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Roles of AMPK in the regulation of cardiac metabolism and cell death An, Ding 2006

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ROLES OF AMPK IN THE REGULATION OF CARDIAC METABOLISM AND CELL DEATH by DING AN B.Sc, Lanzhou University, China 1998 M.Sc, University of British Columbia, Canada 2002 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Pharmaceutical Sciences) THE UNIVERSITY OF BRITISH COLUMBIA June 2006 ©DingAn, 2006 ABSTRACT A s a metabolic switch, A M P activated protein kinase ( A M P K ) is known to regulate energy metabolism. Activation of cardiac A M P K has been observed during ischemia, hypoxia, or exercise. It is unclear whether the same alteration occurs following hypoinsulinemia. Using streptozotocin (STZ) induced Type 1 diabetes, we report for the first time that in the acute (4 days) S T Z diabetic heart, A M P K , but not PPAR-ct , is activated. This activation of A M P K likely increases F A oxidation through phosphorylation and inhibition of acetyl-CoA carboxylase ( A C C ) . In chronic diabetes, augmented plasma lipids and expression of CD36 provide the heart with excess F A . In this condition, PPAR-oc, through its regulation of gene expression, likely contributes to high F A oxidation, while A M P K is normalized. In addition to F A oxidation, A M P K is also known to increase F A uptake through activation of CD36. Given that F A released from lipoprotein is suggested to be the main F A source supply to the heart, and lipoprotein lipase (LPL) is the primary enzyme controlling lipoprotein metabolism, of interest to us was the question of whether A M P K also influences F A delivery through L P L . Using fasting and modulators of A M P K , a strong correlation between this metabolic switch and cardiac L P L activity was established. To test whether (3-adrenergic agonism could influence cardiac A M P K and heparin-releasable L P L , the (3-adrenergic agonist isoproterenol was applied in different models. We found that only during conditions of increased workload and excessive energy expenditure, cardiac A M P K is activated and is highly associated with coronary heparin-releasable L P L activity. Finally, beside its role in the regulation of metabolism, i i recent studies suggest that A M P K can also modulate cell death. Given that A M P K , through elevation of F A oxidation, reduces lipid accumulation, it is unclear whether this process can protect cardiomyocytes against high fat induced cell apoptosis. Our study demonstrates that low doses of metformin reduce high fat induced cardiac cell death, likely through its effects in decreasing ceramide formation and caspase-3 activity. However, through its role in increasing proton accumulation and lactic acidosis, metformin can induce cardiomyocyte cell damage in a caspase-3 independent manner. \ i i i TABLE OF CONTENTS ABSTRACT II TABLE OF CONTENTS IV LIST OF TABLES VIII LIST OF FIGURES IX LIST OF ABBREVIATIONS XI ACKNOWLEDGEMENTS XIII DEDICATION XIV 1. INTRODUCTION 1 1.1. C A R D I A C M E T A B O L I S M 1 1.1.1. Glucose uptake and oxidation 1 1.1.2. FA uptake and utilization 2 a) Lipoprotein lipase (LPL) 3 b) FA transporters 4 c) Acyl-CoA synthase (ACS) 4 d) Peroxisome proliferator activated receptors (PPARs) 5 e) AMP activated protein kinase (AMPK)..... 7 f) Malonyl-CoA Decarboxylase (MCD) 7 1.1.3. Interaction between glucose and FA metabolism 7 1.2. L P L 8 1.2.1. Synthesis and modification of LPL 9 1.2.2. Degradation and secretion of LPL 10 1.2.3. Translocation of LPL to the endothelium 11 1.2.4. Regulation of LPL 12 1.2.5. Physiological roles of LPL 13 1.3. A M P K 14 1.3.1 Structure of AMPK 15 1.3.2. Regulation of AMPK 16 1.3.3. Downstream targets of AMPK 18 1.3.4. Physiological roles of AMPK. 19 1.3.5. Roles of AMPK in the heart 19 1.4. R A T I O N A L E A N D OBJECTIVES 2 0 1.5. FIGURES 2 2 1.6. B I B L I O G R A P H Y - 2 6 iv 2. ACUTE AND CHRONIC STREPTOZOTOCIN DIABETES DIFFERENTIALLY REGULATES CARDIAC PPAR-oc AND AMPK 54 2.1 . INTRODUCTION 54 2.2. M E T H O D S 56 2.2.1. Experimental animals 56 2.2.2. Measurement of cardiac gene expression 56 2.2.3. Western blot analysis 57 2.2.4. Serum measurements 58 2.2.5. Separation and characterization of cardiac lipids 58 2.2.6. Statistical analysis 58 2.3. RESULTS 59 2.3.1. General characteristics 59 2.3.2. Gene expression ofPPAR-CCand its targets in acute and chronic diabetic hearts 59 2.3.3. Gene and protein expression of cardiac MCD following acute and chronic diabetes. 59 2.3.4. Changes in cardiac CD36 gene and protein following acute and chronic diabetes.... 60 2.3.5. Cardiac AMPK and ACC phosphorylation in acute and chronic diabetic hearts 60 2.4. DISCUSSION 61 2.5. T A B L E S A N D FIGURES 64 2.6. B I B L I O G R A P H Y 7 0 3. THE METABOLIC 'SWITCH' AMPK REGULATES CARDIAC HEPARIN-RELEASABLE LPL , 74 3.1. INTRODUCTION 74 3.2. M A T E R I A L S A N D M E T H O D S , 76 3.2.1. Experimental animals 76 3.2.2.. Isolated heart perfusion 76 3.2.3. Coronary lumen LPL activity 76 3.2.4. Western Blotting for AMPK 77 3.2.5. Measurement of cardiac LPL expression 78 3.2.6. Measurement of LPL protein and activity in cardiomyocytes 78 3.2.7. Immunolocalization of LPL 79 3.2.8. Treatments 80 3.2.9. Serum measurements 80 3.2.10. Statistical analysis 81 3.2.11. Materials 81 3.3. RESULTS : 82 3.3.1. Fasting influences cardiac AMPK phosphorylation 82 3.3.2. Augmentation of heparin-releasable LPL persists in vitro in fasted hearts 82 . 3.3.3. Inhibition ofAMPK phosphorylation lowers cardiac LPL 83 3.3.4. Promotion ofAMPK phosphorylation in control hearts recruits LPL to the luminal surface 84 v 3 A DISCUSSION 85 3.5. T A B L E S A N D FIGURES 9 0 3.6. B I B L I O G R A P H Y 98 4. p-ADRENERGIC AGONIST STIMULATION PRODUCES CHANGES IN CARDIAC AMPK AND CORONARY LUMEN LPL ONLY DURING INCREASED WORKLOAD 105 4 .1 . INTRODUCTION 105 4.2. M A T E R I A L S A N D M E T H O D S 108 4.2.1. Experimental animals 108 4.2.2. In vivo hemodynamics 108 4.2.3. Isolated working heart perfusion 108 4.2.4. Isolated Langendorff heart perfusion 109 4.2.5. Coronary lumen LPL activity 109 4.2.6. Isolated cardiomyocytes 110 4.2.7. Cardiac LPL gene expression I l l 4.2.8. Western Blotting for AMPK and ACC 112 4.2.9. Separation and characterization of cardiac lipids 112 4.2.10. Serum measurements 113 4.2.11. Statistical analysis 113 4.2.12. Materials 113 4.3. RESULTS 114 4.3.1. Coronary luminal LPL activity increases following a single dose of ISO given in vivo 114 4.3.2. ISO injected intraperitonally augments cardiac AMPK and ACC phosphorylation .114 4.3.3. Isoproterenol does not influence LPL activity and AMPK phosphorylation in myocytes or Langendorffperfused hearts 115 4.3.4. Increasing workload promotes phosphorylation ofAMPK and ACC and enlarges the coronary lumen LPL pool 116 4.3.5. A ugmentation of substrate supply reduces AMPK and A CC phosphorylation and HR-LPL activity in the isolated perfused working heart 116 4.4. DISCUSSION 118 4.5. T A B L E S A N D FIGURES 123 4.6. B I B L I O G R A P H Y 131 5. METFORMIN INFLUENCES CARDIOMYOCYTE C E L L DEATH BY BOTH CASPASE-3 DEPENDENT AND INDEPENDENT PATHWAYS 141 5.1. INTRODUCTION • 141 5.2. M A T E R I A L S A N D METHODS 143 5.2.1. Experimental animals 143 5.2.2. Cardiomyocyte isolation and culturing 143 5.2.3. LDHrelease • 144 vi 5.2.4. Hoechst 33342 staining 144 5.2.5. Estimation of reactive oxygen species (ROS) 144 5.2.6. Western blot analysis 145 5.2.7. Ceramide assay 145 5.2.8. Caspase 3 activity 146 5.2.9. Rates of glycolysis and palmitic acid oxidation 146 5.2.10. Lactate assay 147 5.2.11. Statistical analysis 147 5.2.12. Materials 147 5.3. RESULTS 149 5.3.1. High fat induced LDH release and consequence of metformin 149 5.3.2. High fat induced apoptosis and consequence of metformin 149 5.3.3. ROS generation following provision of high fat and metformin 149 5.3.4. Effects of metformin on AMPK phosphorylation in cardiomyocytes 150 5.3.5. ACCphosphorylation and FA oxidation in cardiomyocytes incubated with metformin 150 5.3.6. Effects of metformin on LCB1 expression and intracellular cer amide level 150 5.3.7. Effects of metformin on high fat induced Caspase 3 activity 151 5.3.8. Lactate production and pH changes after metformin 151 5.3.9. Effects of metformin on glycolysis 151 5.3.10. Effects of glucose removal on LDH release 152 5.4. DISCUSSION 153 5.5. FIGURES 158 5.6. B I B L I O G R A P H Y 167 CONCLUSIONS AND FUTURE DIRECTION 174 vii LIST OF TABLES Table 2-1 General characteristics of the experimental animals 64 Table 2-2 Cardiac lipids in control and S T Z groups 65 Table 3-1 General characteristics o f the animals 90 Table 4-1 General characteristics of the animals 123 Table 4-2 Cardiac lipids in control and isoproterenol treated groups 124 v i i i LIST OF FIGURES Fig. 1 -1 Glucose utilization in the cardiomyocytes 22 Fig. 1 -2 Control o f F A delivery and utilization in the cardiomyocyte 23 Fig. 1-3 Inhibition o f glucose oxidation by F A utilization 24 Fig. 1-4 L P L synthesis, secretion and transfer 25 Fig. 2-1 Gene expression of P P A R - a , CPT-1 and A C O in 4-day and 6-week S T Z diabetic hearts :...66 Fig. 2-2 Expression of M C D in 4-day and 6-week S T Z diabetic hearts 67 Fig. 2-3 Expression of CD36 in 4-day and 6-week S T Z diabetic hearts 68 Fig. 2-4 Cardiac A M P K and A C C phosphorylation in 4-day and 6-week S T Z diabetic hearts 69 Fig. 3-1 Effect o f fasting on cardiac A M P K phosphorylation 91 Fig. 3-2 Alterations in L P L activity and immunofluorescence in hearts isolated from fasted animals 92 Fig. 3-3 L P L gene expression, protein mass and activity in hearts isolated from fasted animals 93 Fig. 3-4 Consequence of A M P K inhibition on heparin-releasable L P L activity in fasted hearts 94 Fig. 3-5 Consequence o f CPT-1 inhibition on A M P K phosphorylation and heparin-releasable L P L activity in control hearts 95 Fig. 3-6 Effect of inhibiting A T P synthesis on A M P K phosphorylation and heparin-releasable L P L activity 96 Fig. 3-7 Regulation of cardiac L P L by A M P K . . . . . 97 Fig. 4-1 Effects of ISO on coronary luminal L P L activity and gene expression after a single in vivo injection 125 Fig. 4-2 A M P K and A C C 2 8 0 phosphorylation in hearts isolated from animals injected with ISO for 1 o r 4 h r s 126 Fig. 4-3 Heparin releasable L P L activity and A M P K phosphorylation i n Langendorff hearts perfused with ISO 127 Fig. 4-4 Comparison o f heparin releasable L P L activity, A M P K and ACC280 phosphorylation in perfused Langendorff or working hearts 128 Fig. 4-5 Consequence of additional substrate provision in regulating A M P K and ACC280 phosphorylation in isolated working hearts in the absence or presence of ISO ..129 Fig. 4-6 Consequence of additional substrate provision in regulating heparin-releasable L P L in isolated working hearts, in the absence or presence of ISO 130 Fig. 5-1 L D H release and cell apoptosis following incubation with high fat and metformin 158 Fig. 5-2 R O S levels following incubation with high fat and metformin 159 Fig . 5-3 Time dependent phosphorylation of A M P K by metformin 160 ix Fig . 5-4 Time dependent phosphorylation o f A C C by metformin and P A oxidation 161 Fig . 5-5 Apoptotic mediators in isolated cardiomyocytes 162 Fig. 5-6 Lactate release in the cardiomyocyte incubation medium 163 Fig . 5-7 Rate o f cardiomyocyte glycolysis following metformin incubation 164 Fig . 5-8 L D H release in the absence of glucose following high fat and metformin 165 Fig . 5-9 Proposed mechanism o f how metformin influences cardiomyocyte cell death... 166 x LIST OF ABBREVIATIONS A C C Acetyl coenzyme A carboxylase A C O Acetyl coenzyme oxidase A C S Acetyl coenzyme synthase A D P Adenosine diphosphate A I C A R 5-Aminoimidazole-4-carboxamide ribonucleoside A M P Adenosine monophosphate A M P K A M P activated protein kinase A N O V A Analysis of variance Apo Apolipoprotein A r a - A Adenine 9-beta-D-arabinofuranoside B S A Bovine serum albumin C B S Cystothionine P synthase CPT-1 Carnitine palmitoyl transferase-1 D A G Diaglycerol D N A Deoxyribonucleic acid E D T A Ethylenediamine tetraacetic acid E R Endoplasmic reticulum F A Fatty acid F A B P p m Fatty acid binding protein plasma membrane F A S Fatty acid synthase FATP Fatty acid transport protein F2,6BP Fructose 2, 6-bisphosphate g Gram G B D Glycogen binding domain G L U T Glucose transporter H E P E S 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid hr Hour H P L C High performance liquid chromatography H R - L P L Heparin releasable-lipoprotein lipase H S P G Heparan sulphate proteoglycan IgG Immunoglobulin ISO Isoproterenol i.p. Intraperitoneal i.v. Intravenous kg Kilogram K H buffer Krebs-Henseleit buffer L C A D Long chain acyl-CoA dehydrogenase L C B Long chain base xi L D H Lactate dehydrogenase L P L Lipoprotein lipase M Molar M C D Malonyl coenzyme A decarboxylase mg Mi l l ig ram min Minute ml Mil l i l i ter mm Millimeter m M Mil l imolar m R N A Messenger ribonucleic acid m T O R Mammalian target of rapamycin N A D + / N A D H Nicotinamide adenine dinucleotide n M Nanomolar P B S Phosphate buffered saline P D H Pyruvate dehydrogenase P D K Pyruvate dehydrogenase kinase P F K Phosphofructokinase P i Phosphate PGC-1 PPAR-y Coactivator-1 P P A R Peroxisome proliferator activated receptor R N A Ribonucleic acid RPP Rate-pressure product RT-PCR Reverse transcription polymerase chain reaction SDS Sodium dodecyl sulphate S E M Standard error of means SPT Serine palmitoyl- transferase S R E B P Sterol regulatory element-binding protein S T Z Streptozotocin T B S - T Tris buffered saline-Tween T G Triglyceride V L C A D Very long chain acy l -CoA dehydrogenase V L D L Very low density lipoprotein ul Microliter u M Micromolar x i i ACKNOWLEDGEMENTS First, I would like to thank my doctoral supervisor, Dr. Brian Rodrigues for his continual support and guidance throughout this project. H i s trust, encouragement and instruction have allowed me to complete this challenging work. It is a great honor and pleasure to work with him. I also appreciate the members of my supervisory committee: Dr. Christ Mcintosh, Dr. Norbert Haunerland, Dr. Thomas Chang and Dr. Kishor Wasan for their valuable suggestions and encouragement during my doctoral training. I would also like to thank our collaborators, Dr. Roger Brownsey, Dr. Michael Al lard and Dr. Sheila Innis for their instructions and technical supports. I would further like to acknowledge my colleagues and friends, Ashraf, Dake, Sanjoy, Thomas, Gir ish and Jennifer for their friendship and supports. Finally, I would like to take this opportunity to thank my parents, brother, Uncle Y u and his family and Aunt Y ing and her family for their love, supports and understanding. x i i i DEDICATION l b my famity, whose Cove andnunurirtQ made this adpossi6Ce xiv 1. Introduction 1.1. Card iac Metabol i sm 1.1.1. Glucose uptake and oxidation Under normal physiological conditions, glucose is the major carbohydrate utilized by the heart. Glucose metabolism is regulated through multiple steps, including uptake, glycolysis, and pyruvate decarboxylation (Fig. 1-1). Cardiac glucose uptake is dependent on the transmembrane glucose gradient, and the content of sarcolemmal glucose transporters ( G L U T 1 and G L U T 4 ) (95, 130). G L U T 1 mediated glucose uptake is insulin independent, and represents basal cardiac uptake. Compared to G L U T 1 , G L U T 4 is the dominant transporter, and its translocation from an intracellular compartment to the sarcolemmal membrane requires insulin. Other than recruiting G L U T to the sarcolemmal membrane, insulin also influences glucose transport through its regulation o f G L U T gene expression. It should be noted that G L U T mediated glucose uptake could also be stimulated through insulin independent mechanisms. Thus, recent studies have demonstrated that A M P activated protein kinase ( A M P K ) also promotes G L U T 4 redistribution to the sarcolemmal membrane (101, 187). Once inside the cardiomyocyte, glucose is broken down through glycolysis, a sequence of reactions that convert glucose into pyruvate. P F K 1 , the enzyme that catalyzes the generation of fructose 1,6-bisphosphate from fructose 6-phosphate, is a rate-limiting enzyme controlling glycolysis (43, 137). PFK-1 is inhibited by low p H , high intracellular 1 citrate or ATP, and is activated by A D P , A M P , phosphate (pi) and fructose 2, 6-bisphosphate (F2,6BP) (43, 137, 162). F2,6BP is formed from fructose 6-phosphate catalyzed by P F K - 2 (81). Given that P F K - 2 is phosphorylated and activated by insulin (18, 44), glucagon (162) or A M P K (80, 112), stimulation of P F K - 2 through these mechanisms increases F2 ,6BP generation, activates PFK-1 and subsequently promotes glycolysis. In an aerobic heart, glycolysis contributes less than 10% of total A T P generated, with no oxygen consumption. Following glycolysis, pyruvate generated has three destinations: carboxylation to oxaloacetate or malate, reduction to lactate, and most importantly, decarboxylation to acetyl-CoA. To be oxidized, pyruvate is transported into mitochondria and decarboxylated to acetyl-CoA through pyruvate dehydrogenase (PDH), a multienzyme complex. P D H is phosphorylated and inactivated by pyruvate dehydrogenase kinase (PDK) (78, 182), which is inhibited by pyruvate and stimulated by high mitochondrial ace ty l -CoA/CoA and N A D H / N A D ( + ) ratios (23, 76). Ace ty l -CoA then enters the citric acid cycle and is eventually broken down to H2O and CO2 for A T P generation. Oxidation of one glucose provides 30 ATP, with 6 oxygen consumed (2.58 ATP/oxygen atom). 1.1.2. FA uptake and utilization Compared to glucose, F A is the preferred substrate and accounts for approximately 70% of A T P generated in an aerobic heart. F A metabolism includes multiple steps, and can be regulated by both acute and chronic mechanisms, with or without modulation of gene expression (Fig. 1-2). 2 a) Lipoprotein lipase (LPL). A s the heart has a limited capacity to synthesize and store F A , it relies on continuous exogenous supply. F A supplied to the heart from the circulation is from two sources, albumin-bound F A and TG-r ich lipoproteins. It should be noted that the molar concentration of F A in lipoprotein-TG is -10 fold larger than F A bound to albumin (114). L P L is the key enzyme, which hydrolyzes lipoproteins to release F A . Thus, when lipoproteins are hydrolyzed by L P L , a large amount of F A are released, and are believed to be the principle source o f F A supplied to the heart (6, 167). L P L is produced in cardiomyocytes and subsequently secreted onto heparan sulphate proteoglycan (HSPG) binding sites on the myocyte cell surface (26, 48, 51). From here, L P L is transported onto comparable binding sites on the luminal surface of coronary endothelial cells (131). A t these sites, L P L hydrolyzes TG-r ich lipoproteins, such as very low density lipoprotein ( V L D L ) and chylomicrons, to release F A . Perfusion of working hearts with V L D L or chylomicrons, in the absence or presence of F A , has revealed that compared to chylomicrons, V L D L is a poor substrate for L P L . In addition, utilization of chylomicrons was inhibited by free F A , which failed to affect cardiac V L D L utilization. Given the important role that L P L plays in regulating F A delivery, alteration in its level is able to change F A delivery, and subsequent oxidation. Indeed, overexpression o f L P L in the heart or skeletal muscle accelerates F A uptake (100, 184). Conversely, tissue specific knock-out o f L P L in the heart switches the cardiac substrate selection preference to glucose (4, 5). Other than hydrolysis by L P L , lipoproteins can also be transported into the cardiac tissue with help of the lipoprotein receptor, with V L D L uptake through this mechanism being 3 more substantial compared to chylomicrons. In perfused working hearts, inhibition of the lipoprotein receptor significantly reduced F A oxidation, without altering F A uptake, suggesting that lipoprotein receptor mediated F A uptake channels F A towards oxidation, while L P L mediated F A uptake channels F A towards both oxidation and T G storage. b) FA transporters. Although F A can translocate into the cell through passive diffusion across the plasma membrane, F A uptake shows saturation kinetics and is inhibited by protease, which digests F A transporters (3, 110, 111, 164). Thus, F A transporters are also likely required to support this process. In the heart, three F A transporters have been identified, and these include CD36 , F A transport protein (FATP), and F A binding protein plasma membrane ( F A B P P M ) (109). Given that 55-80% of F A transport was blocked using general transporter inhibitors (93, 110), and that overexpression of FATP or CD36 has been found to dramatically increase F A metabolism (34, 83), these F A transporters are believed to play a key role in F A delivery to the cardiac tissue. Regulation of F A transport proteins occurs through different mechanisms. In severe S T Z induced diabetes, expression of cardiac CD36 and F A B P p m were augmented (106), suggesting transcriptional control o f this transporter. A t present, the mechanisms that induce this change have yet to be elucidated. Additionally, as muscle contraction or acute insulin treatment does not change protein, but simply relocates C D 3 6 from an intracellular pool to the sarcolernrnal membrane (107, 108), post-translational regulation of this transporter has also been suggested. c) Acyl-CoA synthase (ACS). A C S catalyzes the esterification of F A to fatty 4 acyl-CoA, the initial step of F A metabolism. Fatty acyl -CoA can be transported into the mitochondria for oxidation, or used for intracellular T G synthesis. The fate of fatty acyl -CoA is influenced by the location of different A C S isoforms, energy demand, and the availability of F A (27). Moreover, A C S not only functions as an enzyme catalyzing esterification, but is also actively involved in controlling F A homeostasis (35). Recent studies have demonstrated that A C S is associated with CD36 or FATP on the cytosolic side of the sarcolemmal membrane (62, 136, 150), suggesting that A C S also influences F A uptake. Indeed, overexpression of A C S in the heart or fibroblast causes dramatically augmented F A uptake and intracellular T G accumulation (35). Under normal conditions, 70-90% of the esterified fatty acid that enters cardiomyocytes is oxidized for A T P generation, while 10-30% is converted to T G . The T G pool is not static, with lipolysis and lipogenesis taking place continuously. In a normal heart, intracellular T G level is constant, indicating a balance of lipogenesis and lipolysis. In situations where F A supply supercedes the cellular oxidative capacity, such as obesity or diabetes, intracellular T G accumulates, and is associated with lipotoxicity. Although T G is unlikely to be a direct mediator of cell apoptosis, its augmented lipolysis expands fatty acyl -CoA levels, which may be a key factor mediating cell apoptosis. Thus, T G is often used as a marker o f lipotoxicity. d) Peroxisome proliferator activated receptors (PPARs). P P A R s are a group of ligand-activated transcriptional factors, belonging to the superfamily o f nuclear receptors. They are activated by either natural ligands like F A , or numerous pharmacological ligands (59). Once activated, PPARs form complexes with retinoid X receptors and bind to the promoter regions of a number of target genes, which encode the proteins involved in controlling F A metabolism (55, 57). Through regulation of expression of these genes, PPARs modulate F A utilization at the transcriptional level. P P A R s have three isoforms: P P A R - a , P P A R - P (or 5), and PPAR-y. P P A R - a is extensively expressed in tissues with high F A metabolism, like the heart (11). Activated by elevated intracellular F A levels, P P A R - a promotes expression o f genes that regulate F A oxidation at various steps, such as F A uptake and binding ( L P L , C D 3 6 , and F A binding protein), F A esterification ( A C S ) , and F A oxidation (CPT-1, A C O , L C A D and V L C A D ) (55, 57, 82). Knocking-out cardiac P P A R - a abolishes fasting induced overexpression of F A metabolic genes, and switches substrate selection from F A to glucose (99, 121). Overexpression of cardiac P P A R - a augments F A uptake and oxidation (56, 58). Taken together, P P A R - a is believed to be the primary regulator of F A metabolism in the heart. Similar to P P A R - a , P P A R - P (or 8) is expressed abundantly in the cardiac tissue (11). Activated by elevated intracellular F A (30), P P A R - P (or 8) augments expression of a group of genes that promote F A utilization (45, 119). Cardiac specific knock-out of P P A R - P also decreases F A oxidative gene expression, and F A oxidation (32). Although the targets of P P A R - a and P P A R - P are partially overlapping (119), their unique roles and interaction remains unclear in the heart. PPAR-y, the third number of the P P A R family, is highly expressed in adipose tissue. Through promoting lipogenic gene expression, PPAR-y controls lipogenesis. Loss and gain of function experiments have demonstrated that PPAR -y is necessary for adipose tissue 6 proliferation and differentiation (10, 169). In isolated cardiomyocytes, the expression of PPAR-y is barely detectable (64), suggesting a limited role for this nuclear receptor in regulating cardiac metabolism. e) AMP activated protein kinase (AMPK). A s an energy sensor, A M P K is activated following a rise in the intracellular A M P / A T P ratio (70). Recent studies have demonstrated that A M P K regulates cardiac metabolism. Details are included in section 1.3.5. f) Malonyl-CoA Decarboxylase (MCD). In addition to A M P K , M C D is also known to promote F A oxidation through its lowering o f malonyl-CoA. M C D catalyses the degradation of malonyl-CoA to acetyl-CoA, leading to reduction of malonyl-CoA (46). This action relieves the inhibition of CPT-1 by malonyl-CoA, and favors F A oxidation. Recent studies have suggested that inhibition of cardiac M C D leads to accumulation of malonyl-CoA and reduced F A oxidation (47). I.1.3. Interaction between glucose and FA metabolism Regulation of glucose and F A metabolism does not occur independently, and numerous studies have reported a 'cross-talk' between the utilization of these substrates (Fig. 1-3) (135, 145, 165). Randle and his co-workers demonstrated that F A impairs basal and insulin stimulated glucose uptake and oxidation, an event that is known as the 'Randle cycle' (135). F A influences glucose utilization at multiple levels. Accumulation of F A impairs insulin mediated glucose uptake through inhibition of insulin receptor substrate (IRS) and protein kinase B (66, 85, 127). Accumulation of F A leads to augmented 7 intracellular FA derivatives, such as fatty acyl CoA, diacylglycerol and ceramide. These FA metabolites activate a serine kinase cascade, which involves protein kinase C-9 and IicB-p, leading to serine phosphorylation of IRS (isoform 1 is main isoform in the heart) (89, 189). In this regard, serine phosphorylation of IRS-1 reduces tyrosine phosphorylation and interferes with its action to phosphorylate and activate PI 3-kinase and protein kinase B (143). Increased intracellular FA also activates PPAR-a. As a consequence, PPAR-a promotes the expression of genes involved in FA oxidation, as well as PDK4, which is known to inhibit PDH and pyruvate flux (181). Moreover, increased acetyl-CoA/free CoA, and NADH/NAD + ratios caused by the high rate of FA oxidation are also known to activate PDK4, leading to inactivation of PDH (23, 76). Augmented acetyl-CoA/free CoA also causes accumulation of citrate in the cytosol, which subsequently inhibits PFK and glycolysis (63, 122). Conversely, inhibition of FA oxidation through elevation of malonyl-CoA levels, or using pharmacological inhibitors, favors glucose oxidation. 1.2. L P L LPL is the primary enzyme that hydrolyzes circulating lipoproteins, such as chylomicrons and very low density lipoproteins (VLDL), to provide FA to tissues (13, 48). It is widely expressed in various tissues, including white and brown adipose tissue, heart, skeletal muscle, mammary gland, adrenal, spleen, small intestine, lung, kidney and brain (24). In the heart, LPL mRNA is only detected in cardiomyocytes, rather than endothelial cells (26). This finding indicates that in the heart, LPL is expressed, synthesized and modified in 8 cardiomyocytes before it translocates to the luminal surface of vascular endothelial cells, where L P L functions to hydrolyze lipoprotein T G (Fig. 1-4). In an attempt to localize L P L protein in the heart, cardiac L P L was labeled with immunogold bound antibody and visualized by electron microscopy (20). This study reported that 78% o f total cardiac L P L is in cardiomyocytes, while 3-6% and 18% are located at capillary endothelium and extracellular space respectively. Within the cardiomyocyte, L P L is localized in the sarcoplasmic reticulum, Golgi sacs and transport vesicles (19, 20). 1.2.1. Synthesis and modification of LPL The human L P L is encoded by a single gene located at chromosome 8p22 (120, 161). Given that expression of L P L shows tissue specificity (90), different mechanisms may control transcription o f L P L in various tissues. Indeed, four transcription initiation sites, two promoter elements and several enhancer motifs have been identified in the 5' upstream region of the L P L gene (42). The mechanisms of regulation of these sites are still not fully understood. Fol lowing transcription, inactive and monomeric proenzyme o f L P L is synthesized in endoplasmic reticulum (ER). This inactive monomer requires glycosylation and a sequence of post-translational processing to form an active homodimer L P L (7, 16, 132). During glycosylation, a lipid-linked oligosaccharide, which is rich in mannose, is added to the arginine residues of the nascent L P L polypeptide (24, 50). This mannose rich oligosaccharide allows formed glycoprotein to be retained in E R for further modifications (172). Thus, three terminal glucose residues are cut off by glucosidase I and II in the E R (24). Subsequently, one mannose is removed by an a-mannosidase, 9 before it translocates to the Golg i complex (24). Once inside this compartment, three more mannoses on this L P L glycoprotein are further removed by mannosidase I (24). A s L P L moves in the Golg i complex, a G l c N A c is added onto glycoprotein, while two additional mannose residues are removed by mannosidase II (24). Subsequently, this glycoprotein of L P L is further modified by transferase, and then packaged into vesicles for secretion. 