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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 C E L L 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 T H E 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 o f 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 ( S T Z ) 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. of A M P K  This activation  likely increases F A oxidation through phosphorylation and inhibition of  acetyl-CoA carboxylase ( A C C ) .  In chronic diabetes, augmented plasma lipids and  expression o f C D 3 6 provide the heart with excess F A .  In this condition, PPAR-oc,  through its regulation o f 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 o f C D 3 6 .  Given that F A released from lipoprotein is suggested  to be the main F A source supply to the heart, and lipoprotein lipase ( L P L ) is the primary enzyme controlling lipoprotein metabolism, of interest to us was the question o f 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 o f 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 o f metabolism,  ii  recent studies suggest that A M P K can also modulate cell death.  Given that A M P K ,  through elevation o f 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 l o w doses o f 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.  \  iii  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  CARDIAC METABOLISM  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  7  1.1.3. 1.2.  Decarboxylase (MCD)  Interaction between glucose and FA metabolism  LPL  7 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.  AMPK  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  20  1.5.  FIGURES  22  1.6.  BIBLIOGRAPHY  -26  iv  2. A C U T E AND CHRONIC STREPTOZOTOCIN DIABETES DIFFERENTIALLY REGULATES CARDIAC PPAR-oc AND A M P K  54  2.1.  INTRODUCTION  54  2.2.  METHODS  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  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  59  60  2.4.  DISCUSSION  61  2.5.  T A B L E S A N D FIGURES  64  2.6.  BIBLIOGRAPHY  70  3. T H E METABOLIC 'SWITCH' A M P K REGULATES CARDIAC HEPARIN-RELEASABLE L P L , 3.1.  INTRODUCTION  3.2.  MATERIALS AND METHODS  74 74 ,  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  3.2.5.  Measurement of cardiac LPL expression  78  77  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.  81  3.3.  Materials  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  Inhibition ofAMPK phosphorylation lowers cardiac LPL  83  . 3.3.3. 3.3.4.  Promotion ofAMPK phosphorylation in control hearts recruits LPL to the luminal surface  84  v  3A  DISCUSSION  85  3.5.  T A B L E S A N D FIGURES  90  3.6.  BIBLIOGRAPHY  98  4. p-ADRENERGIC AGONIST STIMULATION PRODUCES CHANGES IN CARDIAC A M P K AND CORONARY L U M E N L P L ONLY DURING INCREASED WORKLOAD  105  4.1.  INTRODUCTION  105  4.2.  MATERIALS AND METHODS  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  Ill  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.  113  Statistical analysis  4.2.12. Materials 4.3.  113  RESULTS  4.3.1.  114  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 . 1 1 4  4.3.3.  Isoproterenol does not influence LPL activity and AMPK phosphorylation in myocytes or Langendorffperfused hearts  4.3.4.  115  Increasing workload promotes phosphorylation ofAMPK and ACC and enlarges the coronary lumen LPL pool  4.3.5.  116  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.  BIBLIOGRAPHY  131  5. METFORMIN INFLUENCES CARDIOMYOCYTE C E L L DEATH BY BOTH CASPASE-3 DEPENDENT AND INDEPENDENT PATHWAYS 141 5.1.  INTRODUCTION  5.2.  MATERIALS A N D METHODS  •  141 143  5.2.1.  Experimental animals  143  5.2.2.  Cardiomyocyte isolation and culturing  143  5.2.3.  LDHrelease  •  vi  144  5.2.4. Hoechst 33342 staining 5.2.5. Estimation of reactive oxygen species (ROS) 5.2.6. Western blot analysis 5.2.7. Ceramide assay  144 144 145 145  5.2.8. 5.2.9. 5.2.10. 5.2.11. 5.2.12. 5.3.  Caspase 3 activity Rates ofglycolysis and palmitic acid oxidation Lactate assay Statistical analysis Materials  RESULTS  146 146 147 147 147 149  5.3.1. 5.3.2. 5.3.3. 5.3.4. 5.3.5.  High fat induced LDH release and consequence of metformin 149 High fat induced apoptosis and consequence of metformin 149 ROS generation following provision ofhigh fat and metformin 149 Effects of metformin on AMPK phosphorylation in cardiomyocytes 150 ACCphosphorylation and FA oxidation in cardiomyocytes incubated with metformin 150 5.3.6. Effects of metformin on LCB1 expression and intracellular ceramide 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.  