of sarcoplasmic reticular membrane vesicles from 6- and 8-week streptozotocin (STZ)-induced , 6-week STZ-inducedl2-week insulin treated diabetic and agematched control rat hearts . For these experimen ts, actin was used as the reference to correct for variability in protein load and/or transfer. Values shown are means ± S.E.M. for 4 experiment done using two different SRMV preparations . *Denotes significantly different from controls (6- and 8-weeks), 6-week STZ-diabetic and insulin-treated.

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pro tein, RyR 2 from 6- and 8-weeks of untreated diabetes boun d 22.8 and 23.9% less [3H]ryanodine when compared to age-matched controls (223.5 ± 34.6 and 227 .5 ± 18.4 fmol [3H]ryanodine/Jlg RyR2 compared with 289.6 ± 24.1298.9 ± 23.0 fmol [3H]ryanodine/Jlg RyR2 from controls). These differences were significantly different at the 95% confidence lev el and suggest tha t RyR2 becomes dysfunctional with chronic diabetes. When compared with age-matched controls, RyR2 from insulin treated animals also bound significantly less [3H]ryanodine per ug RyR2 (255.6 ± 16.1 compared with 298.9 ± 23.0 fmol [3H]ryanodine/Jlg RyR2, p < 0.05). However, the amount of pH]ryanodine per ug RyR2 was grea ter that the amoun t bound to RyR2 from untreated diabetic animals . These data are consistent with our previous findings in

reticular membrane vesicles from 6- and 8-week streptozotocin (STZ)-induced , 6-week STZ -inducedl2-week insulin treated diabetic and agematched control rat hearts . Data shown are means ± S.E.M. for at least 6 experiments done in duplicate using two different membrane preparations . *Denote s significantly different from controls (6- and 8-week s) and insulin-treated. **Denotes signific antly different from controls (6- and 8weeks), 6-week STZ-diabetic and insulin-treated. 'Denotes significantly different from STZ-d iabetic (6- and 8-weeks) and age-matched controls (6- and 8-weeks) . (B) Measure of functional integrit y of RyR2 from 6and 8-week streptozotocin (STZ)-induced, 6-week STZ-inducedl2-week insulin treated diabetic and age-matched control rat hearts. RyR2 conte nt in 100 ug of SRMV preparations were determined and ['H]ryanodine binding was then normal ized to 1 ug of RyR2. Values shown are means ± S.E.M. for at least 6 experim ents done in duplicate using two different membrane preparations . *Denotes significantly different from controls (6and 8-weeks). ' Denotes significantly different from age-matched controls (6- and 8-week s).

121 110

which we showed that although insulin treatment can minimize the loss in RyR2 expression induced by diabetes, the ability of this protein to bind pH]ryanodine remains diminished.

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C2....... The affinities of ryanodine for RyR2 from 6-and 8-weeks STZ-induced diabetic, 6-week STZ-induced diabetic/2-week insulin-treated and 6-and 8-week age-matched control rat hearts were also determined as another way of assessing the functional integrity of RyR2. As shown in Fig . 8, among the 5 experimental groups, the IC so values as well as the overall shape of the curves were not significantly different (IC so = varied between 2.42-4.44 nM while Kd ranged from 0.370.67 nM) . It should also be pointed out the apparent leftward shift in the displacement curve for RyR2 from 8-weeks STZdiabetic may be reflective of the lower amount of RyR2 protein per 100 ug membrane vesicles (less pH]ryanodine bound so less unlabeled ryanodine required to displace it).

Discussion Heart failure is one of the leading causes of morbidity and mortality among chronic diabetic patients [40-42] . While in a general context it is accepted that such diabetes-induced cardiac complications result from a combination of metabolic, biochemical and structural changes [43], the etiology underling 'diabetic cardiomyopathy' remains poorly understood. Patients with chronic diabetes show severe systolic dysfunction [6,40] and this is likely to be due to changes in the expression and function of numerous proteins involved in regulating/maintaining intracellular calcium homeostasis. In this study, we focussed on the effects of diabetes on expression and function of one of these proteins, namely the ryanodine receptor calcium-release channel (RyR2) and used the STZ-diabetic rat model to investigate it. Data from the present study as well as our previous one [29] show that after 6-weeks of untreated diabetes, expression of RyR2 (both mRNA and protein levels) did not change significantly. However, the ability this protein to bind the specific ligand [3H]ryanodine (index of function) decreases markedly. Increasing the duration of untreated diabetes to 8 weeks, decreased both function and expression of RyR2. Therefore, the principal finding of the present study is that in diabetes, loss in RyR2 function precedes reduction in its expression. We were able to discern these two effects using a low dose of streptozotocin, namely 50 mg STZ/kg for induction of diabetes. While we do not know the exact reason(s), it

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log [Ryanodlne] (nM) Fi g. 8. Affinities of RyR2 from 6- and 8-we ek streptozotocin (STZ)-induced, 6-week STZ-inducedl2-week insulin treated diabetic and agematched control rat hearts for ryanod ine. Equilib rium dissociation constant (Kd) and IC,ovalues were determined by incubating MV proteins (0.1 mgt ml) from 6- and 8-week control s, 6- and 8-week STZ- induced and 4-week STZ-induced diabetic/2-week insulin treated 6-week STZ-induced animals for 2 h at 37°C with 6.7 nM [3H]ryanodine and increasing concentrations of unlabeled ryanodine up to 300 nM. At the end of this time, the vesicles were filtered , washed and [3H]ryanodine bound was determined by liquid scintillation counting. Non-specific binding was simultaneously determined by incubating vesicle s with 111M ryanodine. GraphPad Prism 3.0 was used to draw curve s (non-linear regression) and calculate IC,ovalues . Kd values were determ ined using the Cheng-Prusoff equation. Data shown are means for at least 6 experiments done in duplicate using two different membrane preparations.

is likely that this dosage of STZ prolong the development of diabetic cardiomyopathy. Data from the present study also indicate that while the loss in expression of RyR2 induced by 8-weeks of diabetes could be prevented with 2-weeks of insulin treatment, initiated after 6-weeks of untreated diabetes. The ability of this protein to bind [3H]ryanodine remained significantly less that from age-matched control animals (lower BmaJ Thus, 14 days of insulin treatment was not sufficient to completely reverse loss in RyR2 function induced by 6-weeks of untreated diabetes. A likely explanation for this observation is that the half-life of RyR2 is of the order of days . While we do not have direct evidence on this point, it is nonetheless consistent with the findings of Ferrington et al. [44] who found that the half-life of RyR1 (the cognate skeletal muscle isoform) is 8.3 ± 1.3 days. Thus, the question arises 'as to what changes diabetes may induce that are long lasting and can affect RyR2 ability to bind ['Hlryanodlne?' While we do not know the answer to this question, it is likely to result from diabetes-induced increases in post-translation modifications. Two major types of posttranslation modifications are envisioned. First, increased levels of cellular aldose and ketose sugars induced by diabetes will increase rate of formation of Schiff bases on lysine/ar-

122 ginine residues (non-enzymatic glycation reactions) [45-47]. Over time and through a series of oxidation, reduction and cyclization reactions, Schiff bases can rearrange to form advanced glycation end products (AGEs). The formation of AGEs on RyR2 likely will alter its tertiary structure, and this could result in a decrease in its ability to bind the specific ligand [3H]ryanodine. Also, once formed AGEs are essentially irreversibly bound and are eliminated only when RyR2 itself is degraded. Secondly, it is well known that the metabolic shifts brought about by diabetes increase production of reactive oxygen and nitrogen species [48-50] . These species are also capable of reacting with several amino acid (especially cysteine) residues on RyR2, leading to alterations in its tertiary structure and loss in ability to bind pH]ryanodine. In the present study we found that membrane preparation from 6- and 8-week diabetic rat hearts also contained elevated levels of immuno-reactive PMCA. This difference is unlikely to be due to sample preparation, since vesicles from all five experimental groups were prepared simultaneously using similar conditions/buffers, etc. These data suggest that basal levels of calcium inside the myocytes may increase with diabetes and are consistent with studies reported by Smogorzewski et ai. [51] In conclusion, the present study shows that in early stages of diabetes (up to 6-weeks), the functional integrity of RyR2 becomes compromised. As the syndrome progresses, both function and expression of RyR2 decrease . We also show that while insulin treatment was able to prevent and/or minimize the loss in expression of RyR2, it was not able to reverse the dysfunction . Thus, it is likely that loss in activity and expression of RyR2 may contribute in part to decrease in contractility seen in diabetic rat hearts. Also, our data provide a possible explanation for the increase in congestive heart failure seen among diabetics who are in compliance with insulin and/or oral hypoglycemic therapies.

Acknowledgements This work was supported in part by grants from the National Institutes of Health (HL66898) and the Ralph W. and Grace M. Showalter Trust.

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123 29. Bidasee KR, Dincer UD, Besch HR Jr : Ryanodine receptor dysfunction in hearts of streptozotocin-induced diabetic rats. Mol Pharrnacol 60: 1356-1364,2001 30. Rodrigues B, Cam MC, McNeill JH: Metabolic disturbances in diabetic cardiomyopathy. Mol Cell Biochem 180: 53-57, 1998 31. Bidasee KR, Besch HR Jr, Kwon S, Emmick JT, Besch KT, Gerzon K: CI0-0eq N-(4-azido-5-125iodo salicyloyl)-13-alanyl-13-alanyl ryanodine, a novel photo-affinity ligand for the ryanodine binding site . J Labelled Comp Radiopharrn 34: 33-47, 1994 32. Watanabe AM, Besch HR Jr: Interaction between cyclic adenosine monophosphate and cyclic gunaosine monophosphate in guinea pig ventricular myocardium. Circ Res 37 : 309-317, 1975 33. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J BioI Chern 193: 265-275,1951 34. Meissner G, el-Hashem A: Ryanodine as a functional probe of the skeletal muscle sarcoplasmic reticulum Ca 2+ release channel. Mol Cell Biochem 114: 119-123, 1992 35. Chu A, Diaz-Munoz M, Hawkes MJ, Brush K, Hamilton SL: Ryanodine as a probe for the functional state of the skeletal muscle sarcoplasmic reticulum calcium release channel. Mol Pharrnacol37: 735-741,1990 36. Cheng Y-C, PrusoffWH: Relationship between the inhibition constant (Kl) and the concentration of inhibitor which causes 50% inhibition (150) of an enzymatic reaction. Biochem Pharrnacol22: 3099-3108, 1973 37. Russ M , Reinauer H, Eckel J: Diabetes-induced decrease in the mRNA coding for sarcoplasmic reticulum Ca 2+-ATPase in adult rat cardiomyocytes. Biochem Biophys Res Commun 178: 906-912, 1991 38. Zarain-HerzbergA, Yano K, Elimban V, Dhalla NS : Cardiac sarcoplasmic reticulum Ca 2+-ATPase expression in streptozotocin-induced diabetic rat heart. Biochem Biophys Res Commun 203: 113-120, 1994 39. Dhalla NS, Lui X, Panagia V, Takeda N: Subcellular remodeling and

40 . 41. 42 . 43 . 44.

45.

46 . 47 . 48. 49 . 50. 51. 52.

heart dysfunction in chronic diabetes . J Cardiovasc Res 40 : 239-247, 1998 Uusitupa MI, Mustonen IN, Airaksinen KE: Diabetic heart muscle disease . Ann Med 22 : 377-386, 1990 Fein FS: Diabetic cardiomyopathy. Diabetes Care 13(suppI4): 11691179,1990 Bell DS: Diabetic cardiomyopathy. A unique entity or a complication of coronary artery disease. Diabetes Care 18: 708-714, 1995 Chatham JC, Forder JR, McNeill JH (eds) : The Diabetic Heart . Kluwer Academic Press, Massachusetts, USA , 1996 Ferrington DA, Krainev AG, Bigelow DJ : Altered turnover of calcium regulatory proteins of the sarcoplasmic reticulum in aged skeletal muscle. J Bioi Chern 273 : 5885-5891, 1998 Brownlee M, Cerami A, Vlassara H: Advanced glycosylation end products in tissues and the biochemical basis of diabetic complications. N Engl J Med 319 : 315-321 , 1988 Bunn HF, Higgins PJ : Reactions of monosaccharides with proteins: Possible evolutionary significance. Science 213 : 222-224, 1981 Bucala R, Cerami A: Advanced glycosylation: Chemistry, biology and implications for diabetes and aging . Adv Pharrnacol 23: 1-34, 1992 Wolff SP, Jiang ZY, Hunt JV: Protein glycation and oxidative stress in diabetes mellitus and ageing. Free Radic BioI Med 10: 339-352, 1991 Obereley LW: Free radical and diabetes. Free Radic BioI Med 5: 113124, 1988 Giugliano D, Ceriello A, Paolisso G: Oxidative stress and diabetic vascular complications. Diabetes Care 19: 257-267, 1996 Dhalla NS, Temsah RM, Netticadan T: Role of oxidative stress in cardiovascular diseases. J Hypertens 18: 655-673, 2000 Smogorzewski M, Galfayan V, Massry SG : High glucose concentration causes a rise in [Ca 2+); of cardiac myocytes. Kidney Int 53: 12371243, 1998

Molecular and Cellular Biochemistry 249: 125-128,2003. © 2003 Kluwer Academic Publishers.