1.2.2. Degradation and secretion of LPL Following processing in the Golgi complex, active L P L molecules are sorted for two destinations: delivery to lysosomes for degradation or packaged into secretory vesicles for secretion (24, 172). In adipocytes, the majority of newly synthesized L P L is degraded. Thus, in pulse-chase experiments, 80% of newly synthesized L P L in adipocytes is degraded while only a small fraction is secreted into the culture media (39, 172). In a different study, when protein synthesis was inhibited by cycloheximide, a rapid reduction o f intracellular L P L was observed (ti/2 is about 40 min), suggesting a rapid turnover o f L P L (153, 173). Given that the degradation is reduced by leupeptin, the lysosome is suggested to be a site for degradation (172). Interestingly, a recent study demonstrated that misfolded L P L can also be degraded in E R (15). For secretion, L P L is assembled and packaged into secretory vesicles and their emission from cells shows two mechanisms: constitutive and regulated (9, 139, 140). Constitutive secretion represents a basal release of L P L , when there is not any stimulation to cells. During this process, a low or moderate rate of L P L release has been reported from adipocytes or cardiomyocytes (36, 124, 153, 10 154). Constitutive L P L secretion exists in all cell types, while some types of cells also can release L P L through regulated mechanisms. Following a stimulus, a large number of L P L can be secreted. For example, although heparin does not change L P L synthesis rate, numerous studies have reported that it promotes L P L secretion (13). Associated with augmented secretion, there is reduced L P L degradation in the cells, suggesting that heparin diverts L P L away from degradation (39, 153, 172). 1.2.3. Translocation of LPL to the endothelium Binding on the H S P G o f the external cell surface is not the final destination of L P L . Eventually, L P L translocates to its functional site at the luminal surface of blood vessels (51, 126). Thus, perfusion o f isolated hearts with heparin causes release of L P L in two phases: a rapid and large amount of L P L release within seconds after heparin administration, representing the L P L bound to the luminal side of blood vessels, and a sustained release indicating a constant mobilization of L P L from cardiomyocytes (102-104, 138). The mechanisms for this translocation are still unclear. In co-culture experiments using adipocytes and endothelial cells, secretion of heparanase like compounds from endothelial cells was observed, and was suggested to cleave H S P G on adipocytes, leading to release of oligosaccharide bound L P L (131). Subsequently, L P L transfers from the abluminal to the luminal side of endothelial cells. This transcytosis involves both H S P G and very low density lipoprotein receptor, given that inhibition of these two proteins decreases, while overexpression increases L P L transfer across endothelial cells (86, 123, 149, 185). 11 1.2.4. Regulation of LPL Regulation of L P L is complicated, and involves transcriptional and post-transcriptional mechanisms, and shows tissue-specificity. Post-transcriptional regulation includes modification of L P L protein (14, 15), intracellular L P L degradation and secretion (172), L P L transfer to luminal side of blood vessels (133), and L P L recycling (31, 65). Many of these mechanisms have yet to be completely elucidated. Control of L P L at the transcriptional level has been reported under several physiological conditions. For example, L P L is present in the liver, only in fetal or early postnatal life, with gene expression being turned off with age (128). In macrophages, gene expression of L P L is switched on once the cells are activated (8). Moreover, cold exposure augments L P L expression in brown adipose tissue (29). Several nuclear factors, such as hepatic nuclear factor 3-like proteins, PPAR-cc, P P A R - y and 9-cis retinoic acid receptor, have been implicated in the regulation of L P L gene expression (52, 117, 147, 151). In the heart and adipose tissue, changes in L P L activity can also occur through post-transcriptional mechanisms. During fasting, L P L activity is reduced in adipose tissue without corresponding changes in the levels of m R N A and protein, while re-feeding restores suppressed activity (17). A recent study suggests that fasting activates a gene in adipose tissue whose product prevents L P L from becoming active (17). Conversely, fasting increases cardiac H R - L P L at the coronary lumen by mechanisms that have yet to be determined (98, 142). In Type 1 diabetes induced by 55 mg/kg S T Z , cardiac H R - L P L is 12 also increased without change in total cardiac L P L , suggesting a redistribution o f L P L from cardiomyocytes, interstitial space or within endothelial cells to the coronary lumen (138). Another mechanism o f post-transcriptional regulation is through L P L intracellular degradation. For example, incubation of adipocytes with heparin diverts L P L from degradation to secretion pathways (172). L P L is also regulated through recycling of endothelial bound L P L (148). L P L - H S P G complex at the endothelial lumen can be internalized into endothelial cells, followed by release back to the luminal surface, a process that can be promoted by drop of intracellular p H (148). Finally, apolipoproteins in T G rich lipoprotein and those associated with H D L also influence L P L activity at the lumen. Binding of lipoproteins containing apo CII to L P L enhances lipolysis (125), while binding of lipoproteins containing apo CIII or apo E suppresses L P L activity (1, 2). Interestingly, a recent study has shown that overexpression of HDL-associated apo A V accelerates hydrolysis of T G rich lipoproteins, indicating apo A V is another target that stimulates L P L (115, 129). 1.2.5. Physiological roles of LPL Given that L P L is the primary enzyme in lipoprotein metabolism, it has been implicated in several pathophysiological conditions, such as dyslipidemia, insulin resistance, atherosclerosis, diabetes and obesity (113). In human and animals with insulin resistance, Type 1 or Type 2 diabetes, decreased L P L was observed in adipose tissue (49, 134, 160), which may contribute to hyperlipidemia. Moreover, knock out of whole body L P L or inhibition of L P L by overexpression of apo CIII leads to hypertriglyceridemia (1, 178). 13 Conversely, overexpression of L P L prevents diet or diabetes induced hypertriglyceridemia (92, 157, 159), Taken together, these experiments support a key role for L P L in prevention of hypertriglyceridemia. Additionally, L P L also protects against development of atherosclerosis. Thus, global overexpression of L P L prevents diet-induced atherosclerosis (53, 158). Comparatively, the role of L P L in insulin resistance is more controversial. Global overexpression of L P L improves high fat induced insulin resistance (91). Given that whole body overexpression of L P L increase adipose tissue L P L levels, it may divert circulating lipids to adipose tissue, thereby improving insulin resistance. Interestingly, skeletal muscle specific overexpression o f L P L induces insulin resistance and severe myopathy (54, 175). In the heart, L P L has been demonstrated to play an important role in regulating metabolism. Cardiac specific knock out of L P L switched substrate selection from F A to glucose (4), and chronically provokes cardiac dysfunction (5). Overexpression of cardiomyocyte surface-bound L P L in the heart also leads to l ip id overload and lipotoxic cardiomyopathy, similar to diabetic cardiomyopathy (184). Thus, a balance of cardiac L P L is essential to maintain the normal heart metabolism and function. 1.3. AMPK Through catabolism, A T P is generated by converting A D P to ATP. To drive energy supply, A T P is hydrolyzed to A D P . A s a constant A T P generation and supply is fundamental for a cell to maintain its function, a balance of A T P / A D P turn-over is required (10:1 under normal aerobic conditions) (69). This balance is exquisitely controlled by the cell through 14 several mechanisms. Recent studies have suggested that A M P K plays a key role in maintaining this balance. 1.3.1 Structure of AMPK Mammalian A M P K is a heterotrimer with a, p and X subunits (28, 72). Each of these subunits has multiple isoforms. U p to now, two a isoforms ( a l and a2), two P isoforms (p i and p2) and three X isoforms (XI, X2 and A3) have been identified. Therefore, a total of 12 different combinations of heterotrimer exist, and likely show tissue : specificity. In the heart, a2 and p2, rather than a l and p i , is highly expressed (146). XI and X2 are both expressed the heart, while X3 is only found in skeletal muscle (33). The C - terminus of the a subunit contains the binding sites for P and X subunits, which forms a complex with the other two subunits (38). The N-terminus contains a serine/threonine protein kinase catalytic domain (67). The a subunit also contains several residues that can be phosphorylated, such as Thr l72 , Thr258 and Ser485 (180). Thr l72 is located in the activation loop of the catalytic domain, and its phosphorylation leads to the activation of A M P K . The p subunit contains two conservative regions: KIS and A S C (87). A recent study has indicated that the A S C domain functions as an anchor to connect a and X subunits, whereas K I S domain is a glycogen binding domain ( G B D ) (79). The exact function of G B D is still unclear. Interestingly, A M P K is localized with glycogen, and removal of G B D abolished this co-localization (79). Thus, one hypothesis is that G B D is able to regulate A M P K activity by binding with glycogen. The X subunit has four tandem repeats of cystothionine P synthase (CBS) domains (71). Two C B S form a dimer as a 15 functional unit. Recent studies have revealed that C B S dimers contain a binding site for A M P and ATP, and binding with A M P is essential for the activation o f A M P K (152). 1.3.2. Regulation of AMPK When A M P K is inactive, its regulatory domain on the a unit inhibits the catalytic domain (71). Binding of A M P activates A M P K through three mechanisms. First, A M P allosterically activates A M P K by binding to the X subunit (146). This mechanism only mildly activates A M P K . Moreover, binding with A M P also makes A M P K a better substrate to its upstream kinase, which was recently identified as L K B 1 (73, 155, 179). Following A M P binding, the Thr l72 site of a subunit is phosphorylated by L K B 1 , leading to 50-100 times increase in A M P K activity (75). Indeed, Thr l72 phosphorylation is essential for A M P K activity. Using an antibody that specifically recognizes A M P K with Thr l72 phosphorylated, studies have shown that phosphorylation of Th r l72 mirrors A M P K activity (28). Mutation of this site abolishes activation of A M P K (38, 163). Besides Thr l72 , several other sites in the a subunit are also phosphorylated by L K B 1 (180). However, phosphorylation of these sites does not change the activity of A M P K (180). The roles of phosphorylation in these sites remain unknown. The third mechanism by which A M P activates A M P K is through an indirect process. Binding with A M P also makes A M P K a worse substrate for protein phosphatases, which are known to dephosphorylate Thr l72 and inactivate A M P K (41). Compared to A M P , ATP competitively inhibits A M P K . A high concentration of A T P has been shown to antagonize the A M P activation o f A M P K (74). A s A M P activates A M P K while A T P antagonizes this 16 activation, a rise of A M P / A T P ratio is a more accurate indicator of A M P K activation. Additionally, A M P K can also be regulated through A M P / A T P independent mechanisms. Both leptin and adiponectin, hormones secreted from adipose tissue, activate A M P K in the skeletal muscle and liver, through A T P / A M P independent mechanisms (118, 168, 186). Moreover, insulin antagonizes A M P K activation in the heart during ischemia through an ATP-independent mechanism (12, 61). Given that overexpression o f active A k t in cardiomyocyte suppressed A M P K activity, it is likely that activation of A k t mediates the effect of insulin (94). A M P K is activated during various physiological or pathophysiological conditions. During cellular stresses, such as glucose deprivation, ischemia, hypoxia and oxidative stress, A T P generation is compromised, leading to a rise in A M P / A T P ratio and activation of A M P K (88). Moreover, during physiological conditions, such as exercise and muscle contraction, increased A T P consumption, rather than impaired A T P generation, changes A M P / A T P ratio and stimulates A M P K (88). Thus, following exercise, A M P K activation has been observed in heart, skeletal muscle and liver (37, 141, 174). Additionally, recent studies have demonstrated that anti-diabetic drugs also promote A M P K activity. Metformin, a drug commonly used in the management of Type 2 diabetes, activates A M P K through an unknown mechanism (60, 190). Interestingly, a recent study has demonstrated that L K B 1 knockout in liver abolished the effects of metformin to activate A M P K and lower blood glucose, suggesting L K B 1 is required by metformin for A M P K activation (156). Thiazolidinediones, PPAR-y agonists, also activate A M P K , likely though 17 inhibition of complex 1 in the mitochondrial respiratory chain, and changes in A M P / A T P ratio (25, 60). 1.3.3. Downstream targets of AMPK A M P K acts as a cellular "fuel gauge". Through regulating a number of downstream targets, A M P K maintains cellular energy status by two main events: increasing A T P production and decreasing A T P consuming processes. Thus, upon activation, A M P K rapidly modulates a number o f downstream targets, such as A C C , G L U T 4 and P F K 2 , to promote F A oxidation, glucose uptake and glycolysis (69, 88). Through regulation of targets like A C C 1 and F A S , A M P K reduces energy consumption (69). Besides these acute actions, through regulation of gene and protein expression, A M P K also has long-term effects to modulate energy metabolism. For example, through up-regulating P G C 1 , A M P K promotes expression of numerous genes involved in mitochondria biogenesis (166). Moreover, through down-regulating S R E B P expression, A M P K decreases expression of genes involved in lipogenesis (190). A M P K also influences protein synthesis, which accounts for 20% of energy turnover in growing cells. This effect is mediated through three different mechanisms. First, through phosphorylation and activation o f elongation factor 2, A M P K inhibits protein synthesis (77). Another mechanism of A M P K modulating protein synthesis is through inhibition of target of rapamycin (TOR) pathway, which is a main regulator of protein synthesis (22). Finally, A M P K also affects m R N A stability. Through reducing RNA-binding protein HuR, which is known to stabilize specific m R N A s , A M P K reduces m R N A levels, leading to a decrease in protein translation 18 (176, 177). 1.3.4. Physiological roles of AMPK At the cellular level, AMPK plays an essential role in maintaining intracellular energy balance. Recent studies demonstrated that AMPK also regulates whole body metabolism through its effects on different tissues. In hypothalamus, fasting activates AMPK, leading to augmentation of food intake (118). Inhibition of AMPK in hypothalamus by high levels of glucose, leptin or insulin suppress food intake and weight gain (118). Besides controlling food intake, AMPK also regulates energy expenditure in peripheral tissues. In skeletal muscle and liver, activation of AMPK increases glucose disposal and FA oxidation (116). In the liver, AMPK also inhibits gluconeogenesis (105). In pancreatic P cells, activation of AMPK reduces insulin secretion (40, 170). Taken together, AMPK, through its regulation of food intake and energy expenditure, is believed to play a key role in controlling whole body energy balance. 1.3.5. Roles of AMPK in the heart Cardiac AMPK is activated during pathological conditions, such as ischemia or hypoxia, when the energy generation is hindered (188). Moreover, during physiological conditions, like exercise, increased ATP expenditure also activated AMPK (188). Once stimulated, AMPK switches off energy consuming processes like protein synthesis, whereas ATP generating mechanisms, such as FA oxidation and glycolysis, are turned on (68, 77). In heart and skeletal muscle, AMPK promotes glucose uptake and glycolysis through recruiting GLUT4 to the plasma membrane and activating PFK2. Moreover, AMPK also 19 facilitates F A utilization through its control o f acetyl-CoA carboxylase ( A C C ) (96, 97). A s A C C catalyzes the conversion of acetyl-CoA to malonyl-CoA, A M P K by inhibiting A C C is able to decrease malonyl-CoA and minimize its inhibition o f CPT-1 , the rate limiting enzyme controlling F A oxidation. A M P K has also been implicated i n F A delivery to cardiomyocytes through its regulation of the F A transporter, C D 3 6 (107). Interestingly, recent studies using transgenic mice with a dominant negative form o f A M P K has demonstrated that lack of A M P K does not affect cardiac metabolism under physiological conditions (144, 183). A t present, it is unclear as to what compensatory mechanisms are activated following knock-out o f A M P K . Additionally, it is unknown whether overexpression o f A M P K could affect cardiac l ip id homeostasis and metabolism. 1.4. Rationale and objectives A s a metabolic switch, A M P K is known to promote F A oxidation to maintain the energy balance in cells. Following hypoinsulinemia, as glucose utilization is impaired, the heart switches to use F A . Previous studies have suggested that P P A R - a is implicated in promoting F A utilization following chronic hypoinsulinemia (58). Whether this mechanism is also true following acute hypoinsulinemia and i f A M P K is involved in the elevation o f F A oxidation during this condition are unknown. In addition to its role in modulating F A oxidation, A M P K has also been found to be involved in F A uptake through CD36 (107). Given that F A released from lipoprotein is suggested to be the main F A source supply to the heart, and L P L is the primary enzyme controlling lipoprotein metabolism, o f interest to us was the question o f whether A M P K also influences F A 20 delivery through lipoprotein lipase ( L P L ) . In an attempt to examine whether p-agonists are able to influence cardiac H R - L P L , experiments were carried out in different models. However, the results obtained in these studies were controversial. Whether P-agonist can regulate cardiac L P L and i f A M P K is implicated in this process has yet to be determined. Finally, beside its role in the regulation of metabolism, recent studies suggest that A M P K can also modulate cell death. Thus, in the astrocytes and endothelial cells, activation of A M P K has been suggested to protect high fat or hyperglycemia induced apoptosis (21, 84). During obesity or diabetes, augmented circulating l ipid is one of the main reasons leading to lipotoxicity and cardiomyocytes apoptosis (171). Given that A M P K , through elevation of F A oxidation, reduces l ipid accumulation, it is unclear whether this process can protect cardiomyocytes against high fat induced cell apoptosis. Taken together, my Ph.D. project includes 4 objectives: 1. To examine the regulation of cardiac metabolism by A M P K and P P A R - a following acute and chronic hypoinsulinemia. 2. To determine whether cardiac A M P K could influence H R - L P L at the coronary lumen. 3. To examine whether P-agonist could regulate cardiac L P L , and the potential role of A M P K in this process. 4. To investigate whether metformin (a drug widely used in Type 2 diabetes, and known to activate A M P K ) could regulate high fat induced cardiomyocyte apoptosis. 21 1.5. Figures Fig. 1-1 Glucose utilization in the cardiomyocytes Glucose uptake into cardiomyocytes occurs through G L U T ! and G L U T 4 transporters. Once inside, glucose is broken down through glycolysis, a sequence of reactions that convert glucose into pyruvate. P F K 1 , the enzyme that catalyze the generation of fructose 1,6-bisphosphate from fructose 6-phosphate, is a rate-limiting enzyme controlling glycolysis. P F K 1 is activated by 2, 6-bisphosphate, which is formed from fructose 6-phosphate catalyzed by P F K - 2 . Fol lowing glycolysis, the pyruvate generated is transported into mitochondria and decarboxylated to acetyl-CoA through P D H , a multienzyme complex. P D H is phosphorylated and inactivated by P D K . Ace ty l -CoA then enters citric acid cycle and is eventually broken down to H2O and C 0 2 for A T P generation. P F K , phosphofructokinase, P D H , pyruvate dehydrogenase, P D K , pyruvate dehydrogenase kinase. 22 BLOOD VESSEL CARDIOMYOCYTE Fig. 1-2 Control of FA delivery and utilization in the cardiomyocyte F A , either from adipose tissue or released from TG-r ich lipoproteins through hydrolysis by lipoprotein lipase ( L P L ) , is taken up into the cardiomyocyte by three F A transporters: CD36, F A transport protein (FATP), and F A binding protein plasma membrane ( F A B P P M ) . F A is converted to fatty acy l -CoA, which is transported into mitochondria through CPT1/CPT2. Inside the mitochondria, fatty acyl -CoA undergoes (3-oxidation to generate acetyl-CoA, which is further oxidized in citric acid cycle. The utilization of F A is regulated through different mechanisms. F A , through activation of P P A R - a , increases the expression of a number of enzymes involved in F A oxidation. Malony l -CoA, which is generated through carboxylation o f acetyl-CoA catalyzed by A C C , inhibits CPT-1 and F A oxidation. A M P K , inhibits A C C , relieves its inhibition on CPT-1 and promotes F A oxidation. Similarly, M C D , through decreasing malonyl-CoA by decarboxylating it to acetyl-CoA, enhances CPT-1 and F A oxidation. A C C , acetyl-CoA carboxylase, A M P K , A M P activated kinase, M C D , malonyl-CoA decarboxylase. 23 Glucose Fig. 1-3 Inhibition of glucose oxidation by FA utilization Accumulation of F A impairs insulin mediated glucose uptake through inhibition of insulin receptor substrate and protein kinase B . Increased intracellular F A also activates P P A R - a . A s a consequence, P P A R - a promotes the expression of genes involved in F A oxidation, as wel l as P D K 4 , known to inhibit P D H and pyruvate flux. Increased acetyl-CoA and N A D H caused by the high rate of F A oxidation activate P D K 4 , leading to further inactivation of P D H . Augmented acetyl-CoA also causes accumulation of citrate in the cytosol, which subsequently inhibits P F K 1 , and glycolysis. 24 Lumen Endothelial cell Interstitium Cardiomyocyte Fig. 1-4 LPL synthesis, secretion and transfer After gene transcription, L P L is synthesized and processed in E R and Golgi . Mature L P L is packaged into secretory vesicles for either lysosomal degradation or secretion. Secreted L P L binds to cell surface HSPG, before transferring to the abluminal side of endothelial cells. Subsequently, L P L is transcytosed to the apical surface. L P L at the lumen can be internalized and recycled in endothelial cells. 25 1.6. Bibliography 1. Aalto-Setala K, Fisher EA, Chen X, Chajek-Shaul T, Hayek T, Zechner R, Walsh A, Ramakrishnan R, Ginsberg HN, Breslow JL Mechanism of hypertriglyceridemia in human apolipoprotein (apo) CIII transgenic mice. Diminished very low density lipoprotein fractional catabolic rate associated with increased apo CIII and reduced apo E on the particles. J Clin Invest 90: 1889-1900, 1992. 2. Aalto-Setala K, Weinstock PH, Bisgaier CL, Wu L , Smith JD, Breslow JL Further characterization of the metabolic properties of triglyceride-rich lipoproteins from human and mouse apoC-III transgenic mice. J Lipid Res 37: 1802-1811, 1996. 3. Abumrad NA, Park JH, Park CR Permeation of long-chain fatty acid into adipocytes. Kinetics, specificity, and evidence for involvement of a membrane protein. J Biol Chem 259: 8945-8953, 1984. 4. Augustus A, Yagyu H, Haemmerle G, Bensadoun A, Vikramadithyan RK, Park SY, Kim JK, Zechner R, Goldberg IJ Cardiac-specific knock-out of lipoprotein lipase alters plasma lipoprotein triglyceride metabolism and cardiac gene expression. J Biol Chem 279: 25050-25057, 2004. 5. Augustus AS, Buchanan J, Park TS, Hirata K, Noh HL, Sun J, Homma S, D'Armiento J, Abel ED, Goldberg IJ Loss of lipoprotein l i p a s e T d e r i v e d fatty acids leads to increased cardiac glucose metabolism and heart dysfunction. J Biol Chem 2006. 6. Augustus AS, Kako Y, Yagyu H, Goldberg IJ Routes of F A delivery to cardiac muscle: modulation of lipoprotein lipolysis alters uptake of TG-derived F A . Am J Physiol 26 Endocrinol Metab 284: E331-339, 2003. 7. Auwerx J, Leroy P, Schoonjans K Lipoprotein lipase: recent contributions from molecular biology. Crit Rev Clin Lab Sci 29: 243-268, 1992. 8. Auwerx JH, Deeb S, Brunzell JD, Peng R, Chait A Transcriptional activation of the lipoprotein lipase and apolipoprotein E genes accompanies differentiation in some human macrophage-like cell lines. Biochemistry 27: 2651-2655, 1988. 9. Balch WE Biochemistry of interorganelle transport. A new frontier in enzymology emerges from versatile in vitro model systems. J Biol Chem 264: 16965-16968, 1989. 10. Barak Y, Nelson MC, Ong ES, Jones YZ, Ruiz-Lozano P, Chien KR, Koder A, Evans RM P P A R gamma is required for placental, cardiac, and adipose tissue development. Mol Cell A: 585-595, 1999. 11. Barger PM, Kelly DP P P A R signaling in the control of cardiac energy metabolism. Trends Cardiovasc Med 10: 238-245, 2000. 12. Beauloye C, Marsin AS, Bertrand L , Krause U, Hardie DG, Vanoverschelde JL, Hue L Insulin antagonizes AMP-activated protein kinase activation by ischemia or anoxia in rat hearts, without affecting total adenine nucleotides. FEBS Lett 505: 348-352, 2001. 13. Bensadoun A Lipoprotein lipase. Annu Rev Nutr 11: 217-237, 1991. 14. Ben-Zeev O, Doolittle MH, Davis RC, Elovson J, Schotz M C Maturation of lipoprotein lipase. Expression of full catalytic activity requires glucose trimming but not translocation to the cis-Golgi compartment. J Biol Chem 267: 6219-6227, 1992. 15. Ben-Zeev O, Mao HZ, Doolittle M H Maturation of lipoprotein lipase in the 27 endoplasmic reticulum. Concurrent formation of functional dimers and inactive aggregates. JBiol Chem 277: 10727-10738, 2002. 16. Ben-Zeev O, Stahnke G, Liu G, Davis RC, Doolittle M H Lipoprotein lipase and hepatic lipase: the role of asparagine-linked glycosylation in the expression of a functional enzyme. J Lipid Res 35: 1511-1523, 1994. 17. Bergo M , Wu G, Ruge T, Olivecrona T Down-regulation of adipose tissue lipoprotein lipase during fasting requires that a gene, separate from the lipase gene, is switched on. JBiol Chem 211: 11927-11932, 2002. 18. Bertrand L , Alessi DR, Deprez J, Deak M , Viaene E , Rider M H , Hue L Heart 6-phosphofructo-2-kinase activation by insulin results from Ser-466 and Ser-483 phosphorylation and requires 3-phosphoinositide-dependent kinase-1, but not protein kinase B . JBiol Chem 274: 30927-30933, 1999. 19. Blanchette-Mackie EJ, Dwyer NK, Amende L A Cytochemical studies of lipid metabolism: immunogold probes for lipoprotein lipase and cholesterol. Am J Anat 185: 255-263, 1989. 20. Blanchette-Mackie EJ, Masuno H, Dwyer NK, Olivecrona T, Scow RO Lipoprotein lipase in myocytes and capillary endothelium of heart: immunocytochemical study. Am J Physiol 256: E818-828, 1989. 21. Blazquez C, Geelen MJ, Velasco G, Guzman M The AMP-activated protein kinase prevents ceramide synthesis de novo and apoptosis in astrocytes. FEBS Lett 489: 149-153, 2001. 28 22. Bolster DR, Crozier SJ, Kimball SR, Jefferson LS AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling. J Biol Chem 277: 23977-23980, 2002. 23. Bowker-Kinley M M , Davis Wl, Wu P, Harris RA, Popov K M Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex. Biochem J 3 2 9 ( Pt 1): 191-196, 1998. 24. Braun JE, Severson DL Regulation of the synthesis, processing and translocation of lipoprotein lipase. Biochem J287 ( Pt 2): 337-347, 1992. 25. Brunmair B, Staniek K, Gras F, Scharf N, Althaym A, Clara R, Roden M, Gnaiger E, Nohl H, Waldhausl W, Furnsinn C Thiazolidinediones, like metformin, inhibit respiratory complex I: a common mechanism contributing to their antidiabetic actions? Diabetes 53: 1052-1059, 2004. 26. Camps L, Reina M , Llobera M , Vilaro S, Olivecrona T Lipoprotein lipase: cellular origin and functional distribution. Am J Physiol 258: C673-681, 1990. 27. Carley AN, Severson DL Fatty acid metabolism is enhanced in type 2 diabetic hearts. Biochim Biophys Acta 1734: 112-126,2005. 28. Carling D The AMP-activated protein kinase cascade—a unifying system for energy control. Trends Biochem Sci 29: 18-24, 2004. 29. Carneheim C, Nedergaard J, Cannon B Cold-induced beta-adrenergic recruitment of lipoprotein lipase in brown fat is due to increased transcription. Am J Physiol 254: E155-161, 1988. 29 30. Chawla A, Lee CH, Barak Y, He W, Rosenfeld J, Liao D, Han J, Kang H, Evans RM PPARdelta is a very low-density lipoprotein sensor in macrophages. Proc Natl Acad Sci USA 100: 1268-1273, 2003. 31. Cheng CF, Oosta GM, Bensadoun A, Rosenberg RD Binding of lipoprotein lipase to endothelial cells in culture. JBiol Chem 256: 12893-12898, 1981. 32. Cheng L, Ding G, Qin Q, Huang Y, Lewis W, He N, Evans RM, Schneider MD, Brako FA, Xiao Y, Chen YE, Yang Q Cardiomyocyte-restricted peroxisome proliferator-activated receptor-delta deletion perturbs myocardial fatty acid oxidation and leads to cardiomyopathy. Nat Med 10: 1245-1250, 2004. 33. Cheung PC, Salt IP, Davies SP, Hardie DG, Carling D Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in A M P binding. Biochem J 346 Pt 3: 659-669, 2000. 34. Chiu HC, Kovacs A, Blanton RM, Han X, Courtois M , Weinheimer CJ, Yamada KA, Brunet S, Xu H, Nerbonne JM, Welch MJ, Fettig NM, Sharp T L , Sambandam N, Olson K M , Ory DS, Schaffer JE Transgenic expression of fatty acid transport protein 1 in the heart causes lipotoxic cardiomyopathy. Circ Res 96: 225-233, 2005. 35. Chiu HC, Kovacs A, Ford DA, Hsu FF, Garcia R, Herrero P, Saffitz JE, Schaffer JE A novel mouse model of lipotoxic cardiomyopathy. J Clin Invest 107: 813-822, 2001. 36. Chohan P, Cryer A Lipoprotein lipase activity o f rat cardiac muscle. Changes in the enzyme activity during incubations of isolated cardiac-muscle cells in vitro. Biochem J 1 8 6 : 873-879, 1980. 30 37. Coven DL, Hu X, Cong L, Bergeron R, Shulman GI, Hardie DG, Young L H Physiological role of AMP-activated protein kinase in the heart: graded activation during exercise. Am J Physiol Endocrinol Metab 285: E629-636, 2003. 38. Crute BE, Seefeld K, Gamble J, Kemp BE, Witters L A Functional domains of the alphal catalytic subunit of the AMP-activated protein kinase. J Biol Chem 273: 35347-35354, 1998. 39. Cupp M, Bensadoun A, Melford K Heparin decreases the degradation rate of lipoprotein lipase in adipocytes. J Biol Chem 262: 6383-6388, 1987. 40. da Silva Xavier G, Leclerc I, Varadi A, Tsuboi T, Moule SK, Rutter GA Role for AMP-activated protein kinase in glucose-stimulated insulin secretion and preproinsulin gene expression. Biochem J 371: 761-774, 2003. 41. Davies SP, Helps NR, Cohen PT, Hardie DG 5 ' - A M P inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein phosphatase-2C alpha and native bovine protein phosphatase-2AC. FEBS Lett 311: 421-425, 1995. 42. Deeb SS, Peng RL Structure of the human lipoprotein lipase gene. Biochemistry 28: 4131-4135, 1989. 43. Depre C, Veitch K, Hue L Role of fructose 2,6-bisphosphate in the control of glycolysis. Stimulation of glycogen synthesis by lactate in the isolated working rat heart. Acta Cardiol 48: 147-164, 1993. 