BIBLIOGRAPHY  167  CONCLUSIONS AND FUTURE DIRECTION  vii  174  LIST OF TABLES  Table 2-1  General characteristics o f the experimental animals  64  Table 2-2  Cardiac lipids i n control and S T Z groups  65  Table 3-1  General characteristics o f the animals  90  Table 4-1 Table 4-2  General characteristics o f the animals Cardiac lipids i n control and isoproterenol treated groups  viii  123 124  LIST OF FIGURES Fig. 1 -1  Glucose utilization i n 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 Fig. 2-1  L P L synthesis, secretion and transfer Gene expression o f P P A R - a , CPT-1 and A C O in 4-day and 6-week S T Z diabetic hearts  25 :...66  Fig. 2-2 Expression o f M C D i n 4-day and 6-week S T Z diabetic hearts  67  Fig. 2-3  68  Expression o f C D 3 6 i n 4-day and 6-week S T Z diabetic hearts  Fig. 2-4 Cardiac A M P K and A C C phosphorylation i n 4-day and 6-week S T Z diabetic Fig. 3-1  hearts  69  Effect o f fasting on cardiac A M P K phosphorylation  91  Fig. 3-2 Alterations i n L P L activity and immunofluorescence i n hearts isolated from fasted animals L P L gene expression, protein mass and activity i n hearts isolated from fasted  92  Fig. 3-3  animals Consequence o f A M P K inhibition on heparin-releasable L P L activity i n fasted  93  Fig. 3-4  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 o f inhibiting A T P synthesis on A M P K phosphorylation and heparin-releasable L P L activity  96  Fig. 3-7 Regulation o f cardiac L P L by A M P K . . . . . Fig. 4-1 Effects o f ISO on coronary luminal L P L activity and gene expression after a  97  single in vivo injection 125 Fig. 4-2 A M P K and A C C 2 8 0 phosphorylation i n hearts isolated from animals injected with ISO for 1 o r 4 h r s Fig. 4-3 Heparin releasable L P L activity and A M P K phosphorylation i n Langendorff hearts perfused with I S O  126 127  Fig. 4-4 Comparison o f heparin releasable L P L activity, A M P K and ACC280 phosphorylation i n perfused Langendorff or working hearts Fig. 4-5  128  Consequence o f additional substrate provision in regulating A M P K and ACC280 phosphorylation in isolated working hearts in the absence or presence o f ISO ..129  Fig. 4-6  Consequence o f additional substrate provision in regulating heparin-releasable L P L i n isolated working hearts, i n the absence or presence o f I S O  Fig. 5-1  130  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 i n isolated cardiomyocytes  162  Fig. 5-6  Lactate release i n the cardiomyocyte incubation medium  163  Fig. 5-7  Rate o f cardiomyocyte glycolysis following metformin incubation  164  F i g . 5-8  L D H release i n the absence o f 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 ACC  Acetyl coenzyme A carboxylase  ACO  Acetyl coenzyme oxidase  ACS  A c e t y l coenzyme synthase  ADP  Adenosine diphosphate  AICAR  5-Aminoimidazole-4-carboxamide ribonucleoside  AMP  Adenosine monophosphate  AMPK  A M P activated protein kinase  ANOVA  Analysis o f variance  Apo  Apolipoprotein  Ara-A  Adenine 9-beta-D-arabinofuranoside  BSA  Bovine serum albumin  CBS  Cystothionine P synthase  CPT-1  Carnitine palmitoyl transferase-1  DAG  Diaglycerol  DNA  Deoxyribonucleic acid  EDTA  Ethylenediamine tetraacetic acid  ER  Endoplasmic reticulum  FA  Fatty acid  FABPp  m  Fatty acid binding protein plasma membrane  FAS  Fatty acid synthase  FATP  Fatty acid transport protein  F2,6BP  Fructose 2, 6-bisphosphate  g  Gram  GBD  Glycogen binding domain  GLUT  Glucose transporter  HEPES  4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid  hr  Hour  HPLC  H i g h performance liquid chromatography  HR-LPL  Heparin releasable-lipoprotein lipase  HSPG  Heparan sulphate proteoglycan  IgG  Immunoglobulin  ISO  Isoproterenol  i.p.  Intraperitoneal  i.v.  Intravenous  kg  Kilogram  K H buffer  Krebs-Henseleit buffer  LCAD  L o n g chain a c y l - C o A dehydrogenase  LCB  L o n g chain base  xi  LDH  Lactate dehydrogenase  LPL M  Lipoprotein lipase  MCD  M a l o n y l coenzyme A decarboxylase  mg  Milligram  min  Minute  ml  Milliliter  mm  Millimeter  mM  Millimolar  mRNA  Messenger ribonucleic acid  mTOR  Mammalian target o f rapamycin  NAD /NADH  Nicotinamide adenine dinucleotide  nM  Nanomolar  PBS  Phosphate buffered saline  PDH  Pyruvate dehydrogenase  PDK  Pyruvate dehydrogenase kinase  PFK  Phosphofructokinase  Pi  Phosphate  PGC-1 PPAR  P P A R - y Coactivator-1 Peroxisome proliferator activated receptor  RNA  Ribonucleic acid  RPP  Rate-pressure product  RT-PCR  Reverse transcription polymerase chain reaction  SDS  Sodium dodecyl sulphate  SEM  Standard error o f means  SPT  Serine palmitoyl- transferase  SREBP  Sterol regulatory element-binding protein  Molar  +  STZ  Streptozotocin  TBS-T  Tris buffered saline-Tween  TG  Triglyceride  VLCAD  Very long chain a c y l - C o A dehydrogenase  VLDL  Very low density lipoprotein  ul uM  Microliter Micromolar  xii  ACKNOWLEDGEMENTS  First, I would like to thank m y 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 o f my supervisory committee: Dr. Christ Mcintosh, Dr. Norbert Haunerland, Dr. Thomas Chang and Dr. Kishor Wasan for their valuable suggestions and encouragement during m y doctoral training. I would also like to thank our collaborators, Dr. Roger Brownsey, Dr. Michael Allard and Dr. Sheila Innis for their instructions and technical supports. I would further like to acknowledge m y colleagues and friends, Ashraf, Dake, Sanjoy, Thomas, Girish and Jennifer for their friendship and supports. Finally, I would like to take this opportunity to thank m y parents, brother, Uncle Y u and his family and Aunt Y i n g and her family for their love, supports and understanding.  