The relationship between QTc interval and cardiac autonomic neuropathy in diabetes mellitus Ali Pourmoghaddas 1 and Ali Hekmatnia' Departments of 'Internal Medicine; 2Radiology, Isfahan University of Medical Sciences, Iran

Abstract Cardiovascular complications are the most common causes of mortality and morbidity in diabetic patients . Autonomic neuropathy is one of the complications in diabetic patients, which may also involve cardiovascular system . Autonomic system abnormality may increase QTc interval. On the other hand patients with prolonged QTc interval are prone to ventricular arrhythmias, especially unique torsade-de-point and also sudden cardiac death. This study intends to detect the prevalence of QTc prolongation in diabetic and nondiabetic patients as well as its correlation with diabetic autonomic neuropathy. This study includes 200 diabetic (case group) and 200 non-diabetic patients (control group) with comparable age and gender. Evaluation of autonomic nervous system was carried out in all cases with prolonged QTc interval. Autonomic nervous system evaluation in control group was performed too. The results of the study in the case and the control group were compared. The prevalence of prolonged QTc interval was significantly higher in the case group in comparison with the control group, 8 vs. 2% respectively (p value =0.012, OR =4.3). Sympathetic nervous system evaluation test in cases with QTc interval prolongation and negative exercise test demonstrates abnormal results in more than 50% of case group (OR = 3). Parasympathetic nervous system evaluation tests in case group showed abnormal results in comparison with control group (OR =9). Abnormality of parasympathetic nervous system is more common than (3 fold) abnormality in sympathetic nervous system. With regard to the prolonged QTc interval in the case group in comparison with the control group and abnormal autonomic nervous system function in more than half of the case group, the probability of ventricular arrhythmia, torsade de points , has increased. The mentioned ones are in increased risk of sudden cardiac death. Rendering approaches for decreasing the risk of sudden cardiac death in diabetic patients are seriously recommended. (Mol Cell Biochem 249: 125-128,2003) Key words: QTc interval prolongation, cardiac autonomic neuropathy, diabetes mellitus

Introduction Diabetes mellitus (DM) is the most common metabolic disorder in human that has multiple complications in patients . Cardiovascular complication is the most common complication in type II diabetes mellitus that increases mortality in these patients. This complication is classified into three groups . 1. Atherosclerotic coronary artery disease (CAD) 2. Dilated cardiomyopathy (DCM) 3. Cardiac autonomic neuropathy (CAN)

Coronary artery disease and dilated cardiomyopathy are the significant causes of mortality in these patients; multiple researches showed that the prevalence of sudden death in diabetic patients is more than nondiabetic ones. All causes of mortality were not due to coronary artery disease and dilated cardiomyopathy [1, 2]. Therefore, the probable causes of sudden death in diabetic patients are cardiac autonomic neuropathy and QTc prolongation [9]. The purpose of this research is to determine the prevalence of QTc prolongation in diabetic patients, compared with nondiabetic ones. If QTc interval is increased, correlation of cardiac autonomic neuropathy with QTc prolongation is determined.

Addressfor offprints: A. Pourmoghaddas, Isfahan Unive rsity of Medical Sciences , Isfahan, Iran (E-mail : [email protected])

126

Materials and methods In this study, which is a case -control , 200 diabetic patients (109 females and 91 males) from endocrine and metabolism research center were selected. This research was performed in 1998. The selection method was a random sampling . Then the files of the patients and the electrocardiograms were studied. The control groups were 200 people with the sex and age matched nondiabetic ones, without the history of current drug usage or another cardiovascular disease . Standard 12 lead electrocardiogram at rest position was obtained. The QTc interval in multiple beats and leads was determined by the Bazett's formula [QTc = QTf(R-R)] and then the most prolonged QTc interval was calculated. QTc interval in normal male group was less than 0.42 sec and in normal female group was less than 0.44 sec [5,6]. The QTc more than this criterion was considered as QTc interval prolongation . Color Doppler echocardiography, exercise test, blood calcium and potassium level measuring in all cases with QTc prolongation were obtained. Using above measures, other causes of QTc prolongation such as mitral valve prolapse, dilated cardiomyopathy, ischemic heart disease, hypokalemia and hypocalcaemia were excluded. Current drug usage history was obtained and all cardiac drugs were discontinued 48 h before QTc measurement. In Color Doppler echocardiography, left ventricular ejection fraction and contractility, diastolic function and valvular flow measurements were studied. Exercise test was done with Bruce protocol in standard manner. The criteria for termination of exercise test are appearance of cardiac symptom and receiving to target heart rate (Target heart rate is 220-age) [1, 2]. Exercise test results according to cardiac symptom and severity of exercise ST-T changes during the test were classified into four groups . A - Positive, B - Negative, C - Equivocal, D - Incomplete After above procedures, cardiac autonomic nervous system evaluation in the case group was performed . Parasympathetic functions, which are usually lost before sympathetic functions in diabetic autonomic neuropathy, were evaluated by determining beat-to-beat variation of the heart rate. The heart rate is often high at rest and may be virtually fixed [2, 3]. Sympathetic nervous function can be assessed by determining systolic blood pressure response during standing or diastolic blood pressure response during static exercise [3,4] . Measurement of variations in the electrocardiographic R-R interval has been advocated as the simplest and the most reliable means of testing for autonomic dysfunction. Heart rate response to the Valsalva maneuver was tested by having the subject blow against an aneroid or mercury manometer to 40 mmHg for 15 sec. The test was performed three times with a rest of 1 min in between. An electrocardiogram was taken

continuously during the test [3]. The Valsalva ratio has the longest R-R interval after release as the numerator and the shortest R-R interval during the maneuver as the denominator. Heart rate variation during deep breathing was evaluated by having the patient take six deep breaths per minute with the electrocardiogram running and marked at inspiratory and expiratory points [3]. Maximal and minimal R-R intervals were measured and converted to heart rate. Immediate heart rate response to standing was tested by measuring the R-R interval at the 15th and 30th beats after the patient rises from a supine to an upright posture. The result was reported as the 30th:15th ratio. Blood pressure response to standing was determined by using the fall in systolic blood pressure on standing as the test marker. Blood pressure response to static exercise was tested by sustained handgrip. The blood pressure normally rises during isometric exercise. Three basal diastolic pressures were compared with the highest diastolic pressure developed during sustained handgrip [3].

Results This study was performed in Isfahan endocrine and metabolism research center. The date of study was October to March 1999. The incidence of QTc prolongation in 200 non-diabetic persons was 8% (16 case), normal QTc interval was 92% (184 cases). The incidence of QTc prolongation in 200 non-diabetic persons was 2% (4 persons) and normal QTc interval was 98% (196 persons). According to statistical results P value was 0.012 and odds ratio was 4.3. Normal exercise test responses in 10 diabetic patients with QTc prolongation were seen, but they were abnormal in 4 diabetic patients with QTc prolongation. The rest of patients (2 cases) with QTc prolongation and unable to perform exercise test, (one patients had unstable angina, another patients had leg amputation) were excluded from the study. Exercise test responses in control group with QTc prolongation were normal. In two-dimensional echocardiography of 16 cases with QTc prolongation, 3 patient s had LV dilatation or regional wall motion abnormality (19%). Among those three patients, two patient s had abnormal exercise test response and one patient had normal exercise test response. In Color Doppler echocardiography, mitral valve flow and Ef A ratio (evaluation of diastolic function) were studied. Abnormal mitral valve flow (EfA ratio < 1) in all patients with abnormal exercise test (4 patients) was present. The two other patients with normal exercise test had abnormal mitral valve flow (EfA ratio < 1).

Autonomic nervous system abnormality

After the exclusion of those two patients who were unable to perform exercise test, other patients with QTc prolong a-

127 tion (14 cases) for autonomic nervous system evaluation were classified into two groups. (A) (B)

tem evaluation in 1 patient was abnormal and in 3 patients was normal. Parasympathetic nervous system evaluation in 1 patient was also abnormal but in other patients was normal.

Diabetic patients with QTc prolongation and negative exercise test (10 patients), Diabetic patients with QTc prolongation and positive exercise test (4 patients).

Discussion In the present study 200 diabetic patients were investigated for QTc interval and autonomic neuropathy. These 200 patients were compared with age and sex matched non-diabetic persons . The results of QTc interval and autonomic nervous system evaluation among them were compared. The patients with ischemic heart disease (by exercise test) and dilated cardiomyopathy (by echocardiography) were excluded from the study. Prevalence of prolonged QTc interval in diabetic patients (case group) was 8%, but in control group was 2% (p value = 0.012 and odds ratio = 4.3) Evaluation of autonomic nervous system in case group showed abnormality in parasympathetic nervous system more common than abnormality in sympathetic nervous system. Odds ratio of parasympathetic nervous system abnormality in the case group compared with the control group was 9. Odds ratio of sympathetic nervous system abnormality in the case group compared with the control group was 3. Parasympathetic nervous system abnormality was significantly higher than sympathetic nervous system abnormality. The results of other studies are described below. Kahn et al. have shown evidence of cardiac autonomic neuropathy with QTc prolongation in 17 cases from 30 patients who suffered from insulin dependent diabetes mellitus. QTc interval prolongation occurred with maximum exercise in two patients and in 15 patients at rest. On the other hand, QTc prolongation was exclusively seen in patients with cardiac autonomic neuropathy [7]. Gentile et al. have also described a close relationship between painless MI, sudden cardiac death and diabetic autonomic neuropathy [8]. Ewing in 'Autonomic neuropathy, QTc prolongation and unexpected sudden cardiac death in male diabetic patients ' expressed that 39 diabetic patients with different degrees of autonomic neuropathy, QTc interval were calculated and the patients were

Results of autonomic nervous system evaluation were obtained as below.

Case group results In group A (10 diabetic patients with QTc prolongation and negative exercise test), sympathetic nervous system evaluation in 5 patients was abnormal, while in other 5 patients it was normal (odds ratio = 3). Parasympathetic nervous system evaluation result in 3 patients was abnormal , in 1 patient was normal and in 6 patients was borderline (odds ratio = 9). In group B (4 diabetic patients with QTc prolongation and positive exercise test), sympathetic nervous system evaluation in 1 patient was abnormal, in 2 patients was normal and in one patient was borderline. Parasympathetic nervous system evaluation in one patient was abnormal, in 2 patients was normal and in one patient was borderline. Serum calcium and potassium level in all diabetic patients with prolonged QTc interval were in normal range. Serum calcium and potassium level above 8.5 and 3.5 meg/lit was considered as normal value, respectively.

Control group results In 200 non-diabetic persons, 4 persons had QTc interval prolongation. Exercise test in all 4 persons had normal responses . In echocardiography of 4 persons with QTc prolongation, 1 person had mild mitral valve prolapse without any mitral regurgitation. Other persons with prolonged QTc interval had normal echocardiographic results . Sympathetic nervous sys-

Table 1. Normal and abnonnal values in tests of autonomic function

Test

Test

Normal

Borderline

Abnormal

Parasympathetic (HR response)

I - Valsalva ratio 2 - Deep breathing (Max: min HR) 3 -Standing (30: 15 ratio R-R) 1- Standing (systolic BP) 2- Exercise (diastolic BP)

1.21 15BPM

1.11-1.20 11-14 BPM

1.10 10BPM

1.04

1.01-1.03

1.00

IOmmHg

11-29mmHg

30mmHg

16mmHg

11-15 mmHg

10mmHg

Sympathetic (BP response)

128 followed for 3 years. On follow-up of the diabetic patients, progression of QTc interval prolongation was parallel to deterioration of autonomic neuropathy . Eight of thirteen deaths in these patients had sudden cardiac death [10]. Gonin in 'QTc prolongation as diagnostic tool for assessment of cardiac autonomic neuropathy in diabetic mellitus' measured QTc interval in 73 diabetic patients . Twenty-five patients had prolonged QTc interval and among those cases only 23 had evidences of cardiac autonomic neuropathy [11]. Chambers et al. in 'QTc interval prolongation in diabetic autonomic neuropathy' described that ventricular arrhythmia and sudden cardiac death is associated with QTc interval prolongation . In their investigation sympathetic nervous system was impaired in one third of diabetic patients [12]. Roy et al. in 'Autonomic influence on cardiovascular performance in diabetic subjects' have shown parasympathetic nervous system impairment in all 25 diabetic subjects whom were investigated [13]. Zola et at. in their research emphasized on the abnormality of cardiovascular performance in cardiac autonomic neuropathy in diabetic patients [14]. According to above results and researches, the presence of probable relationship between QTc interval prolongation and cardiac autonomic neuropathy in diabetic patients is suggested and one cause of sudden cardiac death in diabetic patients with autonomic neuropathy is arrhythmic (probably Torsade De Points) . Therefore, calculation of QTc interval is a simple, cheap and safe method for finding diabetic patients with cardiac autonomic neuropathy and finally we recommend prophylactic and therapeutic measures for reduction of sudden cardiac death.

Acknowledgement We are thankful to Mrs. Maryam Bagherzadeh for her sincere collaborations in preparation of this article.