44. Deprez J, Vertommen D, Alessi DR, Hue L, Rider M H Phosphorylation and 31 activation of heart 6-phosphofructo-2-kinase by protein kinase B and other protein kinases of the insulin signaling cascades. J Biol Chem 272: 17269-17275, 1997. 45. Dressel U, Allen T L , Pippal JB, Rohde PR, Lau P, Muscat GE The peroxisome proliferator-activated receptor beta/delta agonist, GW501516, regulates the expression of genes involved in l ip id catabolism and energy uncoupling in skeletal muscle cells. Mol Endocrinol 17: 2477-2493, 2003. 46. Dyck JR, Barr AJ, Barr RL, Kolattukudy PE, Lopaschuk GD Characterization of cardiac malonyl-CoA decarboxylase and its putative role in regulating fatty acid oxidation. Am J Physiol 275: H2122-2129, 1998. 47. Dyck JR, Cheng JF, Stanley WC, Barr R, Chandler MP, Brown S, Wallace D, Arrhenius T, Harmon C, Yang G, Nadzan A M , Lopaschuk GD Malonyl coenzyme a decarboxylase inhibition protects the ischemic heart by inhibiting fatty acid oxidation and stimulating glucose oxidation. Circ Res 94: e78-84, 2004. 48. Eckel RH Lipoprotein lipase. A multifunctional enzyme relevant to common metabolic diseases. N Engl J Med 320: 1060-1068, 1989. 49. Eckel RH, Yost TJ, Jensen DR Alterations in lipoprotein lipase in insulin resistance. Int J Obes Relat Metab Disord 19 Suppl 1: SI6-21, 1995. 50. Elbein AD Glycosidase inhibitors: inhibitors of N-l inked oligosaccharide processing. FasebJS: 3055-3063, 1991. 51. Enerback S, Gimble J M Lipoprotein lipase gene expression: physiological regulators at the transcriptional and post-transcriptional level. Biochim Biophys Acta 1169: 32 107-125, 1993. 52. Enerback S, Ohlsson BG, Samuelsson L, Bjursell G Characterization of the human lipoprotein lipase ( L P L ) promoter: evidence of two cis-regulatory regions, LP-alpha and LP-beta, of importance for the differentiation-linked induction of the L P L gene during adipogenesis. Mol Cell Biol 12: 4622-4633, 1992. 53. Fan J, Unoki H, Kojima N, Sun H, Shimoyamada H, Deng H, Okazaki M, Shikama H, Yamada N, Watanabe T Overexpression o f lipoprotein lipase in transgenic rabbits inhibits diet-induced hypercholesterolemia and atherosclerosis. J Biol Chem 276: 40071-40079,2001. 54. Ferreira LD, Pulawa L K , Jensen DR, Eckel RH Overexpressing human lipoprotein lipase in mouse skeletal muscle is associated with insulin resistance. Diabetes 50: 1064-1068,2001. 55. Finck BN The role of the peroxisome proliferator-activated receptor alpha pathway in pathological remodeling of the diabetic heart. Curr Opin Clin Nutr Metab Care 7: 391-396, 2004. 56. Finck BN, Han X, Courtois M, Aimond F, Nerbonne JM, Kovacs A, Gross RW, Kelly DP A critical role for PPARalpha-mediated lipotoxicity in the pathogenesis of diabetic cardiomyopathy: modulation by dietary fat content. Proc Natl Acad Sci USA 100: 1226-1231,2003. 57. Finck BN, Kelly DP Peroxisome proliferator-activated receptor alpha (PPARalpha) signaling in the gene regulatory control of energy metabolism in the normal and diseased 33 heart. J Mol Cell Cardiol 34: 1249-1257, 2002. 58. Finck BN, Lehman JJ, Leone TC, Welch MJ, Bennett MJ, Kovacs A, Han X, Gross RW, Kozak R, Lopaschuk GD, Kelly DP The cardiac phenotype induced by PPARalpha overexpression mimics that caused by diabetes mellitus. J Clin Invest 109: 121-130, 2002. 59. Forman BM, Chen J, Evans R M Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta. Proc Natl Acad Sci USA 94: 4312-4317, 1997. 60. Fryer LG, Parbu-Patel A, Carling D The Anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways. J Biol Chem 277: 25226-25232, 2002. 61. Gamble J, Lopaschuk GD Insulin inhibition of 5' adenosine monophosphate-activated protein kinase in the heart results in activation of acetyl coenzyme A carboxylase and inhibition of fatty acid oxidation. Metabolism 46: 1270-1274, 1997. 62. Gargiulo C E , Stuhlsatz-Krouper SM, Schaffer JE Localization of adipocyte long-chain fatty acy l -CoA synthetase at the plasma membrane. J Lipid Res 40: 881-892, 1999. 63. Garland PB, Randle PJ, Newsholme EA Citrate as an Intermediary in the Inhibition of Phosphofructokinase in Rat Heart Muscle by Fatty Acids , Ketone Bodies, Pyruvate, Diabetes, and Starvation. Nature 200: 169-170, 1963. 34 64. Gilde AJ, van der Lee KA, Willemsen PH, Chinetti G, van der Leij FR, van der Vusse GJ, Staels B, van Bilsen M Peroxisome proliferator-activated receptor (PPAR) alpha and PPARbeta/delta, but not PPARgamma, modulate the expression of genes involved i n cardiac l ip id metabolism. Circ Res 92: 518-524, 2003. 65. Goldberg IJ Lipoprotein lipase and lipolysis: central roles in lipoprotein metabolism and atherogenesis. J Lipid Res 37: 693-707, 1996. 66. Grundleger M L , Thenen SW Decreased insulin binding, glucose transport, and glucose metabolism in soleus muscle of rats fed a high fat diet. Diabetes 31: 232-237, 1982. 67. Hanks SK, Quinn A M , Hunter T The protein kinase family: conserved features and deduced phylogeny o f the catalytic domains. Science 241: 42-52, 1988. 68. Hardie DG Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endocrinology 144: 5179-5183, 2003. 69. Hardie DG The AMP-activated protein kinase pathway-new players upstream and downstream. J Cell Sci 117: 5479-5487, 2004. 70. Hardie DG, Carling D The AMP-activated protein kinase-fuel gauge of the mammalian cell? Eur J Biochem 246: 259-273, 1997. 71. Hardie DG, Hawley SA AMP-activated protein kinase: the energy charge hypothesis revisited. Bioessays 23:1112-1119,2001. 72. Hardie DG, Scott JW, Pan DA, Hudson ER Management of cellular energy by the AMP-activated protein kinase system. FEBS Lett 546: 113-120, 2003. 35 73. Hawley SA, Boudeau J, Reid JL, Mustard KJ , Udd L , Makela TP, Alessi DR, Hardie DG Complexes between the L K B 1 tumor suppressor, S T R A D alpha/beta and M 0 2 5 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade. J Biol 2: 28, 2003. 74. Hawley SA, Davison M , Woods A, Davies SP, Beri RK, Carling D, Hardie DG Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase. J Biol Chem 271: 27879-27887, 1996. 75. Hawley SA, Selbert MA, Goldstein EG, Edelman A M , Carling D, Hardie DG 5 ' -AMP activates the AMP-activated protein kinase cascade, and Ca2+/calmodulin activates the calmodulin-dependent protein kinase I cascade, v ia three independent mechanisms. J Biol Chem 270: 27186-27191, 1995. 76. Holness MJ, Sugden M C Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation. Biochem Soc Trans 31: 1143-1151, 2003. 77. Horman S, Browne G, Krause U, Patel J, Vertommen D, Bertrand L, Lavoinne A, Hue L, Proud C, Rider M Activation of AMP-activated protein kinase leads to the phosphorylation of elongation factor 2 and an inhibition of protein synthesis. Curr Biol 12: 1419-1423,2002. 78. Huang B, Wu P, Popov K M , Harris RA Starvation and diabetes reduce the amount of pyruvate dehydrogenase phosphatase in rat heart and kidney. Diabetes 52: 1371-1376, 2003. 36 79. Hudson ER, Pan DA, James J, Lucocq JM, Hawley SA, Green KA, Baba O, Terashima T, Hardie DG A novel domain in AMP-activated protein kinase causes glycogen storage bodies similar to those seen in hereditary cardiac arrhythmias. Curr Biol 13:861-866,2003. 80. Hue L , Beauloye C, Marsin AS, Bertrand L , Horman S, Rider M H Insulin and ischemia stimulate glycolysis by acting on the same targets through different and opposing signaling pathways. J Mol Cell Cardiol 34: 1091-1097, 2002. 81. Hue L , Rider M H Role of fructose 2,6-bisphosphate in the control of glycolysis in mammalian tissues. Biochem J 2 4 5 : 313-324, 1987. 82. Huss JM, Kelly DP Nuclear receptor signaling and cardiac energetics. Circ Res 95: 568-578, 2004. 83. Ibrahimi A, Bonen A, Blinn WD, Hajri T, L i X, Zhong K, Cameron R, Abumrad NA Muscle-specific overexpression of FAT/CD36 enhances fatty acid oxidation by contracting muscle, reduces plasma triglycerides and fatty acids, and increases plasma glucose and insulin. JBiol Chem 274: 26761-26766, 1999. 84. Ido Y, Carling D, Ruderman N Hyperglycemia-induced apoptosis in human umbilical vein endothelial cells: inhibition by the AMP-activated protein kinase activation. Diabetes 51: 159-167, 2002. 85. Itani SI, Zhou Q, Pories WJ, MacDonald KG, Dohm GL Involvement of protein kinase C in human skeletal muscle insulin resistance and obesity. Diabetes 49: 1353-1358, 2000. 37 86. Iwasaki T, Takahashi S, Takahashi M , Zenimaru Y, Kujiraoka T, Ishihara M, Nagano M , Suzuki J, Miyamori I, Naiki H, Sakai J, Fujino T, Miller NE, Yamamoto TT, Hattori H Deficiency of the very low-density lipoprotein ( V L D L ) receptors in streptozotocin-induced diabetic rats: insulin dependency of the V L D L receptor. Endocrinology 146: 3286-3294, 2005. 87. Jiang R, Carlson M The Snf l protein kinase and its activating subunit, Snf4, interact with distinct domains o f the Sipl/Sip2/Gal83 component in the kinase complex. Mol Cell Biol 17: 2099-2106, 1997. 88. Kahn BB, Alquier T, Carling D, Hardie DG AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 1: 15-25, 2005. 89! Kim JK, Fillmore JJ, Sunshine MJ, Albrecht B, Higashimori T, Kim DW, Liu ZX, Soos TJ, Cline GW, O'Brien WR, Littman DR, Shulman GI PKC-theta knockout mice are protected from fat-induced insulin resistance. J Clin Invest 114: 823-827, 2004. 90. Kirchgessner TG, LeBoeuf RC, Langner CA, Zollman S, Chang CH, Taylor BA, Schotz MC, Gordon JI, Lusis AJ Genetic and developmental regulation o f the lipoprotein lipase gene: loci both distal and proximal to the lipoprotein lipase structural gene control enzyme expression. JBiol Chem 264: 1473-1482, 1989. 91. Kitajima S, Morimoto M , Liu E, Koike T, Higaki Y, Taura Y, Mamba K, Itamoto K, Watanabe T, Tsutsumi K, Yamada N, Fan J Overexpression of lipoprotein lipase improves insulin resistance induced by a high-fat diet in transgenic rabbits. Diabetologia 38 47: 1202-1209, 2004. 92. Koike T, Liang J , Wang X, Ichikawa T, Shiomi M , Liu G, Sun H, Kitajima S, Morimoto M , Watanabe T, Yamada N, Fan J Overexpression o f lipoprotein lipase in transgenic Watanabe heritable hyperlipidemic rabbits improves hyperlipidemia and obesity. J Biol Chem 279: 7521-7529, 2004. 93. Koonen DP, Glatz JF, Bonen A, Luiken JJ Long-chain fatty acid uptake and FAT/CD36 translocation in heart and skeletal muscle. Biochim Biophys Acta 1736: 163-180, 2005. 94. Kovacic S, Soltys CL, Barr AJ, Shiojima I, Walsh K, Dyck JR A k t activity negatively regulates phosphorylation of AMP-activated protein kinase in the heart. J Biol Chem 278: 39422-39427, 2003. 95. Kraegen EW, Sowden JA, Halstead MB, Clark PW, Rodnick KJ, Chisholm DJ, James DE Glucose transporters and in vivo glucose uptake in skeletal and cardiac muscle: fasting, insulin stimulation and immunoisolation studies o f G L U T 1 and G L U T 4 . Biochem J 295 ( P t 1): 287-293, 1993. 96. Kudo N, Barr AJ, Barr RL, Desai S, Lopaschuk GD High rates of fatty acid oxidation during reperfusion of ischemic hearts are associated with a decrease in malonyl-CoA levels due to an increase in 5'-AMP-activated protein kinase inhibition of acetyl-CoA carboxylase. J Biol Chem 270: 17513-17520, 1995. 97. Kudo N, Gillespie JG, Kung L, Witters LA, Schulz R, Clanachan AS, Lopaschuk GD Characterization o f 5'AMP-activated protein kinase activity in the heart and its role in 39 inhibiting acetyl-CoA carboxylase during reperfusion following ischemia. Biochim Biophys Acta 1301: 67-75, 1996. 98. Ladu MJ, Kapsas H, Palmer WK Regulation of lipoprotein lipase in adipose and muscle tissues during fasting. Am J Physiol 260: R953-959, 1991. 99. Leone TC, Weinheimer CJ, Kelly DP A critical role for the peroxisome proliferator-activated receptor alpha (PPARalpha) in the cellular fasting response: the PPARalpha-null mouse as a model of fatty acid oxidation disorders. Proc Natl Acad Sci U S A 96: 7473-7478, 1999. 100. Levak-Frank S, Radner H, Walsh A , Stollberger R, Knipping G, Hoefler G, Sattler W, Weinstock PH, Breslow JL, Zechner R Muscle-specific overexpression of lipoprotein lipase causes a severe myopathy characterized by proliferation of mitochondria and peroxisomes in transgenic mice. J Clin Invest 96: 976-986, 1995. 101. Li J, Hu X, Selvakumar P, Russell RR, 3 rd , Cushman SW, Holman GD, Young L H Role of the nitric oxide pathway in AMPK-media ted glucose uptake and G L U T 4 translocation in heart muscle. Am J Physiol Endocrinol Metab 287: E834-841, 2004. 102. Liu G, Bengtsson-Olivecrona G, Olivecrona T Assembly of lipoprotein lipase in perfused guinea-pig hearts. Biochem J292 (P t 1): 277-282, 1993. 103. Liu G, Olivecrona T Synthesis and transport of lipoprotein lipase in perfused guinea pig hearts. Am J Physiol 263: H43 8-446, 1992. 104. Liu GQ, Olivecrona T Pulse-chase study on lipoprotein lipase in perfused guinea pig heart. Am J Physiol 261: H2044-2050, 1991. 40 105. Lochhead PA, Salt IP, Walker KS, Hardie DG, Sutherland C 5-aminoimidazole-4-carboxarriide riboside mimics the effects of insulin on the expression of the 2 key gluconeogenic genes P E P C K and glucose-6-phosphatase. Diabetes 49: 896-903, 2000. 106. Luiken JJ, Arumugam Y, Bell RC, Calles-Escandon J, Tandon NN, Glatz JF, Bonen A Changes in fatty acid transport and transporters are related to the severity of insulin deficiency. Am J Physiol Endocrinol Metab 283: E612-621, 2002. 107. Luiken JJ, Coort SL, Willems J, Coumans WA, Bonen A, van der Vusse GJ, Glatz JF Contraction-induced fatty acid translocase/CD36 translocation in rat cardiac myocytes is mediated through AMP-activated protein kinase signaling. Diabetes 52: 1627-1634, 2003. 108. Luiken JJ, Koonen DP, Willems J, Zorzano A, Becker C, Fischer Y, Tandon NN, Van Der Vusse GJ, Bonen A, Glatz JF Insulin stimulates long-chain fatty acid utilization by rat cardiac myocytes through cellular redistribution of FAT/CD36 . Diabetes 51: 3113-3119,2002. 109. Luiken JJ, Schaap FG, van Nieuwenhoven FA, van der Vusse GJ, Bonen A, Glatz JF Cellular fatty acid transport in heart and skeletal muscle as facilitated by proteins. Lipids 34 Suppl: SI69-175, 1999. 110. Luiken JJ, Turcotte LP, Bonen A Protein-mediated palmitate uptake and expression of fatty acid transport proteins in heart giant vesicles. J Lipid Res 40: 1007-1016, 1999. 111. Luiken JJ, van Nieuwenhoven FA, America G, van der Vusse GJ, Glatz JF 41 Uptake and metabolism of palmitate by isolated cardiac myocytes from adult rats: involvement of sarcolemmal proteins. J Lipid Res 38: 745-758, 1997. 112. Marsin AS, Bertrand L, Rider MH, Deprez J, Beauloye C, Vincent MF, Van den Berghe G, Carting D, Hue L Phosphorylation and activation of heart P F K - 2 by A M P K has a role in the stimulation of glycolysis during ischaemia. Curr Biol 10: 1247-1255, 2000. 113. Mead JR, Irvine SA, Ramji DP Lipoprotein lipase: structure, function, regulation, and role in disease. J Mol Med 80: 753-769, 2002. 114. Merkel M , Eckel RH, Goldberg IJ Lipoprotein lipase: genetics, l ip id uptake, and regulation. J Lipid Res 43: 1997-2006, 2002. 115. Merkel M , Loeffler B, Kluger M , Fabig N, Geppert G, Pennacchio LA, Laatsch A, Heeren J Apolipoprotein A V accelerates plasma hydrolysis o f triglyceride-rich lipoproteins by interaction with proteoglycan-bound lipoprotein lipase. J Biol Chem 280: 21553-21560,2005. 116. Merrill GF, Kurth EJ, Hardie DG, Winder WW A I C A riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle. Am J Physiol 273: E l 107-1112, 1997. 117. Michaud SE, Renier G Direct regulatory effect o f fatty acids on macrophage lipoprotein lipase: potential role of PPARs . Diabetes 50: 660-666, 2001. 118. Minokoshi Y, Kim YB, Peroni OD, Fryer LG, Muller C, Carling D, Kahn BB Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 42 415: 339-343,2002. 119. Muoio DM, MacLean PS, Lang DB, L i S, Houmard JA, Way J M , Winegar DA, Corton JC, Dohm GL, Kraus WE Fatty acid homeostasis and induction of l ipid regulatory genes in skeletal muscles of peroxisome proliferator-activated receptor (PPAR) alpha knock-out mice. Evidence for compensatory regulation by P P A R delta. J Biol Chem 277: 26089-26097, 2002. 120. Murthy V, Julien P, Gagne C Molecular pathobiology o f the human lipoprotein lipase gene. Pharmacol Ther 70: 101-135, 1996. 121. Nagao M , Parimoo B, Tanaka K Developmental, nutritional, and hormonal regulation of tissue-specific expression of the genes encoding various acyl -CoA dehydrogenases and alpha-subunit o f electron transfer flavoprotein in rat. J Biol Chem 268: 24114-24124, 1993. 122. Newsholme EA, Randle PJ Regulation of glucose uptake by muscle. 7. Effects o f fatty acids, ketone bodies and pyruvate, and of alloxan-diabetes, starvation, hypophysectomy and adrenalectomy, on the concentrations of hexose phosphates, nucleotides and inorganic phosphate in perfused rat heart. Biochem J93: 641-651, 1964. 123. Obunike JC, Lutz EP, Li Z, Paka L, Katopodis T, Strickland DK, Kozarsky KF, Pillarisetti S, Goldberg IJ Transcytosis of lipoprotein lipase across cultured endothelial cells requires both heparan sulfate proteoglycans and the very low density lipoprotein receptor. JBiol Chem 276: 8934-8941, 2001. 124. Olivecrona T, Chernick SS, Bengtsson-Olivecrona G, Garrison M, Scow RO 43 Synthesis and secretion of lipoprotein lipase in 3T3-L1 adipocytes. Demonstration of inactive forms of lipase in cells. JBiol Chem 262: 10748-10759, 1987. 125. O'Looney P, Irwin D, Briscoe P, Vahouny GV Lipoprotein composition as a component in the lipoprotein clearance defect in experimental diabetes. J Biol Chem 260: 428-432, 1985. 126. Otarod JK, Goldberg IJ Lipoprotein lipase and its role in regulation of plasma lipoproteins and cardiac risk. Curr Atheroscler Rep 6: 335-342, 2004. 127. Paz K, Hemi R, LeRoith D, Karasik A, Elhanany E , Kanety H, Zick Y A molecular basis for insulin resistance. Elevated serine/threonine phosphorylation of IRS-1 and IRS-2 inhibits their binding to the juxtamembrane region of the insulin receptor and impairs their ability to undergo insulin-induced tyrosine phosphorylation. J Biol Chem 272: 29911-29918, 1997. 128. Peinado-Onsurbe J, Staels B, Deeb S, Ramirez I, Llobera M , Auwerx J Neonatal extinction o f liver lipoprotein lipase expression. Biochim Biophys Acta 1131: 281-286, 1992. 129. Pennacchio LA, Olivier M , Hubacek JA, Cohen JC, Cox DR, Fruchart JC, Krauss RM, Rubin E M A n apolipoprotein influencing triglycerides in humans and mice revealed by comparative sequencing. Science 294: 169-173, 2001. 130. Pessin JE, Bell GI Mammalian facilitative glucose transporter family: structure and molecular regulation. Annu Rev Physiol 54: 911-930, 1992. 131. PHIarisetti S, Paka L , Sasaki A, Vanni-Reyes T, Yin B, Parthasarathy N, Wagner 44 WD, Goldberg IJ Endothelial cell heparanase modulation o f lipoprotein lipase activity. Evidence that heparan sulfate oligosaccharide is an extracellular chaperone. J Biol Chem 272: 15753-15759, 1997. 132. Previato L, Parrott CL, Santamarina-Fojo S, Brewer HB, Jr. Transcriptional regulation of the human lipoprotein lipase gene in 3T3-L1 adipocytes. J Biol Chem 266: 18958-18963, 1991. 133. Pulinilkunnil T, Rodrigues B Cardiac lipoprotein lipase: metabolic basis for diabetic heart disease. Cardiovasc Res 69: 329-340, 2006. 134. Pykalisto OJ, Smith PH, Brunzell JD Determinants o f human adipose tissue lipoprotein lipase. Effect o f diabetes and obesity on basal- and diet-induced activity. J Clin Invest 56: 1108-1117, 1975. 135. Randle PJ, Garland PB, Hales CN, Newsholme EA The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances o f diabetes mellitus. Lancet 1: 785-789, 1963. 136. Richards MR, Harp JD, Ory DS, Schaffer JE Fatty acid transport protein 1 and long-chain acyl coenzyme A synthetase 1 interact in adipocytes. J Lipid Res 47: 665-672, 2006. 137. Rider MH, Bertrand L , Vertommen D, Michels PA, Rousseau GG, Hue L 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: head-to-head with a bifunctional enzyme that controls glycolysis. Biochem J '381: 561-579, 2004. 138. Rodrigues B, Cam MC, Jian K, Lim F, Sambandam N, Shepherd G Differential 45 effects of streptozotocin-induced diabetes on cardiac lipoprotein lipase activity. Diabetes 46: 1346-1353, 1997. 139. Rose JK, Doms RW Regulation o f protein export from the endoplasmic reticulum. Annu Rev Cell Biol 4: 257-288, 1988. 140. Rothman JE, Orci L Movement of proteins through the Golg i stack: a molecular dissection of vesicular transport. Faseb J 4: 1460-1468, 1990. 141. Ruderman NB, Park H, Kaushik VK, Dean D, Constant S, Prentki M , Saha A K A M P K as a metabolic switch in rat muscle, liver and adipose tissue after exercise. Acta Physiol Scand 178: 435-442, 2003. 142. Ruge T, Bergo M , Hultin M, Olivecrona G, Olivecrona T Nutritional regulation of binding sites for lipoprotein lipase in rat heart. Am J Physiol Endocrinol Metab 278: E211-218, 2000. 143. Rui L , Aguirre V, Kim JK, Shulman GI, Lee A, Corbould A, Dunaif A, White MF Insulin/IGF-1 and TNF-alpha stimulate phosphorylation of IRS-1 at inhibitory Ser307 via distinct pathways. J Clin Invest 107: 181-189, 2001. 144. Russell RR, 3rd, L i J, Coven DL, Pypaert M, Zechner C, Palmeri M , Giordano FJ, Mu J, Birnbaum MJ, Young L H AMP-activated protein kinase mediates ischemic glucose uptake and prevents postischemic cardiac dysfunction, apoptosis, and injury. J Clin Invest IH: 495-503, 2004. 145. Saha AK, Vawas D, Kurowski TG, Apazidis A, Witters LA, Shafrir E, Ruderman NB M a l o n y l - C o A regulation in skeletal muscle: its link to cell citrate and the 46 glucose-fatty acid cycle. Am J Physiol 272: E641-648, 1997. 146. Sambandam N, Lopaschuk GD AMP-activated protein kinase ( A M P K ) control of fatty acid and glucose metabolism in the ischemic heart. Prog Lipid Res 42: 238-256, 2003. 147. Sartippour MR, Renier G Differential regulation of macrophage peroxisome proliferator-activated receptor expression by glucose : role of peroxisome proliferator-activated receptors in lipoprotein lipase gene expression. Arterioscler Thromb Vase Biol 20: 104-110, 2000. 148. Saxena U, Klein MG, Goldberg IJ Metabolism of endothelial cell-bound lipoprotein lipase. Evidence for heparan sulfate proteoglycan-mediated internalization and recycling. J 5/0/CAe/w 265: 12880-12886, 1990. 149. Saxena TJ, Klein MG, Goldberg IJ Transport of lipoprotein lipase across endothelial cells. Proc Natl Acad Sci USA 88: 2254-2258, 1991. 150. Schaffer JE Fatty acid transport: the roads taken. Am J Physiol Endocrinol Metab 282: E239-246, 2002. 151. Schoonjans K, Peinado-Onsurbe J, Lefebvre A M , Heyman RA, Briggs M , Deeb S, Staels B, Auwerx J PPARalpha and PPARgamma activators direct a distinct tissue-specific transcriptional response via a P P R E in the lipoprotein lipase gene. Embo J 15: 5336-5348, 1996. 152. Scott JW, Hawley SA, Green KA, Anis M, Stewart G, Scullion GA, Norman DG, Hardie DG C B S domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations. J Clin Invest 113: 274-284, 2004. 47 153. Semb H, Olivecrona T Mechanisms for turnover of lipoprotein lipase in guinea pig adipocytes. Biochim Biophys Acta 921: 104-115, 1987. 154. Severson DL, Lee M , Carroll R Secretion of lipoprotein lipase from myocardial cells isolated from adult rat hearts. Mol Cell Biochem 79: 17-24, 1988. 155. Shaw RJ, Kosmatka M , Bardeesy N, Hurley RL, Witters LA, DePinho RA, Cantley L C The tumor suppressor L K B 1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci USA 101: 3329-3335, 2004. 156. Shaw RJ, Lamia KA, Vasquez D, Koo SH, Bardeesy N, Depinho RA, Montminy M, Cantley L C The kinase L K B 1 mediates glucose homeostasis in liver and therapeutic effects of metformin. Science 310: 1642-1646, 2005. 157. Shimada M , Ishibashi S, Gotoda T, Kawamura M , Yamamoto K, Inaba T, Harada K, Ohsuga J, Perrey S, Yazaki Y, et al. Overexpression of human lipoprotein lipase protects diabetic transgenic mice from diabetic hypertriglyceridemia and hypercholesterolemia. Arterioscler Thromb Vase Biol 15: 1688-1694, 1995. 158. Shimada M , Ishibashi S, Inaba T, Yagyu H, Harada K, Osuga JI, Ohashi K, Yazaki Y, Yamada N Suppression of diet-induced atherosclerosis in low density lipoprotein receptor knockout mice overexpressing lipoprotein lipase. Proc Natl Acad Sci U SA 93: 7242-7246, 1996. 159. Shimada M , Shimano H, Gotoda T, Yamamoto K, Kawamura M , Inaba T, Yazaki Y, Yamada N Overexpression of human lipoprotein lipase in transgenic mice. 48 Resistance to diet-induced hypertriglyceridemia and hypercholesterolemia. J Biol Chem 268: 17924-17929, 1993. 160. Simsolo RB, Ong JM, Saffari B, Kern PA Effect of improved diabetes control on the expression o f lipoprotein lipase in human adipose tissue. J Lipid Res 33: 89-95, 1992. 161. Sparkes RS, Zollman S, Klisak I, Kirchgessner TG, Komaromy MC, Mohandas T, Schotz M C , Lusis AJ Human genes involved in lipolysis o f plasma lipoproteins: mapping of loci for lipoprotein lipase to 8p22 and hepatic lipase to 15q21. Genomics 1: 138-144, 1987. 162. Stanley WC, Recchia FA, Lopaschuk GD Myocardial substrate metabolism in the normal and failing heart. Physiol Rev 85: 1093-1129, 2005. 163. Stein SC, Woods A, Jones NA, Davison MD, Carling D The regulation of AMP-activated protein kinase by phosphorylation. Biochem J 345 Pt 3: 437-443, 2000. 164. Stremmel W Fatty acid uptake by isolated rat heart myocytes represents a carrier-mediated transport process. J Clin Invest 81: 844-852, 1988. 165. Taegtmeyer H, Hems R, Krebs HA Utilization of energy-providing substrates in the isolated working rat heart. Biochem J 1 8 6 : 701-711, 1980. 166. Terada S, Goto M, Kato M , Kawanaka K, Shimokawa T, Tabata I Effects of low-intensity prolonged exercise on PGC-1 m R N A expression in rat epitrochlearis muscle. Biochem Biophys Res Commun 296: 350-354, 2002. 167. Teusink B, Voshol PJ, Dahlmans VE, Rensen PC, Pijl H, Romijn JA, Havekes L M Contribution of fatty acids released from lipolysis of plasma triglycerides to total 49 plasma fatty acid flux and tissue-specific fatty acid uptake. Diabetes 52: 614-620, 2003. 168. Tomas E, Tsao TS, Saha AK, Murrey HE, Zhang Cc C, Itani SI, Lodish HF, Ruderman NB Enhanced muscle fat oxidation and glucose transport by A C R P 3 0 globular domain: acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation. Proc Natl Acad Sci US A 99: 16309-16313,2002. 169. Tontonoz P, Hu E, Spiegelman B M Stimulation of adipogenesis in fibroblasts by P P A R gamma 2, a lipid-activated transcription factor. Cell 79: 1147-1156, 1994. 170. Tsuboi T, da Silva Xavier G, Leclerc I, Rutter GA 5'-AMP-activated protein kinase controls insulin-containing secretory vesicle dynamics. J Biol Chem 278: 52042-52051, 2003. 171. Unger RH, Orci L Lipoapoptosis: its mechanism and its diseases. Biochim Biophys 4 ^ 1585:202-212,2002. 172. Vannier C, Ailhaud G Biosynthesis of lipoprotein lipase in cultured mouse adipocytes. II. Processing, subunit assembly, and intracellular transport. J Biol Chem 264: 13206-13216, 1989. 173. Vannier C, Amri EZ, Etienne J, Negrel R, Ailhaud G Maturation and secretion of lipoprotein lipase in cultured adipose cells. I. Intracellular activation o f the enzyme. J Biol Chem 260: 4424-4431, 1985. 174. Vawas D, Apazidis A, Saha AK, Gamble J, Patel A, Kemp BE, Witters LA, Ruderman NB Contraction-induced changes in acetyl-CoA carboxylase and 5'-AMP-activated kinase in skeletal muscle. J Biol Chem 272: 13255-13261, 1997. 50 175. Voshol PJ, Jong MC, Dahlmans VE, Kratky D, Levak-Frank S, Zechner R, Romijn JA, Havekes L M In muscle-specific lipoprotein lipase-overexpressing mice, muscle triglyceride content is increased without inhibition of insulin-stimulated whole-body and muscle-specific glucose uptake. Diabetes 50: 2585-2590, 2001. 176. Wang W, Fan J, Yang X, Furer-Galban S, Lopez de Silanes I, von Kobbe C, Guo J, Georas SN, Foufelle F, Hardie DG, Carling D, Gorospe M AMP-activated kinase regulates cytoplasmic H u R . Mol Cell Bio! 22: 3425-3436, 2002. 177. Wang W, Yang X, Lopez de Silanes I, Carling D, Gorospe M Increased A M P : A T P ratio and AMP-activated protein kinase activity during cellular senescence linked to reduced H u R function. JBiol Chem 278: 27016-27023, 2003. 178. Weinstock PH, Bisgaier CL, Aalto-Setala K, Radner H, Ramakrishnan R, Levak-Frank S, Essenburg AD, Zechner R, Breslow JL Severe hypertriglyceridemia, reduced high density lipoprotein, and neonatal death in lipoprotein lipase knockout mice. M i l d hypertriglyceridemia with impaired very low density lipoprotein clearance in heterozygotes. J Clin Invest 96: 2555-2568, 1995. 179. Woods A, Johnstone SR, Dickerson K, Leiper FC, Fryer LG, Neumann D, Schlattner U , Wallimann T, Carlson M , Carling D L K B 1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr Biol 13: 2004-2008, 2003. 180. Woods A, Vertommen D, Neumann D, Turk R, Bayliss J, Schlattner U , Wallimann T, Carling D, Rider M H Identification o f phosphorylation sites in AMP-activated protein kinase ( A M P K ) for upstream A M P K kinases and study of their 51 roles by site-directed mutagenesis. J Biol Chem 278: 28434-28442, 2003. 181. Wu P, Inskeep K, Bowker-Kinley M M , Popov K M , Harris RA Mechanism responsible for inactivation of skeletal muscle pyruvate dehydrogenase complex in starvation and diabetes. Diabetes 48: 1593-1599, 1999. 182. Wu P, Sato J, Zhao Y, Jaskiewicz J, Popov K M , Harris RA Starvation and diabetes increase the amount o f pyruvate dehydrogenase kinase isoenzyme 4 in rat heart. Biochem J329 ( P t 1): 197-201, 1998. 183. Xing Y, Musi N, Fujii N, Zou L, Luptak I, Hirshman MF, Goodyear LJ , Tian R Glucose metabolism and energy homeostasis in mouse hearts overexpressing dominant negative alpha2 subunit of AMP-activated protein kinase. J Biol Chem 278: 28372-28377, 2003. 184. Yagyu H, Chen G, Yokoyama M, Hirata K, Augustus A, Kako Y, Seo T, Hu Y, Lutz EP, Merkel M , Bensadoun A, Horama S, Goldberg IJ Lipoprotein lipase (LpL) on the surface of cardiomyocytes increases lipid uptake and produces a cardiomyopathy. J Clin Invest 111:419-426, 2003. 185. Yagyu H, Lutz EP, Kako Y, Marks S, Hu Y, Choi SY, Bensadoun A, Goldberg IJ Very low density lipoprotein ( V L D L ) receptor-deficient mice have reduced lipoprotein lipase activity. Possible causes of hypertriglyceridemia and reduced body mass with V L D L receptor deficiency. J Biol Chem 277: 10037-10043, 2002. 186. Yamauchi T, Kamon J, Minokoshi Y, Ito Y, Waki H, Uchida S, Yamashita S, Noda M, Kita S, Ueki K, Eto K, Akanuma Y, Froguel P, Foufelle F, Ferre P, Carling D, 52 Kimura S, Nagai R, Kahn BB, Kadowaki T Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat Med 8: 1288-1295, 2002. 187. Yang J, Holman GD Insulin and contraction stimulate exocytosis, but increased AMP-activated protein kinase activity resulting from oxidative metabolism stress slows endocytosis of G L U T 4 in cardiomyocytes. JBiol Chem 280: 4070-4078, 2005. 188. Young L H , Li J, Baron SJ, Russell RR AMP-activated protein kinase: a key stress signaling pathway in the heart. Trends Cardiovasc Med 15: 110-118, 2005. 189. Yuan M , Konstantopoulos N, Lee J, Hansen L, Li ZW, Karin M , Shoelson SE Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta. Science 293: 1673-1677, 2001. 190. Zhou G, Myers R, L i Y, Chen Y, Shen X, Fenyk-Melody J, Wu M, Ventre J, Doebber T, Fujii N, Musi N, Hirshman MF, Goodyear L J , Moller DE Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 108: 1167-1174,2001. 