xiii  DEDICATION  l b myfamity, whose Cove andnunurir made this adpossi6Ce  xiv  1. Introduction 1.1. C a r d i a c M e t a b o l i s m 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  glycolysis, and pyruvate decarboxylation (Fig. 1-1). on  the transmembrane  Cardiac glucose uptake is dependent  glucose gradient, and the content  transporters ( G L U T 1 and G L U T 4 ) (95, 130).  uptake,  o f sarcolemmal glucose  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. stimulated  It should be noted that G L U T mediated glucose uptake could also be through  demonstrated  insulin  independent  mechanisms.  Thus, recent  that A M P activated protein kinase ( A M P K )  studies  also promotes  have  GLUT4  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. generation o f fructose  1,6-bisphosphate  P F K 1 , the enzyme that catalyzes the  from fructose 6-phosphate,  is a rate-limiting  enzyme controlling glycolysis (43, 137). P F K - 1 is inhibited by l o w 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). P F K - 2 (81).  F 2 , 6 B P is formed from fructose 6-phosphate catalyzed by  G i v e n that P F K - 2 is phosphorylated and activated by insulin (18, 44),  glucagon (162) or A M P K (80, 112), stimulation o f P F K - 2 through these mechanisms increases F 2 , 6 B P generation, activates P F K - 1 and subsequently promotes glycolysis.  In  an aerobic heart, glycolysis contributes less than 10% o f 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 ( P D H ) , a multienzyme complex.  P D H is phosphorylated and inactivated by pyruvate dehydrogenase kinase  ( P D K ) (78, 182), which is inhibited by pyruvate and stimulated by high mitochondrial a c e t y l - C o A / C o A and N A D H / N A D ( + ) ratios (23, 76).  A c e t y l - C o A 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 A T P , 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% o f A T P generated i n an aerobic heart.  F A metabolism includes multiple steps, and can be  regulated by both acute and chronic mechanisms, with or without modulation o f 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 T G - r i c h lipoproteins.  It should be  noted that the molar concentration o f F A i n lipoprotein-TG is - 1 0 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 o f 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 ( H S P G ) 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 o f coronary endothelial cells (131).  At  these sites, L P L hydrolyzes T G - r i c h lipoproteins, such as very low density lipoprotein ( V L D L ) and chylomicrons, to release F A . chylomicrons, in the  Perfusion o f working hearts with V L D L or  absence or presence o f F A , has  chylomicrons, V L D L is a poor substrate for L P L .  revealed that compared  to  In addition, utilization o f chylomicrons  was inhibited by free F A , which failed to affect cardiac V L D L utilization.  G i v e n the  important role that L P L plays in regulating F A delivery, alteration i n its level is able to change F A delivery, and subsequent oxidation.  Indeed, overexpression o f L P L i n the heart  or skeletal muscle accelerates F A uptake (100, 184).  Conversely, tissue specific knock-out  o f L P L i n 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 o f 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). also likely required to support this process.  Thus, F A transporters are  In the heart, three F A transporters have been  identified, and these include C D 3 6 , F A transport protein (FATP), and F A binding protein plasma membrane ( F A B P P M ) (109).  G i v e n that 55-80% o f F A transport was blocked  using general transporter inhibitors (93, 110), and that overexpression o f FATP or C D 3 6 has been found to dramatically increase F A metabolism (34, 83), these F A transporters are believed to play a key role i n F A delivery to the cardiac tissue. Regulation o f F A transport proteins occurs through different mechanisms.  In severe S T Z  induced diabetes, expression o f cardiac C D 3 6 and F A B P p m were augmented (106), suggesting transcriptional control o f this transporter. induce this change have yet to be elucidated.  A t present, the mechanisms that  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 o f this transporter has also been suggested. c)  Acyl-CoA  synthase  (ACS).  A C S catalyzes the esterification o f F A to fatty  4  acyl-CoA, the initial step o f F A metabolism.  Fatty a c y l - C o A can be transported into the  mitochondria for oxidation, or used for intracellular T G synthesis.  The fate o f fatty  a c y l - C o A is influenced by the location o f different A C S isoforms, energy demand, and the availability o f 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 C D 3 6 or F A T P on the cytosolic side of the sarcolemmal membrane (62, 136, 150), suggesting that A C S also influences F A uptake.  