References I . Williams GH, Lilly LS , Seely EW: The heart in endocrine disorder. In: E. Braunwald, D.P. Zipes, P. Libby (eds). Heart Disease, 6th edn . WB. Saunders and Company, USA, 2001 , pp 1901-1902 2. Zein JZ, Sonnenblick EH : Endocrine disea se and cardiovascular system. In : R.W. Alexander, R.C. Schlant, V. Fu ster (ed s). The Heart. (Hur st' s) McGraw Hill, USA , 1998, pp 2121-2122 3. Unger RH, Fo ster DW : Di abetes mellitus. In : J .D . Wil son , D.W Fo ster, H.M. Kronenberg, P.R. Larsen (eds). Will iam's Textbook of Endocrinology. WB. Saunders and Company, USA, 1998, pp 1024-1027 4. Jasp ak JB, Green AJ: The neuropathies of diabete s. In: LJ. Degroot, M. Besser, H.G Burger (eds). Endocrinology. Saunders Co ., USA, 1995, pp 1551-1552 5. Foster DW : Diabetes mellitus. In: A.S. Fauci, E. Braunwald, K.Z. Isselb acher (eds). Harrison's Principles ofInternal Medicine. McGraw Hill Co., USA , 1998, pp 2076-2077 6. Sherwin RS: Diabetes mellitu s. In: GN. Gill , J.P. Kokko , GL. Mandell, R.K. Ockner, T.W. Smith (eds). Cecil Textbook of Medicine. Saunders Co., USA , 1996 , pp . 1274-1275 7. Kahn JK, Sisson JC , Vinik AI: QT interval prolongation and sudden cardi ac in diabetic autonomic neuropathy. J Clin Endocrinol Metab 64: 75 1- 754, 1987 8. Gentile S, Marmo R, Costume A, Per sico M, Bronzino P, Contaldi P, Stroffolini T: Diab etic neuropathies, autonomic neuropathy, peripheral sympathetic innervations and the cardiovascular system . Minerva Med 75: 1053- 106 1, 1984 9. Kahn JK, Sis son Je Vinik AI : Prediction of sudden cardiac death in diab etic autonomic neuropathy. J Nucl Med 29 : 1605-1606, 1988 10. Ewing DJ, Boland 0 , Neil son JM, Cho CG, Clarke BF : Autonomic neuropathy, QT interval lengthening and unexpected deaths in male diabetic patients. Diabetologia 34: 182-185, 1991 II . Gonin 1M, Kadrofskc MM, Schmaltz S, Bastyr EJ, Vinik AI: Corrected Q-T interval prolong ation as diagnostic tool for assessment of cardi ac autonomic neuropathy in diabetes mellitus. Diabetes Care 13: 68-71, 1990 12. Chambers JB , Sampson MJ, Sprigings DC , Jackson G: QT prolongation on the electrocardiogram in diabetic autonomic neuropathy. Diabet Med 7: 105-110, 1990 13. Roy TM , Peterson HR, Snider HL, Cyrus J, Broadstone VL, Fell RD, Rothchild AH, Samols E, Pfeifer MA : Autonomic influence on cardiovascular performance in diabetic subjects. Am J Med 87: 382-388, 1989 14. Zola B, Kahn JK, Juni JE, Aaron IV: Abn ormal cardiac function in diabeti c patient s with autonomic neuropathy in the absence of ischemic heart di sease. J Clin Endocrinol Metab 63 : 208, 1986

Molecular and Cellular Biochemistry 249: 129-140,2003. © 2003 Kluwer Academic Publishers.

Antioxidants decreases the intensification of low density lipoprotein in vivo peroxidation during therapy with statins Vadim Z. Lankin,' AlIa K. Tikhaze,' Valery V. Kukharchuk,' Galina G. Konovalova,' Oleg I. Pisarenko,' Alexander I. Kaminnyi, 1 Konstantin B. Shumaev' and Yury N. Belenkov' I Cardiology Research Complex, Miasnikov's Institute of Clinical Cardiocogy ; 2Institute of Experimental Cardiology, 3rd Cherepkovaskaya, Moscow, Russia

Abstract The oxidative modification of low density lipoprotein (LDL) is thought to play an important role in atherogenesis. Drugs of 13hydroxy-l3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) family are usually used as a very effective lipid-lowering preparations but they simultaneously block biosynthesis of both cholesterol and ubiquinone Q IO (coenzyme Q), which is an intermediate electron carrier in the mitochondrial respiratory chain. It is known that reduced form of ubiquinone QlOacts in the human LDL as very effective natural antioxidant. Daily per as administration of HMG-CoA reductase inhibitor simvastatin to rats for 30 day had no effect on high-energy phosphates (adenosin triphosphate, creatine phosphate) content in liver but decreased a level of these substances in myocardium. We study the Cu2+-mediated susceptibility of human LDL to oxidation and the levels of free radical products of LDL lipoperoxidation in LDL particles from patients with atherosclerosis after 3 months treatment with natural antioxidants vitamin E as well as during 6 months administration ofHMG-CoA reductase inhibitors such as pravastatin and cerivastatin in monotherapy and in combination with natural antioxidant ubiquinone Q IO or synthetic antioxidant probucol in a double-blind placebo-controlled trials. The 3 months of natural antioxidant vitamin E administration (400 mg daily) to patients did not increase the susceptibility of LDL to oxidation. On the other hand, synthetic antioxidant probucol during long-time period of treatment (3-6 months) in low-dose (250 mg daily) doesn't change the lipid metabolism parameters in the blood of patients but their high antioxidant activity was observed. Really, after oxidation of probucol-contained LDL by C-15 animallipoxygenase in these particles we identified the electron spin resonance signal of probucol phenoxyl radical that suggests the interaction of LDL-associated probucol with lipid radicals in vivo. We observed that 6 months treatment of patients with pravastatine (40 mg daily) or cerivastatin (0.4 mg daily) was followed by sufficiently accumulation ofLDL lipoperoxides in vivo . In contrast, the 6 months therapy with pravastatin in combination with ubiquinone Q IO (60 mg daily) sharply decreased the LDL initiallipoperoxides level whereas during treatment with cerivastatin in combination with probucol (250 mg daily) the LDL lipoperoxides concentration was maintained on an invariable level. Therefore, antioxidants may be very effective in the prevention of atherogenic oxidative modification of LDL during HMG-CoA reductase inhibitors therapy. (Mol Cell Biochem 249: 129-140,2003) Key words : HMG-CoA-reductase inhibitors, vitamin E, ubiquinone QIO' probucol, LDL free radical peroxidation, lipohydroperoxides

Addressfor offprints : V. Lankin, Cardiology Research Complex, Miasnikov's Institute of Clinical Cardiocogy, 3rd Cherepkovaskaya 15A, 121552 Moscow, Russia (E-mail : [email protected])

130 'QH + HO-Asc-OH

Introduction High blood level of cholesterol, especially low density lipoprotein (LDL) cholesterol, has been associated with an increased risk of atherosclerosis development [1]. Statins, the inhibitors of key enzyme of cholesterol biosynthesis i.e, 13hydroxy-Bsmethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor are widely used as cholesterol-lowering drugs in the prophylaxis and therapy of atherosclerosis [2] . These preparations reduce plasma content of cholesterol in patients with coronary heart disease but cause some delayed adverse effects. Inhibitors of HMG-CoA reductase suppress cholesterol synthesis not only in the liver, but also in other organs, in particular in the brain, which may have an undesirable effect because brain membranes are enriched with cholesterol and it is intensively synthesized in this tissue [3]. Furthermore, HMG-CoA reductase inhibitors depre sses the synthesis not only of cholesterol, but also of the isoprenoid lateral chain of ubiquinone Q IO (coenzyme Q) [4] as one can see in the scheme (Fig. 1). Ubiquinone Q IO is present in all human and animal tissues and is involved in variou s metabolic processes, including electron transfer associated with adenosine triphosphate (ATP) synthesis in the respiratory chain of mitochondria [4]. Thus, statin induced inhibition of ubiquinone QlObiosynthesis in tissues has adverse consequences, in particular, impaired energy supply to skeletal muscles leading to myopathy [5, 6]. Negative effects of HMG-CoA reductase inhibitors extend further. There is evidence that oxidized LDL are involved in atherogenesis [7]. Oxidized LDL rapidly accumulated by monocytes-macrophages of the vascular wall which are transformed into foam cells forming the areas of lipoidosis in vascular wall [7]. Until present, the major natural antioxidant a-tocopherol (vitamin E) was believed to play the major role in the antioxidant protection of circulating LDL involved in its transport in the body. Recent studies demonstrated that ubiquinone Q IO (Q), a component of LDL, is a more potent antioxidant then a-tocopherol if it is readily transformed into the corresponding phenol (QHz) and may reduce tocopheroxyl radical (a-TO·) which formed after interaction of a-tocopherol (a-TOH) with lipid peroxyl (LO z· ) or lipid alkoxyl (LO·) radicals [8]: ｌｏｺ

ＧＫ｡Ｍｔｏｈｾｌｯｲ

a-TOH + 'QH,

a-TO' + 'QH ｾ

a-TOH + Q.

During this reactions from ubiphenol Q IO (QH z) formed corresponding ubisemiquinone radical CQH) the reduction of which proceeds after interaction with ascorbic acid (HO-AscOH) [9]:

HO-Asc-O· + QHz-

It is known that different enzymes are participated in the ascorbic acid free radical i.e. semidehydroascorbate (HOAsc-O') reduction in the organism [l0-12]. This mechanism ofLDL protection has a considerable biological significance, because the inhibition ofLDL oxidation involves the expenditure of ubiquinone Q j which synthesized in the body, but not essential vitamin E. At the same time , the content of ubiquinone Q IO in LDL considerably decreases in patients with atherosclerosis treated with HMGCoA reductase inhibitors due to suppression of this compound biosynthesis [4, 13, 14]. These data suggest that HMG-CoA reductase inhibitors may promote the LDL oxidation by reducing the content of ubiquinone QlO' a natural protector against free radical oxidation. In this article we tested this hypothesis and substantiated combined use of natural (such as ubiquinone QIO) or synthetic (such as probucol) antioxidants and drugs from HMG-CoA reductase inhibitors family for the correction ofLDL oxidation during cholesterol-lowered therapy.

Acetyl-CoA

ｾ

HMG-CoA

ｾ

I

Mevalonate

HMG-CoA reductase inhibitors ..

Mevalonyl-PP

!

Isopentenyl-PP

ｾ

Geranyl-PP

ｾ

Famesyl-PP ［

a-TO' + QHz ｾ

ｾ

CoQ

ＮＭ ｾ 10

Squalene

1

Dolychol

Cholesterol Fig. 1. Scheme of chole sterol and ubiquinone Q10 biosynthe sis suppression by HMO-eoA reductase inhibitors (statin s).

131

Materials and methods The alteration of the high-energy phosphates contents in tissues of rats after I-month HMG-CoA reductase inhibitor simvastatin administration Experiments were performed on male Wistar rats having weight 240 ± 20 g which received 24 mg/kg daily for 30 days of simvastatin ('Zokor' , Merck Sharp and Dohme) in 0.5 ml water suspension (experimental group, n = 10) or 0.5 ml distilled water daily (control group, n = 10) through a oesophageal tube. The rats were anesthetized with urethane and tissue samples (the heart and the liver) were taken by a Wollenberg forceps cooled in liquid nitrogen. These samples were homogenized in cold 6% HCI0 4 (10 ml/g tissue) on an ice bath using an Ultra-Turrax T-25 tissue desintegrator (IKALabortechnik). Proteins were precipitated by centrifugation and supernatants were neutralized with 5 M K2C03 to pH 7.4. The dry weight of samples was estimated by weighting of precipitates after extraction with HCI0 4 and drying them to a constant weight at 110°C for 12 h. The contents of adenosine triphosphate (ATP) and creatine phosphate (CrP) in tissue extracts were measured spectrophotometrically using glucose-6-phosphate dehydrogenase, hexokinase, and creatine kinase [15] . Adenosine diphosphate (ADP) content was estimated enzymatically using pyruvate kinase and lactate dehydrogenase [16] . Creatine (Cr) concentration in tissues was measured by the reaction with a-naphthol and diacetyl [17]. The measurements were performed using a Yanaco2000 spectrophotometer. The total creatine content (LCr) was calculated as: LCr =CrP + Cr. The concentrations of adenine nucleotides, CrP, and Cr in tissues were expressed in umol/g dry weight.

Treatment ofpatients with vitamin E The 32 men aged 55 ± 4.1 with coronary heart disease and hypercholesterolemia IIa and lIb types (total cholesterol 6.2 ± 0.34) were treated in out-patient conditions with vitamin E (as a-tocopherol acetate, 'Slovakofarma') in daily dose 400 mg during 3 months. In the period of observation the patients received no another antioxidant preparations.

Treatment ofpatients with different doses of synthetic antioxidant probucol The investigation were conducted with 28 men aged 51 ± 1.3 with coronary heart disease and hypercholesterolemia IIa and lIb types treated at the dispensary of Russian Cardiology Research Complex. Three months before examination the patients received no lipotropic drugs and 1 month before

examination they followed a hypolipidemic diet. The patients received 250 (125 x 2) or 1000 mg (500 x 2) probucol preparation ( 'Phenbutol', Akrikhin company, Russia) every day during 6 months.

Treatment ofpatients with HMG-CoA reductase inhibitor pravastatin and ubiquinone QJO preparation A double-blind placebo-controlled trial was performed on 20 men (49 ± 2.5 years) with chronic coronary heart disease and type IIa and lIb hyperlipidemia (total plasma cholesterol 7.2 ± 0.4 mmolll) which were treated at Russian Cardiology Research Complex. The patients received no lipotropic drugs for 3 months before examination and followed a low-cholesterol diet for 2 months before the therapy. The 10 patients were treated during 6 months with HMG-CoA reductase inhibitor pravastatin ('Lipostat', Bristol-Mayers Squibb) in daily dose of 40 mg and placebo of ubiquinone Q 10' The other 10 patients were treated during 6 months with HMG-CoA reductase inhibitor pravastatin ('Lipostat' , Bristol-Myers Squibb) and natural antioxidant ubiquinone QIO preparation ('Bioquinone', Phrama Nord) in daily doses of 40 and 60 mg respectively.