53 2. Acute and chronic streptozotocin diabetes differentially regulates cardiac PPAR-a and AMPK 2.1. Introduction Cardiac tissue acquires its energy mostly from metabolism of two substrates, glucose and F A (19). Glucose uptake is insulin dependent, and once inside cells, glucose is broken down into pyruvate, which enters the mitochondria for A T P production. Glucose oxidation provides the heart with approximately 30% of its energy requirements (18). Compared to glucose, F A is the preferred substrate, and when supplied at physiological levels, contributes around 70% of the A T P necessary for normal heart function. Exogenous F A supply depends on L P L mediated hydrolysis of circulating lipoprotein triglyceride, and membrane transporters like CD36. Once inside the cardiomyocyte, C P T facilitates F A transfer into the mitochondria, for eventual oxidation. F A uptake and oxidation are regulated at various steps by a number of genes under the control of a nuclear receptor, P P A R - a (9). Following activation, P P A R - a regulates the expression of genes such as fatty acyl C o A synthase (17), CPT-1 (5), and A C O (23). The "fuel gauge" A M P K also plays a key role in the regulation of F A utilization. In the heart, A M P K facilitates F A utilization through its control of A C C (14). A s A C C catalyzes the conversion of acetyl-CoA to malonyl-CoA, A M P K by inhibiting A C C is able to decrease malonyl-CoA and minimize its inhibition of F A oxidation. A M P K has also been implicated in F A delivery to cardiomyocytes through its regulation of the F A transporter, 54 C D 3 6 ( 1 6 ) a n d L P L ( l ) . Following chronic diabetes, as glucose transport and utilization are impaired, the heart switches to excessive use of F A for energy production (25). This cardiac adaptation is achieved by activation of P P A R - a , and up-regulation of an array o f genes involved in F A uptake and oxidation (9). Although chronic diabetes is believed to increase cardiac P P A R - a (9), it is unclear whether the same alteration occurs acutely in diabetes (4-day). Moreover, it is unknown whether diabetes influences cardiac A M P K . The objective of the present study was to determine cardiac P P A R - a and A M P K following acute and chronic diabetes. We demonstrate that cardiac A M P K is activated in acute S T Z diabetes, whereas following chronic diabetes, the P P A R - a cascade is turned on. 55 2.2. Methods 2.2.1. Experimental animals The investigation conforms to the guidelines for the care and use of laboratory animals published by the U S National Institutes o f Health and the University o f Brit ish Columbia (animal care certificate A00-0291). Adult male Wistar rats (250-280 g) were obtained from the U B C A n i m a l Care Unit and supplied with a standard laboratory diet (PMI Feeds, Richmond, V A ) , and water ad libitum. Rats were randomly divided into nondiabetic control ( C O N ) and diabetic (STZ) groups. Halothane-anesthetized rats were injected with S T Z (55 mg/kg IV, Sigma Chemical Co) or an equivalent volume (1 mL/kg) of saline. A l l STZ-treated rats displayed hyperglycemia (>13 mmol/L) 24 hours after injection. Although these animals are insulin deficient, they do not require insulin supplementation for survival, and do not develop ketoacidosis. Diabetic rats were maintained for either 4-day (acute) or 6-week (chronic) after STZ.injection. Where indicated, some acute diabetic rats were injected with a rapid-acting insulin (8 U , Humulin R, E l i L i l l y Canada Inc.) into the tail vein, maintained for three hours (time required for establishment of sustained euglycemia), and hearts removed (STZ+I). 2.2.2. Measurement of cardiac gene expression Gene expression was measured in the indicated groups using RT-PCR. Briefly, total R N A from hearts (100 mg) was extracted using Trizol (Invitrogen). After spectrophotometric quantification and resolving o f R N A integrity using a formaldehyde agarose gel, reverse transcription was carried out using an oligo (dT) primer and superscript II R T (Invitrogen). 56 c D N A of P P A R - a , CPT-1 , A C O , M C D and CD36 were amplified using specific primers: 5' - G A C A A G G C C T C A G G A T A C C A - 3 ' (left) and 5' - A A A C G G A T T G C A T T G T G T G A - 3 ' (right) for P P A R - a (M88592); 5' - T A T G T G A G G A T G C T G C T T C C - 3 ' (left) and 5 ' - C T C G G A G A G C T A A G C T T G T C - 3 ' (right) for CPT-1 (L07736); 5' - G C C C T C A G C T A T G G T A T T A C - 3 ' (left) and 5' - A G G A A C T G C T C T C A C A A T G C - 3 ' (right) for A C O (J02752); 5 ' - G C C T G G T A C C T T T A C G G T G A - 3 ' (left) and 5 ' - G C T A C C A G G C T G A G G A T C T G - 3 ' (right) for M C D (NM_053477) ; 5' - C T C T G A C A T T T G C A G G T C C A - 3 ' (left) and 5' - C A C A G G C T T T C C T T C T T T G C - 3 ' (right) for CD36 (NM_031561). The P-actin (J00691) gene was amplified as an internal control using 5' - C G T A A A G A C C T C T A T G C C A A - 3 ' (left) and 5 ' - A G C C A T G C C A A A T G T C T C A T - 3 ' (right). The linear range was found to be between 15-40 cycles. The amplification parameters were set at: 94°C for 1 min, 56-58°C for 1 min and 72°C for 1 min, for a total of 28-35 cycles. The P C R products were electrophoresed on a 1.7% agarose gel containing ethidium bromide. Expression levels were represented as the ratio of signal intensity of the respective genes relative to f3-actin. 2.2.3. Western blot analysis On immediate removal o f hearts from control, diabetic or insulin treated rats, 50 mg of heart tissue was ground under liquid nitrogen and homogenized. Western blot was carried out as described previously (1). Samples were diluted, boiled with sample loading dye, and 50 pg protein used in SDS-polyacrylamide gel electrophoresis. After transfer, membranes were blocked in 5% skim milk in Tris-buffered saline containing 0.1% 57 Tween-20. Membranes were incubated with rabbit AMPK-oc , phospho-AMPK (Thr-172), phospho-ACC, CD36 and M C D antibodies and subsequently with secondary goat anti-rabbit HRP-conjugated antibody, and visualized using an E C L detection kit. 2.2.4. Serum measurements Blood samples were removed from animals and centrifuged immediately to collect serum that was stored at -20°C until assayed. Diagnostic kits were used to measure glucose, T G (Sigma), and non-esterified fatty acid (Wako). 2.2.5. Separation and characterization of cardiac lipids. Total cardiac lipids were extracted and solubilized in chloroform-methanol-acetone-hexane (4:6:1:1 vol/vol/vol/vol). Separation o f T G and F A was achieved using H P L C (Waters 2690 Alliance H P L C , Mil ford , M A ) equipped with an auto-sampler and column heater. F A were quantified as their respective methyl esters using heptadecaenoic acid (17:0) as the internal standard with a Varian 3400 G L C equipped with a flame ionization detector, a Varian Star data system, and a SP-2330 capillary column (30 m x 0.25 mm; Supelco, Bellefonte, PA). Value of cardiac F A and T G were expressed as micrograms per milligram protein. 2.2.6. Statistical analysis. One or two-way A N O V A followed by the Tukey or Bonferroni tests, or t-test were used to determine differences between group mean values. The level of statistical significance was set at P< 0.05. 58 2.3. Results 2.3.1. General characteristics Four days following S T Z injection, diabetic rats showed significantly lower body weight and higher blood glucose compared to control animals (Table 2-1). N o change in serum (Table 2-1) or cardiac (Table 2-2) F A or T G was observed in these acutely diabetic rats. Similar to acute diabetes, six week diabetic rats showed significantly lower body weight, and higher blood glucose compared to control animals (Table 2-1). Serum T G in these chronically diabetic animals was the highest, with no change in serum F A (Table 2-1). Cardiac T G and F A significantly increased in six-week diabetic hearts (Table 2-2). 2.3.2. Gene expression of PPAR-a and its targets in acute and chronic diabetic hearts Previous studies have demonstrated that in chronic diabetic hearts, activation of P P A R - a promoted F A utilization through increasing expression of a number of genes (9). In the present study, no significant change in cardiac P P A R - a (Fig. 2-1 A ) , C P T - l (Fig. 2- IB) , and A C O (Fig. 2-1C) gene expression was observed following acute diabetes. To confirm the results of previous studies, cardiac gene expression was also determined in chronic diabetic animals. Although gene expression of P P A R - a (Fig. 2-1 A ) did not change, its downstream targets, CPT-1 (Fig. 2-IB) and A C O (Fig. 2-1C) showed increased expression. 2.3.3. Gene and protein expression of cardiac MCD following acute and chronic diabetes Another target of P P A R - a , M C D , catalyzes the degradation o f malonyl -CoA to acetyl-CoA, thereby promoting F A oxidation ( 8 ) . In 4-day diabetic hearts, no change in M C D gene 59 (Fig. 2-2A) and protein (Fig. 2-2B) was observed. Following chronic diabetes, and consistent with previous studies, augmented expression of cardiac gene (Fig. 2-2C) and M C D protein (Fig. 2-2D) were evident. 2.3.4. Changes in cardiac CD36 gene and protein following acute and chronic diabetes A s a F A transporter, C D 3 6 facilitates cardiac F A uptake. Previous studies have reported that CD36 protein increases in both intracellular and plasma membrane obtained from chronic diabetic hearts, an effect likely due to increased expression of CD36 (15). In the present study, no changes of gene expression (Fig. 2-3A) and protein (Fig. 2-3B) were observed in the acute diabetic heart. Interestingly, both CD36 gene expression (Fig. 2-3C) and protein (Fig. 2-3D) were higher in chronic diabetic hearts compared to control. 2.3.5. Cardiac AMPK and ACC phosphorylation in acute and chronic diabetic hearts A M P K is phosphorylated and activated following an increase in A M P / A T P or creatine/phosphocreatine ratios. Extracellular hormones such as insulin (13) and adiponectin (22) can also regulate A M P K . Once activated, A M P K phosphorylates and inactivates A C C . Measurement of A M P K and A C C revealed that both A M P K (Fig. 2-4A) and A C C (Fig. 2-4B) phosphorylation were significantly increased in 4-day diabetic hearts, an outcome that was reversed by insulin (Fig. 2-4A and 2-4B). This activation was likely cardiac specific, as liver and skeletal muscle from acutely diabetic animals showed no change in A M P K phosphorylation (data not shown). Interestingly, following 6-week of diabetes, there was no difference in A M P K and A C C phosphorylation between control and diabetic rat hearts (Fig. 2-4C and 2-4D). 60 2.4. Discussion Cardiac tissue is versatile in choosing substrates for energy generation. During diabetes, as glucose uptake,. glycolysis and oxidation are impaired, the heart switches to predominantly using F A to ensure sufficient production of A T P (25). Although this metabolic switching can be regulated by multiple mechanisms, P P A R - a plays a key role (9). Once activated, and through binding of a co-activator, P P A R - a augments the expression of genes involved in F A uptake and oxidation (3, 24). Thus, using "gain and loss of function" strategies, cardiac specific overexpression (9) or knock out (2, 6) of P P A R - a brings about enhanced or reduced F A uptake and oxidation respectively. Chronic diabetes is known to activate P P A R - a (9). To investigate whether P P A R - a is activated and contributes to high F A oxidation previously demonstrated in 4-day acute diabetic hearts (10), we measured gene expression of cardiac P P A R - a and its targets, CPT-1 and A C O . Interestingly, acute diabetes did not change expression of these genes. Using real-time P C R , a previous study has also reported that following 1 week of STZ, no change in expression of P P A R - a and CPT-1 were observed (7). Another target of P P A R - a , M C D , catalyzes the degradation o f malonyl-CoA to acetyl-CoA, thereby promoting F A oxidation (8). Previous studies have reported higher cardiac M C D in chronic diabetic animals (20). In 4-day diabetic animals, no change of M C D gene and protein levels were observed. Given that augmented F A and its metabolites activate P P A R - a (12), we assessed the F A transporter CD36 , and plasma and cardiac F A and TG. Acute diabetes did not change gene and protein levels o f CD36 , or plasma and cardiac lipids, and could explain the 61 absence of P P A R - a activation. Taken together, our results indicate that mechanisms other than P P A R - a activation promote F A oxidation in these 4-day S T Z hearts. A s a "fuel gauge", A M P K regulates F A metabolism. In the present study, our data for the first time revealed activation of A M P K (as evidenced by elevated A M P K and A C C phosphorylation) in hearts, but not skeletal muscle and liver, from acutely diabetic rats. The underlying mechanism for this activation remains unclear. A M P K can be activated by both A T P dependent and independent mechanisms. Indeed, insulin is known to inhibit cardiac A M P K in an ATP-independent manner (4, 13), and level of this hormone declines within 24 hours o f S T Z injection (21). A s a single injection of insulin normalized both A M P K and A C C phosphorylation, our results suggest that following acute diabetes, reduction in insulin may be a key regulator of cardiac A M P K . To examine whether A M P K stays activated in chronic diabetes under these conditions, A M P K and A C C phosphorylation were measured in 6-week S T Z diabetic hearts. Cardiac A M P K and A C C phosphorylation remained unchanged in these diabetic animals compared with control. The mechanism behind this lack of activation of cardiac A M P K in chronic diabetes is currently unknown. Interestingly, chronic diabetic hearts demonstrated increased expression of both CD36 gene and protein, consistent with a previous study (15). A s overexpression of CD36 increases the rate of F A uptake (11), our data suggest that transcriptional modulation of this F A transporter likely increases F A delivery to the chronic diabetic heart. Indeed, associated with augmented expression o f CD36, increased cardiac T G and F A were 62 observed. Previous studies have demonstrated that augmented intracellular F A activates P P A R - a , with subsequent overexpression of genes involved in F A utilization (9). In the present study, although gene expression of P P A R - a did not change, expression of CPT-1 and A C O , targets o f P P A R - a , increased. Additionally, augmented expression o f both M C D gene and protein was observed in these chronic diabetic hearts. Overall, our data suggests that P P A R - a and not A M P K may contribute towards high F A oxidation in 6-week diabetic hearts. In summary, our results indicate that acute and chronic diabetes has different effects on cardiac A M P K and P P A R - a . In the acute S T Z diabetic heart, A M P K , rather than P P A R - a , is activated. This activation of A M P K likely increases F A oxidation through phosphorylation and inhibition of A C C . Given that glucose utilization is compromised following hypoinsulinemia, this acute adaptation would ensure adequate cardiac energy production. In chronic diabetes, augmented plasma lipids and expression o f CD36 provides the heart with excess F A . In this condition, P P A R - a , through its regulation of gene expression, contributes to high F A oxidation. 63 2.5. Tables and figures Table 2-1. General characteristics of the experimental animals ACUTE CHRONIC Control STZ Control STZ Body Weight (g) 3 1 9 ± 2 298 ± 4 * 494 ± 7 440 ± 8 * Blood Glucose (mM) 5.8 ± 0 . 5 22.3 ± 1.1 * 5.7 ± 0 . 3 25.9 ± 1 . 0 * Serum free fatty acid (mM) 0.32 ± 0.04 0.34 ± 0 . 0 6 0.40 ± 0.03 0.58 ± 0 . 1 9 Serum triglyceride (mM) 0.49 ± 0.03 0.51 ± 0 . 0 7 0.46 ± 0.04 1.11 ± 0.12 # Following 4-day (acute) or 6-week (chronic) of S T Z diabetes, animals from all groups were killed, blood collected and serum separated for measurement of various parameters. Values are mean ± S E for 6 animals in each group. 'Significantly different from control; "significantly different from all other groups, P < 0.05. 64 Table 2-2. Cardiac lipids in control and S T Z groups Control STZ (Acute) STZ (Chronic) Cardiac T G (ng/mg Protein) 4.9 + 1.8 6.2+ 1.8 31.2 ± 9.3* Cardiac FFA(Lig/mg Protein) 1.7 + 0.3 2.2 + 0.1 3.1+0.4* Following 4-day (acute) or 6-week (chronic) of S T Z diabetes, animals from all groups were killed. Cardiac lipids were extracted and separated using H P L C . Values are mean ± SE for 3 animals in each group. *Significantly different from control. 65 CON STZ CON STZ A CPT-1 • |3 Acute Chronic Fig. 2-1 Gene expression of PPAR-a, CPT-1 and ACO in 4-day and 6-week STZ diabetic hearts Hearts from control and 4-day and 6-week STZ diabetic animals were removed and immediately snap frozen in liquid nitrogen. Gene expression of PPAR-a (A), CPT-1 (B) and A C O (C) were measured using rt-PCR. Data are means ± SE; n=4. Two-way ANOVA followed by the Bonferroni test was used to determine differences between means. 'Significantly different from corresponding control groups, P < 0.05; #Significantly different from corresponding acute diabetic group, P < 0.05, 66 A C U T E A B Fig. 2-2 Expression of MCD in 4-day and 6-week STZ diabetic hearts Hearts from control and 4-day (A , B) and 6-week (C, D) S T Z diabetic animals were removed and immediately snap frozen in liquid nitrogen. M C D gene expression was measured by R T - P C R and protein determined by Western Blotting. Data are means ± SE; n=4. t-test was used to determine differences between means. 67 A C U T E A B Fig. 2-3 Expression of CD36 in 4-day and 6-week STZ diabetic hearts Hearts from control, 4-day (A , B ) and 6-week (C, D) S T Z diabetic animals were removed and immediately snap frozen in liquid nitrogen. Gene expression of CD36 was measured using rt-PCR. Protein level was evaluated by Western Blotting. Data are means ± SE; n=4. t-test was used to determine differences between means. Significantly different from control group, P < 0.05. 68 A C U T E A B Fig. 2-4 Cardiac AMPK and ACC phosphorylation in 4-day and 6-week STZ diabetic hearts Hearts from control, 4-day ( A , B ) and 6-week (C, D) S T Z diabetic rats, or diabetic animals treated with insulin were removed and immediately snap frozen in liquid nitrogen. A M P K - a (total and phosphorylated) and phospho-ACC were measured using Western Blotting. Data are means ± SE ; n=4. One-way A N O V A followed by the Tukey test was used to determine differences between means. Significantly different from other groups, P < 0 . 0 5 . 69 2.6. Bibliography 1. An D, Pulinilkunnil T, Qi D, Ghosh S, Abrahani A, Rodrigues B The metabolic "switch" A M P K regulates cardiac heparin-releasable lipoprotein lipase. Am J Physiol Endocrinol Metab 288: E246-253, 2005. 2. Aoyama T, Peters J M , Iritani N, Nakajima T, Furihata K, Hashimoto T, Gonzalez FJ Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha). J Biol Chem 273: 5678-5684, 1998. 3. Barger PM,Kelly DP P P A R signaling in the control o f cardiac energy metabolism. Trends Cardiovasc Med 10: 238-245, 2000. 4. Beauloye C, Marsin AS, Bertrand L, Krause U, Hardie DG, Vanoverschelde JL, Hue L Insulin antagonizes AMP-activated protein kinase activation by ischemia or anoxia in rat hearts, without affecting total adenine nucleotides. FEBS Lett 505: 348-352, 2001. 5. Brandt JM, Djouadi F, Kelly DP Fatty acids activate transcription o f the muscle carnitine palmitoyltransferase I gene in cardiac myocytes v ia the peroxisome proliferator-activated receptor alpha. J Biol Chem 273: 23786-23792, 1998. 6. Campbell F M , Kozak R, Wagner A, Altarejos JY, Dyck JR, Belke DD, Severson DL, Kelly DP, Lopaschuk GD A role for peroxisome proliferator-activated receptor alpha (PPARalpha ) in the control o f cardiac malonyl-CoA levels: reduced fatty acid oxidation rates and increased glucose oxidation rates in the hearts o f mice lacking PPARalpha are associated with higher concentrations of malonyl-CoA and reduced expression of 70 malonyl-CoA decarboxylase. J Biol Chem 277: 4098-4103, 2002. 7. Depre C, Young M E , Ying J, Ahuja HS, Han Q, Garza N, Davies PJ, Taegtmeyer H Streptozotocin-induced changes in cardiac gene expression in the absence of severe contractile dysfunction. J Mol Cell Cardiol 32: 985-996, 2000. 8. Dyck JR, Barr AJ, Barr RL, Kolattukudy PE, Lopaschuk GD Characterization of cardiac malonyl-CoA decarboxylase and its putative role in regulating fatty acid oxidation. Am J Physiol 215: H2122-2129, 1998. 9. Finck BN, Lehman JJ, Leone TC, Welch MJ, Bennett MJ, Kovacs A, Han X, Gross RW, Kozak R, Lopaschuk GD, Kelly DP The cardiac phenotype induced by PPARalpha overexpression mimics that caused by diabetes mellitus. J Clin Invest 109: 121-130,2002. 10. Ghosh S, Qi D, An D, Pulinilkunnil T, Abrahani A, Kuo K H , Wambolt RB, Allard M , Innis SM, Rodrigues B Br ief episode of STZ-induced hyperglycemia produces cardiac abnormalities in rats fed a diet rich in n-6 P U F A . Am J Physiol Heart Circ Physiol 287: H2518-2527, 2004. 11. Ibrahimi A, Bonen A, Blinn WD, Hajri T, Li X, Zhong K, Cameron R, Abumrad NA Muscle-specific overexpression o f FAT/CD36 enhances fatty acid oxidation by contracting muscle, reduces plasma triglycerides and fatty acids, and increases plasma glucose and insulin. J Biol Chem 274: 26761-26766, 1999. 12. Kliewer SA, Sundseth SS, Jones SA, Brown PJ, Wisely GB, Koble CS, Devchand P, Wahli W, Willson T M , Lenhard JM, Lehmann J M Fatty acids and eicosanoids 71 regulate gene expression through direct interactions with peroxisome proliferator-activated receptors alpha and gamma. Proc Natl Acad Sci USA 94: 4318-4323, 1997. 13. Kovacic S, Soltys CL, Barr AJ, Shiojima I, Walsh K, Dyck JR Akt activity negatively regulates phosphorylation of AMP-activated protein kinase in the heart. J Biol Chem 278: 39422-39427, 2003. 14. Kudo N, Gillespie JG, Kung L, Witters LA, Schulz R, Clanachan AS, Lopaschuk GD Characterization of 5'AMP-activated protein kinase activity in the heart and its role in inhibiting acetyl-CoA carboxylase during reperfusion following ischemia. Biochim Biophys Acta 1301: 67-75, 1996. 15. Luiken JJ, Arumugam Y, Bell RC, Calles-Escandon J, Tandon NN, Glatz JF, Bonen A Changes in fatty acid transport and transporters are related to the severity of insulin deficiency. Am J Physiol Endocrinol Metab 283: E612-621, 2002. 16. Luiken JJ, Coort SL, Willems J, Coumans WA, Bonen A, van der Vusse GJ, Glatz JF Contraction-induced fatty acid translocase/CD36 translocation in rat cardiac myocytes is mediated through AMP-activated protein kinase signaling. Diabetes 52: 1627-1634, 2003. 17. Martin G, Schoonjans K, Lefebvre A M , Staels B, Auwerx J Coordinate regulation of the expression of the fatty acid transport protein and acyl-CoA synthetase genes by PPARalpha and PPARgamma activators. JBiol Chem 272: 28210-28217, 1997. 18. Randle PJ, Garland PB, Hales CN, Newsholme EA The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1: 72 785-789, 1963. 19. Rodrigues B,McNeill JH The diabetic heart: metabolic causes for the development of a cardiomyopathy. Cardiovasc Res 26: 913-922, 1992. 20. Sakamoto J, Barr RL, Kavanagh K M , Lopaschuk GD Contribution of malonyl-CoA decarboxylase to the high fatty acid oxidation rates seen in the diabetic heart. Am J Physiol Heart Circ Physiol 278: H I 196-1204, 2000. 21. Sambandam N, Abrahani MA, St Pierre E, Al-Atar O, Cam M C , Rodrigues B Localization of lipoprotein lipase in the diabetic heart: regulation by acute changes in insulin. Arterioscler Thromb Vase Biol 19: 1526-1534,1999. 22. Shibata R, Ouchi N, Ito M, Kihara S, Shiojima I, Pimentel DR, Kumada M, Sato K, Schiekofer S, Ohashi K, Funahashi T, Colucci WS, Walsh K Adiponectin-mediated modulation of hypertrophic signals in the heart. Nat Med 10: 1384-1389, 2004. 23. Varanasi TJ, Chu R, Huang Q, Castellon R, Yeldandi AV, Reddy JK Identification of a peroxisome proliferator-responsive element upstream of the human peroxisomal fatty acyl coenzyme A oxidase gene. J Biol Chem 271: 2147-2155, 1996. 24. Vega RB, Huss JM, Kelly DP The coactivator PGC-1 cooperates with peroxisome proliferator-activated receptor alpha in transcriptional control o f nuclear genes encoding mitochondrial fatty acid oxidation enzymes. Mol Cell Biol 20: 1868-1876, 2000. 25. Wall SR,Lopaschuk GD Glucose oxidation rates in fatty acid-perfused isolated working hearts from diabetic rats. Biochim Biophys Acta 1006: 97-103, 1989. 73 3 . The metabolic 'switch' AMPK regulates cardiac heparin-releasable LPL 3.1. Introduction Cardiac tissue acquires most of its energy from metabolism of two major substrates, glucose and F A (33). Entry of glucose into the heart is largely insulin dependent and once inside, it is converted into pyruvate, acetyl-CoA is produced and A T P is ultimately generated (31). Compared to glucose, F A is the preferred substrate consumed by the heart, contributing approximately 70% A T P when supplied at physiological levels. F A is transported into mitochondria as acyl-CoA, which undergoes P-oxidation to release acetyl-CoA. In addition to entering the T C A cycle to produce ATP, acetyl-CoA can also be converted to malonyl-CoA under the influence of A C C (11). Ma lony l -CoA is an allosteric inhibitor of CPT-1 , the enzyme that manages the transport of long-chain fatty acyl-CoA from the cytosol to the mitochondria, where they are oxidized. The heart has no measurable potential to synthesize F A and thus is dependent upon exogenous supply, a process that is accelerated by transporters like fatty acid transport protein, CD36 and fatty acid binding protein plasma membrane (24). Exogenous sources of F A include the plasma free F A fraction or F A released during hydrolysis of TG-r ich lipoproteins; the latter is considered to be the principal source of F A for cardiac utilization (2,41). L P L is the rate-limiting enzyme for lipoprotein T G breakdown. L P L synthesized in cardiomyocytes is secreted as an active enzyme and binds to myocyte cell surface HSPG. Subsequently, the enzyme is translocated onto comparable H S P G binding sites on the luminal side of the vessel wall where T G lipolysis occurs (6,7,38,39). 74 The "fuel gauge" A M P K regulates cellular metabolism (14). During metabolic stresses associated with energy depletion like ischemia (when manufacture of A T P is hindered) or exercise (when A T P expenditure is augmented), changes in intracellular A M P / A T P levels promote Threonine (Thrl72) phosphorylation and activation of A M P K , an important regulator of both l ipid and carbohydrate metabolism (13,17). Once stimulated, A M P K switches off energy consuming processes like T G and protein synthesis whereas A T P generating mechanisms are turned on. Thus, in heart and skeletal muscle, phosphorylated A M P K stimulates glucose uptake by inducing G L U T 4 recruitment to the plasma membrane (16,21,35) and subsequent glycolysis through activation of 6-phosphofructo-2-kinase (25). More importantly, through its control of A C C , A M P K facilitates F A utilization. A s A C C catalyzes the conversion of acetyl-CoA to malonyl-CoA, A M P K by inhibiting A C C is able to decrease malonyl -CoA and minimize its inhibition of F A oxidation (19,20). The majority o f studies examining A M P K regulation of F A utilization have focused on F A oxidation. More recently, A M P K has also been implicated in F A delivery to cardiomyocytes through its regulation of CD36 (23). Given the importance of L P L in providing hearts with F A (2,41), the objective of the present study was to investigate i f activation of A M P K influences L P L at its functionally relevant location, the coronary lumen. We demonstrate that following A M P K phosphorylation, heparin-releasable L P L activity is amplified providing an additional mechanism whereby this metabolic "switch" could regulate cellular energy. 75 3.2. Materials and Methods 3.2.1. Experimental animals The investigation conforms to the guide for the care and use of laboratory animals published by the U S National Institutes of Health and the University of British Columbia. Adult male Wistar rats (220-240 g) were obtained from the U B C Animal Care Unit and supplied with a standard laboratory diet (PMI Feeds, Richmond, V A ) , and water ad libitum. Where indicated, some rats were fasted for 16 hours (6 P M to 10 A M ) . During fasting, food was withdrawn from the animals, but they had free access to water. 3.2.2. Isolated heart perfusion Hearts were isolated and perfused as described previously (32). Briefly, rats were anesthetized with 65 mg/kg sodium pentobarbital and the hearts carefully excised. Rats were not injected with heparin before ki l l ing because it displaces L P L bound to H S P G on the capillary endothelium. Consequently, it was necessary to cannulate the heart quickly to avoid clotting o f blood in the coronary arteries. Following cannulation of the aorta, hearts were secured by tying below the innominate artery and perfused retrogradely with Krebs-Henseleit 4-(2-hydroxyethyl)-l-piperazine-ethanesul-phonic acid (HEPES) buffer containing 10 m M glucose (pH 7.4). Perfusion fluid was continuously gassed with 95% 02/5%) CO2. The rate of coronary flow (7-8 ml/min) was controlled by a peristaltic pump. 3.2.3. Coronary lumen LPL activity To measure coronary endothelium-bound L P L , the perfusion solution was changed to buffer containing 1% fatty acid free B S A and heparin (5 U/ml) . This concentration of 76 heparin can maximally release cardiac L P L from its H S P G binding sites. The coronary effluent (perfusate that drips down to the apex of the heart) was collected in timed fractions (10 sec) over 5 or 10 min where indicated, and assayed for L P L activity by measuring the hydrolysis of a sonicated [ H] triolein substrate emulsion (34). Retrograde perfusion of whole hearts with heparin results in a discharge of L P L that is rapid (within 0.5 to 1 min; suggested to represent L P L located at or near the endothelial cell surface) followed by a prolonged slow release (that is considered to originate from the myocyte cell surface) (32). A s we were primarily concerned with examining A M P K regulation of L P L at coronary lumen, only peak L P L activities are illustrated. L P L activity is expressed as nanomoles oleate released per hour per milliliter. Subsequent to L P L displacement with heparin, ' hearts were rapidly removed, washed with Krebs buffer, frozen in l iquid nitrogen, and stored at - 8 0 ° C for Western Blot assay of A M P K . 3.2.4. Western Blotting for AMPK A M P K phosphorylation increases its activity approximately 50-100 fold. To determine total and phosphorylated A M P K - o c , whole cell homogenates were isolated as described previously (1). Briefly, hearts were ground under l iquid nitrogen and 50 mg homogenized. After centrifugation at 5,000 g for 20 min, the protein content of the supernatant was quantified using a Bradford protein assay. Samples were diluted, boiled with sample loading dye, and 50 u.g used in SDS-polyacrylamide gel electrophoresis. After transfer, membranes were blocked in 5% skim milk in Tris-buffered saline containing 0.1% Tween-20. Membranes were incubated either with rabbit A M P K - a or phosopho-AMPK 77 (Thr-172) antibody, and subsequently with secondary goat anti-rabbit HRP-conjugated antibody, and visualized using an ECL®detect ion kit. 3.2.5. Measurement of cardiac LPL expression L P L gene expression was measured in the indicated groups using R T - P C R . Briefly, total R N A from hearts (100 mg) was extracted using Trizol (Invitrogen). After spectrophotometric quantification and resolving of R N A integrity using a formaldehyde agarose gel, reverse transcription was carried out using an oligo (dT) primer and superscript II R T (Invitrogen). c D N A was amplified using L P L ( N M O 1 2 5 9 8 ) specific primers (8); 5' - A T C C A G C T G G G C C T A A C T T T - 3 ' (left) and 5 ' - A A T G G C T T C T C C A A T G T T G C - 3 ' (right). The p-actin gene (NM_031144) was amplified as an internal control using 5' - T G G T G G G T A T G G G T C A G A A G G - 3 ' (left) and 5 ' - A T C C T G T C A G C G A T G C C T G G G - 3 ' (right). The linear range was found to be between 15-30 cycles. The amplification parameters were set at: 94°C for 1 min, 58°C for 1 min and 72°C for 1 min, for a total o f 30 cycles. The P C R products were electrophoresed on a 1.7% agarose gel containing ethidium bromide. Expression levels were represented as the ratio of signal intensity for L P L m R N A relative to p-actin m R N A . 