Indeed, overexpression o f A C S i n the heart or fibroblast causes dramatically  augmented F A uptake and intracellular T G accumulation (35). Under normal conditions, 70-90% o f 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 o f 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 o f cell apoptosis, its augmented lipolysis expands fatty a c y l - C o A levels, w h i c h 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 o f  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, P P A R s form complexes with retinoid X receptors and bind to the  promoter regions o f a number o f target genes, which encode the proteins involved in controlling F A metabolism (55, 57).  Through regulation o f expression o f these genes,  P P A R s modulate F A utilization at the transcriptional level. P P A R - a , P P A R - P (or 5), and PPAR-y.  P P A R s have three isoforms:  P P A R - a is extensively expressed i n 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 o f F A metabolic genes, and switches substrate selection from F A to glucose (99, 121). augments F A uptake and oxidation (56, 58).  Overexpression o f cardiac P P A R - a  Taken together, P P A R - a is believed to be the  primary regulator o f F A metabolism i n the heart. expressed abundantly i n the cardiac tissue (11).  Similar to P P A R - a , P P A R - P (or 8) is  Activated by elevated intracellular F A (30),  P P A R - P (or 8) augments expression o f a group o f genes that promote F A utilization (45, 119).  Cardiac specific knock-out o f P P A R - P also decreases F A oxidative gene expression,  and F A oxidation (32).  Although the targets o f P P A R - a and P P A R - P are partially  overlapping (119), their unique roles and interaction remains unclear i n the heart.  PPAR-y,  the third number o f the P P A R family, is highly expressed i n adipose tissue.  Through  promoting lipogenic gene expression, PPAR-y controls lipogenesis. function experiments have demonstrated  Loss and gain o f  that P P A R - y is necessary for adipose  6  tissue  proliferation and differentiation (10, 169).  In isolated cardiomyocytes, the expression o f  P P A R - y is barely detectable (64), suggesting a limited role for this nuclear receptor i n regulating cardiac metabolism. e)  AMP activated protein kinase (AMPK).  following  a rise in the  A s an energy sensor, A M P K is activated  intracellular A M P / A T P  ratio  demonstrated that A M P K regulates cardiac metabolism.  (70).  Recent  studies  have  Details are included i n 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 o f m a l o n y l - C o A to acetyl-CoA, leading to reduction o f m a l o n y l - C o A (46). This action relieves the inhibition o f CPT-1 by malonyl-CoA, and favors F A oxidation. Recent studies have suggested that inhibition o f 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 o f glucose and F A metabolism does not occur independently, and numerous studies have reported a 'cross-talk' between the utilization o f 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 o f F A  impairs insulin mediated glucose uptake through inhibition o f insulin receptor substrate (IRS)  and protein kinase B (66, 85, 127).  Accumulation o f 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 o f 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 i n 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 i n cardiomyocytes, while 3-6% and 18% are located at capillary endothelium and extracellular space respectively.  Within the cardiomyocyte, L P L is localized i n the  sarcoplasmic reticulum, G o l g i 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 o f L P L shows tissue specificity (90), different mechanisms may control transcription o f L P L i n various tissues.  Indeed, four transcription initiation sites,  two promoter elements and several enhancer motifs have been identified i n the 5' upstream region o f the L P L gene (42). fully understood. synthesized  in  The mechanisms o f regulation o f these sites are still not  Following transcription, inactive and monomeric proenzyme o f L P L is endoplasmic  reticulum  (ER).  This  inactive  monomer  requires  glycosylation and a sequence o f post-translational processing to form an active homodimer L P L (7, 16, 132).  During glycosylation, a lipid-linked oligosaccharide, which is rich i n  mannose, is added to the arginine residues o f the nascent L P L polypeptide (24, 50).  This  mannose rich oligosaccharide allows formed glycoprotein to be retained i n 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  9  a-mannosidase,  before it translocates to the G o l g i complex (24).  Once inside this compartment, three  more mannoses on this L P L glycoprotein are further removed by mannosidase I (24).  As  L P L moves i n the G o l g 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 o f 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 i n the G o l g i 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 o f newly synthesized L P L is degraded.  Thus, i n pulse-chase experiments, 80% o f newly synthesized L P L i n 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).  