Treatment ofpatients with HMG -CoA reductase inhibitor cerivastatin and synthetic antioxidant probucol A double-blind placebo-controlled trial was performed on 32 men (53 ± 5 years) with chronic coronary heart disease and type IIa and lIb hyperlipidemia (total plasma cholesterol 7.4 ± 1.1 mmolll) subjected to out-patient treatment at Russian Cardiology Research Complex. The patients received no lipotropic drugs for 3 months before examination and followed a low-cholesterol diet for 2 months before the therapy. The 16 patients were treated during 6 months with HMG-CoA reductase inhibitor cerivastatin ('Lipobay', Bayer) in daily dose of 0.4 mg and placebo of probucol. The other 16 patients were treated during 6 months with HMG-CoA reductase inhibitor cerivastatin ('Lipobay ', Bayer) and synthetic antioxidant probucol (' Alcolex', ICN Pharmaceuticals, Inc.) in daily doses of 0.4 and 250 mg respectively. The patients took probucol and placebo of probucol in two equal doses with 8-h interval.

Low density lipoproteins preparation and its in vitro peroxidation For monthly LDL control, venous blood was obtained on an empty stomach and stabilized with 1 mg/ml EDTA. Plasma was centrifuged twice in a NaBr density gradient for 2 h at 42,000 rpm in a Beckman L-8 ultracentrifuge (angle 50Ti

132 rotor) at 4°C [18]. Thereafter, the plasma was dialyzed for 16 h at 4°C against 1000 volumes of phosphate buffered saline. The LDL preparations obtained by this technique were free from other plasma proteins and were identical in particles size and lipid composition to lipoproteins isolated by the standard method of Lindgren [19]. Protein content was determined according to Lowry et at. and LDL concentration was adjusted to 50 ug protein/ml with 50 mM K,Na-phosphate buffer pH 7.4 containing 0.154 M NaCl. Oxidation of LDL was induced with 3 x lO-sM CuS04 and accumulation of lipohydroperoxides (conjugated diens) was measured on a Hitachi 220A UV-spectrophotometer at 233 nm at fixed time intervals [20]. On the other in vitro experiments LDL from healthy donors without hyperlipidemia were oxidized in the presence of exogenous probucol added to the incubation medium as an ethanol solution (2% final ethanol concentration). Kinetic curves were reconstructed and the duration of lag-phase of oxidation was calculated. In some experiments, LDL (2 mg protein/ml) from patients receiving 250 mg/day probucol during 3 months were oxidized by C-I5 lipoxygenase from rabbit reticulocyte s [21, 22] to a hydroperoxide concentration of 0.5 umol/mg protein and then LDL lipoperoxides were decomposed with hemin using to lipid alkoxyl radical. Electron spine resonance (ESR) spectra were recorded on a Varian E-l 09E spectrometer at 25°C under anaerobic conditions [23]. The content of lipoperoxides in LDL was determined by Fe" oxidation with lipid hydroperoxides using xylenol orange as Fe 3+ indicator and triphenylphosphine for reduction of organic hydroperoxides [24].The tert-butyl hydroperoxide was used as a standard.

Lipid analysis The contents of total cholesterol were estimated on a Kone Progress chemical analyzer by enzymatic method with using Boehringer assay kits in Laboratory of Clinical Chemistry of Russian Cardiology Research Complex. Cholesterol level in LDL was calculated after plasma cholesterol concentration and cholesterol level in high density lipoprotein (HDL) estimation.

cals. Adenosine triphosphate, adenosine diphosphate, creatine phosphate, creatine, a-naphthol, diacetyl, probucol, triphenylphosphine, bovine albumin, Folin phenol reagent, urethane, HCI0 4, EDTA, NaBr, NaCl, ｾｈｐｏ 4and NaH zP04 were from Sigma Chemicals. Xylenol orange sodium salt was from Aldrich; tert-butyl hydroperoxide was from Merck. Methanol, ethanol, CuS04'5HP and (NH4\Fe(S04) Z'6HP were purchased from Reachim Company (Russia) and were analytical grade or better. All reagent solutions were prepared fresh just before experiments.

Statistics Results are expressed as mean ± S.E.M. Statistical analysis between two groups were performed with an unpaired Student t-test. Probability values of p < 0.05 were considered to be significant.

Results The changes of the high-energy phosphates content in heart of rats after I -month HMG-CoA reductase inhibitor simvastatin administration In rats treated with HMG-CoA reductase inhibitor simvastatin the contents of ATP, ADP, CrP, and Cr in the liver did not differ from the control (Table 1).ADP concentration andATP/ ADP ratio did not differ between experimental and control groups of animals (Table 1). At the same time, the contents of ATP,CrP, and Cr in the myocardium decreased by 13, 18, and 19% respectively after l-month simvastatin treatment compared to the control (Table 1). The total content of myocardial Cr (LCr) reflecting the integrity of cardiomyocyte sarcolemma decreased in simvastatin-treated animals, which attests to the development of myocardial dysfunction [17]. Thus, the HMG-CoA reductase inhibitor decreased the conTable 1. Effects of simvastatin on adenine nucleotides, creatine phosphate and creatine content in rat liver and heart (umol/g dry weight, M ± m) Parameter

Liver (n = 10) Control Treatment

Heart (n = 10) Control Treatment

ATP ADP

7.37 ± 0.42 6.38 ± 0.16 1.16 ± 0.07 0.21 ± 0.04 0.59 ± 0.02 0.80 ± 0.05 0.35 ± 0.40

19.39 ± 0.23 4.10 ± 0.22 4.79 ± 0.26 24.49 ± 0.65 33.97 ± 2.26 59.22 ± 2.20 0.75 ± 0.05

Enzyme preparations and chemical reagents Animal C-I5 lipoxygenase was isolated from rabbit reticulocytes and purified by ion-exchange chromatography on a DEAE-Sephadex Aso column followed by preparative isoelectrofocusing as described [21, 22]. Glucose-6-phosphate dehydrogenase, hexokinase, creatine kinase, pyruvate kinase, L-lactate dehydrogenase were purchased from Sigma Chemi-

ATP/ADP

CrP Cr ECr CrP/Cr

8.86 ± 0.56 6.56 ± 0.26 1.38 ± 0.12 0.25 ± 0.03 0.66 ± 0.03 0.91 ± 0.02 0.38 ± 1.00

*p < 0.05 compared to the control.

16.79 ± 0.99* 4.31 ± 0.15 4.25 ± 0.40 20.05 ± 0.75* 27.39 ± 1.00* 47.45 ± 1.63* 0.73 ± 0.03

133 tent of high-energy phosphates in the myocardium, but not in the liver. This was probably related to a short period of observations in our experiments, but it should be taken into account that in clinical practice statins are used for 3-6 months period of treatment.

The influence vitamin E (400 mg per day) administration to patients on the susceptibility of their plasma LDL to free radical Cu2+-mediated oxidation Figure 2 shows the results of LDL oxidazibility study after administration of 400 mg daily vitamin E in the form of utocopherol acetate to patients with coronary heart disease and hypercholesterolemia during 3 months. As can see from Fig. 2, the susceptibility of plasma LDL to free radical Cu't-mediated oxidation is not different sufficiently after long-time vitamin E administration to patients.

The influence of different doses of exogenous probucol on the susceptibility ofLDLfrom normal people to free radical Cu2+ -mediated oxidation The supplementation of exogenous synthetic antioxidant probucol in a concentration range of 10-50 ｾ ｍ effectively inhibits Cu't-mediated free radical oxidation of unsaturated phospholipids in native LDL from normal men (Fig. 3). Increasing the level of probucol in the incubation medium to 100 flM results in complete inhibition of LDL oxidation. Since the dynamic of probucol concentration changes in blood plasma after its administration to patients was studied in detail earlier by means of high performance liquid chromatography method, we calculated that the mean probucol concentration in the plasma produced by two doses of 125 mg taken at 8-h interval is about 25 ｾ [25]. Thus, even the minimum probucol doses used in our study in vitro must be very effective for LDL protection against in vivo oxidative modification in the circulation.

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Fig. 8. The cholesterol level in human LDL which were isolated from blood

Fig . 9. The LDL lipoperoxides levels in the blood plasma of patients with

plasma of patients with atherosclerosis before and after 6-months administration of HMG-CoA reductase inhibitor pravastatin (40 mg daily) or pravastatin (40 mg daily) in combination with natural antioxidant ubiquinone Q IO (60 mg daily) . The LDL cholesterol content were calculated after total cholesterol and HDL cholesterol estimation by enzymatic method . The data are presented as the mean ± S.E.M. (n = 10 for each group of patients), *p < 0.05, NS - not sufficiently different.

atherosclerosis after 6-months treatment with HMG-CoA-reductase inhibitor pravastatin (40 mg daily) or pravastatin (40 mg daily) in combination with natural antioxidant ubiquinone Q IO (60 mg daily). The content oflipohydroperoxides in isolated LDL was determined by ferrous ion oxidation in presence of xylenol orange in conjunction with triphenylphosphine. The data (mean ± S.E.M.) are presented as relative units (control points was taken as 1 for each group , n = 10 for each group of patients), *p < 0.05 .

cholesterol (Fig. 8), and increased the concentration of HDL cholesterol (not shown). The content of lipid peroxides in LDL increased during monotherapy with pravastatin, but decreased after combined treatment with pravastatin and ubiquinone QIO (Fig. 9). Pravastatin alone and in combination with ubiquinone QlOreduced the content ofMDA in LDL, but these changes were less pronounced (data not shown). These data suggest that HMG-CoA reductase inhibitors intensify the LDL oxidation in vivo, while ubiquinone Q IO inhibits this process. Therefore, ubiquinone Q IO would be appropriate for use in combined therapy of patients with coronary heart disease and hypercholesterolemia for preventing adverse effects of statins, which promote atherogenic oxidative modification of LDL.

coronary heart disease in the daily dose 250 mg in opposition to high probucol dose (1000 mg per day) have not influence on the lipid metabolism parameters in the plasma of patients such as total cholesterol, LDL cholesterol, and HDL cholesterol. On the other hand, probucol administration to patients in daily dose 250 mg effectively preserved the plasma LDL from lipohydroperoxide accumulation in vivo (Fig. 6). As it appears from the above consideration, we used in this clinical trial probucol in daily dose 250 mg for maximal manifestation of this drug antioxidative activity without some influence on the blood lipid content. After groups randomization the levels of total cholesterol, LDL cholesterol, and HDL cholesterol in the groups of patients treated with cerivastatin + probucol or cerivastatin + placebo sufficiently not change and were 8.1 ± 0.92; 5.7 ± 0.88; 1.3 ± 0.43 or 7.4 ± 0.86; 5.2 ± 0.86; 1.3 ± 0.48 correspondingly. Therapy with cerivastatin alone during 6 months decreased LDL cholesterollevel on 38% and in the group of patients treated with combination of cerivastatin + probucol - on 49% (Fig. 10, not sufficiently different). The same results were received after investigation of total cholesterol level which was decreased in the group of patients treated with cerivastatin along during 6 months therapy on 25% and in group of patients treated with combination of cerivastatin + probucol - on 39%

The changes of lipohydroperoxides level in LDLfrom blood plasma ofpatients with coronary heart disease treated long-time with HMG-CoA reductase inhibitor cerivastatin alone or in combination with low dose of synthetic antioxidant probucol

In the preliminary study [25] and in this work (Fig. 7) we found that long-time probucol administration to patients with

137 Before treatment

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(not shown, not sufficiently different). Thus , probucol in daily dose 250 mg sufficiently not increased the cholesterol-lowering effect of cerivastatin, but this may be connected with low number of patient in our study. The lowering of cholesterol content during therapy with cerivastatin was more higher then after long-time treatment with other HMG -CoA reductase inhibitor pravastatin just as we expected on the base of literature data (compare [30] and Figs 8 and 10). If our hypothesis is correct and HMG-CoA reductase inhibitors simultaneously depressed in vivo both cholesterol and ubiquinone Q IO biosynthesis, during treatment of patients with cerivastatin we must observed more higher level ofLDL free radical peroxidation then after pravastatin administration. Really, as it was shown on Fig . 11, cerivastatin administration drastically increased the LDL lipoperoxides level by third month of treatment (more then 5 times) and similar very high level of LDL lipoperoxides remains the same during following period of observation (Fig . 11). It should be note, that during 6 months treatment of patients with pravastatin (Fig. 9) the LDL lipoperoxides level increased only by 30%. On the other hand, in group of patients treated with combination of cerivastatin + probucol the LDL lipoperoxides content did

2

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Time of therapy, months

cerivastatin cerivastatin + placebo + probucol

F 10 The cholesterol level in human LDL which were isolated from blood plasma of patients with athero sclero sis before and after 6-months administration of HMG-CoA reducta se inhibitor cerivastatin (0.4 mg daily) or ceri vastatin (0.4 mg dail y) in combination with synthetic antioxidant probucol (250 mg daily) . The LDL chole sterol content were calculated after total cholesterol and HDL cholesterol estimation by enzymatic method. The data are presented as the mean ± S.E.M. (n 16 for each group of patients), *p < 0.05, NS - not sufficiently different.