3.2.6. Measurement of LPL protein and activity in cardiomyocytes A s endothelial cells cannot synthesize L P L , it is manufactured and processed in myocytes and then translocated across the interstitial space onto H S P G binding sites on the luminal surface of endothelial cells (38,39). L P L protein and activity measurement was done using isolated cardiomyocytes. Ventricular calcium-tolerant myocytes were prepared by a 78 previously described procedure (29,30). Cardiac myocytes were suspended at a final cell density of 0.4 x 10 6 cells/mL, medium separated by centrifugation, and L P L protein and activity assayed in cell pellets. For Western Blot analysis, 25 u,g total protein was size fractionated in a SDS-polyacrylamide gel, and blotted onto a nitrocellulose membrane. After blocking overnight at 4°C, the membrane was transferred to a solution of 1:1000 diluted primary antibody (5D2, a monoclonal mouse anti bovine L P L generously provide by Dr. J. Brunzell, University of Washington, Seattle, WA) , and kept for 2 h at room temperature with gentle shaking. After washing with TBS-T, the membrane was treated with 1:3000 diluted secondary antibody (sheep anti-mouse IgG HRP-conjugated) for 1 h at room temperature and visualized using an ECL® detection kit. Measurement of L P L activity in the cell pellet was carried out by a previously described method (32). 3.2.7. Immunolocalization of LPL Hearts were removed and placed in 10% formalin for 24 hours. After formalin-fixation and paraffin embedding, 3 pm sections were cut on silane-coated glass slides. Immunostaining was carried out as described before (30). Briefly, after deparaffinization and rehydration, slides were treated with 5% (v/v) heat-inactivated rabbit plasma to block nonspecific binding. Slides were then incubated with chicken anti-bovine L P L antibody (1:400 dilution) overnight. Following washing with T B S , slides were incubated with biotinylated rabbit anti-chicken IgG (Chemicon Corp., 1:150 dilution) and Streptavidin-conjugated Cy3 fluorescent probe (1:1000 dilution) for 1 hour respectively. 79 Slides were visualized using a Bio-Rad Confocal Microscope. 3.2.8. Treatments Adenine 9-P-D-arabinofuranoside (Ara-A), a precursor of Ara-ATP, is a competitive inhibitor of A M P K . Insulin is also known to inhibit A M P K phosphorylation. Thus, fasted hearts were perfused for 1 hour with A r a - A or insulin, and A M P K phosphorylation and heparin-releasable L P L activity measured. To determine whether promotion of A M P K phosphorylation can influence cardiac L P L , isolated control hearts were perfused with perhexiline (1-10 u M ) , both in the presence or absence of glucose for 45 minutes, and cardiac L P L activity subsequently measured. Perhexiline, an anti-anginal agent, has been described to inhibit CPT-1 and myocardial consumption of F A , and in preliminary experiments, in the absence o f glucose, has been shown to activate the phosphorylation of A M P K . To deplete intracellular A T P and induce metabolic stress and activation of A M P K , oligomycin (1-5 u M ) , an inhibitor of the mitochondrial electron transport chain, was perfused through control hearts for 8-30 minutes, and heparin-releasable cardiac L P L activity subsequently measured. Oligomycin up to 300 f i M has been used previously in cardiomyocytes to activate A M P K (23). 3.2.9. Serum measurements Blood samples were removed from animals and centrifuged immediately to collect serum that was stored at -20°C until assayed. Diagnostic kits were used to measure glucose, T G (Sigma), and non-esterified fatty acid (Wako). 80 3.2.10. Statistical analysis Wherever appropriate, one-way ANOVA followed by the Tukey or Bonferroni tests or the unpaired and paired Student's Mest was used to determine differences between group mean values (as indicated in the specific figure legends). The level of statistical significance was set at P< 0.05. 3.2.11. Materials Oligomycin, adenine 9-beta-D-arabinofuranoside (Ara-A) and perhexiline were purchased from Sigma. Total AMPK-a and Phospho-AMPK-a antibodies were obtained from Cell Signaling (Beverly, MA). ECL® detection kit was obtained from Amersham. 81 3.3. Results 3.3.1. Fasting influences cardiac AMPK phosphorylation Previous studies have reported A M P K phosphorylation in the liver from fasted animals (43). In the present study, overnight (16 h) fasting reduced serum glucose and triglyceride levels, with no affect on F F A (Table 3-1). Following fasting, an approximately 3-fold increase of A M P K phosphorylation was observed on immediate removal of the heart (Fig. 3-1). Interestingly, perfusion with Krebs buffer for 1 hour further augmented A M P K phosphorylation, but only in hearts from fasted animals (Fig. 3-1). 3.3.2. Augmentation of heparin-releasable LPL persists in vitro in fasted hearts A M P K is known to promote cardiac F A oxidation (37). We considered whether the increase in A M P K phosphorylation is also related to increased F A delivery to the heart via L P L action at its functionally relevant location, the coronary lumen. Results from this and a previous study (36) showed that overnight fasting for 16 hours caused a 2.5-fold increase in L P L activity at the coronary lumen (Fig. 3-2A, left panel). Immunohistochemical studies of myocardial sections were performed to complement our observation that the augmented L P L in fasted hearts was mainly localized at the endothelial cells. Whereas L P L immunofluorescence was found throughout the control and fasted myocardium, capillary blood vessels in the fasted heart demonstrated a more intense L P L immunoreactivity compared with control (arrows, F ig . 3-2B). Overall, our data suggest a correlation between A M P K phosphorylation and amplified endothelial L P L in fasted hearts. 82 To determine whether this increase in L P L is a consequence o f changes in gene expression, L P L m R N A was measured. Interestingly, changes in luminal L P L activity were independent of shifts in m R N A levels (Fig. 3-3A). Additionally, given that following fasting, L P L protein (Fig. 3-3B) and activity (Fig. 3-3C) remain unchanged in cardiomyocytes, the predominant cel l type in the heart responsible for L P L synthesis and processing, our data suggest that L P L increase at the coronary lumen is likely via posttranslational mechanisms. Given that A M P K phosphorylation increased further in fasted hearts perfused for 1 hour, we evaluated whether there would be an additional increase or maintenance of L P L over this period, in vitro. Thus, hearts from control and fasted animals were perfused with normal Krebs buffer (minus heparin) for 1 hour followed by heparin perfusion for 10 min. Interestingly, following the one-hour perfusion, fasted hearts demonstrated no further increase in L P L activity (Fig. 3-2A, right panel). Abi l i ty of these hearts to maintain their high heparin-releasable L P L in vitro suggested either an increased recruitment from myocytes or decreased displacement from the coronary lumen. To evaluate this further, during the 60 min perfusion with heparin free buffer in the recirculating mode, L P L activity in the buffer reservoir (total volume 30 ml) was determined at various intervals. Interestingly, fasted hearts showed greater basal release of L P L throughout the perfusion (Fig. 3-2, inset). 3.3.3. Inhibition ofAMPK phosphorylation lowers cardiac LPL A s in vitro, hearts from.fasted animals maintain an augmented A M P K phosphorylation and 83 L P L activity, we hypothesized that inhibition of A M P K phosphorylation should decrease L P L activity. A r a - A , a precursor of Ara-ATP, is a competitive inhibitor of A M P K (27). Perfusion of fasted hearts for 1 hour with A r a - A decreased A M P K phosphorylation (Fig. 3-4A), had no affect on the augmented basal enzyme release (1.6 fold higher than control at 60 minutes), but decreased heparin-releasable L P L activity (Fig. 3-4B). Because insulin is also known to inhibit A M P K phosphorylation (18), fasted hearts were perfused for 1 hour with insulin. Similar to A r a - A , insulin reduced both A M P K phosphorylation (Fig. 3-4A) and luminal L P L activity (Fig. 3-4B). 3.3.4. Promotion of AMPK phosphorylation in control hearts recruits LPL to the luminal surface. In the presence of glucose, perfusion of isolated control hearts with perhexiline did not change either A M P K phosphorylation (Fig. 3-5A) or L P L activity (Fig. 3-5B). However, removal of glucose from the perfusion buffer significantly augmented both A M P K phosphorylation and luminal L P L activity (Fig. 3-5A and B) . With oligomycin, there was a rapid and concentration dependent increase in both A M P K phosphorylation (Fig. 3-6A), and coronary luminal L P L (Fig. 3-6B). Both perhexiline and oligomycin were without effect on L P L m R N A , and protein or activity in cardiomyocytes (data not shown). 84 3.4. Discussion A M P K is the switch that activates pathways that produce A T P while turning off ATP consuming processes (13,26). Thus during fasting, when glucose supply is inadequate and its oxidation is compromised due to P D K 4 overexpression (28,40), A M P K activity is augmented in the liver (37). The present study for the first time demonstrates that fasting also increases cardiac A M P K phosphorylation. A s activation o f A M P K is known to exaggerate F A utilization in the ischemic reperfused heart (19), our results suggest that a similar relationship between A M P K activity and F A oxidation must also exist following fasting. Indeed, rates of cardiac oleate oxidation do increase following fasting (45). Given the role of A M P K in regulating F A oxidation, it is conceivable that it could also influence F A delivery. Provision of F A to the heart involves: a) release from adipose tissue and transport to the heart (22), b) breakdown of endogenous cardiac T G stores, c) internalization of whole lipoproteins, and d) hydrolysis of circulating TG-r i ch lipoproteins to F A by lipoprotein lipase ( L P L ) positioned at the endothelial surface of the coronary lumen (7), and A M P K has been implicated in some of these processes. Thus, activation of A M P K is known to augment F A uptake in the contracting isolated cardiomyocyte through the recruitment o f fatty acid transporters to the plasma membrane (23). Moreover, although its role in cardiac T G lipolysis is unknown, at least in adipocytes, A M P K is suggested to mediate the lipolytic effect of adrenergic stimulation (44). Given that L P L mediated hydrolysis o f lipoproteins was recently suggested to be the principal source of F A for cardiac utilization (2,41), we examined its relationship to A M P K activity. In 85 agreement with previous studies, we observed an increase o f heparin-releasable L P L activity following overnight fasting, and immunostaining revealed that most of the L P L protein was located at the coronary lumen. Our data suggests that A M P K phosphorylation may play a role in increasing cardiac functional L P L . A s changes in luminal L P L activity were independent of shifts in L P L m R N A or alterations in protein and activity in cardiomyocytes, our data imply that this L P L increase at the coronary lumen is likely via posttranslational mechanisms. Other studies have also established that the fasting-induced changes in cardiac L P L activity are likely through posttranslational mechanisms that do not involve alterations in m R N A levels, protein synthesis or specific activity o f the protein (10). Moreover, at least in adipose tissue, down-regulation of L P L during fasting is also post-translational, and involves a shift from active to inactive forms of the lipase (5). To further examine this relationship, hearts from fasted animals were perfused with Krebs buffer for 1 hour (in the absence of heparin), and heparin-releasable L P L activity subsequently determined. After the 1 hour perfusion, only fasted hearts showed further increase in A M P K phosphorylation. Given that the fasted heart prefers F A as an energy substrate for A T P generation, it is likely that this added effect on A M P K phosphorylation is due to lack of albumin-bound F A and circulating lipoproteins in the perfusate. It should be noted that even though A M P K phosphorylation increased with time in vitro, heparin releasable L P L activity at the coronary lumen did not expand further. A s previous studies have demonstrated that the coronary lumen of the rat heart has a finite number of L P L binding sites and that under normal conditions only a fraction of these binding sites are 86 occupied by L P L (29), our present data is indicative of a model which suggests that once A M P K activation fills these sites (following overnight fasting) with the enzyme, no further increase of L P L is possible. Interestingly, throughout the 1-hour perfusion, fasted hearts showed greater basal release of L P L . Given that this increased basal L P L release was not followed by a decline in heparin releasable L P L activity, our data suggest that in vitro, it is likely that the fasted heart has higher enzyme transfer from the myocyte to the coronary lumen. Following ischemia and reperfusion, cardiac A M P K activity is activated, an effect that is prevented by insulin (3). Additionally, in skeletal muscle, stimulation of A M P K by A I C A R has been shown to be normalized by A r a - A (27). In our study, these agents were effective in lowering A M P K phosphorylation in the fasted heart. More importantly, in these hearts, insulin and A r a - A also reduced the endothelial bound L P L pool. Our data suggest that in vitro, the fasted heart is able to maintain its high L P L activity through A M P K , and inhibition of A M P K phosphorylation can lower this high enzyme activity. A more direct examination o f the relationship between A M P K and L P L was realized using compounds that activate A M P K . One mechanism by which A M P K is stimulated includes modulation of intracellular A M P concentrations. It should be noted that as the effect of A M P can be antagonized by high concentrations of ATP, a higher A M P / A T P ratio is more efficient in activating A M P K than a rise in A M P alone (15). Another method that can induce A M P K activation is through inhibition of A T P production that in turn increases intracellular generation of A M P (12). Perhexiline, a CPT-1 blocker that is widely used in 87 the treatment of ischemia, inhibits F A oxidation and switches the heart to utilize glucose. Given that F A is a major substrate contributing approximately 70% A T P in heart, we predicted that perhexiline would activate A M P K through impaired A T P generation. However, perfusion o f control hearts with perhexiline was without effect on cardiac A M P K or L P L activity, suggesting that metabolic switching to utilize glucose may be sufficient to prevent A T P depletion. To test this idea, glucose was removed from our perfusion media when using perhexiline. Interestingly, this adjustment increased A M P K phosphorylation and was able to recruit L P L to the endothelial cell. Oligomycin, an A T P synthase inhibitor, has been shown to markedly change the A M P / A T P ratio and A M P K activity in isolated perfused hearts (25) and cardiomyocytes (23). In our study, oligomycin increased A M P K phosphorylation and heparin-releasable L P L activity in a dose-dependent manner. Given the rapid (8 minutes) augmentation of L P L following oligomycin, and in the absence of any change in L P L m R N A , or protein and activity in cardiomyocytes, our data suggest that the regulation of L P L by A M P K is likely through posttranslational mechanisms. One such mechanism could include increase in secretion of L P L from the myocyte to the coronary lumen. In this regard, despite the high basal release of L P L to the medium from isolated fasted hearts, heparin-releasable L P L activity remained high, suggesting augmented L P L transfer from myocyte to coronary lumen. However, even though enzyme transfer may have increased, L P L protein and activity remain unchanged in myocytes from fasted animals suggesting that reduced intracellular lysosomal degradation may also be occurring concomitantly. In adipocytes, stimulation of L P L secretion is known to reduce enzyme 88 degradation (9,42). Additionally, A T P depletion in C H O cells following incubation with 2-deoxy-D-glucose drastically reduces L P L degradation rate (4). A t present, the target for A M P K phosphorylation that controls L P L secretion and degradation is not known. In summary, using fasting and modulators of A M P K , a strong correlation between this metabolic switch and cardiac L P L activity was established. Given the rapidity by which oligomycin duplicates the above relationship, our data suggest that in addition to its direct role in promoting F A oxidation, increased A M P K recruitment of L P L from its major storage site, the cardiomyocyte, to the coronary lumen, and decreased intracellular degradation could represent an immediate compensatory response by the heart to guarantee F A supply (Fig. 3-7). 89 3.5. Tables and figures Table 3-1. General characteristics of the animals Control Fasted Body Weight (g) 243 ± 3 236 ± 4 Serum Glucose (mM) 6.1 ± 0 . 2 3.5 ± 0 . 2 * Serum FFA (mM) 0.67 ± 0 . 1 0 0.53 ± 0.04 Serum Triglyceride (mM) 1.81 ± 0 . 0 7 0.53 ± 0.04 * Values are mean±SE for 6 animals. Following an overnight fast, animals were killed, blood collected and serum separated for measurement of various parameters. * Significantly different from control, P < 0.05. 90 Control Fasted Control Fasted Phospho-AMPK-a • - ,,.„„„„.. .,,,..,-.,„„ . m 1 1 1 m,n„« I 1 Control Fasted Fig. 3-1 Effect of fasting on cardiac AMPK phosphorylation Following fasting for 16 hours, total or phosphorylated A M P K were determined, either immediately upon removal of the heart, or following a 1 hour perfusion with Krebs buffer. Total and phospho-AMPK-a were measured using Western Blotting, and either rabbit A M P K - a or phospho-AMPK (Thr-172) antibodies. Data are means±SE of four different hearts in each group. 'Significantly different from control, "Significantly different from all other groups, P O . 0 5 . 91 Fig. 3-2 Alterations in LPL activity and immunofluorescence in hearts isolated from fasted animals On immediate removal of hearts from control and overnight fasted rats, LPL activity was determined after perfusion with heparin. In a separate experiment, hearts from the two groups were first perfused for 1 h with Kreb's buffer in the recirculating mode (and in the absence of heparin). During the 60 min perfusion, basal L P L activity was determined in the buffer reservoir over time (inset). Subsequently, L P L was displaced by heparin, and activity determined. Results are the means±SE of 4 rats in each group. The lower panel (B) is representative photograph showing the effect of fasting on LPL immunofluorescence as visualized by fluorescent microscopy. Heart sections were fixed, incubated with the polyclonal chicken antibody against bovine L P L followed by incubations with biotinylated rabbit anti-chicken IgG and streptavidin-conjugated Cy3 fluorescent probe respectively. C, control; F, fasted. * Significantly different from control, PO.05. 92 Control Fasted Fig . 3-3 LPL gene expression, protein mass and activity in hearts isolated from fasted animals Rats were fasted for 16 hours; during fasting, food was withdrawn from the animals, but they had free access to water. L P L gene expression in the whole heart was measured using R T - P C R (A). L P L protein (B) and activity (C) measurements were done using isolated cardiomyocytes. Data are means±SE o f three different hearts in each group. 9 3 A Fasted + Ara-A + Insulin Phospho-AMPK-a Control + Ara-A + Insulin + Ara-A + Insulin Fig. 3-4 Consequence of AMPK inhibition on heparin-releasable LPL activity in fasted hearts A M P K phosphorylation in fasted hearts was inhibited using A r a - A (500 uM) or insulin (5 U/ml) perfusion for 1 hour. Subsequently, L P L was displaced by heparin, and activity determined (panel B) , or total and phospho-AMPK-ct measured using Western Blotting (panel A ) . Data are means±SE of four different hearts in each group. 'Significantly different from control, P O . 0 5 . 94 + ~ Glucose - + Perhexiline Phospho-AMPK-a AMPK-a 3.0 - | + + - Glucose - + + Perhexiline B + + — Glucose — -f- + Perhexiline Fig . 3-5 Consequence of CPT-1 inhibition on AMPK phosphorylation and heparin-releasable LPL activity in control hearts Hearts from control animals were perfused either in the presence or absence o f perhexiline (5 m M ) , with or without glucose, for 45 minutes. Subsequently, L P L was displaced by heparin, and activity determined (panel B) , or A M P K measured using Western Blotting (panel A ) . Data are means±SE of four different hearts in each group. 'Significantly different from control, P<0.05. 95 Control 1 3 5 Oligomycin (uM) Control 1 3 5 Oligomycin (pJ\fl) Control 1 3 5 Ol igomycin (pM) Fig. 3-6 Effect of inhibiting ATP synthesis on AMPK phosphorylation and heparin-releasable LPL activity Hearts from control animals were perfused either in the presence or absence of oligomycin (1-5 uM) for 8 minutes. Subsequently, LPL was displaced by heparin, and activity determined (panel B), or AMPK measured using Western Blotting (panel A). Data are means±SE of four different hearts in each group. Significantly different from control, P<0.05. 96 LPL expression t Translation and glycosylation t Processing t Vesicle *- I— AMPK j Degradation Blood vessel Interstitial space Cardiomyocyte Fig. 3-7 Regulation of cardiac LPL by AMPK Following synthesis and processing of LPL in myocytes, the enzyme is either transported to lysosomes for degradation, or delivered onto cell-surface HSPG binding sites. From here, LPL is transferred onto similar binding sites on the luminal surface of endothelial cells. At this location, the enzyme hydrolyzes the triglyceride core of circulating lipoproteins to FFA's, which are then transported to the heart for ATP generation. During metabolic stress, AMPK-mediated recruitment of LPL to the coronary lumen could represent an immediate compensatory response by the heart to guarantee FA supply. One such mechanism by which AMPK does this is through increased secretion/transfer of LPL from the myocyte to the coronary lumen. Additionally, AMPK may potentially reduce intracellular lysosomal degradation. 97 3.6. Bibliography 1. Atkinson L L , Fischer MA, Lopaschuk GD. Leptin activates cardiac fatty acid oxidation independent of changes in the AMP-activated protein kinase-acetyl-CoA carboxylase-malonyl-CoA axis. JBiol Chem 277:29424-29430, 2002. 2. Augustus AS, Kako Y, Yagyu H, Goldberg IJ. Routes of F A delivery to cardiac muscle: modulation of lipoprotein lipolysis alters uptake of TG-derived F A . Am J Physiol 284:331-339,2003. 3. Beauloye C, Marsin AS, Bertrand L, Krause U, Hardie DG, Vanoverschelde JL, Hue L . Insulin antagonizes AMP-activated protein kinase activation by ischemia or anoxia in rat hearts, without affecting total adenine nucleotides. FEBS Lett 505:348-352, 2001. 4. Ben-Zeev O, Mao HZ, Doolittle MH. Maturation of lipoprotein lipase in the endoplasmic reticulum. Concurrent formation of functional dimers and inactive aggregates. JBiol Chem 277:10727-38, 2002. 5. Bergo M , Wu G, Ruge T, Olivecrona T. Down-regulation o f adipose tissue lipoprotein lipase during fasting requires that a gene, separate from the lipase gene, is switched on. J B i o l Chem. 277:11927-11932, 2002. 6. Blanchette-Mackie EJ, Dwyer NK, Amende LA. Cytochemical studies of lipid metabolism: immunogold probes for lipoprotein lipase and cholesterol. Am J Anat 185:255-263,1989. 98 7. Blanchette-Mackie EJ, Masuno H, Dwyer NK, Olivecrona T, Scow RO. Lipoprotein lipase in myocytes and capillary endothelium o f heart: immunocytochemical study. Am J Physiol 256:818-828, 1989. 8. Brault D, Noe L , Etienne J, Hamelin J, Raisonnier A, Souli A, Chuat JC, Dugail I, Quignard-Boulange A, Lavau M , Galibert, F. Sequence of rat lipoprotein lipase-encoding c D N A . Gene 121:237-46,1992. 9. Cupp M , Bensadoun A, Melford K. Heparin decreases the degradation rate of lipoprotein lipase in adipocytes. J B i o l Chem. 262:6383-6388, 1987. 10. Doolittle M H , Ben-Zeev O, Elovson J, Martin D, Kirchgessner T G The response of lipoprotein lipase to feeding and fasting. Evidence for posttranslational regulation. J B i o l Chem.265:4570-4577, 1990. 11. Dyck JR, Barr AJ, Barr RL, Kolattukudy PE, Lopaschuk GD. Characterization of cardiac malonyl-CoA decarboxylase and its putative role in regulating fatty acid oxidation. Am J Physiol 275:2122-2129, 1998. 12. Frederich M , Balschi JA. The relationship between AMP-act ivated protein kinase activity and A M P concentration in the isolated perfused rat heart. J Biol Chem 277:1928-1932, 2002. 13. Hardie DG, Carling D. The AMP-activated protein kinase-fuel gauge of the mammalian cell?" Eur J Biochem 246:259-273, 1997. 14. Hardie DG, Hawley SA. AMP-activated protein kinase: the energy charge hypothesis revisited. Bioessays 23: 1112-1119, 2001. 99 15. Hardie DG. Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endocrinology 144:5179-5183, 2003. 16. Hayashi T, Hirshman MF, Fujii N, Habinowski SA, Witters LA, Goodyear LJ . Metabolic stress and altered glucose transport: activation of AMP-activated protein kinase as a unifying coupling mechanism. Diabetes 49:527-531, 2000. 17. Hong SP, Leiper FC, Woods A, Carling D, Carlson M . Activation of yeast Snfl and mammalian AMP-activated protein kinase by upstream kinases. Proc Natl Acad Sci USA 100:8839-8843,2003. 18 Kovacic S, Soltys CL, Barr AJ, Shiojima I, Walsh K, Dyck JR. Ak t activity negatively regulates phosphorylation of AMP-activated protein kinase in the heart. J Biol Chem 278:39422-39427, 2003. 19. Kudo N, Barr AJ, Barr RL, Desai S, Lopaschuk GD. H igh rates of fatty acid oxidation during reperfusion of ischemic hearts are associated with a decrease in malonyl-CoA levels due to an increase in 5'-AMP-activated protein kinase inhibition of acetyl-CoA carboxylase. JBiol Chem 270:17513-17520, 1995. 20. Kudo N, Gillespie JG, Kung L, Witters LA, Schulz R, Clanachan AS, Lopaschuk GD. Characterization of 5'AMP-activated protein kinase activity in the heart and its role in inhibiting acetyl-CoA carboxylase during reperfusion following ischemia. Biochim Biophys Acta 1301:67-75, 1996. 100 21. Kurth-Kraczek EJ, Hirshman MF, Goodyear L J , Winder WW. 5' AMP-activated protein kinase activation causes G L U T 4 translocation in skeletal muscle. Diabetes 48:1667-1671, 1999. 22. Lopaschuk GD, Belke DD. Gamble J, Itoi T, Schonekess BO. Regulation o f fatty acid oxidation in the mammalian heart in health and disease. Biochim Biophys Acta nu-.iei-ne, 1994. 23. Luiken JJ, Coort SL, Willems J, Coumans WA, Bonen A, van der Vusse GJ, Glatz JF. Contraction-induced fatty acid translocase/CD36 translocation in rat cardiac myocytes is mediated through AMP-activated protein kinase signaling. Diabetes 52:1627-1634, 2003. 24. Luiken JJ, Turcotte LP, Bonen A. Protein-mediated palmitate uptake and expression of fatty acid transport proteins in heart giant vesicles. J Lipid Res 40:1007-1016, 1999. 25. Marsin AS, Bertrand L, Rider MH, Deprez J, Beauloye C, Vincent MF, Van den Berghe G, Carling D, Hue L. Phosphorylation and activation o f heart P F K - 2 by A M P K has a role in the stimulation of glycolysis during ischemia. Curr Biol 10:1247-1255, 2000. 26. Muoio DM, Seefeld K, Witters LA, Coleman RA. AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target. Biochem J 338:783-791, 1999. 101 27. Musi N, Hayashi T, Fujii N, Hirshman MF, Witters LA, Goodyear LJ . AMP-activated protein kinase activity and glucose uptake in rat skeletal muscle. Am J Physiol 280:677-684, 2001. 28. Pilegaard H, Saltin B, Neufer PD. Effect of short-term fasting and refeeding on transcriptional regulation o f metabolic genes in human skeletal muscle. Diabetes 52:657-662, 2003. 29. Pulinilkunnil T, Abrahani A, Varghese J, Chan N, Tang I, Ghosh S, Kulpa J, Allard M, Brownsey R, Rodrigues B. Evidence for rapid "metabolic switching" through lipoprotein lipase occupation of endothelial binding sites. J Mol Cell Cardiol. 35:1093-1103,2003. 30. Pulinilkunnil T, Abrahani A, Varghese J, Chan N, Tang I, Ghosh S, Kulpa J, Allard M, Brownsey R, Rodrigues B. Evidence for rapid "metabolic switching" through lipoprotein lipase occupation of endothelial-binding sites. J M o l Ce l l Cardiol. 35:1093-103,2003. 31. Randle PJ, Hales CN, Garland PB, Newsholme EA. The glucose fatty acid cycle: its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1: 785-789,1963. 32. Rodrigues B, Cam M C , Jian K, Lim F, Sambandam N, Shepherd G. Differential effects of streptozotocin-induced diabetes on cardiac lipoprotein lipase activity. Diabetes 46:1346-1353, 1997. 102 33. Rodrigues B, McNeill, JH. The diabetic heart: metabolic causes for the development of a cardiomyopathy. Cardiovasc Res 26: 913-922, 1992. 34. Rodrigues B, Spooner M , Severson DL. Free fatty acids do not release lipoprotein lipase from isolated cardiac myocytes or perfused hearts. Am J Physiol 262:216-223, 1992. 35. Russell RR 3rd, Bergeron R, Shulman GI, Young L H . Translocation of myocardial GLUT-4 and increased glucose uptake through activation of A M P K by AICAR. Am J Physiol 277:643-649, 1999. 36. Sambandam N, Abrahani MA, St Pierre E, Al-Atar O, Cam MC, Rodrigues B. Localization of lipoprotein lipase in the diabetic heart: regulation by acute changes in insulin. Arterioscler Thromb Vase Biol 19:1526-1534, 1999. 37. Sambandam N, Lopaschuk GD. AMP-activated protein kinase (AMPK) control of fatty acid and glucose metabolism in the ischemic heart. Prog Lipid Res 42:238-256, 2003. 38. Saxena U, Klein MG, Goldberg IJ. Transport of lipoprotein lipase across endothelial cells. Proc Natl Acad Sci USA 88:2254-2258, 1991. 39. Stins MF, Maxfield FR, Goldberg IJ. Polarized binding of lipoprotein lipase to endothelial cells. Implication for its physiological actions. Arterioscler Thromb 12:1437-1446, 1992. 40. Sugden M C , Greenwood GK, Smith ND, Holness MJ. Peroxisome-proliferator-activated receptor-alpha (PPARalpha) deficiency leads to 103 dysregulation of hepatic l ipid and carbohydrate metabolism by fatty acids and insulin. Biochem 7364:361-368, 2002. 41. Teusink B, Voshol PJ, Dahlmans VE, Rensen PC, Pijl H, Romijn JA, Havekes L M . Contribution o f fatty acids released from lipolysis o f plasma triglycerides to total plasma fatty acid flux and tissue-specific fatty acid uptake. Diabetes 52:614-620, 2003. 