G i v e n 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 l o w or moderate  rate o f 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 o f cells also  can release L P L through regulated mechanisms. L P L can be secreted.  Following a stimulus, a large number o f  F o r 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 i n 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 o f L P L . Eventually, L P L translocates to its functional site at the luminal surface o f blood vessels (51, 126). phases:  Thus, perfusion o f isolated hearts with heparin causes release o f L P L in two  a rapid and  large  amount  o f L P L release  within  seconds  after  heparin  administration, representing the L P L bound to the luminal side o f blood vessels, and a sustained release indicating a constant mobilization o f 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 o f heparanase like compounds from endothelial cells was observed, and was suggested to cleave H S P G on adipocytes, leading to release o f oligosaccharide bound L P L (131).  Subsequently, L P L transfers from the  abluminal to the luminal side o f endothelial cells.  This transcytosis involves both H S P G  and very l o w density lipoprotein receptor, given that inhibition o f 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 o f L P L is complicated, and involves transcriptional and post-transcriptional mechanisms,  and  shows  tissue-specificity.  Post-transcriptional regulation  includes  modification o f L P L protein (14, 15), intracellular L P L degradation and secretion (172), L P L transfer to luminal side o f blood vessels (133), and L P L recycling (31, 65).  Many of  these mechanisms have yet to be completely elucidated. Control  o f L P L at the  physiological conditions.  transcriptional level has  been  reported  under  several  For example, L P L is present i n the liver, only in fetal or early  postnatal life, with gene expression being turned off with age (128). gene expression o f L P L is switched on once the cells are activated (8). exposure augments L P L expression in brown adipose tissue (29).  In macrophages, Moreover, cold  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 o f 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 i n adipose tissue  without corresponding changes i n the levels o f 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 i n 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 degradation.  o f post-transcriptional  regulation  is through  L P L intracellular  For example, incubation o f adipocytes w i t h heparin diverts L P L from  degradation to secretion pathways (172). endothelial bound L P L (148).  L P L is also regulated through recycling o f  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 o f 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 o f lipoproteins containing apo CII to L P L enhances lipolysis (125), while  binding o f lipoproteins containing apo CIII or apo E suppresses L P L activity (1, 2). Interestingly, a recent study has shown that overexpression o f HDL-associated apo A V accelerates hydrolysis o f 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,  atherosclerosis, diabetes and obesity (113).  such  as  dyslipidemia,  insulin  resistance,  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 o f whole body L P L or  inhibition o f L P L by overexpression o f apo CIII leads to hypertriglyceridemia (1, 178).  13  Conversely, overexpression o f L P L prevents diet or diabetes induced hypertriglyceridemia (92, 157, 159),  Taken together, these experiments  prevention o f hypertriglyceridemia. of  atherosclerosis.  Thus,  atherosclerosis (53, 158). controversial. (91).  global  support a key role for L P L in  Additionally, L P L also protects against development overexpression  of  L P L prevents  diet-induced  Comparatively, the role o f L P L i n insulin resistance is more  Global overexpression o f L P L improves high fat induced insulin resistance  Given that whole body overexpression o f 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 i n regulating metabolism.  Cardiac specific knock out o f 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 i n the heart also leads to lipid overload and lipotoxic cardiomyopathy, similar to diabetic cardiomyopathy (184).  Thus, a balance o f 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. A T P is hydrolyzed to A D P .  To drive energy supply,  A s a constant A T P generation and supply is fundamental for a  cell to maintain its function, a balance o f 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 i n  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). subunits has multiple isoforms.  Each o f these  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 o f heterotrimer exist, and likely show tissue specificity. :  the heart, a 2 and p2, rather than a l and p i , is highly expressed (146).  