1

11. The LDL lipoperoxides levels in the blood plasma of patients with atherosclero sis after 6-months treatment with HMG-CoA-reductase inhibitor cerivastatin (0.4 mg daily) or cerivastatin (0.4 mg daily) in combination with synthetic antioxidant probucol (250 mg daily). The content of Iipohydroperoxides in isolated LDL was determined by ferrou s ion oxidation in presence of xylenol orange in conjunction with triphenylphosphine. The data (mean ± S.E.M.) are presented as relative units (control points was taken as 1 for each group, n = 16 for each group of patient s), *p < 0.05.

not differ from the initial level and practically unchanged during all observation period (Fig. 11). In that way 250 mg dose of probucol per day very effectively protected the LDL from in vivo lipoperoxidation even during treatment patients with powerful HMG-CoA reductase inhibitor as cerivastatin which induced free radical LDL oxidation in the greater degree then other drug from the same family - pravastatin.

Discussion For the first time the hypothesis about important role of free radical lipid peroxidation in the etiology and pathogenesis of atherosclerosis was put forward by our scientific group [3134]. In particular, we observed accumulation oflipoperoxides in the liver, blood and aorta of animals with experimental hypercholesterolemia as well as in the same tissues in the human atherosclerosis [33-36]. On the other hand, we have found a sharp decrease in the enzymatic systems activity which utilizes reactive oxygen species and lipid peroxides (such as superoxide dismutase and glutathione peroxidase) in the hepatocytes, blood cells and aortic intima cells during

138 atherogenesis [32-34, 37]. On the base of these studies it was assumed that the decreasing of antioxidative enzymes activity may be a direct cause of intensification of free radical lipoperoxidation in the blood and other tissues during atherosclerosis [33, 34, 37]. We also found that LDL are most susceptible to free radical oxidation in vitro than other classes of lipoproteins [38]. Next came the suggestion that oxidized LDL are most atherogenic and accumulated in the aorta induced vascular wall injury [7]. At the present time the interventions that block oxidative modification of LDL are currently under intensive study [7]. If oxidative modification of LDL results in enhanced uptake by macrophages, use of an appropriate antioxidant should protect LDL from oxidation, decrease the rate of LDL uptake by macrophage foam cells and slow the development of fatty streaks in the arterial wall. A number of studies have evaluated the role of antioxidants in preventing oxidative modification of LDL [7]. In our investigation we studied the influence of the vitamin E administration on the copper-mediated oxidizability of plasma LDL from patients with atherosclerosis. So far as LDL is the main transport form of natural antioxidant a-tocopherol we were surprised to find that during 4-months vitamin E supplementation in the daily dose 400 mg did not increase the oxidation resistance of LDL (Fig. 2). These observations are consistent with the view that mainly natural antioxidant of LDL may be not a-tocopherol but reduced form of ubiquinone Q IO - ubiquinol Q IO [8]. As we found the treatment of patients with the synthetic antioxidant probucol in the daily dose 250 mg in opposition to vitamin E sharply increase the lag time of LDL oxidation in vitro (Fig. 5). Using one quarter of the usual dose of probucol - 250 mg per day - we observed the same inhibition of lipohydroperoxides accumulation in the LDL of patients with cardiosclerosis in vivo as using of high probucol dose 1000 mg per day (Fig. 6). We isolated the LDL from blood plasma of patients with atherosclerosis after 3 months probucol administration in daily dose 250 mg and oxidized this probucol-contained LDL by C-15 animallipoxygenase. After decomposition of enzymatically accumulated acyl-lipohydroperoxides in LDL phospholipids [21,22] by hemin with corresponding alkoxyl radicals formation we identified in these particles the electron spin resonance signal of probucol phenoxyl radical (Fig. 4) These our findings suggest the possibility of LDL-associated probucol interaction with lipid radicals in vivo. Several studies have demonstrated that intensive lowering of serum cholesterol or LDL cholesterol may retard progression of coronary atherosclerosis [I] .At present the suppressors of key enzyme of cholesterol biosynthesis - HMG-CoA-reductase inhibitors used in the clinical conditions as one of the more effective lipid-lowering drugs [2]. It should be note, that HMG-CoA-reductase inhibitors must depress not only cholesterol but also ubiquinone Q IO biosynthesis [4, 14] so far as

biosynthesis both of this substances involved a common precursor (Fig. I). Because ubiquinone QlOact in the organisms as electron carrier in respiratory chain of mitochondria, the inhibition of this substance biosynthesis must lead to depression of high-energy phosphates level in tissues . Really, as it was shown in our experiments in the myocardium of rats treated with HMG-CoA-reductase inhibitor simvastatin the contents of ATP and CrP are considerably sufficiently decreased (Table I). This results are consistent with the data obtained by Wills et at. [39] on rats receiving a standard diet containing 400 mg lovastatin per kg of food ad libitum for 4 weeks. Taking into account the weight of animals and daily food consumption, we conclude that the daily dose of lovastatin in this experiment was similar to the therapeutic dose of simvastatin used in our experiments. Clinical studies showed that lovastatin and simvastatin in therapeutic doses produce similar effects on lipid metabolism and decrease the content ofLDL cholesterol in patients [30]. Hence, lovastatin and simvastatin in similar doses should be equally potent in inactivating HMG-CoA reductase and inhibiting ubiquinone Q IO biosynthesis in animal tissues. Myocardial ubiquinone Q IO content in rats receiving lovastatin for I month decreased by 14% [39] , that is consistent with the reduction of myocardial ATP and CrP concentrations in rats treated with simvastatin in our experiment (Table I) . Our findings and data of other authors [4, 13, 14] indicate that HMG-CoA reductase inhibitor suppresses biosynthesis of ubiquinone QIO' decreases its content in the myocardium [39] and, therefore, impairs energy supply to the heart. Our results are confirmed by published data that l-month lovastatin therapy not only decreases blood ubiquinone Q10 content in patients, but also impairs energy-dependent myocardial functions: stroke volume, cardiac output, and contractile index decreased [4]. These data indicate that long-term administration of statins to patients with coronary heart disease and hypercholesterolemia suppresses ubiquinone Q IO biosynthesis in myocytes [39], causes myopathy [5, 6], and impairs myocardial energy supply [4], which decreases the efficiency of therapy . On the other hand, the data available in the literature indicate the decreasing of ubiquinone Q IO level in the LDL of patients with hypercholesterolemia during treatment with HMG-CoA-reductase inhibitors such as lovastatin , pravastatin and other preparations from this drugs family [4,13,14].There are also some experiments suggesting that ubiquinone Q10 in reduced form is an important antioxidant in human LDL [8]. The suggestion has not been verified and we study the level of lipoperoxides in LDL from patients with atherosclerosis during long-time treatment with drugs from HMG-CoA reductase inhibitors family in monotherapy as well as in combination with natural or synthetic antioxidants such as ubiquinone Q IO and probucol in double-blind placebo controlled trials (Figs 9 and II). The treatment of patients with inhibitor of cholesterol and ubiquinone Q IO biosynthesis pravastatin alone in

139 daily dose 40 mg during 6 months followed by accumulation of LDL lipohydroperoxides (Fig. 9). On the other hand, the 6 months administration the same dose of pravastatin in combination with natural antioxidant ubiquinone QlOin daily dose 60 mg sharply decreased even the LDL initiallipoperoxides level (Fig . 9). In another part of our study the 6 months therapy with 0.4 mg daily of other HMG-CoA reductase inhibitor cerivastatin, which is more effective than pravastatin as cholesterol-lowering drug (Figs 8 and 10), sharply increased the level of LDL lipohydroperoxides (Fig. 11). At the same time, administration of cerivastatin in combination with synthetic antioxidant probucol in daily dose 250 mg not produces the increase of the lipohydroperoxide level in the LDL during all time the observation (Fig. 11). There is evidence that antioxidative effect of probucol may be connected not only with direct antioxidant action of this drug (Fig. 4), but also with indirect activation of natural preventive systems responsible for enzymatic detoxification of active oxygen species and lipoperoxides during probucol administration [40,41]. It is very demonstrative that after our preliminary communication on this article subject [42] data which are consistent with the our view on the conference 'Free radicals, nitric oxide, and inflammation' (Antalya, Turkey, 2001) was presented [43]. In this work it was demonstrated that administration ofHMG-CoA reductase inhibitor atorvastatin (40 mg daily) to patients with type I diabetes melitus during 12 weeks sufficiently increase the susceptibility of plasma LDL to free radical peroxidation in vitro. As it appears from the above, consideration for prevention of atherogenic oxidative modification of LDL in the blood of patients with atherosclerosis during treatment with cholesterol-lowering drugs from HMG-CoA-reductase inhibitors family it is necessary to use in the combination with antioxidants. The most attractive conclusion is that not only natural antioxidant ubiquinone QIO' but non-toxic synthetic antioxidant probucol in low dose also can act in the LDL as a trap for lipid free radicals and may be effective in the prevention of LDL peroxidation during atherogenesis and during cholesterol-lowering therapy.

Acknowledgements This study was partly supported by the Russian Foundation for Basic Research (grant no. 00-04-49100) . The authors wish to thank Drs Violetta Kaminnaya, ViktorTutunov, Irina Studneva, Lena Nagler, Antonina Kozachenko, Svetlana Gurevich, Gulnara Izmailova, and Tatiana Kotkina for collaboration in performing some studies. We wish to thank also Nursiania Saberova for excellent technical assistance. The pravastatin (lipostate) was kindly provided by Bristol-Mayers Squibb, ubiquinone QlOpreparation (bioquinone) as well as placebo of ubiquinone Q lO - by Pharma Nord (Denmark), cerivastatin

(lipobay) - by Bayer, probucol (alcolex) - by ICN Pharmaceuticals Inc. respectively.

References 1. Steinberg D, Witztum JL: Lipoproteins and atherogenesis - current concepts . JAMA 264: 3047-3052, 1990 2. Davignon J, Laaksonen R: Low-density lipoprotein-independent effects of statins . Curr Opin Lipidol 10: 543-559, 1999 3. Gibbons GF, Mitropoulos KA, Myant NB: In: Biochemistry of Cholesterol. Biomedical Press , Amsterdam, 1982, pp 109-130 4. Folkers K, Langsjoen P, Willis R, Richardson P, Xia LJ, Ye CQ , Tamagawa H: Lovastatin decreases coenzyme Q levels in humans . Proc Natl Acad Sci USA 87: 8931-8934, 1990 5. Laaksonen R, Jokelainen K, Sahi T, Tikkanen MJ, Himberg JJ: Decreases in serum ubiquinone concentrations do not result in reduced levels in muscle tissue during short-term simvastatin treatment in humans. Clin Pharmacol Ther 57: 62-66, 1995 6. Martinez-Garcia FA, Martin-Fernandez J, Molto JM, Villaverde R, Morales A, Fernandez-Barreiro A: Myopathy caused by inhibitors of hydroxymethylglutaryl-coenzyme A reductase. Rev Neurol 25: 869871,1997 7. Steinberg D: Role of oxidized LDL and antioxidants in atherosclerosis. In: J.B. Longenecker et al. (eds). Nutrition and Biotechnology in Heart Disease and Cancer. Plenum Press, New York, 1995, pp 39-48 8. Stocker R, Bowry VW, Frei B: Ubiquinol-l0 protect human low density lipoprotein more efficiently against lipid peroxidation than does a-tocopherol. Proc Natl Acad Sci USA 88: 1646-1650, 1991 9. Cadenas E: Mechanisms of oxygen activation and reactive oxygen species detoxification. In: S. Ahmad (ed) . Oxidative Stress and Antioxidant Defenses in Biology. Chapman and Hall, New York, 1995, pp 1-61 10. Arrigoni 0, Dipierro S, Borraccino G: Ascorbate free radical reductase, a key enzyme of the ascorbic acid system . FEBS Lett 125: 242244,1981 11. Maellaro E, Del Bello B, Sugherini L, Santucci A, Comporti M, Casini AF: Purification and characterization of glutathione-dependent dehydroascorbate reductase from rat liver. Biochem J 301: 471476, 1994 12. Del Bello B, MaellaroE, Sugherini L, Santucci A, Comporti M, Casini AF: Purification of NADPH-dependent dehydroascorbate reductase from rat liver and its identification with 3a-hydroxysteroid dehydrogenase . Biochem J 304: 385-390, 1994 13. Mortensen S, Leth A, Agner E, Rohde M: Dose-related decrease of serum coenzyme Q-I0 during treatment with HMG-CoA reductase inhibitors . Mol Aspects Med 18: SI37-S144, 1997 14. Palomaki A, Malminiemi K, Sovakivi T, Malminiemi 0 : Ubiquinone supplementation during lovastatin treatment: Effect on LDL oxidation ex vivo . J Lipid Res 39: 1430-1437,1998 15. Lamprecht W, Stein P, Henz F, Weisser H: Creatine phosphate determination with creatine kinase, hexokinase, and glucose-6-phosphate dehydrogenase. In: H.U. Bergmeyer (ed) . Methods of Enzymatic Analysis, vol. 4. Academic Press, New York, 1974, pp 1777-1781 16. Pisarenko 01, Tskitishvily OV, Studneva 1M, Serebryakova LI, Korchazhkina OV: Metabolic effects of ischemic preconditioning and adenosine receptor blockade in dogs . Ann NY Acad Sci 793: 85-97, 1996 17. Reimer KA, Hill ML, Jennings RB: Prolonged depletion of ATPand of adenine nucleotide pool due to delayed resynthesis of adenine nucleotides following reversible myocardi al ischemic injury in dogs. J Mol Cell Cardiol13: 229-239, 1981