42. Vannier C, Ailhaud G. Biosynthesis of lipoprotein lipase in cultured mouse adipocytes. II. Processing, subunit assembly, and intracellular transport. J B io l Chem.264:13206-13216,1989. 43. Witters LA, Gao G, Kemp BE, Quistorff B. Hepatic 5'-AMP-activated protein kinase: zonal distribution and relationship to acetyl-CoA carboxylase activity in varying nutritional states. Arch Biochem Biophys 308:413-419, 1994. 44. Yin W, Mu J, Birnbaum MJ. Role of AMP-activated protein kinase in cyclic AMP-dependent lipolysis in 3T3-L1 adipocytes. JBiol Chem 278:43074-43080, 2003. 45. Young ME, Guthrie PH, Razeghi P, Leighton B, Abbasi S, Patil S, Youker KA, Taegtmeyer H. Impaired long-chain fatty acid oxidation and contractile dysfunction in the obese Zucker rat heart. Diabetes 51:2587-2595, 2002. 104 4. ^-adrenergic agonist stimulation produces changes in cardiac AMPK and coronary lumen LPL only during increased workload 4.1. Introduction Cardiac contractility requires an uninterrupted supply . o f ATP. Under normal physiological conditions, the heart utilizes two major substrates, glucose and F A (51). Compared to glucose, F A is the preferred substrate, and accounts for approximately 70% of A T P production. However, the heart has the ability to choose its substrates depending on their availability and the prevailing physiological (e.g., exercise) or pathophysiological (e.g., ischemia) conditions. A M P K is likely a key player in modulating this substrate selection. Thus, during exercise (when A T P expenditure is augmented) or ischemia (when manufacture of A T P is hindered), changes in intracellular A M P / A T P levels promote threonine (Thrl72) phosphorylation and activation o f A M P K (22, 25). Upon stimulation, A M P K switches off energy consuming processes like T G and protein synthesis, whereas A T P generating mechanisms are turned on (21, 26). In heart and skeletal muscle, phosphorylated A M P K stimulates glucose uptake (23, 55) and subsequent glycolysis through the activation of 6-phosphofructo-2-kinase (38). More importantly, through its control of A C C , A M P K facilitates F A oxidation (31,32). A M P K has also been implicated in F A delivery to cardiomyocytes through its regulation of the F A transporter, CD36 (36). Finally, results from our laboratory have demonstrated a strong correlation between activation of cardiac A M P K and increases in coronary lumen L P L activity (2). 105 L P L is a rate-limiting enzyme for hydrolysis of T G rich lipoproteins, thus regulating the supply of F A to meet the metabolic demands of different tissues. It is synthesized in myocytes and subsequently transported onto H S P G binding sites on the myocyte cell surface (13, 15). Through mechanisms that are not completely understood, L P L is then transported onto H S P G binding sites on the luminal surface o f the capillary endothelium (43). A t this location, the enzyme plays a crucial role in hydrolysis o f T G rich lipoproteins to F A , which are transported to the heart and used, either for energy production, or for re-synthesis of TG. Recently, LPL-mediated hydrolysis of circulating T G was suggested to be the principal source o f F A for cardiac utilization (4, 65). In addition to its role as a lipolytic enzyme, L P L also mediates a non-catalytic bridging function that allows it to bind simultaneously to both lipoproteins and specific cell surface proteins, facilitating cellular uptake of lipoproteins (40, 56, 63). Through its role in T G hydrolysis, L P L activity directly affects the level of circulating lipoprotein-TG. For example, in transgenic rabbits with global overexpression of L P L , attenuation of hypertriglyceridemia was observed, an effect suggested to contribute toward amelioration o f insulin resistance and obesity (28). Contrary to systemic overexpression, tissue-specific overexpression of L P L in skeletal muscle and heart is associated with insulin resistance in these tissues as well as severe myopathy, characterized by both muscle fiber degeneration and extensive proliferation of mitochondria and peroxisomes (27, 34, 67). In a more recent study using genetically engineered mice that specifically overexpressed non-transferable cardiomyocyte surface-bound L P L , l ipid oversupply and deposition were 106 observed, along with excessive dilatation and impaired left ventricular systolic function (cardiomyopathy) (69). In vascular smooth muscle, overexpression of L P L brings about F A loading and vascular dysfunction (16). Heart failure, coupled with conditions such as coronary artery disease and hypertension, is often associated with activation of the sympathetic nervous system, and higher levels of circulating catecholamines (14, 17). Although the P-adrenergic agonist isoproterenol had no influence on myocyte cell surface or intracellular LPL(60) , or L P L measured in whole heart homogenates (12), its influence on L P L at cardiac coronary lumen is unknown. Given the importance of L P L in cardiac and vascular pathology, the objective of the present study was to investigate whether P-adrenergic agonist stimulation influences L P L at its functionally relevant location, the coronary lumen. We demonstrate that isoproterenol does indeed augment luminal L P L , likely secondary to an increase in workload and-excessive energy expenditure, rather than a direct effect. 107 4.2. Materials and Methods 4.2.1. Experimental animals The investigation conforms to the guide for the care and use of laboratory animals published by the U S National Institutes of Health and the University of British Columbia. Adult male Wistar rats (220-240 g) were obtained from the U B C Animal Care Unit and supplied with a standard laboratory diet (PMI Feeds, Richmond, V A ) , and water ad libitum. A l l of the experiments were conducted between 9:00 to 13:00 hours. 4.2.2. In vivo hemodynamics Rats were anesthetized with 65 mg/kg sodium pentobarbital, and a cannula inserted into the carotid artery to measure systemic blood pressure and heart rate. Anesthesia is known to cause cardio depression. Following stabilization, animals were either injected i.p. with saline or 10 pg/kg of the P-adrenergic agonist, Isoproterenol (ISO). Blood pressure and heart rate were recorded every 20 min for 1 hr subsequent to ISO. 4.2.3. Isolated working heart perfusion Hearts were isolated and perfused as described previously (1). Briefly, rats were anesthetized using 2-3% halofhane, and the hearts carefully excised. Rats were not injected with heparin before ki l l ing because it displaces L P L bound to H S P G on the capillary endothelium. Consequently, it was necessary to cannulate the heart quickly to avoid clotting of blood in the coronary arteries. Following cannulation of the aorta, hearts were secured by tying below the innominate artery, and perfused retrogradely with Krebs-Henseleit ( K H ) buffer containing 10 m M glucose (as the only substrate), and 2 m M 108 calcium (pH 7.4, 37°C). Perfusion fluid was continuously gassed with 95% 02 /5% C O 2 . After 10 min, hearts were then switched to a working mode (perfusion through the left atria), with a left atrial preload of 11.5 mmHg, and an aortic afterload of 80 mmHg, for 15 min. Heart function (rate pressure product, RPP) was measured using a Direcwin physiograph (Raytech). Where indicated, insulin (100 m U / L ) , palmitate (0.4 m M ) prebound to fatty acid-free albumin, or ISO (10 u M ) were then added (together or separately) to the K H buffer. A t the end of 15 min, heart perfusion was switched from the working mode back to the Langendorff method, and L P L released by the addition of heparin to the K H buffer (heparin-releasable L P L , H R - L P L ) . 4.2.4. Isolated Langendorff heart perfusion Hearts were isolated and perfused as described previously (46, 50). Briefly, rats were anesthetized, and the hearts carefully excised. Following cannulation of the aorta, hearts were perfused retrogradely with K H buffer containing 10 m M glucose (as the only substrate). The rate o f coronary flow (7-8 ml/min) was controlled by a peristaltic pump. Where indicated, ISO (10 u M ) was then added to the K H buffer. 4.2.5. Coronary lumen LPL activity To measure coronary endothelium-bound L P L , the perfusion solution was changed to buffer containing 1% fatty acid free B S A , and heparin (5 U/ml) . This concentration of heparin can maximally release cardiac L P L from its H S P G binding sites (44, 50). The coronary effluent (perfusate that drips down to the apex of the heart) was collected in timed fractions (10 sec) over 5 or 10 min where indicated, and assayed for L P L activity by 109 measuring the hydrolysis of a sonicated [ 3 H] triolein substrate emulsion (52). Retrograde perfusion of whole hearts with heparin results in a discharge of L P L that is rapid (within 0.5 to 1 min; suggested to represent L P L located at or near the endothelial cell surface) followed by a prolonged slow release (considered to represent enzyme that originates from the myocyte cell surface). A s we were primarily concerned with examining regulation of L P L at coronary lumen, only peak L P L activities are illustrated. L P L activity is expressed as nanomoles oleate released per hour per milliliter. Subsequent to L P L displacement with heparin, hearts were rapidly removed, washed with Krebs buffer, frozen in liquid nitrogen, and stored at - 8 0 ° C for Western Blot assay o f A M P K and A C C . 4.2.6. Isolated cardiomyocytes Ventricular calcium-tolerant myocytes were prepared by a previously described procedure (44-46, 50). Briefly, myocytes were made calcium-tolerant by successive exposure to increasing concentrations of calcium. Our method of isolation yields a highly enriched population of calcium-tolerant myocardial cells that are rod-shaped in the presence of 1 m M C a 2 + , with clear cross striations. Intolerant cells are intact but hypercontract into vesiculated spheres. Yie ld of myocytes was determined microscopically using an improved Neubauer hemocytometer. Myocyte viability was assessed as the percentage of elongated cells with clear cross striations that excluded 0.2% trypan blue. Cardiac myocytes were suspended at a final cell density of 0.4x10 6 cells/ml, incubated with or without ISO (10 u M ) at 37°C for 1 hr, and basal L P L activity in the medium and cell pellet (after centrifugation) measured. To release surface-bound L P L activity, heparin (5 U/ml) 110 was then added to the myocyte suspension. After incubation for 10 min, an aliquot of cell suspension was removed, medium separated by centrifugation in an Eppendorf microcentrifuge (1 min, 10,000 g), and assayed for L P L activity. The total cellular L P L activity was measured by sonicating (Vibra Cel l™ sonicator at a frequency of 40 H z for 2x30 s) the cell pellets after resuspending them in 0.2 m l of 50 m M N H 4 C 1 buffer (pH 8.0) containing 0.125% (v/v) Triton X-100. After sonication, the volume was adjusted to 1 ml using a sucrose buffer (0.25 M sucrose, 1 m M E D T A , 1 m M dithiothreitol, 10 m M H E P E S , p H 7.4). Assay for cell sonicate L P L activity was done using 20 pi o f the cell sonicate and heparin (2 U/ml) . 4.2.7. Cardiac LPL gene expression L P L gene expression was measured in the indicated groups using R T - P C R (2, 44). Briefly, total R N A from hearts (100 mg) was extracted using Trizol (Invitrogen). After spectrophotometric quantification and resolving of R N A integrity using a formaldehyde agarose gel, reverse transcription was carried out using an oligo (dT) primer and superscript II R T (Invitrogen). c D N A was amplified using L P L (NM_012598) specific primers (6), 5' - A T C C A G C T G G G C C T A A C T T T - 3 ' (left) and 5 ' - A A T G G C T T C T C C A A T G T T G C - 3 ' (right). The p-actin gene ( N M 031144) was amplified as an internal control using 5' - T G G T G G G T A T G G G T C A G A A G G - 3 ' (left) and 5 ' - A T C C T G T C A G C G A T G C C T G G G - 3 ' (right). The linear range was found to be between 15-30 cycles. The amplification parameters were set at: 94°C for 1 min, 58°C for 1 min and 72°C for 1 min, for a total of 30 cycles. The P C R products were 111 electrophoresed on a 1.7% agarose gel containing ethidium bromide. Expression levels were represented as the ratio of signal intensity for L P L m R N A relative to p-actin m R N A . 4.2.8. Western Blotting for AMPK and ACQ A M P K phosphorylation increases its activity approximately 50-100 fold (53, 54, 58). Activated A M P K phosphorylates and inactivates A C C (53, 54, 58). To determine total and phosphorylated A M P K - a and A C C , whole cell homogenafes were isolated as described previously (3). Briefly, hearts were ground under liquid nitrogen, and 50 mg homogenized. After centrifugation at 5,000 g for 20 min, the protein content of the supernatant was quantified using a Bradford protein assay. Samples were diluted, boiled with sample loading dye, and 50 pg used in SDS-polyacrylamide gel electrophoresis. After transfer, membranes were blocked in 5% skim milk in Tris-buffered saline containing 0.1% Tween-20. Membranes were incubated either with rabbit A M P K - a , phospho-AMPK (Thr-172) or phospho-ACC (Ser-79) antibodies, and subsequently with secondary goat anti-rabbit HRP-conjugated antibodies, and visualized using E C L detection. Measuring the phospho form of A M P K is a surrogate for estimation of its activity. 4.2.9. Separation and characterization of cardiac lipids Total cardiac lipids were extracted and solubilized in chloroform:methanol:acetone:hexane (4:6:1 : lv/v/v/v) . Separation of triglycerides and F A was achieved using H P L C (Waters 2690 Alliance H P L C , Mi l ford , M A ) equipped with an auto-sampler and column heater. F A were quantified as their respective methyl esters using heptadecaenoic acid (17:0) as the 112 internal standard with a Varian 3400 G L C equipped with a flame ionization detector, a Varian Star data system, and a SP-2330 capillary column (30 m x 0.25 mm PA). Value of cardiac F A and triglycerides were expressed as pg/mg protein. 4.2.10. Serum measurements Blood samples were removed from animals and centrifuged immediately to collect serum which was then stored at -20°C until assayed. Diagnostic kits were used to measure glucose, T G (Sigma), and non-esterified fatty acid (Wako). 4.2.11. Statistical analysis Wherever appropriate, one-way A N O V A followed by the Tukey or Bonferroni tests or the unpaired and paired Student's t-test was used to determine differences between group mean values (as indicated in the specific figure legends). The level of statistical significance was set at P < 0.05. 4.2.12. Materials ISO was purchased from Sigma. Total A M P K - a , P h o s p h o - A M P K - a and Phospho-ACC antibodies were obtained from Cel l Signaling (Beverly, M A ) . E C L detection kit was obtained from Amersham. 113 4.3. Results 4.3.1. Coronary luminal LPL activity increases following a single dose of ISO given in vivo Following ISO administration, rate pressure product increased significantly when compared to control (Table 4-1). Changes in plasma parameters following 1 hr of ISO included a significant increase in serum free FA, with no effect on either glucose or triglycerides (Table 4-1). To determine whether ISO controls L P L (at its functionally relevant location, the coronary lumen), hearts isolated from ISO treated animals were perfused retrogradely with heparin. Compared to saline injected rats, there was a substantial increase in L P L activity (-300%) at the vascular lumen following 1 hr of ISO (Fig. 4-1, upper panel). This increase in L P L activity was maintained for an additional 3 hr (Fig. 4-1, upper panel). Notably, alterations in luminal L P L activity were independent of shifts in mRNA levels (Fig. 4-1, lower panel). 4.3.2. ISO injected intraperitonally augments cardiac AMPK and ACC phosphorylation As changes in luminal L P L activity were independent of shifts in L P L mRNA, the LPL increase at the coronary lumen is likely via posttranscriptional mechanisms. Interestingly, upon injection of ISO, the increase in L P L was also associated with phosphorylation of A M P K , both at 1 and 4 hr after administration (Fig 4-2, upper panel). Once activated, A M P K phosphorylates and inactivates A C C . As A C C catalyzes the conversion of acetyl-CoA to malonyl-CoA, A M P K , by phosphorylating A C C , is able to decrease malonyl-CoA and minimize its inhibition of FA oxidation (53, 54, 58). In the heart, 114 ACC280 is the predominant isoform. Following 1 h of ISO, phosphorylation of ACC280 increased; although A C C 2 8 0 phosphorylation appeared higher at 4 hr, no statistical difference could be identified (P=0.0896) (Fig. 4-2, lower panel). Interestingly, ISO induced a time dependent drop in cardiac TG, without affecting cardiac F F A (Table 4-2). 4.3.3. Isoproterenol does not influence LPL activity and AMPK phosphorylation in myocytes or Langendorffperfused hearts Previous studies have shown that incubation o f cardiomyocytes with ISO for 30 minutes had no effect on either basal or H R - L P L activity (60). In the present study, incubation with this p-adrenergic agonist was extended to 1 hr, and no effect was observed on basal (unpublished observation), heparin-releasable (control, 847 ± 94; ISO, 814 ± 155; nmol/hr/10 6 cells), or cell sonicate (control, 2287 ± 317; ISO, 2142 ± 354; nmol/hr/10 6 cells) L P L activity. To determine whether ISO influences L P L in the intact heart, we perfused hearts retrogradely with this p-adrenergic agonist. Retrograde perfused hearts allow for adequate coronary perfusion with limited expenditure o f energy (these hearts do not beat against an afterload). Similar to myocytes, ISO did not change H R - L P L in Langendorff isolated hearts (Fig. 4-3). Overall, our data suggest that specifically in these preparations in vitro, ISO does not have a direct effect in regulating L P L activity. In a recent report, we described that recruitment of L P L to the coronary lumen can be regulated by A M P K (2). The relationship between A M P K phosphorylation and L P L activity was evaluated in isolated myocytes and Langendorff perfused hearts. We demonstrate that 115 ISO did not affect A M P K phosphorylation in both myocytes (as measured by densitometry; control, 0.97 ± 0.02; ISO, 0.88 ± 0.04; units), or retrograde perfused hearts (Fig. 4-3, inset) (as measured by densitometry; control, 1.0 ± 0.1; ISO, 1.03 ± 0.14; units). 4.3.4. Increasing workload promotes phosphorylation of AMPK and ACC and enlarges the coronary lumen LPL pool Given that the ISO induced increase in rate pressure product was associated with phosphorylation of A M P K and A C C , and coronary luminal L P L activity in vivo, and that myocytes or retrograde perfused hearts do not represent a physiological contractile circumstance, we considered whether simply increasing workload by switching from Langendorff to the isolated perfused working heart would induce similar changes. Compared to retrograde perfusion, the working heart perfused only with glucose (but in the absence of insulin) demonstrated higher phospho-AMPK (Fig. 4-4, inset) (as measured by densitometry; Langendorff heart, 0.91 ± 0.15; Working heart, 2.15 ± 0.6; units, P < 0.05) and phospho-ACC28o (Fig. 4-4, inset) (as measured by densitometry; Langendorff heart, 0.9 ± 0.08; Working heart, 1.4 ± 0.04; units, P < 0.05), and H R - L P L activity (Fig. 4-4). These results suggest that when increasing workload augments cardiac mechanical performance and energy expenditure, L P L is recruited to the coronary lumen. 4.3.5. Augmentation of substrate supply reduces AMPK and ACC phosphorylation and HR-LPL activity in the isolated perfused working heart Glucose entry into the heart is predominantly insulin dependent (7). Compared to glucose, 116 F A is the preferred substrate, and when supplied at physiological levels, contributes approximately 70% of the A T P necessary for normal heart function (47, 48). Interestingly, provision of insulin and albumin-bound palmitic acid to the working heart reduced phosphorylation of A M P K (Fig. 4-5, upper panel) and ACC2so(Fig. 4-5, lower panel), and H R - L P L activity (Fig. 4-6) without changing the rate pressure product (glucose only, 25,400 ± 2000; insulin+palmitate, 24,700 ± 300). In these hearts, introduction of ISO to the buffer perfusate increased rate pressure product (control, 24,700 ± 300; ISO, 33,600 ± 900, P < 0.05), and reversed the effects on A M P K and A C C 2 8 0 phosphorylation and L P L activity (Fig. 4-5 & 4-6). Taken together, our results suggest that the ISO-induced increase in A M P K and A C C phosphorylation occurs through indirect mechanisms. 117 4.4. Discussion F A delivery and utilization by the heart involves: i) release from adipose tissue and transport to the heart after complexing with albumin (35), ii) provision through the breakdown of endogenous cardiac T G stores (42, 57), i i i) internalization of whole lipoproteins (20, 61, 64), and iv) hydrolysis of circulating TG-r ich lipoproteins to F A by L P L positioned at the endothelial surface o f the coronary lumen (5). It should be noted that the molar concentration o f F A bound to albumin is —10 fold less than that of F A in lipoprotein-TG (39) and recently, LPL-mediated hydrolysis of lipoproteins to F A was suggested to be the principal source o f F A for cardiac utilization (4). L P L is synthesized in cardiomyocytes and subsequently secreted onto H S P G binding sites on endothelial cells in the coronary lumen (13, 15, 39, 41). Thus, even though the majority o f enzyme is located in myocytes, vascular endothelial-bound L P L likely determines the rate of plasma lipoprotein-TG clearance (41). Previously, incubation of cardiomyocytes with ISO for 30 minutes did not change either H R - L P L or cellular L P L activity (60). We have confirmed these findings even after incubation with ISO was prolonged for 1 hr. A s cardiomyocytes are quiescent, we extended these experiments to an isolated retrograde perfused heart, and demonstrated a similar absence o f effect of this P-adrenergic agonist on L P L . The above models are not truly representative o f a heart beating in vivo. In the only study that examined the influence of ISO in the intact animal, L P L activity was measured in whole heart homogenates (12), which does not distinguish between the heparin-releasable (localized on capillary endothelial cells) and cellular (that represents a storage form of the enzyme) pools 118 of L P L . Our results, for the first time, suggest that a single injection of ISO, given in vivo, can increase cardiac H R - L P L . Through its interaction with cell surface adrenergic receptors, p-adrenergic agonists increase intracellular messengers such as adenosine 3', 5'-cyclic monophosphate, which leads to increased adipose and cardiac tissue lipolysis, and heart rate and contractility (49, 68). Indeed, plasma free F A were augmented, as well as heart function, following a single injection of ISO. It is interesting that despite an increase in H R - L P L and plasma F F A , measurement o f intracellular lipids revealed a drop in T G with no change in F A . These data imply that circulating free F A are either inefficiently transported into the myocyte, or excessively utilized. Increasing evidence has demonstrated that p-adrenergic agonists increase glucose, glycogen and F A utilization (10, 19, 59). Additionally, ISO is known to augment T G lipolysis in quiescent myocytes, an effect that was independent of its inotropic response (30). Given that H R - L P L only increased following in vivo administration of ISO, it is likely that under conditions of excessive cardiac workload and energy expenditure, L P L is recruited to the coronary lumen. Interestingly, increasing cardiac workload following exercise is also associated with enhanced H R - L P L (18, 66). Overall, these data suggest that the influence of ISO on L P L is not via a direct mechanism, but is likely through its effects on regulating cardiac workload and energy expenditure. The "fuel gauge" A M P K regulates cellular metabolism (53, 54, 58). During ischemia or exercise, changes in intracellular A M P / A T P levels promote activation o f A M P K , an important regulator of both l ipid and carbohydrate metabolism (22, 25). Thus, in heart 119 and skeletal muscle, phosphorylated A M P K stimulates glycolysis through activation of 6-phosphofructo-2-kinase and facilitates F A utilization via control o f A C C (31, 32). A M P K also regulates substrate uptake. For example A M P K induces glucose transport by recruiting G L U T 4 to the plasma membrane (33, 55). In addition, A M P K has also been implicated in F A delivery to cardiomyocytes through its regulation of CD36 (36). More recently, we have also reported that A M P K can regulate cardiac L P L by facilitating movement of this enzyme to the coronary lumen (2). A s A M P K phosphorylation was only observed following the administration of ISO in vivo, and not in the isolated non-beating myocytes or retrograde non-working hearts, it appears that activation of A M P K is secondary to augmented workload and energy expenditure. Although intracellular A T P remains unchanged in skeletal muscle during exercise, increased energy expenditure augments A M P , leading to a change in the A M P / A T P ratio and subsequent activation of A M P K (8, 62). Similar effects on A M P K activation in the heart were also observed following increased heart rate and contractility during exercise (11). A s A M P K activation phosphorylates and inhibits ACC280, and likely controls the increases in L P L at the coronary lumen, our data imply that following ISO, with its associated increase in energy expenditure, F A delivery and oxidation are increased to maintain cellular ATP. In Langendorff hearts, retrograde perfusion through the aorta allows for adequate coronary perfusion that maintains normal beating, but does not generate pressure-volume work as observed in vivo. In contrast, in the working heart, buffer enters through the left atrium, fills the left ventricle, and is expelled against an afterload. Under these conditions, energy 120 expenditure in the Langendorff heart is l ikely lower compared to the working heart, and probably explains why A M P K and H R - L P L remains unchanged even after 1 hr perfusion with glucose as the sole substrate. In contrast, in the working heart perfused in this manner, A T P generation is l ikely not matched to A T P expenditure, and A M P K would be expected to increase. Indeed, in the working heart, A M P K and ACC280 phosphorylation, and H R - L P L activity were higher compared to the Langendorff heart. Whether this augmentation of A M P K phosphorylation is due to insufficient A T P production or increased A M P accumulation is unknown. In an attempt to decrease A M P K and A C C phosphorylation and increase A T P generation, we provided the working heart with insulin (known to decrease A M P K phosphorylation in an Ak t dependent manner) (29) and albumin-bound F A , in addition to glucose. Interestingly, although we were able to lower both A M P K and ACC280 phosphorylation, and H R - L P L activity, it is unclear whether these effects are a consequence o f changes in energy status or the direct effects o f insulin. Under these in vitro conditions, provision of ISO was able to duplicate the effects seen when this p-adrenergic agonist was given in vivo. Overall, these experiments further validate the idea that following increased workload and high energy expenditure, A M P K activation not only promotes F A oxidation, but likely, through its effect on H R - L P L , also provides the hearts with F A . Interestingly, in a different study that perfused isolated mouse hearts, basal (constitutive) L P L release was higher from working compared to Langendorff hearts (37). In summary, our data suggest that ISO can influence H R - L P L , only during conditions 121 of increased workload and excessive energy expenditure, and is highly associated with phosphorylation of A M P K . Whether this increase in L P L following ISO can induce cardiovascular damage is currently unknown. However, L P L mediated lipoprotein hydrolysis can provide excessive F A that may affect the biology of the vessel wall (9, 16). Indeed, when endothelial cell monolayers are incubated with increasing concentrations of FA, the permeability of the monolayer is enhanced allowing albumin to traverse faster across the vessel wal l (24). Additionally, tissue-specific overexpression of L P L is associated with severe myopathy (27, 34, 67), and promotion of atherosclerosis (16). Thus, a novel mechanism by which excessive P-adrenergic agonists could induce cardiovascular complications is through their control of H R - L P L . ! 122 ( 4.5. Tables and figures Table 4-1. General characteristics of the animals Control ISO Blood Glucose (mM) 5.7 ± 0.2 5.4 ± 0 . 2 Serum free fatty acid (mM) 0.34 ± 0 . 0 4 0.49 ± 0.05 * Serum triglyceride (mM) 1.4 ± 0 . 2 1.3 ± 0 . 1 Systolic pressure (mm Hg) 1 1 6 ± 4 131 ± 10 Heart rate (Beats/min) 421 ± 1 6 • 4 7 4 ± 1 8 * Rate pressure product 49192 ± 3 1 2 1 62039 ± 5 3 7 8 * Values are mean ± S E for 6 animals in each group. Rats were anesthetized with 25 mg/kg sodium pentobarbital, and a cannula inserted into the carotid artery to measure systemic blood pressure and heart rate. Following stabilization, animals were either injected i.p. with saline or 10 p.g/kg o f the p-adrenergic agonist, ISO. Blood pressure and heart rate were recorded every 20 min for 1 hr, and averages are presented. For measurement of serum parameters, blood was collected at termination (1 h after ISO). * Significantly different from control, P < 0.05. 123 Table 4-2. Cardiac lipids in control and Isoproterenol treated groups Triglyceride Free fatty acid (p,g/mg protein) (u,g/mg protein) Control 1.7 ± 0 . 2 4.6 ± 0 . 5 l h r 1.1 ± 0 . 1 * 5.0 ± 0 . 6 Isoproterenol 4hr 0.7 ± 0 . 1 * 3.4 ± 0 . 1 Values are mean ± S E for 4 animals in each group. 1 or 4 hrs following in vivo injection o f ISO, hearts were collected and cardiac T G and free F A serarated using H P L C . * Significantly different from control, P < 0.05. 124 Control jUir 4hr ISO P-actin LPL Control ISO Fig . 4-1 Effects of ISO on coronary luminal LPL activity and gene expression after a single in vivo injection Rats were injected i.p. with either saline or ISO (10 p.g/kg), and animals killed after 1 or 4 hrs. O n immediate removal o f hearts, L P L activity was determined after perfusion with heparin. Data are means ± S E o f five different hearts in each group. 'Significantly different from control, P O . 0 5 (upper panel). One hr following injection of ISO, animals were ki l led and hearts frozen in liquid nitrogen. L P L gene expression in the whole heart was measured using RT-PCR. Data are means ± S E o f three different hearts in each group (lower panel). 125 C o n t r o l 1 h r 4 h r ISO C o n t r o l 1 hr 4 h r ISO Fig. 4-2 AMPK and ACC280 phosphorylation in hearts isolated from animals injected with ISO for 1 or 4 hrs Following ISO injection for 1 or 4 hours, hearts were removed and snap frozen in liquid nitrogen. A M P K - a (total and phosphorylated) (upper panel) and phospho-ACC28o (lower panel) were measured using Western Blotting, and rabbit A M P K - a , phospho-AMPK (Thr-172), or phospho-ACC28o (Ser-79) antibodies. Data are means ± SE of five different hearts in each group. Significantly different from control, P < 0.05. 