In  XI and X2 are both  expressed the heart, while X3 is only found in skeletal muscle (33). The C - terminus o f the a subunit contains the binding sites for P and X subunits, which forms a complex with the other two subunits (38). serine/threonine protein kinase catalytic domain (67).  The N-terminus contains a  The a subunit also contains several  residues that can be phosphorylated, such as T h r l 7 2 , Thr258 and Ser485 (180).  T h r l 7 2 is  located i n the activation loop o f the catalytic domain, and its phosphorylation leads to the activation o f A M P K .  The p subunit contains two conservative regions: K I S 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). function o f G B D is still unclear.  The exact  Interestingly, A M P K is localized with glycogen, and  removal o f 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 o f cystothionine P synthase ( C B S ) 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 o f A M P activates A M P K  through three mechanisms.  allosterically activates A M P K by binding to the X subunit (146). mildly activates A M P K .  First, A M P  This mechanism only  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 T h r l 7 2 site o f a subunit is phosphorylated by L K B 1 , leading to 50-100 times increase i n A M P K activity (75). essential for A M P K activity.  Indeed, T h r l 7 2 phosphorylation is  Using an antibody that specifically recognizes A M P K with  T h r l 7 2 phosphorylated, studies have shown that phosphorylation o f T h r l 7 2 mirrors A M P K activity (28).  Mutation o f this site abolishes activation o f A M P K (38, 163).  Besides  T h r l 7 2 , several other sites i n the a subunit are also phosphorylated by L K B 1 (180). However, phosphorylation o f these sites does not change the activity o f A M P K (180). The roles o f phosphorylation i n these sites remain unknown.  The third mechanism by  which A M P activates A M P K is through an indirect process.  B i n d i n g with A M P also  makes  AMPK  dephosphorylate  a worse Thrl72  substrate for protein phosphatases, and  competitively inhibits A M P K .  inactivate  AMPK  (41).  which  are known to  Compared to  A M P , ATP  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 o f A M P / A T P ratio is a more accurate indicator o f 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 i n 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 i n the heart during ischemia through an ATP-independent mechanism (12, 61).  Given that overexpression o f active A k t i n  cardiomyocyte suppressed A M P K activity, it is likely that activation o f A k t mediates the effect o f 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 i n heart, skeletal muscle and liver (37, 141, 174). studies  have  demonstrated  that anti-diabetic  drugs  also  Additionally, recent  promote  AMPK  activity.  Metformin, a drug commonly used in the management o f 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 i n liver abolished the effects o f 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, P P A R - y  agonists,  17  also activate  AMPK,  likely  though  inhibition o f complex 1 i n the mitochondrial respiratory chain, and changes i n 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 o f 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 o f  targets like A C C 1 and F A S , A M P K reduces energy consumption (69).  Besides these  acute actions, through regulation o f 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 o f numerous genes involved i n mitochondria biogenesis (166). Moreover, through down-regulating S R E B P expression, A M P K decreases expression of genes involved i n lipogenesis (190).  A M P K also influences protein synthesis, which  accounts for 20% o f energy turnover i n growing cells. three different mechanisms. factor 2, A M P K  This effect is mediated through  First, through phosphorylation and activation o f elongation  inhibits protein synthesis (77).  Another mechanism o f  AMPK  modulating protein synthesis is through inhibition o f target o f rapamycin ( T O R ) pathway, which is a main regulator o f protein synthesis (22). stability.  Finally, A M P K also affects m R N A  Through reducing R N A - b i n d i n g protein H u R , 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 i n protein translation  18  (176, 177). 1.3.4.  Physiological  roles of AMPK  At the cellular level, A M P K 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, A M P K 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. 19  Moreover, AMPK also  facilitates F A utilization through its control o f acetyl-CoA carboxylase ( A C C ) (96, 97).  As  A C C catalyzes the conversion o f acetyl-CoA to malonyl-CoA, A M P K by inhibiting A C C is able to decrease m a l o n y l - C o A and minimize its inhibition o f C P T - 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 o f 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 o f A M P K does not affect cardiac metabolism under physiological conditions (144, 183). activated  following  A t present, it is unclear as to what compensatory mechanisms are knock-out  of A M P K .  