140 18. Tertov VV, Kaplun VV,Dvoryantsev SN, Orekhov AN: Apolipoprotein B-bound lipids as a marker for evaluation of low density lipoprotein oxidation in vivo. Biochem Biophys Res Commun 214: 608-613, 1995 19. Lindgren RF: Preparative ultracentrifugallaboratory procedure suggestions for lipoprotein analy sis. In: E.G. Perkin s (ed) . Analysis of Lipids and Lipoproteins. American Oil Chemical Society, New York, 1975, pp 204---224 20. Tikhaze AK, Lankin VZ, Kolycheva SN, Konovalova GG. Shumaev KB, Kozachenko AI, Gurevich SM, Zharova EA, Smirnov LD: Does trimetazidine act as antioxidant? Bull Exp Bioi Med 126: 1132-1134, 1998 21. Lankin VZ, Gordeeva NT, Osis YuG,Vikhert AM, Schewe T, Rapoport SM: Animallipoxygenases: Change in activity oflipoxygenase from reticulocytes upon interaction with blood plasma lipoprote ins. Biochemistry (Moscow) 48: 782-788, 1983 22. Lankin VZ, Kuhn H, Hiebsch C, Schewe T, Rapoport S, Tikhaze AK, Gordeeva NT: On the nature of the stimulation of the lipoxygenase from rabbit reticulocytes by biological membranes. Biomed Biochim Acta 44:655-664,1985 23. Shumaev KB, Ruuge EK, Dmitrov sky AA, Bykhovsky VYa, Kukharchuk VV: Effect oflipid peroxidation products and antioxidants on the formation of probucol radical in low density lipoproteins. Biochemistry (Moscow) 62: 657-660, 1997. 24. Nourooz-Zadeh J, Tajaddini-Sarmadi J, Wolf SP: Measurement of pla sma hydroperoxide concentrations by the ferrou s oxidation xylenol orange assay in conjunction with triphenylphosphine. Analyt Biochem 220: 403-409, 1994 25. TikhazeAK, Lankin VZ, Konovalova GG,Shumaev KB, Kaminnyi AI, Kozachenko AI, Gurevich SM, Nagler LG, Zaitseva TM, Kukharchuk VV: Antioxidant probucol as an effective scavenger of lipid radicals in low density lipoproteins in vivo and in vitro. Bull Exp Bioi Med 128: 818-821, 1999 26. Kuzuya M, Kuzuya F: Probucol as an antioxidant and antiatherogenic drug . Free Radic Bioi Med 14: 67-77, 1993 27. Cristol LS, Jialal I, Grundy SM: Effect of low-dose probucol therapy on LDL oxidation and the plasma lipoprote in profile in male volunteers. Atherosclerosis 97: 11-20, 1992 28. Dujovne CA, Harris WS, Gerrond LLC: Comparison of effects of probucol vs. vitamin E on ex vivo oxidation susceptibility of lipoproteins in hyperlipoproteinemia . Am J Cardiol 74: 38-42, 1994 29. Rodes J, Cote G, Lesperance J, Bourassa M, Doucet S, Bilodeau L, Bertrand OF, Hagel F, Gallo R, Tardif JC: Prevention of restenosis after angioplasty in small coronary arteries with probucol. Circulation 97: 429-436, 1998 30. Dujovne CA: New lowering drugs and new effects of old drugs . CUIT Opin Lipidol 8: 362-368, 1997

31. Lankin VZ, Tikhaze AK, Kotelevtseva NV: Lipid peroxides and atherosclerosi s. Kardiologiia (Cardiology) 16: 23-30, 1976 [Article in Russian; abstract in English] 32. Lankin VZ: Lipid peroxide s and atherosclerosis. Hypothesis: The role of cholesterol and free radical lipid peroxidation in altering cell membrane properties during hypercholesterolemia and atherosclerosis. Kardiologiia (Cardiology) 20: 42-48, 1980 [Article in Russian; abstract in English] 33. Lankin VZ: Atherosclerosis as a free radical pathology . Excerpta Med (Int Congr Ser) G98: 385-388, 1992 34. Lankin VZ: Free radicallipoperoxidation during atherosclerosis. Free Radic Bioi Med 16: 8, 1994 35. Belkner J, Wiesner R, Kuhn H, Lankin VZ: The oxygenation of cholesterol esters by the reticulo cyte lipoxygenase. FEBS Lett 279: 110114, 1991 36. Kuhn H, Belkner J, Wiesner R, Schewe T, Lankin VZ, Tikhaze AK: Structure elucidation of oxygenated lipids in human atherosclerotic lesions. Eicosanoids 5: 17-22,1992 37. Lank in VZ, Vikhert AM, Kosykh VA, Tikhaze AK , Galakhov IE, Orekhov AN, Repin VN: Enzymatic detoxication of superoxide anionradical and lipoperoxides in intima and media of atherosclerotic aorta. Biomed Biochim Acta 43 : 797-802, 1984 38. Osis YuG,Formaziuk VE, Lankin VZ, Dudin a EI, Vikhert AM, Vladimirov YuA: The chemiluminescence of different classes lipoproteins from human blood serum. Vopr Med Khim (Problems of Medical Chemistry) 28: 122-126, 1982 [Article in Russian ; abstract in English] 39. Wills RA, Folkers K, Lan Tucker J, Chun-Qu Y,Li-Jun X, Tamagawa H: Lovastatin decreases coenzyme Q levels in rats. Proc NatiAcad Sci USA 87: 8928-8930, 1990 40. Tikhaze AK, Lankin VZ, Mikhin VP, Revenko VM, Lupanov VP: The antioxidant probucol as a regulator of the intensity of free radical lipid peroxidation processes in the blood of patients with coronary atherosclerosis. Ter. Arkh . (Therapeutic Arch ive) 69: 35-41 , 1997 [Article in Russian; abstract in English] 41. Kumar D, Palace V, Danelisen I, Jugdutt BI, Singal PK: Probucol induced antioxidant confers protection again st ischemia-reperfusion injury. J Mol Cell Cardiol. 33: A62, 2001 42 . Lankin VZ, Tikhaze AK, .Konovalova GG. Kukharchuk VV: HMGCoA reductase inhibitors induced the LDL oxidation. J Mol Cell Cardiol 33: A65, 2001 43. Mannuel y Kennoy B, Vertommen J, Vinckx M, De Leeuw L: Effects of atorvastatin and and vitamin E on lipid peroxidation in patients with type I diabetes melitus. In: Abstr. Book Int. Conf. 'Free Radicals, Nitric Oxide, and Inflammation: Molecular, Biochemical, and Clinical Aspects ' . NATO-ASI, Anthalya (Turkey), 2001, p 83

Molecular and Cellular Biochemistry 249: 141-149, 2003 . © 2003 Kluwer Academic Publishers.

Native and minimally oxidized low density lipoprotein depress smooth muscle matrix metalloproteinase levels David Wilson,' Hamid Massaeli,' Grant N. Pierce? and Peter Zahradka 1 'Institute of Cardiovascular Sciences; 2Division of Stroke and Vascular Disease, St. Boniface General Hospital Research Centre, and Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada

Abstract Vascular lesion development is associated with an accumulation of extracellular matrix proteins within the vessel wall. Matrix metalloproteinases (MMPs) degrade these proteins . Conversely, oxidized low density lipoprotein (LDL) is implicated in atherogenesis through, amongst other cellular effects, a stimulation of the deposition of collagen within the vascular lesion. The present study investigated the potential for an interaction between oxidized LDL and MMP levels. Within the vessel wall fibroblasts, smooth muscle, endothelial and infiltrating cells have been reported to secrete MMPs into the extracellular space to effect remodeling of the extracellular matrix . A consequence of angioplasty and atherosclerotic disease is the loss of endothelial cells or endothelial function, respectively. We have investigated the effects of chronic incubation of cultured vascular smooth muscle cells from rabbit thoracic aorta with oxidized LDL and its influence on MMP levels in the extracellular space. Our data indicate that a low concentration of minimally oxidized LDL (0.005 mg/mL) significantly depressed the levels of MMP-2 and MMP-9 present in the culture medium. Native LDL exerted the same effect but exhibited reduced potency. The effects were not attributable to cytotoxicity exerted by the oxidized LDL. The reduction in MMP secretion into the extracellular medium was a result of decreased enzyme synthesis within the smooth muscle cell. Our results demonstrate that an important atherogenic moiety, oxidized LDL, can reduce MMP activity and hence has the potential to increase the deposition of extracellular matrix proteins within SMC-rich vascular lesions . (Mol Cell Biochem 249: 141-149,2003) Key words : experimental, vasculature, cellular, lipoproteins, oxidized low density lipoprotein, smooth muscle, matrix metalloproteinases, extracellular matrix, atherosclerosis, rabbit

Introduction Atherosclerosis is associated with hyperlipidemia and increases in lipid and extracellular matrix (ECM) accumulation within the vessel wall. Increases in fibrous collagen elements in the atherosclerotic lesion are believed to function to support the increased circumferential stress associated with lipid accumulation and necrosis within the medial layer. Recently, Matthys et al. [l] have established that endogenously applied oxLDL or native LDL in vivo profoundly increases intimal thickening , collagen accumulation and polymorphonuclear cell infiltration. OxLDL has also been shown to stim-

ulate collagen synthesis and deposition [2, 3]. However, the mechanism responsible for this effect is unclear. Matrix metalloproteinases (MMPs) also have a role in remodeling collagen and extracellular matrix elements [4-6]. MMP accumulation has been observed in smooth muscle cells in atherosclerotic lesions coincident with regions of circumferential stress [7], as well as throughout the body of restenotic and atherosclerotic lesions [8-10]. Since oxLDL and MMPs have been implicated in the changes in extracellular matrix accumulation that are associated with the development of vascular lesions, we were interested in examining the effects of oxLDL on MMP activity. It would be of value

Address for offprints: P. Zahradka or UN. Pierce, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, Manitoba, Canada, R2H 2A6 (E-mail: [email protected];[email protected])

142 to understand if oxLDL has the ability to modulate MMP activity and thereby alter vascular ECM protein levels. Recently, lipid lowering therapy has been shown to alter MMP activity and increase collagen content in rabbit atheroma [11]. Clearly then, lipids may modulate MMP activity but the underlying mechanism remains unknown. Smooth muscle cells playa key role in the development of vascular lesions. However, the MMPs present in both restenotic and atherosclerotic lesions are localized to both vascular smooth muscle (VSMC) and infiltrating cells [8, 12]. Furthermore, infiltrating cells in lesions have been shown to be sources of cytokines that are involved in stimulating autocrine and paracrine MMP production [13, 14]. As well, macrophages are a major source of several MMPs [15]. For these reasons, it has been difficult to distinguish the contribution of SMC-derived MMPs to lesion progression. The purpose of the present study was to determine the capacity of oxLDL to alter VSMC MMP production prior to involvement of infiltrating cells . We therefore chose to isolate and examine the effects of oxLDL on MMPs using a rabbit vascular smooth muscle cell culture system. We were also interested in establishing whether prolonged exposure to a cholesterolenriched diet altered circulating plasma MMP levels in rabbits.

Materials and methods Materials Dulbecco's Modified Eagle Medium (DMEM) , fetal bovine serum, penicillin-streptomycin, trypsin-EDTA and culture dishes were purchased from Gibco BRL. MMP-9 antibody was obtained from Santa Cruz Biotechnology Inc. Super signal HRP detection reagents were purchased from Pierce Chemical Co. Reflection autoradiographic film was purchased from Dupont NEN. Tris and glycerol were purchased from ICN Pharmaceuticals, Inc . Sodium dodecyl sulfate (SDS), Coomassie Blue R-250, and PVDF membrane were purchased from BioRad Laboratories. Acrylamide, glycine, CaC12 , Triton X-100, and the MTT assay kit were purchased from Boehringer Mannheim, Indianapolis, Indiana. Bromophenol blue, dithiobisnitrobenzoic acid, anti-mouse Cy3, gelatin , phenylmethylsulfonyl fluoride, thimerosal, transferrin, selenium, ascorbate, insulin, cholesterol oxidase, cholesterol esterase, horse-radish peroxidase (HRP) conjugated to anti-mouse and goat IgG, and monoclonal antibodies to aactin, smooth muscle myosin and caldesmon were obtained from Sigma-Aldrich, Oakville, Ont., Canada. The Live/Dead Viability Assay kit was purchased from Molecular Probes, Eugene, OR, USA.

Low density lipoprotein isolation and oxidation LDL (density 1.019-1.063 g/ml) was prepared by sequential ultracentrifugation of serum derived from 0.5% cholesterol fed rabbits, as described [16] . Dithiobisnitrobenzoic acid (1.5 mmollL), phenylmethylsulfonyl fluoride (2 mmol/L), and thimerosal (0.08 mg/rnL) were added to the plasma to inhibit lecithin: cholesterol acyl transferase, proteolysis and bactericides, respectively [16]. The LDL fraction was exten sively dialyzed against 0.15 mollL NaCl, 0.1 mmollL EDTA (pH 7.4), filter sterilized (0.2 um pore size) and stored at 4°C in the dark. The protein content of LDL was determined by Lowry's method [17], and cholesterol (free and esterified) was measured enzymatically as described [18]. The absence of LDL oxidation during isolation or prior to its use in experiments was confirmed by an absence of malondialdehyde (MDA) reactive products and oxidized cholesterol [16]. Native LDL was diluted with 150 mmollL NaCl solution (pH 7.4) and oxidized by incubation with a solution of 50 umol/ L FeC13 and 0.25 mmollL ADP for a period of 3 h at 37°C. The extent of LDL oxidation was evaluated by (i) measurement of thiobarbituric acid reactive substances (TBARS) [18], (ii) electrophoretic mobility on agarose gels (using the Chiron Diagnostic Lipoprotein System), and (iii) depletion of the LDL a-tocopherol content as measured by HPLC [19].