126 Phospho-AMPK-a Total A M P K - a 500 400 « =5 300 i o E 200 X c ns a> D_ 100 H o - 1 Control ISO Fig. 4-3 Heparin releasable LPL activity and AMPK phosphorylation in Langendorff hearts perfused with ISO To determine whether ISO influences L P L in the intact heart, we perfused hearts retrogradely with this P-adrenergic agonist (10 uM) for 1 hr. Subsequently, L P L from control or ISO perfused hearts was displaced by heparin, and activity determined. Data are means ± SE of four different isolated hearts in each group. Subsequently to displacement of LPL, hearts were removed and snap frozen in liquid nitrogen. A M P K - a (total and phosphorylated) (inset) was measured using Western Blotting. Data are means±SE of four different isolated hearts in each group. 127 Phospho-AMPK-a Total AMPK-a Phospho-ACC ^ 1600 O E 1200 > o < a : i ro a. 800 400 Fig. 4-4 Comparison of heparin releasable LPL activity, AMPK and ACC280 phosphorylation in perfused Langendorff or working hearts Langendorff (no pressure-volume work) or working hearts (beating against an afterload) were perfused for 15 min with glucose as the sole substrate. Subsequently, LPL was displaced by heparin, and activity determined. Data are means ± SE of three different hearts in each group. 'Significantly different from control, P < 0.05. Subsequently, AMPK-a (total and phosphorylated) and phospho-ACC2so (inset) were measured using Western Blotting. Data are means ± SE of three different hearts in each group. 128 + + + Glucose + + P A » + + Insul in + ISO Fig. 4-5 Consequence of additional substrate provision in regulating AMPK and ACC280 phosphorylation in isolated working hearts in the absence or presence of ISO Working hearts were perfused either with glucose or with buffer containing glucose, insulin and palmitic acid. In an additional group, ISO was added to buffer containing al l o f the above components. ,Subsequently, A M P K - a (total and phosphorylated) (upper panel) and phospho-ACC 28o (lower panel) were measured using Western Blotting. Data are means ± SE of three different hearts in each group. 'Significantly different from control, P < 0.05. 129 Fig. 4-6 Consequence of additional substrate provision in regulating heparin-releasable LPL in isolated working hearts, in the absence or presence of LSO Working hearts were perfused either with glucose or with buffer containing glucose, insulin and palmitic acid. In an additional group, ISO was added to buffer containing all o f the above components. Subsequently, L P L was displaced by heparin, and activity determined. Data are means ± SE of three different hearts in each group. Significantly different from control, P < 0.05. 130 4.6. Bibliography 1. Allard MF, Henning SL, Wambolt RB, Granleese SR, English DR, and Lopaschuk GD. Glycogen metabolism in the aerobic hypertrophied rat heart. Circulation ,96:676-682,1997. 2. An D, Pulinilkunnil T, Qi D, Ghosh S, Abrahani A, and Rodrigues B. The metabolic "switch" A M P K regulates cardiac heparin-releasable lipoprotein lipase. Am J Physiol Endocrinol Metab 288: E246-253, 2005. 3. Atkinson L L , Fischer MA, and Lopaschuk GD. Leptin activates cardiac fatty acid oxidation independent of changes in the AMP-activated protein kinase-acetyl-CoA carboxylase-malonyl-CoA axis. J Biol Chem 277: 29424-29430, 2002. 4. Augustus AS, Kako Y, Yagyu H, and Goldberg IJ. Routes of F A delivery to cardiac muscle: modulation of lipoprotein lipolysis alters uptake of TG-derived F A . Am J Physiol Endocrinol Metab 284: E331-339, 2003. 5. Blanchette-Mackie EJ , Masuno H, Dwyer NK, Olivecrona T, and Scow RO. Lipoprotein lipase in myocytes and capillary endothelium o f heart: immunocytochemical study. AmJ Physiol 256: E818-828, 1989. 6. Brault D, Noe L, Etienne J, Hamelin J, Raisonnier A, Souli A, Chuat JC, Dugail I, Quignard-Boulange A, Lavau M , and et al. Sequence o f rat lipoprotein lipase-encoding c D N A . Gene 121: 237-246, 1992. 7. Brownsey RW, Boone AN, and Allard MF. Actions of insulin on the mammalian heart: metabolism, pathology and biochemical mechanisms. Cardiovasc Res 34: 3-24, 131 1997. 8. Chen ZP, Stephens TJ , Murthy S, Canny BJ, Hargreaves M, Witters LA, Kemp BE, and McConell GK. Effect of exercise intensity on skeletal muscle AMPK signaling in humans. Diabetes 52: 2205-2212, 2003. 9. Christon RA. Mechanisms of action of dietary fatty acids in regulating the activation of vascular endothelial cells during atherogenesis. Nutr Rev 61: 272-279, 2003. 10. Collins-Nakai RL, Noseworthy D, and Lopaschuk GD. Epinephrine increases ATP production in hearts by preferentially increasing glucose metabolism. Am J Physiol 267: H1862-1871, 1994. 11. Coven DL, Hu X, Cong L, Bergeron R, Shulman GI, Hardie DG, and Young LH. Physiological role of AMP-activated protein kinase in the heart: graded activation during exercise. Am J Physiol Endocrinol Metab 285: E629-636, 2003. 12. Deshaies Y, Geloen A, Paulin A, Marette A, and Bukowiecki LJ . Tissue-specific alterations in lipoprotein lipase activity in the rat after chronic infusion of isoproterenol. Horm Metab Res 25: 13-16, 1993. 13. Eckel RH. Lipoprotein lipase. A multifunctional enzyme relevant to common metabolic diseases. N Engl J Med 320: 1060-1068, 1989. 14. Elam M, Sverrisdottir YB, Rundqvist B, McKenzie D, Wallin BG, and Macefield V G Pathological sympathoexcitation: how is it achieved? Acta Physiol Scand 111: 405-411,2003. 15. Enerback S and Gimble JM. Lipoprotein lipase gene expression: physiological 132 regulators at the transcriptional and post-transcriptional level. Biochim Biophys Acta 1169: 107-125,1993. 16. Esenabhalu V E , Cerimagic M , Malli R, Osibow K, Levak-Frank S, Frieden M , Sattler W, Kostner G M , Zechner R, and Graier WF. Tissue-specific expression of human lipoprotein lipase in the vascular system affects vascular reactivity in transgenic mice. Br J Pharmacol 135: 143-154, 2002. 17. Floras JS. Sympathetic activation in human heart failure: diverse mechanisms, therapeutic opportunities. Acta Physiol Scand 177: 391-398, 2003. 18. Goldberg DI, Rumsey WL, and Kendrick ZV. Exercise activation of (myocardial lipoprotein lipase in male and estrogen-treated female rats. Metabolism 33: 964-969, 1984. 19. Goodwin GW, Ahmad F, and Taegtmeyer H. Preferential oxidation of glycogen in isolated working rat heart. J Clin Invest 97: 1409-1416, 1996. 20. Haffner SM. Lipoprotein disorders associated with type 2 diabetes mellitus and insulin resistance. Am J Cardiol 90: 55i-61i, 2002. 21. Hardie D G Minireyiew: the AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endocrinology 144: 5179-5183, 2003. 22. Hardie DG and Carling D. The AMP-activated protein kinase—fuel gauge of the mammalian cell? Eur J Biochem 246: 259-273, 1997. 23. Hayashi T, Hirshman MF, Fujii N, Habinowski SA, Witters LA, and Goodyear LJ. Metabolic stress and altered glucose transport: activation of AMP-activated protein kinase as a unifying coupling mechanism. Diabetes 49: 527-531, 2000. 133 24. Hennig B, Shasby DM, Fulton AB, and Spector AA. Exposure to free fatty acid increases the transfer o f albumin across cultured endothelial monolayers. Arteriosclerosis 4: 489-497, 1984. 25. Hong SP, Leiper FC, Woods A, Carling D, and Carlson M . Activation of yeast Snfl and mammalian AMP-activated protein kinase by upstream kinases. Proc Natl Acad Sci USA 100: 8839-8843, 2003. 26. Herman S, Browne G, Krause U, Patel J, Vertommen D, Bertrand L, Lavoinne A, Hue L, Proud C, and Rider M . Activation of AMP-activated protein kinase leads to the phosphorylation of elongation factor 2 and an inhibition of protein synthesis. Curr Biol 12: 1419-1423,2002. 27. Kim JK, Fillmore JJ, Chen Y, Yu C, Moore IK, Pypaert M , Lutz EP, Kako Y, Velez-Carrasco W, Goldberg IJ, Breslow JL, and Shulman GI. Tissue-specific overexpression of lipoprotein lipase causes tissue-specific insulin resistance. Proc Natl Acad Sci USA9Z: 7522-7527, 2001. 28. Kitajima S, Morimoto M , Liu E , Koike T, Higaki Y, Taura Y, Mamba K, Itamoto K, Watanabe T, Tsutsumi K, Yamada N, and Fan J. Overexpression of lipoprotein lipase improves insulin resistance induced by a high-fat diet in transgenic rabbits. Diabetologia 47: 1202-1209, 2004. 29. Kovacic S, Soltys CL, Barr AJ, Shiojima I, Walsh K, and Dyck JR. Ak t activity negatively regulates phosphorylation of AMP-activated protein kinase in the heart. J Biol Chem 278: 39422-39427, 2003. 134 30. Kryski A, Jr., Kenno KA, and Severson DL. Stimulation of lipolysis in rat heart myocytes by isoproterenol. Am J Physiol 248: H208-216, 1985. .31. Kudo N, Barr AJ, Barr RL, Desai S, and Lopaschuk GD. High rates of fatty acid oxidation during reperfusion of ischemic hearts are associated with a decrease in malonyl-CoA levels due to an increase in 5'-AMP-activated protein kinase inhibition of acetyl-CoA carboxylase. J Biol Chem 270: 17513-17520, 1995. 32. Kudo N, Gillespie JG, Kung L , Witters LA, Schulz R, Clanachan AS, and Lopaschuk GD. Characterization of 5'AMP-activated protein kinase activity in the heart and its role in inhibiting acetyl-CoA carboxylase during reperfusion following ischemia. Biochim Biophys Acta 1301:67-75, 1996. 33. Kurth-Kraczek EJ, Hirshman MF, Goodyear L J , and Winder WW. 5' AMP-activated protein kinase activation causes G L U T 4 translocation in skeletal muscle. Diabetes 48: 1667-1671, 1999. 34. Levak-Frank S, Radner H, Walsh A, Stollberger R, Knipping G, Hoefler G, Sattler W, Weinstock PH, Breslow JL, and Zechner R. Muscle-specific overexpression of lipoprotein lipase causes a severe myopathy characterized by proliferation o f mitochondria and peroxisomes in transgenic mice. J Clin Invest 96: 976-986, 1995. 35. Lopaschuk GD, Belke DD, Gamble J, Itoi T, and Schonekess BO. Regulation of fatty acid oxidation in the mammalian heart in health and disease. Biochim Biophys Acta 1213: 263-276, 1994. 36. Luiken JJ, Coort SL, Willems J, Coumans WA, Bonen A, van der Vusse GJ, and 135 Glatz JF. Contraction-induced fatty acid translocase/CD36 translocation in rat cardiac myocytes is mediated through AMP-activated protein kinase signaling. Diabetes 52: 1627-1634,2003. 37. Mardy K, Belke DD, and Severson DL. Chylomicron metabolism by the isolated perfused mouse heart. Am J Physiol Endocrinol Metab 281: E357-364, 2001. 38. Marsin AS, Bertrand L, Rider MH, Deprez J, Beauloye C, Vincent MF, Van den Berghe G, Carling D, and Hue L. Phosphorylation and activation of heart P F K - 2 by A M P K has a role in the stimulation of glycolysis during ischaemia. Curr Biol 10: 1247-1255, 2000. 39. Merkel M, Eckel RH, and Goldberg IJ. Lipoprotein lipase: genetics, l ipid uptake, and regulation. J Lipid Res 43: 1997-2006, 2002. 40. Mulder M , Lombardi P, Jansen H, van Berkel TJ, Frants RR, and Havekes L M . L o w density lipoprotein receptor internalizes low density and very low density lipoproteins that are bound to heparan sulfate proteoglycans via lipoprotein lipase. J Biol Chem 268: 9369-9375,1993. 41. Otarod JK and Goldberg IJ. Lipoprotein lipase and its role in regulation of plasma lipoproteins and cardiac risk. Curr Atheroscler Rep 6: 335-342, 2004. 42. Paulson DJ and Crass MF, 3rd. Endogenous triacylglycerol metabolism in diabetic heart. Am J Physiol 242: H1084-1094, 1982. 43. Pillarisetti S, Paka L, Sasaki A, Vanni-Reyes T, Yin B, Parthasarathy N, Wagner WD, and Goldberg IJ. Endothelial cell heparanase modulation o f lipoprotein lipase 136 activity. Evidence that heparan sulfate oligosaccharide is an extracellular chaperone. J Biol Chem 272: 15753-15759, 1997. 44. Pulinilkunnil T, Abrahani A, Varghese J , Chan N, Tang I, Ghosh S, Kulpa J, Allard M, Brownsey R, and Rodrigues B. Evidence for rapid "metabolic switching" through lipoprotein lipase occupation of endothelial-binding sites. J Mol Cell Cardiol 35: 1093-1103,2003. 45. Pulinilkunnil T, An D, Yip P, Chan N, Qi D, Ghosh S, Abrahani A, and Rodrigues B. Palmitoyl lysophosphatidylcholine mediated mobilization of L P L to the coronary luminal surface requires P K C activation. J Mol Cell Cardiol 37: 931-938, 2004. 46. Pulinilkunnil T, Qi D, Ghosh S, Cheung C, Yip P, Varghese J, Abrahani A, Brownsey R, and Rodrigues B. Circulating triglyceride lipolysis facilitates lipoprotein lipase translocation from cardiomyocyte to myocardial endothelial lining. Cardiovasc Res 59: 788-797, 2003. 47. Randle PJ, Garland PB, Hales CN, and Newsholme EA. The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances o f diabetes mellitus. Lancet 1: 785-789, 1963. 48. Randle PJ, Priestman DA, Mistry S, and Halsall A. Mechanisms modifying glucose oxidation in diabetes mellitus. Diabetologia 37 Suppl 2: S155-161, 1994. 49. Robidoux J, Martin TL, and Collins S. Beta-adrenergic receptors and regulation of energy expenditure: a family affair. Annu Rev Pharmacol Toxicol 44: 297-323, 2004. 50. Rodrigues B, Cam MC, Jian K, Lim F, Sambandam N, and Shepherd G. 137 Differential effects of streptozotocin-induced diabetes on cardiac lipoprotein lipase activity. Diabetes 46: 1346-1353, 1997. 51. Rodrigues B, Cam M C , and McNeill JH. Myocardial substrate metabolism: implications for diabetic cardiomyopathy. J Mol Cell Cardiol 27: 169-179, 1995. 52. Rodrigues B, Spooner M , and Severson DL. Free fatty acids do not release lipoprotein lipase from isolated cardiac myocytes or perfused hearts. Am J Physiol 262: E216-223, 1992. 53. Ruderman N and Prentki M . AMP kinase and malonyl-CoA: targets for therapy of the metabolic syndrome. Nat Rev Drug Discov 3: 340-351, 2004. 54. Ruderman NB, Saha AK, and Kraegen EW. Minireview: malonyl CoA, AMP-activated protein kinase, and adiposity. Endocrinology 144: 5166-5171, 2003. 55. Russell RR, 3rd, Bergeron R, Shulman GI, and Young L H . Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR. Am J Physiol 277: H643-649, 1999. 56. Rutledge JC and Goldberg IJ. Lipoprotein lipase (LpL) affects low density lipoprotein (LDL) flux through vascular tissue: evidence that LpL increases LDL accumulation in vascular tissue. J Lipid Res 35: 1152-1160, 1994. 57. Saddik M and Lopaschuk GD. Triacylglycerol turnover in isolated working hearts of acutely diabetic rats. Can J Physiol Pharmacol 72: 1110-1119, 1994. 58. Saha AK and Ruderman NB. Malonyl-CoA and AMP-activated protein kinase: an expanding partnership. Mol Cell Biochem 253: 65-70, 2003. 138 59. Schiffelers SL, van Harmelen VJ, de Grauw HA, Saris WH, and van Baak MA. Dobutamine as selective beta(l)-adrenoceptor agonist in in vivo studies on human thermogenesis and lipid utilization. J Appl Physiol 87: 977-981, 1999. 60. Severson DL, Carroll R, Kryski A, Jr., and Ramirez I. Short-term incubation of cardiac myocytes with isoprenaline has no effect on heparin-releasable or cellular lipoprotein lipase activity. Biochem J248: 289-292, 1987. 61. Steiner G. Diabetes and atherosclerosis—a lipoprotein perspective. Diabet Med 14 Suppl 3: S38-44, 1997. 62. Stephens TJ, Chen ZP, Canny BJ, Michell BJ, Kemp BE, and McConell GK. Progressive increase in human skeletal muscle A M P K a l p h a 2 activity and A C C phosphorylation during exercise. Am J Physiol Endocrinol Metab 282: E688-694, 2002. 63. Takahashi S, Suzuki J, Kohno M, Oida K, Tamai T, Miyabo S, Yamamoto T, and Nakai T. Enhancement of the binding of triglyceride-rich lipoproteins to the very low density lipoprotein receptor by apolipoprotein E and lipoprotein lipase. J Biol Chem 270: 15747-15754, 1995. 64. Tannock LR and Chait A. Lipoprotein-matrix interactions in macrovascular disease in diabetes. Front Biosci 9: 1728-1742, 2004. 65. Teusink B, Voshol PJ, Dahlmans VE, Rensen PC, Pijl H, Romijn JA, and Havekes L M . Contribution of fatty acids released from lipolysis o f plasma triglycerides to total plasma fatty acid flux and tissue-specific fatty acid uptake. Diabetes 52: 614-620, 2003. 139 66. Vaziri ND, Liang K, and Barton CH. Effect of increased afterload on cardiac lipoprotein lipase and V L D L receptor expression. Biochim Biophys Acta 1436: 577-584, 1999. 67. Voshol PJ, Jong M C , Dahlmans VE, Kratky D, Levak-Frank S, Zechner R, Romijn JA, and Havekes L M . In muscle-specific lipoprotein lipase-overexpressing mice, muscle triglyceride content is increased Without inhibition of insulin-stimulated whole-body and muscle-specific glucose uptake. Diabetes 50: 2585-2590, 2001. 68. Xiao RP, Zhu W, Zheng M , Chakir K, Bond R, Lakatta EG, and Cheng H. Subtype-specific beta-adrenoceptor signaling pathways in the heart and their potential clinical implications. Trends Pharmacol Sci 25: 358-365, 2004. 69. Yagyu H, Chen G, Yokoyama M, Hirata K, Augustus A, Kako Y, Seo T, Hu Y, Lutz EP, Merkel M , Bensadoun A, Homma S, and Goldberg IJ. Lipoprotein lipase (LpL) on the surface of cardiomyocytes increases lipid uptake and produces a cardiomyopathy. J Clin Invest 111:419-426, 2003. 140 5. Metformin influences cardiomyocyte cell death by both caspase-3 dependent and independent pathways 5.1. Introduction Heart disease is a leading cause of death in diabetic patients, with coronary vessel disease and atherosclerosis being primary reasons for the increased incidence o f cardiovascular dysfunction (26, 41). However, a number of diabetic patients also suffer from a specific impairment of heart muscle (termed diabetic cardiomyopathy), a condition also evident in rodent models o f chronic diabetes (20, 46). Several factors have been put forward to explain the development of diabetic cardiomyopathy including an increased stiffness of the left ventricular wal l , and abnormalities o f various proteins that regulate ion flux, specifically intracellular calcium (5, 14). More recently, the view that diabetic cardiomyopathy could also occur as a consequence of apoptosis, a process of cell death that occurs subsequent to the activation of a genetically • programmed, energy-dependent mechanism, has been put forward (6, 50). The heart and other non-adipose tissues have inadequate ability to handle excess fatty acid (FA). For this reason, an increase in intracardiac F A concentration can overwhelm F A oxidation. In this situation, F A accumulate and can, either by themselves, or via their channeling towards the production of second messengers like ceramide, provoke apoptosis (10, 16). Enlargement of the intracardiac F A pool during diabetes may similarly overpower the tissue capacity for utilization, and the resultant accumulation of cardiotoxic 141 lipid can be implicated in cardiac myocyte apoptosis (45, 50). Metformin, a drug widely used in the treatment of Type 2 diabetes, decreases both plasma glucose and lipids. Recently, A M P K has been implicated in mediating the effects of metformin (31, 49), in a L K B 1 dependent manner (38). Knocking-out L K B 1 or inhibition of A M P K abolishes the clinical effects of metformin (38, 49). Once activated, A M P K stimulates glucose uptake and subsequent glycolysis through translocation of glucose transporter 4 (GLUT-4) and activation of 6-phosphbfructo-2-kinase (PFK2) (15, 23, 29). Recent studies have reported that metformin, through activation of A M P K , potentiates insulin signaling downstream targets, such as protein kinase B and atypical protein kinase C , leading to improved insulin signaling with elevated glucose uptake (24, 28). Moreover, through its control of lipoprotein lipase, CD36, and acetyl-CoA carboxylase ( A C C ) , A M P K facilitates F A delivery and oxidation (1 ,21 , 27). In addition to its effect on substrate metabolism, metformin is also known to regulate endothelial cell death (9). Whether metformin regulates cardiomyocyte cell death is unknown. Given the role o f metformin in the regulation o f l ipid metabolism, we hypothesized that it would prevent high F A induced cardiomyocyte cell death. 142 5.2. Materials and methods 5.2.1. Experimental animals The investigation conforms to the guide for the care and use of laboratory animals published by the U S National Institutes of Health and the University o f British Columbia (animal care certificate A00-0291). Adult male Wistar rats (250-280 g) were obtained from the U B C Animal Care Unit and supplied with a standard laboratory diet (PMI Feeds, Richmond, V A ) , and water ad libitum. 5.2.2. Cardiomyocyte isolation and culturing Ventricular myocytes were prepared by a previously described procedure (36). Briefly, myocytes were made calcium-tolerant by successive exposure to increasing concentrations of calcium. Our method of isolation yields a highly enriched population of calcium-tolerant myocardial cells that are rod-shaped (over 80%) in the presence of 1 mmol/1 C a 2 + , with clear cross striations. Intolerant cells are intact but hypercontract into vesiculated spheres. Yie ld o f myocytes was determined microscopically using an improved Neubauer hemocytometer. Myocyte viability was assessed as the percentage of elongated cells with clear cross striations that excluded 0.2% trypan blue. Cardiomyocytes were plated on laminin-coated 6 wel l culture plates to a density of 100,000 cells/well. Cells were maintained using Media-199, in the presence or absence of 1 mmol/1 albumin bound palmitic acid (1:2), and incubated at 37 °C under an atmosphere of 95%02-5%C02 for 16 hours. Where indicated, metformin (1-5 mmol/1) was added to 143 the culture medium, and myocytes kept for either 1 or 16 hours. In some experiments, myocytes were incubated with metformin for 16 hrs in the absence of glucose. 5.2.3. LDH release Lactate dehydrogenase ( L D H ) is a cellular enzyme released upon membrane damage. Thus, L D H release is recognized as a marker of cell damage or death (8). L D H released into the myocyte incubation media was estimated using an assay kit (Sigma, St. Louis, Mo.) . In brief, L D H reduces nicotinamide adenine dinucleotide (NAD+), which then converts a tetrazolium dye to a soluble, colored formazan derivative; this was measured using a spectrophotometer (490 nm). 5.2.4. Hoechst 33342 staining To study high fat induced apoptosis and the influence of metformin on this process, cells were examined for morphological evidence of apoptosis using the fluorescent DNA-binding dye, Hoechst 33342. Cells were stained with 5 pg/mL Hoechst 33342, and viewed on a Zeiss I M fluorescence microscope (x400). Cells were scored as apoptotic i f they exhibited unequivocal nuclear chromatin condensation and/or fragmentation. To quantify apoptosis, 500 nuclei from different isolations (3 different isolations were prepared) were randomly picked and examined, and the results presented as apoptotic cells per 1000 cells. 5.2.5. Estimation of reactive oxygen species (ROS) The redox-sensitive dye 5-(and-6)- chloromethyl-2',7'-dichlorodihydrofluoresceindiacetate acetyl ester ( C M - H 2 D C F D A ) was used to assess R O S levels. C M - H 2 D C F D A is a 144 cell-permeant indicator that is oxidized in the presence of R O S such as H2O2 whereby it emits green fluorescence. Following 16 hrs incubation, the medium was discarded and wells were loaded with C M - H 2 D C F D A (5 u M for 30 min at 37°C). Subsequently, cells were washed and incubated for an additional 45 min after which green fluorescence was measured in a fluorimeter at 485/530-nm wavelengths. 5.2.6. Western blot analysis Western blot was carried out as described previously (1). Briefly, 50 mg of heart tissue was ground under liquid nitrogen, and homogenized. After centrifugation at 5,000 g for 20 min, the protein content of the supernatant was quantified using a Bradford protein assay. Samples were diluted, boiled with sample loading dye, and 50 pg used in SDS-polyacrylamide gel electrophoresis. After transfer, membranes were blocked in 5% skim milk in Tris-buffered saline containing 0.1% Tween-20. Membranes were incubated with rabbit A M P K - a , phosopho-AMPK (Thr-172), phospho-ACC, i N O S , and long chain base biosynthesis protein 1 ( L C B 1 ) antibodies, and subsequently with secondary goat anti-rabbit or goat anti-mouse HRP-conjugated antibodies, and visualized using an E C L detection kit. 5.2.7. Ceramide assay Intracellular ceramide was estimated using a D A G kinase assay as described before (30). Briefly, following 16 hrs incubation, phospholipids were extracted using chloroform/methanol from same number of cells in different groups (1:2, v/v). Samples 145 were then incubated with reaction mixture containing 5 mmol/1 imidazole/HCL, p H 6.6, 1 mmol/1 D E T A P A C , p H 6.6, 50 mmol/1 N a C l , 12.5 mmol/1 M g C l 2 , 1 mmol/1 E G T A , 10 mmol/1 dithiothreitol, 1 mmol/1 ATP, [y- 3 2 P]ATP, D A G kinase (15.5 munits/assay), 2.5 % octyl-(3-D-glucoside, 1 mmol/l cardiolipin. Following incubation, lipids were extracted with 470 pi of chloroform/methanol/10 mmol/1 HC1 (15:30:2. v/v/v) containing 20 pg/ml of phosphatidate as a carrier. Samples were separated on plastic thin layer plates of silica gel 60 (Merck) by developing with chloroform/methanol/NH40H (65:35:7.5; v/v/v), drying and then developing i n chloroform/methanol/acetic acid/acetone/water (10:2:3:4:1 by volume). P-labeled ceramide-1-phosphate was identified with authentic standards and a Radio-imager. 5.2.5. Caspase 3 activity Activity o f cardiac caspase-3 was determined using a fluorescent kit (EnzChek Caspase-3 Assay K i t , Molecular Probes). Briefly, myocytes were lysed and protein extracted by centrifugation at 5,000 rpm for 5 min. 50 p i of protein were added to an equal volume of reaction buffer that contained 50 umol/1 of the respective substrate, and incubated at room temperature for 30 min. The enzyme-catalyzed release of aminomethylcoumarin was quantified in a fluorimeter at 380/450-nm wavelengths. Protein was determined using a Bradford assay. Caspase 3 activity is presented as activity per mg protein for each sample. 5.2.9. Rates of glycolysis and palmitic acid oxidation 3 3 To measure rates of glycolysis and F A oxidation, 5- H-glucose or H-palmitic acid were 146 added separately into the incubation medium. After 16 hrs incubation, medium samples were collected. 3 H20 generated from glycolysis or F A oxidation was separated from 5- 3H-glucose or 3 H-palmitic acid by previously described methods (25). Radioactivity was measured by liquid scintillation counting. Protein was quantified using a Bradford protein assay. Results were expressed as pmol/min/mg protein. 5.2.10. Lactate assay Glycolysis results in the formation of protons and pyruvate. Bu i ld up of these metabolites augments lactic acid generation, a process catalyzed by L D H (35). Therefore, lactate accumulation is recognized as a marker of acidosis. Lactate released into the myocyte incubation medium was determined using a kit from Sigma. Briefly, 100 p i of culture medium was incubated with 1 ml reaction medium at 30 °C for 10 min, and lactate concentration measured using a spectrophotometer. 5.2.11. Statistical analysis One-way A N O V A followed by the Tukey tests was used to determine differences between group mean values. The level o f statistical significance was set at P < 0.05. 5.2.12. Materials Total A M P K - a and Phospho -AMPK-a , and A C C antibodies were obtained from Cel l Signaling (Beverly, M A ) . An t i - iNOS antibody was obtained from Santa Cruz Biotech. A n t i - L C B l was obtained from B D Sciences. Medium 199 was obtained from Sigma. D M E M was obtained from Invitrogen. 3 2 P , 5- 3H-glucose and 3 H-palmitic acid were 147 obtained from N e w England Nuclear. E C L detection kit was obtained from Amersham. 148 5.3. Results 5.3.1. High fat induced LDH release and consequence of metformin Myocytes incubated with palmitic acid for 16 hours augmented L D H release, an effect that was partly blunted by low concentration of metformin (Fig. 5-1 A ) . Unexpectedly, 5 mmol/1 metformin dramatically increased L D H release in myocytes incubated with high fat (Fig. 5-1 A ) . In the absence of high fat, metformin alone was without effect on L D H release (data not shown). 5.3.2. High fat induced apoptosis and consequence of metformin Fig. 5-1 B and C describe evidence o f apoptosis and the effect of metformin. In the control group, less than 50 out of 1000 cells were scored as apoptotic. . High fat significantly increased apoptosis (241/1000 cells). Introducing low concentrations of metformin into the medium significantly lowered the number of apoptotic cells (1 mmol/1, 170/1000; 2 mmol/1, 185/1000). With a higher concentration of metformin (5 mmol/1), a larger number of cells (635/1000) underwent apoptosis. 5.3.3. ROS generation following provision of high fat and metformin R O S generated in the mitochondria is known to induce oxidative stress and apoptosis. In the present study, incubation of cardiomyocytes with 1 mmol/1 palmitic, acid for 16 hrs decreased R O S levels compared to control (without palmitic acid) (Fig. 5-2). Introducing low concentration of metformin (1 or 2 mmol/1) into the medium increased R O S to the level similar to control (Fig. 5-2). In contrast, the lowest level of R O S was observed when high concentration (5 mmol/1) of metformin was used (Fig. 5-2). 149 5.3.4. Effects of metformin on AMPK phosphorylation in cardiomyocytes Previous studies have reported that metformin activates A M P K in skeletal muscle, liver and cardiomyocytes (7, 31, 49). In the present study, following one-hour incubation with metformin, an approximately two-fold increase in A M P K phosphorylation was observed (Fig. 5-3A). N o difference in A M P K phosphorylation was observed between groups treated with 1, 2 and 5 mmol/1 metformin. Increasing the incubation time of metformin to 16 hours further increased A M P K phosphorylation (~4-6-fold) (Fig. 5-3B), with 5 mmol/1 metformin demonstrating the highest level of A M P K activation. 5.3.5. ACC phosphorylation and FA oxidation in cardiomyocytes incubated with metformin A M P K phosphorylates and inhibits A C C , the key enzyme that controls generation of malonyl-CoA from acetyl-CoA. A s malonyl-CoA decreases F A oxidation through inhibition of CPT-1 , phosphorylation of A C C relieves the inhibition of CPT-1 , favoring F A oxidation. In the current study, metformin increased A C C phosphorylation in cardiomyocytes within 1 hour (Fig. 5-4A). Prolonging the incubation time to 16 hours further increased A C C phosphorylation (Fig. 5-4B). Coupled to this phosphorylation of A C C , metformin increased palmitic acid oxidation rate, with 5 mmol/1 having the highest effect (Fig. 5-4C). 5.3.6. Effects of metformin on LCB1 expression and intracellular cer amide level High fat increases ceramide, a pro-apoptotic mediator, through activation of serine palmitoyltransferase (SPT) (39). Incubation of cardiomyocytes with palmitic acid (1 150 mmol/1) for 16 hours increased L C B 1 protein, a subunit of SPT (Fig. 5-5A), and intracellular ceramide (Fig. 5-5B). Introduction of metformin into the culture media reduced the high fat induced L C B 1 protein expression (Fig. 5-5A), and ceramide levels (Fig.5-5B). 5.3.7. Effects of metformin on high fat induced Caspase 3 activity Accumulation of ceramide induces apoptosis in a caspase-3 dependent manner (43, 44). Incubation of cardiomyocytes with palmitic acid (1 mmol/1) for 16 hours increased caspase activity (Fig. 5-5C). Consistent with the lowering of ceramide, introduction of metformin into the cardiomyocyte incubation medium also reduced the high fat mediated increases in caspase 3 activity (Fig. 5-5C). 