Additionally,  it  is  unknown  whether  overexpression o f A M P K could affect cardiac lipid 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 i n cells.  Following hypoinsulinemia, as glucose utilization is impaired, the heart  switches to use F A . promoting  Previous studies have suggested that P P A R - a is implicated in  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 i n the elevation o f F A oxidation during this condition are unknown.  In addition to its role i n  modulating F A oxidation, A M P K has also been found to be involved i n F A uptake through C D 3 6 (107).  G i v e n 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 i n 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 i n this process has yet to be determined. Finally, beside its role in the regulation o f metabolism, recent studies suggest that A M P K can also modulate cell death.  Thus, i n the astrocytes and endothelial cells, activation o f  A M P K has been suggested to protect high fat or hyperglycemia induced apoptosis (21, 84). During obesity or diabetes, augmented circulating lipid is one o f the main reasons leading to lipotoxicity and cardiomyocytes apoptosis (171).  G i v e n 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.  Taken together, m y Ph.D. project  includes 4 objectives: 1. To examine the regulation o f 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 o f A M P K i n this process.  4.  To investigate whether metformin (a drug widely used i n 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 o f reactions that convert glucose into pyruvate.  P F K 1 , the  enzyme that catalyze the generation o f 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 .  F o l l o w i n g 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 .  A c e t y l - C o A then enters citric acid cycle and is eventually broken down to H2O and C 0 for 2  A T P generation.  P F K , phosphofructokinase,  pyruvate dehydrogenase kinase.  22  P D H , pyruvate  dehydrogenase, P D K ,  BLOOD VESSEL  CARDIOMYOCYTE  Fig. 1-2 Control of FA delivery  and utilization  in the cardiomyocyte  F A , either from  adipose tissue or released from T G - r i c h lipoproteins through hydrolysis by lipoprotein lipase ( L P L ) , is taken up into the cardiomyocyte by three F A transporters: C D 3 6 , F A transport protein (FATP), and F A binding protein plasma membrane ( F A B P ) . P M  F A is  converted to fatty a c y l - C o A , which is transported into mitochondria through C P T 1 / C P T 2 . Inside the mitochondria, fatty a c y l - C o A undergoes (3-oxidation to generate acetyl-CoA, which is further oxidized i n citric acid cycle. different mechanisms.  The utilization o f F A is regulated through  F A , through activation o f P P A R - a , increases the expression o f a  number o f enzymes involved i n F A oxidation.  M a l o n y l - C o A , w h i c h is generated through  carboxylation o f acetyl-CoA catalyzed by A C C , inhibits CPT-1 and F A oxidation. ,  inhibits A C C , relieves its inhibition on CPT-1 and promotes F A oxidation.  AMPK 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 , m a l o n y l - C o A decarboxylase.  23  Glucose  Fig. 1-3 Inhibition  of glucose oxidation by FA utilization  Accumulation o f F A impairs  insulin mediated glucose uptake through inhibition o f 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 o f genes involved i n F A oxidation, as w e l 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 o f P D H .  Augmented  acetyl-CoA also causes accumulation o f 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 synthesized and processed i n E R and Golgi.  After gene transcription,  Mature L P L is packaged into secretory  vesicles for either lysosomal degradation or secretion.  Secreted L P L binds to cell surface  H S P G , before transferring to the abluminal side o f endothelial cells. transcytosed to the apical surface.  L P L is  Subsequently, L P L is  L P L at the lumen can be internalized and recycled i n  endothelial cells.  25  1.6. Bibliography 1.  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Exogenous F A supply depends on L P L mediated hydrolysis o f circulating lipoprotein triglyceride, and membrane transporters like C D 3 6 .  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 o f genes under the control o f a nuclear receptor, P P A R - a (9).  Following activation, P P A R - a regulates the  expression o f genes such as fatty acyl C o A synthase (17), CPT-1 (5), and A C O (23). "fuel gauge" A M P K also plays a key role i n the regulation o f F A utilization. A M P K facilitates F A utilization through its control o f A C C (14).  The  In the heart,  A s A C C catalyzes the  conversion o f a c e t y l - C o A to malonyl-CoA, A M P K by inhibiting A C C is able to decrease malonyl-CoA and minimize its inhibition o f F A oxidation.  AMPK  has also been  implicated i n F A delivery to cardiomyocytes through its regulation o f the F A transporter,  54  CD36(16)andLPL(l). Following chronic diabetes, as glucose transport and utilization are impaired, the heart switches to excessive use o f F A for energy production (25).  