Culture of rabbit vascular smooth muscle cells An explant technique originally developed for the enrichment of smooth muscle cells from porcine coronary artery while excluding fibroblasts [20] was used to generate primary cultures from normal rabbit thoracic aorta. The aorta from a male New Zealand White rabbit (2.5-3 kg body wt) was isolated and cleaned of excess fat and connective tissue. The endotheliallayer was scraped off. The aorta was cut into 2-3 mm sections and transferred to a culture dish with growth media (20% fetal bovine serum in Dulbecco's Modified Eagle Medium, DMEM) and 5% antibiotic-antimycotic (lOOx: 10,000 U/ml penicillin G, 10,000 ug/ml streptomycin sulfate, 25 ｾｧｬ ml amphotericin). The explants were incubated in a humidified incubator equilibrated with 5x CO 2 and maintained at 37°C. To induce differentiation , the smooth muscle cells were placed (for 4 days) into serum-free media supplemented with transferrin (5 ug/ml.), selenium (1 nmol/L), ascorbate (200 umol/L), and insulin (10 nmol/L), This period of quiescence was crucial for full development of contractile proteins in the cultured vascular smooth muscle cells (VSMCs) [20] . Confirmation of smooth muscle cell phenotype and purity of VSMC cultures were identified by immunohistochemical staining with monoclonal antibodies specific against smooth muscle a-actin, myosin and h-caldesmon [21].

143

Experimental conditions for treatment of VSMC with LDL oroxLDL The smooth muscle cells used in our experimental protocol were from either first or second passage. VSMC were grown to 50% confluence in 6, 12 or 96 well dishes . These cells were placed in quiescence medium for 4 days prior to presentation of oxLDL. At the end ofthis period, cells were approximately 70-80% confluent. In an independent experiment (Fig. 3), cells were placed in quiescence medium for four days and placed in 5% serum in the presence of oxLDL. Over the treatment period, medium was withdrawn and replaced with fresh medium (and treatment) every 24 h. Each experimental condition was replicated 4 times and repeated at least twice. Groups were exposed to different concentrations of LDL or oxLDL. The lipoprotein fraction was changed daily with the culture medium. Culture medium was removed and aliquoted at each 24 hour interval from the various treatments and stored at -70°C.

Cell cytotoxicity The cytotoxicity of different concentrations ofLDL or oxLDL were assessed by two methods: (i) lactate dehydrogenase (LDH) released into the culture media; (ii) MTT assay (Roche Diagnostics, Montreal, Canada). For the LDH assays, the VSMC were passaged and seeded in 12-well tissue culture plates. These cells were then placed, as described above, in 1 ml of phenol red-free Dulbecco's Modified Eagle Medium. A 500 ul aliquot of media was collected from each well every day for the LDH assay (as described by Gutman and Wahlefeld [22]) . The results were expressed as a percent of total LDH release. To obtain total cellular LDH levels, cells were permeabilized with a solution of 0.5% Triton X-lOO, 3 mmollL sodium cholate, and 0.1 mollL Tris-HCI pH 6.6 at the end of the experiment. At 3 and 6 days, cells that had been incubated in 96 well format culture plates with oxLDL treatment were used for the determination of cell viability using the MTT assay [23], which is considered a reliable index of mitochondrial function.

protein. Gels were incubated in buffer (0.05 mollL Tris-HCI pH 8.0, 0.005 mollL CaCl z' 0.1 mollL PMSF) at 37°C for 12 h (PMSF was included to inhibit serine proteases in the culture medium). Following Coomassie Blue R-250 staining, MMP activity was detectable as lytic activity (clear zones) in an otherwise blue gel (inclusion of MMP inhibitors ortho-phenanthroline or GM600 1 (10-4mollL) prevented the formation of lytic bands, confirming the metalloproteinase nature of these enzymes). Gels were scanned using a BioRad GS670 imaging densitometer, high tran smittance reflecting increased protein levels. Following from Kleiner and Setler-Stevenson [25], the protein load and incubation time was adjusted to be in the linear range (l0-200 pg) . The identity of MMP-2 and MMP9 was confirmed using Western analysis, and molecular mass markers were subsequently used to distinguish the location of MMP-2 (72 kDa) and MMP-9 (92 kDa).

Western blot analysis of cellular MMP-9 VSMC were grown in 24-well culture dishes and treated. The cells were subsequently lysed with SDS sample buffer (0.5 mollL Tris-HCI pH 6.8, 2.0% SDS, 10% glycerol, 0.001 % bromophenol blue with 5% l3-mercaptoethanol as reducing agent), and 15 ug of protein was loaded per well of a 7.5% polyacrylamide gel. Following transfer to PVDF membrane, the relative quantity ofMMP-9 was established using an antiMMP-9 antibody (diluted 1:100) and HRP chemiluminescence detection, followed by densitometry (BioRad GS670 Imaging Densitometer).

Effect of cholesterol enriched diet on serum MMP levels One month old rabbits were fed standard chow either unmodified or with 0.5% cholesterol enrichment (Purina test diet, Richmond, IN, USA) . At 1, 2 and 3 months after placement on a cholesterol enriched diet, 5 mL of blood was collected in heparinized vials . Blood was centrifuged (3000 rpm, 4°C, for 10 min), and the serum stored at -70°C in 0.5 ml aliquots. Serum was diluted 10-fold in SDS sample buffer without reducing agent prior to gelatin zymography.

Zymographic determination matrix metalloproteinase (MMP) levels

Statistical analysis

Culture medium was diluted 1:1 in SDS sample buffer (0.5 moll L Tris-HCI pH 6.8, 2.0% SDS, 10% glycerol, 0.001 % bromophenol blue without reducing agent, and 7 ul was loaded per well of a 7.5% polyacrylamide gel containing 0.1 % gelatin [24]. Gels were washed in glycine-Triton buffer (0.025 mollL glycine pH 8.3, 2.5% Triton X-lOO) twice for 10 min each at 4°C to remove the SDS and permit partial renaturation of the

Controls loaded in each gel were used to compensate for differences in staining intensity (gelatin-SDS PAGE) between the gels . Although decreased staining represents increased lytic activity, MMP activity is reported as positive values, which were achieved by a mathematical transformation involving subtraction from the background in each gel. Data were quantified and graphically represented as means ±

144 S.E.M. Unpaired Student's r-tests were used to compare treatment means versus control. Differences were considered significant when p < 0.05.

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Effect ofoxLDL on MMPs Quiescent rabbit aortic SMCs were exposed to a range of oxLDL concentrations (0.005-0.05 mg/mL) for 3 days. These concentrations are relatively low and well below those typically used by other investigators [26-28] . Furthermore, the oxidized LDL used in our experiments was minimally modified . We observed a modest increase in electrophoretic mobility on agarose gel, a 20% decrease in a -tocopherol content and a significant increase (8 nmol/mg) in TBARS, which is significantly lower than is obtained with copper-mediated oxidation of LDL (- 50 nmol/mg increase in TBARS [29]) . MMP-2 and MMP-9 were measured in samples of the secreted extracellular fluid (culture medium), the physiologically relevant site of MMP activity. Low concentrations of oxLDL (0.005 mg/mL) significantly attenuated both MMP2 and MMP-9 activity in these samples (Fig. 1). It was noted that the magnitude of the observed changes varied between experiments, but the pattern was nevertheless identical. Slight variations in LDL oxidation likely account for these differences. Despite a reduction in MMP levels subsequent to three days exposure to low levels of oxLDL, total protein levels remained constant at 0.31 ± 0.01, 0.34 ± 0.03, 0.33 ± 0.02, and 0.34 ± 0.02 mg/ml for control , 0.005, 0.025, and 0.05 mgt mL oxLDL, respectively. Over the course of an extended six day experiment, MMP-2 activity as measured by zymography was reduced to 6.2 ± 4.2% of control, and MMP-9 activity was undetectable. In experiments carried out on cells in the presence of 5.0% serum and oxLDL, low levels of oxLDL (0.005 and 0.025 mg/mL) also produced a significant reduction in MMP-2 and MMP-9 levels (Fig. 2).

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that 3 or 6 days exposure of VSMCs to minimally oxLDL (0.005-0.025 mg/mL) is not cytotoxic.

Influence ofLDL oxidative state on MMP s Contribution ofoxWL cytotoxicity to changes in MMP levels The observed reduction in MMP activity could be due to cytotoxic effects of the oxLDL. If oxLDL were cytotoxic, one would expect a release of intracellular enzymes like LDH into the extracellular fluid . Very slight, 5 and 12%, release of total cell homogenate LDH were detected over the 6 day experimental period when cells were exposed to 0.005 and 0.025 mgt mL oxLDL, respectively (Fig. 3a). The MTT assay was employed as an alternative method of determining cell viability and indicated that 0.005 mg/mL oxLDL slightly reduce (- 10%) mitochondrial activity at three days (p < 0.05) but increased activity at six days (Fig. 3b). These results indicate

Qualitatively, the effect of native LDL was similar to that of oxLDL over the effective concentration range (Fig. 4). Low levels of native LDL over a concentration range of 0.0050.05 mg/mL caused a significant reduction ofMMP-9 activity. However, there was a greater decrease in MMP-9 at low concentrations of oxidized LDL.

Effect of oxLDL on cellular MMP content The decrease in MMP activity detected in the medium surrounding the cells after exposure to oxLDL may be due to

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an inhibition of the release of the enzyme from the cell. Alternatively, oxLDL may cause a depression in cellular production of the enzyme. Cells were exposed to a range of oxLDL concentrations (0.005-0.05 mg/mL) for 3 days. There was a striking reduction in cellular MMP-9 compared to control as determined by Western blotting at all oxLDL concentrations examined (Fig. 5). These data suggest that a broad range of oxLDL suppresses the synthesis of MMPs by smooth muscle cells.

Circulating serum MMP levels in cholesterol fed rabbits One month old rabbits fed a high cholesterol diet for 1 month through 3 months exhibited a small reduction «5%) in circulating serum derived MMP-9. In contrast, serum MMP-2

Fig . 3. Determination of oxLDL cytotoxicity. (Panel a) Measurement of lactate dehydrogenase (LDH) release from rabbit vascular smooth muscle cells. Relative LDH activity was compared using a colourimetric assay after exposure of cells to 0.005 and 0.025 mg/ml oxLDL over 6 days . There was no detectable LDH in the absence oftreatrnent. Data are means ± S.E.M. of 4 replicates per treatment normalized to control cells at day 6. (Panel b) Influence of oxLDL on mitochondrial activity from isolated rabbit vascu lar smooth muscle cells . Relative mitochondrial activity was compared using the colourimetric MIT assay at various concentrations of oxLDL (0.005, 0.25 and 0.05 mg/mL) at days 3 (_) and 6 (e) of culture. Data are means ± S.E.M. from 6 replicates per treatment. *p < 0.05 vs. control.

levels increased by - 15% at after 1,2 and 3 months on an atherogenic diet (Fig . 6).

Discussion The data presented in this report demonstrate that administration of oxLDL to isolated vascular smooth muscle cells results in a decrease in MMP production. Both MMP-2 and MMP-9 levels were reduced by oxLDL, although MMP-9 appears to be more sensitive at early time points. Interestingly, the reduction in secreted MMP levels appears to involve a decrease in the cellular content of the enzyme, presumably resulting from either reduced synthesis or increased turnover. Although the mechanism responsible for the change in MMPs remains unresolved, it may relate to changes in secre-

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OxLDL (lJg/ml) Fi g. 5. MMP -9 in cytosolic extra cts of rabb it vascular smooth muscle cell s exposed to oxid ized LDL. Western blotting was used to compare the MMP 9 content of control cell s and cell s exposed to 0.005, 0.025 and 0.05 mglml oxLDL. Rel ative band inten sities of dupl icate samples from a single replicate are shown in the inset. Data repre sent s mean s ± S.E.M. from 4 replicates per treatmen t. *p < 0.05 vs. control.

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Fig . 6. Effect of athero genic diet on serum MMP levels. Gelatin zyrnography was used to assay MMP s in serum samples obtained from rabbits maintained on a cholesterol diet (0.5% cholestero l enrichment) for 4, 8 and 12 weeks. Raw data in the form of an overexposed film is presented in panel A. Quantific ation of band intensities by densitometry for MMP-9 and MMP2 is presented in panel s B and C, respectively. Data are means of2 animal s per treatment.