5.3.8. Lactate production and pH changes after metformin A M P K activation enhances glucose uptake and glycolysis through stimulation of G L U T - 4 and P F K - 2 respectively (15, 23, 29). Uncoupling of glycolysis from glucose oxidation leads to lactate production and proton accumulation (18). In the current study, high fat did not change either lactate or p H (control: 7.40 ± 0.01; high fat: 7.38 ± 0.02) in the medium following 16 hours incubation. Although low concentrations of metformin increased lactate (Fig. 5-6) and decreased p H (1 mmol/1-7.26 ± 0.02; 2 mmol/1-7.25 + 0.03, P O . 0 5 ) , myocytes incubated with 5 mmol/1 metformin showed the highest lactate concentration (Fig. 5-6) and lowest p H (7.16 ± 0.03, P<0.05) in the medium. 5.3.9. Effects of metformin on glycolysis A M P K activation is known to stimulate glycolysis through phosphorylation and activation 15.1 of P F K - 2 . Provision of 1 mmol/1 palmitic acid significantly reduced the rate o f glycolysis in cardiomyocytes (Fig. 5-7). Metformin dramatically increased glycolysis in a dose dependent manner (Fig. 5-7). 5.3.10. Effects of glucose removal on LDH release We used glucose free medium and repeated our incubations with palmitic acid, in the absence or presence of metformin. Absence of glucose normalized p H (data not shown) and prevented the metformin (5 mmol/1) induced increase in L D H release (Fig. 5-8). In fact, in this glucose free medium, the release of L D H with metformin (5 mmol/1) was similar to control. 152 5.4. Discussion Metformin, a drug widely used in the treatment of Type 2 diabetes, lowers plasma glucose without causing either hypoglycemia or weight gain. Recently, activation of A M P K has been implicated in mediating this effect on glucose, either through a reduction in hepatic glucose production, or an increase in glucose uptake and utilization (31, 49). Through activation of A M P K , metformin also enhances F A uptake and oxidation (37). Whether metformin, as a consequence of its modulated metabolism, influences cardiomyocyte cell death remained unknown. In the present study, metformin per se has no effect on cell death. A t low concentrations, this agent reduced high fat induced apoptotic cells and L D H release, whereas a high concentration of metformin dramatically amplified both the apoptotic cell number and L D H secretion. Our data suggest that the dual effects of metformin on high fat induced cell death are dose dependent. R O S plays a key role in increasing mitochondrial cytochrome c release and inducing apoptosis. In endothelial cells, metformin has been shown to reduce hyperglycemia induced R O S generation and oxidative stress (11, 22). Therefore, we examined whether the same mechanisms are also involved in protecting cardiomyocytes against high fat induced cell death. Surprisingly, incubation of cardiomyocytes with 1 mmol/1 palmitic acid lowered, rather than increased, R O S levels. This observation is consistent with previous studies, which suggest that high concentration of palmitic acid decreases mitochondrial membrane potential, and the ability of the mitochondria to produce ROS (17, 42). Provision of low concentration of metformin (1 or 2 mmol/1) restored R O S to control 153 levels, likely due to the effect of metformin in promoting F A oxidation and electron flow through the mitochondrial electron transport chain (42). In contrast, in the presence of palmitic acid, cardiomyocytes treated with 5 mmol/1 metformin exhibited the lowest R O S level. One explanation is that following 16 hrs treatment, 5 mmol/1 metformin induces apoptosis in over 60% of cardiomyocytes, likely leading to impaired oxidative metabolism and R O S generation. Overall, our data suggest that metformin prevents high fat induced apoptosis in cardiomyocytes through a R O S independent mechanism. In the heart, when supply of F A exceeds tissue oxidative capacity, accumulation of lipids decreases cardiolipin synthesis (33), and induces myofibrillar degeneration (10), leading to lipotoxicity and apoptosis. Given that exogenous d-ceramide mimicks the deleterious effects of F A , and that inhibition of ceramide formation blocks these toxic effects caused by high fat, several studies have suggested that de novo ceramide formation plays an important contributory role in apoptosis (32, 40, 50). Direct interaction with cytochrome C followed by its release from the mitochondria and activation of caspase is one mechanism proposed to explain this effect of ceramide in propagating apoptosis (12). In the present study, we demonstrate that metformin prevented the high fat induced activation of caspase 3, likely through its inhibition of ceramide synthesis. Ceramide levels can increase as a consequence of SPT mediated de novo synthesis, in the presence of excessive fatty acyl C o A . Given the role of A M P K in decreasing the expression of SPT (3), activation of A M P K by metformin would be expected to decrease ceramide formation. Moreover, through activation of A M P K and inhibition of A C C , metformin also promotes 154 palmitic acid oxidation. It is possible that this increased F A oxidation could divert F A from ceramide generation to energy consumption, thus contributing to reduced ceramide levels. Interestingly, several studies have shown that activation of A M P K protects endothelial cells or astrocytes against hyperglycemia or high fat induced cell death, likely through lowering ceramide generation or caspase 3 activity (3, 19). In the current study, myocytes incubated with metformin demonstrated higher A M P K phosphorylation, and inhibition of high fat induced L C B - 1 (a subunit of SPT) expression and intracellular ceramide levels. Our data suggest that the beneficial effects of low doses of metformin in preventing cell death are likely through its modulation of ceramide formation and caspase 3 activation. It should be noted that although A M P K could mediate the protective effects of metformin against high fat induced cell death, further studies are required to substantiate this hypothesis. Additionally, whether metformin can also prevent cell death by mechanisms other than ceramide formation remains to be determined. It should be noted that despite the drop in ceramide and caspase 3 activity, high concentrations of metformin elicited substantial apoptosis. These data suggested that mechanisms other than lipotoxicity could be playing a central role in mediating these injurious effects of metformin. Metformin has been associated with lactic acidosis (2, 4). In the present study, as 5 mmol/1 metformin caused the highest change in lactate and p H measured in the cardiomyocyte incubation medium, lactic acidosis could be the major reason in explaining this toxic effect o f metformin. Metformin, associated with its activation o f A M P K , stimulates glucose uptake by 155 inducing G L U T 4 recruitment to the plasma membrane, and subsequent glycolysis through activation of P F K - 2 (15, 23, 29, 47). Metformin has also been implicated in F A oxidation through activation of A M P K and inhibition of A C C (48). Indeed, measurement of glycolysis and F A oxidation demonstrated a dose dependent increase in utilization of these substrates in myocytes treated with metformin. It should be noted that F A oxidation is known to inhibit glucose oxidation; acetyl-coA generated from F A can inhibit the pyruvate dehydrogenase complex, a rate-limiting enzyme in pyruvate oxidation (13, 34). Thus, even though metformin promotes glycolysis, glucose oxidation is likely compromised, leading to dissociation between glycolysis and glucose oxidation. In this situation, the protons and lactate generated from glycolysis accumulate, decrease intracellular p H , leading to intracellular calcium overload and cell death. To confirm that it is protons generated from glycolysis that dramatically increase apoptosis, glucose was removed from the culture media. Absence of glucose prevented the metformin (5 mmol/1) induced increase in L D H release. In fact, in this glucose free medium, the release of L D H with metformin was similar to control. Overall, even though high doses o f metformin significantly reduced ceramide and caspase 3 activity, its protective effects against high fat induced cell death is l ikely masked by its ability to augment proton production. A limitation of the present study was the use of quiescent non-beating myocytes. These cells have a lower overall oxidative capacity compared to the beating heart, likely leading to excessive lactate and proton accumulation. Additionally, in vivo, the lactate and protons generated are diluted and removed by body fluids, a situation that is unlikely in 156 our in vitro cell culture. Whether the above limitations explain the L D H release even with low concentrations of metformin is yet to be determined. In summary, our study demonstrates that low doses of metformin reduce high fat induced cardiac cell death, l ikely through its effects i n decreasing ceramide formation and caspase 3 activity (Fig. 5-9). However, through its role in increasing proton accumulation and lactic acidosis, metformin can induce cardiomyocyte cell damage in a caspase 3 independent manner (Fig. 5-9). 157 5.5. Figures A MET(mmol/l) Fig. 5-1 LDH release and cell apoptosis following incubation with high fat and metformin Cardiomyocytes were incubated with 1 mmol/1 palmitic acid, in the presence or absence of metformin (1-5 mmol/1). Following 16 hours, L D H released into the incubation medium was measured (A). Cells were also examined for morphological evidence of apoptosis using the fluorescent DNA-binding dye Hoechst 33342 (B & C). Data are means ± SE; n=3-8 myocyte preparations from different animals. One-way A N O V A followed by the Tukey test was used to determine differences between means. Significantly different from control, P < 0.05. "Significantly different from the high fat treated group, P < 0.05. ®Significantly different from all other groups, P < 0.05. 158 Fig . 5-2 ROS levels following incubation with high fat and metformin Cardiomyocytes were incubated with 1 mmol/1 palmitic acid, in the presence or absence of metformin (1-5 mmol/1). Following 16 hours, R O S levels were measured. Data are means ± SE; n=4 myocyte preparations from different animals. One-way A N O V A followed by the Tukey test was used to determine differences between means. Significantly different from control, P < 0.05. "Significantly different from the high fat treated group, P < 0.05. ®Significantly different from all other groups, P < 0.05. 159 B MET (mmol/1) CON 1 2 5 C O N 1 2 S_ MET (mmol/1) Fig. 5-3 Time dependent phosphorylation of AMPK by metformin Cardiomyocytes were treated with metformin (1-5 mmol/1) for either 1 (A) or 16 (B) hours. Protein was extracted and A M P K - a (total and phosphorylated) was measured using Western Blotting. Data are means ± S E ; n=4 myocyte preparations from different animals. One-way A N O V A followed by the Tukey test was used to determine differences between means. 'Significantly different from control, P < 0.05. @ Significantly different from all other groups, P < 0.05. 160 A B MET (mmol/l| CON P-ACC I p M H MET (mmol/1) C O N 1 P - A C C « — _ _ , immm* t t 8 5 < **9 TT -o . « 4 II o 2 •= is 2 tf) O E 2 1 hr X m MET (mmol/1) MET (mmol/1) 150 - \ I 120 100 MET (mmol/l) F ig . 5-4 Time dependent phosphorylation of ACC by metformin and PA oxidation Cardiomyocytes were treated with metformin (1-5 mmol/1) for either 1 or 16 hours. Protein was extracted and phosphorylated A C C was measured using Western Blotting (A). P A oxidation was measured using Tritium-labeled P A (B). Data are means ± SE ; n=4 myocyte preparations from different animals. One-way A N O V A followed by the Tukey test was used to determine differences between means. Significantly different from other groups, P < 0.05. ®Significantly different from all the other groups, P < 0.05. 161 MET (mmol/1) CON PA 1 2 5 * I I CON 1 2 5 MET (mmol/l) MET (mmol/l) CON PA 1 2 6 P-Ceramide -f_ ~~ , „— —». l.'i.j. '""*••" * I ICON 1 2 5 MET (mmol/l) c 1 2 5 MET (mmol/l) Fig. 5-5 Apoptotic mediators in isolated cardiomyocytes Cardiomyocytes were incubated with l mmol/l palmitic acid, in the presence or absence of metformin (1-5 mmol/1). Following 16 hrs, cardiac LCB1 expression was examined by Western Blot (A). Ceramide levels were measured using a DAG kinase assay (B). Caspase 3 activity was determined using an assay kit (C). Data are means ± SE; n=3-4 myocyte preparations from different animals. One-way ANOVA followed by the Tukey test was used to determine differences between means. 'Significantly different from control, P < 0.05. "Significantly different from the high fat treated group, P < 0.05. 162 2 5 MET (mmol/1) Fig. 5-6 Lactate release in the cardiomyocyte incubation medium Cardiomyocytes were incubated with 1 mmol/1 palmitic acid, in the presence or absence of metformin (1-5 mmol/1). Following 16 hrs, lactate released in the culture medium was measured using an assay kit. Data are means ± SE; n=8 myocyte preparations from different animals. One-way ANOVA followed by the Tukey test was used to determine differences between means. 'Significantly different from control, P < 0.05. ®Significantly different from all other groups, P < 0.05. 163 MET(mmol/l) Fig. 5-7 Rate of cardiomyocyte glycolysis following metformin incubation Cardiomyocytes were incubated with 1 mmol/1 palmitic acid, in the presence or absence of metformin (1-5 mmol/1). Following 16 hrs of incubation, glycolysis was measured using H-glucose. Data are means ± SE; n=4 myocyte preparations from different animals. One-way ANOVA followed by the Tukey test was used to determine differences between means. Significantly different from control, P < 0.05. Significantly different from the high fat treated group, P < 0.05. @Significantly different from all other groups, P < 0.05. 164 M E T (mmol/1) Fig. 5-8 LDH release in the absence of glucose following high fat and metformin Cardiomyocytes were incubated in a glucose free medium containing 1 mmol/1 palmitic acid, in the presence or absence of metformin (1-5 mmol/1). Following 16 hrs, L D H was measured. Data are means ± SE; n=4 myocyte preparations from different animals. One-way A N O V A followed by the Tukey test was used to determine differences between means. 'Significantly different from control, P < 0.05. #Significantly different from the high fat treated group, P < 0.05. 165 M Glucose FA ^ = MET/AMPK =L Glucose Glycolysis f H + +Pyruvate ACC - t FA Oxidation t Cell Damage \ Pyruvate Oxidation F A I Ceramide I — LCB1 I I Met/AMPK Caspase 3 V Apoptosis Mitochondria Cardiomyocyte Fig . 5-9 Proposed mechanism of how metformin influences cardiomyocyte cell death Metformin, likely through activation of A M P K , decreases ceramide formation and caspase 3 activity, thereby preventing high fat induced L D H release. However, as metformin also promotes F A oxidation, the high glycolysis is not matched by glucose oxidation (high fat oxidation inhibits glucose oxidation). This causes dissociation of glycolysis from glucose oxidation, leading to lactic acidosis, and caspase 3 independent cell death. 166 5.6. Bibliography 1. An D, Pulinilkunnil T, Qi D, Ghosh S, Abrahani A and Rodrigues B The metabolic "switch" A M P K regulates cardiac heparin-releasable lipoprotein lipase. Am J Physiol Endocrinol Metab 288: E246-253, 2005. 2. Bailey CJ and Turner RC Metformin. N Engl J Med 334: 574-579, 1996, 3. Blazquez C, Geelen MJ, Velasco G and Guzman M The AMP-activated protein kinase prevents ceramide synthesis de novo and apoptosis in astrocytes. FEBS Lett 489: 149-153,2001. 4. Brown JB, Pedula K, Barzilay J, Herson M K and Latare P Lactic acidosis rates in type 2 diabetes. Diabetes Care 21: 1659-1663, 1998. 5. Cai L and Kang YJ Ce l l death and diabetic cardiomyopathy. Cardiovasc Toxicol 3: 219-228, 2003. 6. Cai L , L i W, Wang G, Guo L, Jiang Y and Kang Y J Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome C-mediated caspase-3 activation pathway. Diabetes 51: 1938-1948, 2002. 7. Chan AY, Soltys C L , Young ME, Proud C G and Dyck JR Activation o f AMP-activated protein kinase inhibits protein synthesis associated with hypertrophy in the cardiac myocyte. JBiol Chem 279: 32771-32779, 2004. 8. Das A, Xi L and Kukreja RC Phosphodiesterase-5 inhibitor sildenafil preconditions adult cardiac myocytes against necrosis and apoptosis. Essential role of nitric oxide signaling. JBiol Chem 280: 12944-12955, 2005. 167 9. Detaille D, Guigas B, Chauvin C, Batandier C, Fontaine E , Wiernsperger N and Leverve X Metformin prevents high-glucose-induced endothelial cell death through a mitochondrial permeability transition-dependent process. Diabetes 54: 2179-2187, 2005. 10. Dyntar D, Eppenberger-Eberhardt M , Maedler K, Pruschy M , Eppenberger HM, Spinas GA and Donath MY Glucose and palmitic acid induce degeneration of myofibrils and modulate apoptosis in rat adult cardiomyocytes. Diabetes 50: 2105-2113, 2001. 11. Gallo A, Geolotto G, Pinton P, Iori E, Murphy E , Rutter GA, Rizzuto R, Semplicini A and Avogaro A Metformin prevents glucose-induced protein kinase C-beta2 activation in human umbilical vein endothelial cells through an antioxidant mechanism. Diabetes 54: 1123-1131, 2005. 12. Ghafourifar P, Klein SD, Schucht O, Schenk U, Pruschy M , Rocha S and Richter C Ceramide induces cytochrome c release from isolated mitochondria. Importance of mitochondrial redox state. J Biol Chem 274: 6080-6084,1999. 13. Hansford RG and Cohen L Relative importance o f pyruvate dehydrogenase interconversion and feed-back inhibition in the effect o f fatty acids on pyruvate oxidation by rat heart mitochondria. Arch Biochem Biophys 191: 65-81, 1978. 14. Hardin NJ The myocardial and vascular pathology of diabetic cardiomyopathy. Coron Artery Dis 7: 99-108, 1996. . 15. Hayashi T, Hirshman MF, Fujii N, Habinowski SA, Witters L A and Goodyear L J Metabolic stress and altered glucose transport: activation o f AMP-activated protein kinase as a unifying coupling mechanism. Diabetes 49: 527-531, 2000. 168 16. Hickson-Bick DL, Buja M L and McMillin JB Palmitate-mediated alterations in the fatty acid metabolism of rat neonatal cardiac myocytes. J Mol Cell Cardiol 32: 511-519, 2000. 17. Hickson-Bick DL, Sparagna GC, Buja L M and McMillin JB Palmitate-induced apoptosis in neonatal cardiomyocytes is not dependent on the generation of R O S . Am J Physiol Heart Circ Physiol 282: H656-664, 2002. 18. Hopkins TA, Dyck JR and Lopaschuk GD AMP-activated protein kinase regulation of fatty acid oxidation i n the ischaemic heart. Biochem Soc Trans 31: 207-212, 2003. 19. Ido Y, Carling D and Ruderman N Hyperglycemia-induced apoptosis in human umbilical vein endothelial cells: inhibition by the AMP-activated protein kinase activation. Diabetes 51: 159-167, 2002. 20. Jackson CV, McGrath GM, Tahiliani AG, Vadlamudi RV and McNeill JH A functional and ultrastructural analysis of experimental diabetic rat myocardium. Manifestation o f a cardiomyopathy. Diabetes 34: 876-883, 1985. 21. Kudo N, Barr AJ, Barr RL, Desai S and Lopaschuk GD High rates of fatty acid oxidation during reperfusion of ischemic < hearts are associated with a decrease in malonyl-CoA levels due to an increase in 5'-AMP-activated protein kinase inhibition of acetyl-CoA carboxylase. J C / z e m 270: 17513-17520, 199.5. 22. Kukidome D, Nishikawa T, Sonoda K, Imoto K, Fujisawa K, Yano M , Motoshima H, Taguchi T, Matsumura T and Araki E Activation of AMP-activated protein kinase reduces hyperglycemia-induced mitochondrial reactive oxygen species 169 production and promotes mitochondrial biogenesis in human umbilical vein endothelial cells. Diabetes 55: 120-127, 2006. 23. Kurth-Kraczek EJ, Hirshman MF, Goodyear L J and Winder WW 5' AMP-activated protein kinase activation causes G L U T 4 translocation in skeletal muscle. Diabetes 48: 1667-1671, 1999. 24. Longnus SL, Segalen C, Giudicelli J, Sajan MP, Farese RV and Van Obberghen E Insulin signalling downstream o f protein kinase B is potentiated by 5'AMP-activated protein kinase in rat hearts in vivo. Diabetologia 48: 2591-2601, 2005. 25. Lopaschuk GD and Barr RL Measurements of fatty acid and carbohydrate metabolism in the isolated working rat heart. Mol Cell Biochem 172: 137-147, 1997. 26. Lteif AA, Mather K J and Clark C M Diabetes and heart disease an evidence-driven guide to risk factors management in diabetes. Cardiol Rev \ 1: 262-274, 2003. 27. Luiken JJ, Coort SL, Willems J, Coumans WA, Bonen A, van der Vusse GJ and Glatz JF Contraction-induced fatty acid translocase/CD36 translocation i n rat cardiac myocytes is mediated through AMP-activated protein kinase signaling. Diabetes 52: 1627-1634, 2003. 28. Luna V, Casauban L, Sajan MP, Gomez-Daspet J, Powe JL, Miura A, Rivas J, Standaert M L and Farese RV Metformin improves atypical protein kinase C activation by insulin and phosphatidylinositol-3,4,5-(P04)3 in muscle of diabetic subjects. Diabetologia 49: 375-382, 2006. 29. Marsin AS, Bertrand L, Rider MH, Deprez J, Beauloye C, Vincent MF, Van den 170 Berghe G, Carling D and Hue L Phosphorylation and activation of heart P F K - 2 by A M P K has a role in the stimulation of glycolysis during ischaemia. Curr Biol 10: 1247-1255, 2000. 30. Martin A, Duffy PA, Liossis C, Gomez-Munoz A, O'Brien L, Stone JC and Brindley DN Increased concentrations of phosphatidate, diacylglycerol and ceramide in ras- and tyrosine kinase (fps)-transformed fibroblasts. Oncogene 14: 1571-1580, 1997. 31. Musi N, Hirshman MF, Nygren J, Svanfeldt M , Bavenholm P, Rooyackers O, Zhou G, Williamson JM, Ljunqvist O, Efendic S, Moller DE, Thorell A and Goodyear L J Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes. Diabetes 51: 2074-2081, 2002: 32. Obeid L M and Hannun YA Ceramide: a stress signal and mediator of growth suppression and apoptosis. J Cell Biochem 58: 191-198, 1995. 33. Ostrander DB, Sparagna GC, Amoscato AA, McMillin JB and Dowhan W Decreased cardiolipin synthesis corresponds with cytochrome c release in palmitate-induced cardiomyocyte apoptosis. JBiol Chem 276: 38061-38067, 2001. 34. Randle PJ, Garland PB, Hales CN and Newsholme EA The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances o f diabetes mellitus. Lancet I: 785-789, 1963. 35. Robergs RA, Ghiasvand F and Parker D Biochemistry of exercise-induced metabolic acidosis. Am J Physiol Regul Lntegr Comp Physiol 287: R502-516, 2004. 36. Rodrigues B, Cam M C , Jian K, Lim F, Sambandam N and Shepherd G 171 Differential effects of streptozotocin-induced diabetes on cardiac lipoprotein lipase activity. Diabetes 46: 1346-1353, 1997. 37. Ruderman NB, Saha A K and Kraegen EW Minireview. malonyl C o A , AMP-activated protein kinase, and adiposity. Endocrinology'144: 5166-5171, 2003. 38. Shaw RJ, Lamia KA, Vasquez D, Koo SH, Bardeesy N, Depinho RA, Montminy M and Cantley L C The kinase L K B 1 mediates glucose homeostasis in liver and therapeutic effects of metformin. Science 310: 1642-1646, 2005. 39. Shimabukuro M , Higa M, Zhou YT, Wang MY, Newgard CB and Unger RH Lipoapoptosis in beta-cells o f obese prediabetic fa/fa rats. Role of serine palmitoyltransferase overexpression. J Biol Chem 273: 32487-32490, 1998. 40. Shimabukuro M , Zhou YT, Levi M and Unger RH Fatty acid-induced beta cell apoptosis: a link between obesity and diabetes. Proc Natl Acad Sci USA 95: 2498-2502, 1998. 41. Sowers JR, Epstein M and Frohlich ED Diabetes, hypertension, and cardiovascular disease: an update. Hypertension 37: 1053-1059, 2001. 42. Sparagna GC, Jones C E and Hickson-Bick DL Attenuation o f fatty acid-induced apoptosis by low-dose alcohol in neonatal rat cardiomyocytes. Am J Physiol Heart Circ Physiol 287: H2209-2215, 2004. 43. Takeda Y, Tashima M , Takahashi A, Uchiyama T and Okazaki T Ceramide generation in nitric oxide-induced apoptosis. Activation of magnesium-dependent neutral sphingomyelinase via caspase-3. J Biol Chem 274: 10654-10660, 1999. 172 44. Unger RH and Orci L Lipoapoptosis: its mechanism and its diseases. Biochim Biophys Acta 1585: 202-212, 2002. 45. Unger R H and Zhou Y T Lipotoxicity o f beta-cells in obesity and in other causes of fatty acid spillover. Diabetes 50 Suppl 1: S118-121, 2001. 46. Vadlamudi RV and McNeill JH Effect o f experimental diabetes on rat cardiac c A M P , phosphorylase, and inotropy. Am J Physiol 244: H844-851,1983. 47. Yang J and Holman GD Long-term metformin treatment stimulates cardiomyocyte glucose transport through an AMPK-dependent reduction in G L U T 4 endocytosis. Endocrinology 2006. 48. Zang M, Zuccollo A, Hou X, Nagata D, Walsh K, Herscovitz H, Brecher P, Ruderman NB and Cohen RA AMP-activated protein kinase is required for the lipid-lowering effect o f metformin in insulin-resistant human HepG2 cells. J Biol Chem 279:47898-47905,2004. 49. Zhou G, Myers R, L i Y, Chen Y, Shen X, Fenyk-Melody J , Wu M , Ventre J, . Doebber T, Fujii N, Musi N, Hirshman MF, Goodyear L J and Moller DE Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 108: 1167-1174,2001. 50. Zhou YT, Grayburn P, Karim A, Shimabukuro M , Higa M , Baetens D, Orci L and Unger RH Lipotoxic heart disease in obese rats: implications for human obesity. Proc Natl Acad Sci USA 97: 1784-1789, 2000. 173 6. Conclusions and future direction For the first time, we report that A M P K , but not P P A R - a , is activated in the acute S T Z diabetic heart. The mechanisms that mediate this activation in A M P K are currently unknown. Insulin, through activation o f P K B , has been demonstrated to inhibit A M P K . In the current study, given that plasma insulin levels decreased following induction of diabetes, lack of insulin could be one potential mechanism the leads to the activation of cardiac A M P K . This activation of A M P K likely increases F A oxidation through phosphorylation and inhibition of A C C . Given that glucose utilization is compromised following hypoinsulinemia, this acute adaptation would ensure adequate cardiac energy production. In chronic diabetes, augmented plasma lipids and expression o f CD36 provides the heart with excess F A . Associated with increased plasma T G and intracellular F F A and TG, cardiac A M P K and A C C phosphorylation were normalized by mechanisms that have yet to be resolved. Interestingly, long-chain acy l -CoA esters are known to inhibit phosphorylation of A M P K by its upstream kinase L K B 1 . Moreover, augmented expression o f protein phosphatase 2C has been observed in hearts of Z D F rats, associated with a high rate of F A supply, which reduces A M P K phosphorylation. Thus, accumulation of F A or its derivatives in cardiomyocyte from chronic S T Z diabetic hearts could be one potential mechanism that leads to reduced A M P K phosphorylation compared to acute diabetic hearts. In this condition, P P A R - a , through its regulation of gene expression, l ikely contributes to high F A oxidation. Using fasting and modulators of A M P K , a strong correlation between this metabolic 174 switch and cardiac L P L activity was established. Our data suggest that in addition to its direct role in promoting FA oxidation, increased A M P K recruitment of L P L from its major storage site, the cardiomyocyte, to the coronary lumen could represent an immediate compensatory response by the heart to guarantee FA supply. The mechanisms that mediate this process are currently unclear. Given that A M P K has been implicated in triggering glucose transporter GLUT4 and fatty acid transporter CD36 translocation to the plasma membrane, through its influence on cytoskeleton reorganization, it is possible that, through similar mechanism, A M P K also enhances L P L vesicle translocation to the plasma membrane, which could lead to increased L P L secretion. Using isolated cardiomyocytes or perfused Langendorff hearts, we demonstrated that, unlike adipocytes, the P-adrenergic agonist isoproterenol has no direct effect on cardiac A M P K and LPL. However, using in vivo models or perfused working hearts, we found that isoproterenol could influence cardiac A M P K and L P L , only during conditions of increased workload and excessive energy expenditure. Finally, we determined that low doses of metformin reduce high fat induced cardiac cell death, likely through its effects in decreasing ceramide formation and caspase 3 activity. However, through its role in increasing proton accumulation and lactic acidosis, metformin can induce cardiomyocyte cell damage in a caspase 3 independent manner. Thus, A M P K may influence high fat induced cardiomyocyte apoptosis secondary to its regulation of metabolism. In clinical studies, over dose of metformin has been associated with lactic acidosis. However, it is controversial whether therapeutic doses of metformin increase the 175 risk of lactic acidosis, especially in patients who have renal or cardiac disease, or who are 70 years of age, or older. My study provides molecular evidence that metformin can induce lactate production and acidosis in cardiomyocytes under high fat conditions. It should be noted that as this study was performed in vitro, higher concentrations of metformin were used, beyond those administered clinically, or in vivo studies. Hence, caution should be used when extrapolating these results to a clinical setting. Nevertheless, our results suggest that even though metformin can be beneficial in reducing the high fat induced increase in cardiovascular disease, overdosing may promote detrimental cardiovascular effects. Overall, my studies indicate that A M P K plays an important role in the regulation of cardiac metabolism and cell death. It is still controversial whether these effects of A M P K on cardiac tissue are beneficial or harmful. For example, during ischemia and reperfusion, activation of A M P K has been suggested to be detrimental (due to its role in promoting FA oxidation and oxygen demand) or protective (due to its role in providing heart with energy generation). In Wolff-Parkinson-White Syndrome, constitutive activation of A M P K is associated with high glycogen accumulation and the development of hypertrophic cardiomyopathy. In my studies, we also found duel effects of metformin/AMPK on cardiomyocytes death. Indeed, metformin is only harmful to cardiomyocytes when a high concentration, together with a high concentration of fatty acid, is used. Therefore, whether A M P K is beneficial or harmful could be dependent on different physiological or pathological conditions or the degree of A M P K activation. Future studies could focus on the following issues: 176 1. Although activation o f A M P K was observed in acute, but not chronic S T Z diabetic hearts, the mechanisms for this dual effect of diabetes on A M P K is unknown. Given that A M P K is activated through both A T P dependent or independent mechanisms, future studies should focus on identifying which potential mechanism(s) control cardiac A M P K during diabetes. 2. Although a strong correlation o f cardiac A M P K and L P L was established, the mechanisms underlying this effect is not clear, and needs further investigation. These include post-translational regulation of L P L like degradation, secretion, transfer and recycling. 3. In isolated cardiomyocytes, metformin influences high fat induced apoptosis. Whether the same effects also occur in intact animals is unknown, and needs to be determined. 177 

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