This study is the first to examine the effect of oxLDL on the MMP content of smooth muscle. While these finding s are consistent with reports ofVSMC MMP -9 being more sensitive to modulation by cytokines than MMP-2 [13, 30], Xu and colle agues [31] have recently reported oxLDL effectively upregulates MMP-9 in macrophages. Thi s contradiction is likely a result of cell specific responses rel ated to oxLDL concentration . The change in MMP-9 levels reported by Xu et al. [31] required> 50 ug/ml oxLDL, whereas the reduction we have observed occur with con siderably lower level s (5 ug/ml). In fact, the increase in MMPs at higher oxLDL concentrations may represent the effect of enhanced gene expres sion in respon se to cellular injury (see Fig. 3) which offsets the decrease obtained at the lower concentrations. Furthermore, such a deviation in response could explain the reduced circulating MMP level s we observed in the cholesterol fed rabbit compared to published reports that described an increase in MMP level s. Minor changes in serum MMP-9 levels were observed in the circulating serum of cholesterol fed

147 rabbits consistent with our cell culture data, however, serum MMP-2 levels increased when animals were exposed to an atherogenic diet. In our study, the atherogenic diet consisted of the addition of 0.5% cholesterol to juvenile rabbits (beginning at one month of age) . This regimen results in the development of atherosclerotic lesions by the fourth month (Pierce, unpublished results). In contrast to our data, others have observed an elevation in serum MMPs subsequent to supplementation of the diet with higher (1.0-2.0%) cholesterollevels [34]. It is likely that the age and relatively low cholesterol content in the diet in the present study may account for the reduced severity of vascular lesion formation and also the relative changes in MMPs. In support of this argument, mature rabbits fed a 1% cholesterol diet have increased vascular MMP secretion as early as eight weeks after onset of the consumption of an atherogenic diet [34], and this is consistent with the appearance of infiltrating macrophages in the vascular lesions. Reports in both restenotic and atherosclerotic lesions have indicated that infiltrating cells contribute significantly to the levels of MMP-9 in vascular lesions and may, via cytokine activation, stimulate VSMC to secrete additional MMP-2 and MMP-9 [13, 30]. Our observation that VSMC MMP- 2 levels were not significantly reduced by oxLDL suggests the effects are not simply a metabolic effect. The mechanism whereby these lipids decrease MMP production within the smooth muscle cell is unclear at present. For several reasons, these data have pathological relevance for restenosis and atherosclerosis and their associated ailments: heart disease and stroke. First, we employed relatively low concentrations of oxLDL. The 0.005 mg/ml concentration that produced a striking decrease of MMPs is well below concentrations of oxLDL commonly used in other studies [26-28]. Second, the oxLDL used in this study is not completely degraded by the oxidation process employed . The FeADP method used to oxidize the LDL produces a minimally oxidized form of oxLDL. The LDL exhibits relatively small change s in electrophoretic mobility, a 21% depletion in vitamin E content and the generation of oxidized lipids like conjugated dienes after incubation with the Fe-ADP (data not shown). Third, the study was designed to more closely approximate the in situ vascular condition. OxLDL is thought to be trapped within the vascular wall during initial vascular lesion development [35]. It must be recognized that vascular lesion development is a gradual proce ss, with clinically relevant effects accumulating over time. It is entirely appropriate, therefore, to examine the effects of oxLDL over extended periods of time instead of acutely as is done in most studies. Fourth, the MMPs examined (MMP-2 and MMP-9) have been shown to be important in remodeling the extracellular matrix under conditions of restenosis or atherosclerosis [36-38].

The present data may have further significance regarding ECM accumulation in vascular lesions . ECM deposition in the vascular wall is an important process involved in both restenosis and atherosclerosis [39]. It is increa singly evident that MMP activity is critical in remodeling collagen and other extracellular matrix elements [5, 6, 39, 40] . Administration of oxLDL has been shown to exacerbate neointimal formation and ECM deposition within the vessel [1]. Since rabbit VSMC have been reported to not produce MMP -l , which is necessary for the degradation of fibrillar collagens, it is unlikely that smooth muscle cells alone are involved in remodelling fibrillar collagens. In vivo, in the presence of infiltrating neutrophils which produce MMP-8 with the capacity to degrade fibrillar collagens, reduction in MMP-2 and MMP-9 as a consequence of oxLDL would be expected to reduce clearance of fibrillar collagen fragments by VSMCs. Basement membrane collagen (collagen IV) is a known substrate for MMP-2 and MMP-9 and it is conceivable that a reduction in MMP levels by oxLDL would result in an altered basement membrane composition. The reported accumulation of proteoglycan and glycosaminoglycan in vascular lesions [39] may in part be due to reduced ECM degradation as a consequence of oxLDL induced depression in MMP levels . Furthermore, infiltrating macrophages are known to stimulate MMP production [12, 40] and are involved in the uptake and removal ofLDL and oxLDL in vivo. The macrophage may therefore function to colonize the vasculature in an effort to remodel the ECM and minimize the effect of oxLDL on the vascular wall. This concept of macrophages assisting the vascular smooth muscle cells in remodelling the vascular wall is supported by the recent findings of Xu and colleagues who reported that oxLDL increases MMP-9 and inhibits TIMP-l [31]. Conversely, oxLDL also increases collagen deposition within the vessel [2]. This is thought to be achieved primarily by inducing collagen synthesis within fibroblasts and smooth muscle cells. However, our data demonstrate that an alternative mechanism may also contribute. During the genesis of a vascular lesion, oxLDL may inhibit the synthesis of MMPs within the vascular smooth muscle cell enabling the development of an ECM rich lesion .

Acknowledgements Operating grants from the Canadian Institutes of Health Research (to G.P. and P.Z.) supported this study. H. Massaeli was a Trainee of the Heart and Stroke Foundation of Canada and D. Wilson was a recipient of a University of Manitoba Graduate Studentship. G.N. Pierce is a Senior Scientist of the Medical Research Council of Canada. The technical support of Alejandro Austria is also gratefully acknowledged.

148

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Molecular and Cellular Biochemistry 249: 151-155,2003. © 2003 Kluwer Academic Publishers.

Low matrix metaUoproteinase levels precede vascular lesion formation in the JCR:LA-cp rat David Wilson.l-' Hamid Massaeli.P James C. Russell," Grant N. Pierce'-' and Peter Zahradka'" 'Institute of Cardiovascular Sciences; 2Division of Stroke and Vascular Disease, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba; 3Department of Physiology, University ofManitoba, Winnipeg, Manitoba; "Department of Surgery, University ofAlberta, Edmonton, Alberta, Canada

Abstract Clinically significant occlusive vascular lesions contain more extracellular matrix (ECM) proteins and lipid deposition than healthy vascular tissue. The events leading to this condition remain unresolved. One possibility is that ECM deposition may exceed ECM degradation which would contribute to the expansion of the vascular lesion. Utilizing lean (+/?) and insulin-resistant , corpulent (cp/cp) JCR:LA-cp rats, which are predisposed to develop vascular lesions, we have compared the matrix metalloproteinase (MMP) profile prior to the development of significant vascular lesions. Analysis of serum MMPs revealed that cp/cp rats have lower circulating levels than (+/?) controls. This is observed prior to the development of any noticeable atherosclerotic lesions . It also occurs as the hyperinsulinemia and insulin resistance is first developing in these rats. Female corpulent animals , which are less prone to develop vascular lesions, also exhibit a depressed serum MMP profile of a similar magnitude to their male counterparts. Primary vascular smooth muscle cells isolated from cp/cp animals also showed a reduction in secreted MMP compared with cells derived from +/? lean controls . We conclude that reduced MMP levels could lead to increased ECM accumulation and thus contribute to early vascular lesion formation. (Mol Cell Biochem 249: 151-155,2003) Key words: matrix metalloproteinase, smooth muscle, JCR :LA-cp rat, corpulent gene

Introduction Late atherosclerotic lesions typically contain a lipid rich core bounded by a fibrous cap. Infiltrating macrophages and activated smooth muscle cells have been implicated in lipid rich foam cell formation which contributes significantly to the mass of the late atherosclerotic lesion [1]. More recently, matrix metalloproteinases (MMPs) have also been localized to both macrophages and smooth muscle cells in late lesions [2]. MMP activity is involved in vascular smooth muscle cell (SMC) migration and intimal lesion formation [2-4] . In addition, MMPs have been implicated in remodeling of the extracellular matrix [5] leading to increased extracellular matrix (ECM) accumulation and perhaps reduced circumferential stress [6] or even plaque [7]. Presently, the majority of studies focusing on MMPs and vascular lesion development have

centred around established late lesions [8]. However, early vascular lesions from cholesterol fed rabbit aorta also contain increased MMP levels [8]. In addition, a number of studies have shown a correlation in the elevation of serum MMP levels with an increased incidence of aneurismal disease [9]. In the present study, we employed the JCR:LA corpulent rat model which is prone to develop severe cardiovascular disea se. Typically, animals homozygous for the corpulent gene (cp/cp) develop severe vascular lesions whereas lean animals (cp/+,+/+) do not. Corpulent females develop less severe vascular lesions than males [10]. The vascular lesions have been associated with the occurrence of spontaneous myocardial infarctions. The JCR:LA-cp animals are particularly valuable because of their metabolic profile. The rats are hyperlipidemic, hyperinsulinemic, glucose intolerant and insulin-resistant. Utilization of a genetic insulin-resistant rat

Address/or offprints: P. Zahradka or GN . Pierce, 51. Boniface General Hospital Research Centre , 35 1 Tache Avenue, Winnipeg , Manitoba, Canada (E-mail : [email protected];gpierce@ sbrc .ca)

152 model that is known to produce vascular lesions in all of the corpulent animals enables us to examine the changes in MMP profile prior to vascular lesion formation without the need for dietary interventions. Evidence is presented that suggests a reduction in MMP levels precedes vascular lesion formation in the JCR:LA-cp rat.

Materials and methods Culture ofJCR:LA-cp vascular smooth muscle cells JCR:LA-cp SMCs were obtained from aortic tissue explants from lean and corpulent rats and grown in 5% fetal bovine serum as described [11]. To induce differentiation, SMCs were placed (for 3 days) into serum-free supplemented media [11]. Over the treatment period, the medium was withdrawn and replaced with fresh medium every 48 h. Each experimental condition was replicated 6 times. Culture medium was removed and aliquoted at each 48 h interval and stored at -70°C. All animals were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care.

Serum collection Heparinized tubes were used to collect 2 mL of whole blood from corpulent and lean JCR :LA-cp rats at the time of sacrifice. Whole blood was centrifuged at 3,000 x g for 5 min at 4°C. Serum was collected and stored at -70°C for not more than 1 week .

Statistical analysis Controls loaded in each zymography gel were used to compensate for differences in staining intensity between the gels. Although decreased staining represents increased lytic activity, MMP activity is reported as positive values, which were achieved by a mathematical transformation involving subtraction from the background in each gel. Data were quantified and graphically represented as means ± S.E.M. Unpaired Student's r-test were used to compare treatment means versus control. The data satisfied the minimum assumptions required for parametric statistics, and the value of all power tests was above 83% . Differences were considered significant when p < 0.05.

Results Serum samples were pooled from male and female JCR:LAcp rats that were three months of age. MMP content was assessed by zymography. It was observed that corpulent animals exhibited reductions in serum MMP-2 and MMP-9 levels compared with age matched lean controls (Fig. 1). Zymography was chosen for its ability to detect both latent and active forms of the enzymes. Western blotting was used to identify the specific MMPs (but could not be used to distinguish between latent and active bands) and to confirm the direction of any changes in MMP amounts. The presence of multiple bands, however, made quantification by Western blotting difficult, and this fact was the basis for not employ-

Zymographic determination of matrix metalloproteinase (MMP) levels 20

Serum was diluted 1:10 with saline prior to dilution 1:1 in SDS sample buffer (0.5 M Tris-HCI pH 6.8, 20% SDS, 10% glycerol, 0.001 % bromophenol blue without reducing agent), while conditioned culture medium was diluted 1:I in SDS sample buffer, and 7 III was loaded per well of a 7.5% polyacrylamide gel containing 0.1 % gelatin [12]. One control sample was loaded per gel to enable normalization of staining between gels. Gels were washed in glycine -Triton buffer (0.025 M glycine pH 8.3, 2.5% Triton X-lOO) twice for 10 min each at 4°C to remove the SDS and permit partial renaturation of the protein . Gels were incubated in buffer (0.05 M Tris-HCI pH 8.0, 5 mM CaCI2 , 0.1 M PMSF) at 37°C for 12 h. Following Coomassie Blue R-250 staining, lytic activity representing MMPs was detectable as clear zones in an otherwise blue gel. Molecular mass markers and Western analysis were used to distinguish the location ofMMP-2 (72 kDa) and MMP-9 (92 kDa).

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153 ing ELISA (an antibody based assay) to monitor MMP activity. Serum samples were also obtained from one month old lean and corpulent JCR :LA-cp rats and analyzed by gelatin zyrnography. In this particular group of animals, we were unable to satisfactorily discriminate between latent and active MMPs and consequently we report the level of pooled MMP (latent and active form). Serum samples from corpulent JCR :LA-cp rats at one month of age exhibited a significant reduction (60%) in serum MMP-2 levels compared with their corresponding age matched lean controls (Fig. 2). Similarly, corpulent animals demon strated a reduction (p < 0.05) in serum MMP-9 levels compared with age matched lean controls . The sex of the animal may represent an important variable underlying the differences in MMP activity. Thus, we partitioned the lean and corpulent 3 month old rats into male and female groups . Both corpulent and lean males demonstrated a consistent trend toward lower serum MMP levels than age matched corpulent or lean females (Fig. 3). Corpulent females had significantly less serum MMP-2 than the lean female group (Fig. 3A). Similarly, serum MMP-2 levels were lower (- 60%) in the three month corpulent males compared with 3 month lean males . MMP-9 levels also exhibited a trend for males to have lower MMP levels than females (Fig. 3B). There was a significant reduction in MMP-9 in the corpulent female group compared with the lean female group. Similarly, corpulent males had significantly lower MMP-9 levels than lean males. It was unclear if the serum MMP data reflected a similar phenomenon in the vascular cells. Therefore, a vascular explant technique [11] was used to prepare a homogenous popu-

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Fig . 2. Comparison of serum matrix metalloproteinase (MMP) levels in 35

day old corpulent versus lean JCR :LA-cp rats. The relative amount of serum MMP-2 and MMP-9 was determined by gelatin zymography . Data are means ± S.E.M. from 5 lean and 5 corpulent animals (mixed sexes). *p < 0.05 vs. corresponding lean values .

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