This cardiac adaptation is  achieved by activation o f P P A R - a , and up-regulation o f an array o f genes involved i n 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 o f 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 i n 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 o f laboratory animals published by the U S National Institutes o f 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 A n i m a l Care Unit and supplied with a standard laboratory diet ( P M I Feeds, Richmond, V A ) , and water ad libitum.  Rats were randomly divided into nondiabetic  control ( C O N ) and diabetic ( S T Z ) groups.  Halothane-anesthetized rats were injected with  S T Z (55 mg/kg IV, Sigma Chemical Co) or an equivalent volume (1 mL/kg) o f 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 , H u m u l i n 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 R T - P C R . from hearts (100 mg) was extracted using Trizol (Invitrogen).  Briefly, total R N A  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 o f P P A R - a , C P T - 1 , A C O , M C D and C D 3 6 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  PPAR-a  (M88592);  5' - T A T G T G A G G A T G C T G C T T C C - 3 '  5'-CTCGGAGAGCTAAGCTTGTC-3' 5' - G C C C T C A G C T A T G G T A T T A C - 3 ' (right)  for  ACO  (J02752);  (left)  5' - C T C T G A C A T T T G C A G G T C C A - 3 '  control  using  and  (right) (left)  and  min  CPT-1  and  (L07736);  5' - A G G A A C T G C T C T C A C A A T G C - 3 '  for  MCD  (left)  and  (NM_053477)  ;  5' - C A C A G G C T T T C C T T C T T T G C - 3 '  The P-actin (J00691) gene was amplified as an internal  5' - C G T A A A G A C C T C T A T G C C A A - 3 '  5 ' - A G C C A T G C C A A A T G T C T C A T - 3 ' (right). 15-40 cycles.  for  5'-GCCTGGTACCTTTACGGTGA-3'  5'-GCTACCAGGCTGAGGATCTG-3'  (right) for C D 3 6 (NM_031561).  (right)  (left)  (left)  and  The linear range was found to be between  The amplification parameters were set at: 94°C for 1 min, 56-58°C for 1  and 72°C for 1 min, for a total o f 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 o f signal intensity o f 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 o f heart tissue was ground under liquid nitrogen and homogenized. out as described previously (1).  Western blot was carried  Samples were diluted, boiled with sample loading dye,  and 50 pg protein used i n SDS-polyacrylamide gel electrophoresis.  After  transfer,  membranes were blocked i n 5% skim milk i n Tris-buffered saline containing 0.1%  57  Tween-20.  Membranes were incubated with rabbit A M P K - o c , p h o s p h o - A M P K (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  B l o o d 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 i n 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 , Milford, 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 m m ; Supelco, Bellefonte, P A ) . Value o f 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. was set at P< 0.05.  58  The level o f statistical significance  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 i n serum  (Table 2-1) or cardiac (Table 2-2) F A or T G was observed i n 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 i n these  chronically diabetic animals was the highest, with no change i n 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 o f P P A R - a promoted F A utilization through increasing expression o f a number o f 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 - I B ) , and A C O (Fig. 2-1C) gene expression was observed following acute diabetes.  To confirm the  results o f previous studies, cardiac gene expression was also determined i n chronic diabetic animals.  Although gene expression o f P P A R - a  (Fig. 2-1 A )  did not change,  its  downstream targets, CPT-1 (Fig. 2 - I B ) 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 o f P P A R - a , M C D , catalyzes the degradation o f m a l o n y l - C o A to acetyl-CoA, thereby promoting F A oxidation ( 8 ) .  In 4-day diabetic hearts, no change i n 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 o f 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  A s a F A transporter, C D 3 6 facilitates cardiac F A uptake.  acute and chronic diabetes  Previous studies have reported  that C D 3 6 protein increases i n both intracellular and plasma membrane obtained from chronic diabetic hearts, an effect likely due to increased expression o f C D 3 6 (15).  In the  present study, no changes o f gene expression (Fig. 2-3A) and protein (Fig. 2-3B) were observed i n the acute diabetic heart.  Interestingly, both C D 3 6 gene expression (Fig. 2-3C)  and protein (Fig. 2-3D) were higher in chronic diabetic hearts compared to control. 2.3.5.  Cardiac AMPK  AMPK  is phosphorylated  creatine/phosphocreatine  and ACC phosphorylation and  ratios.  activated  following  an  increase  Extracellular hormones  adiponectin (22) can also regulate A M P K . inactivates A C C .  in acute and chronic diabetic hearts in A M P / A T P  such as insulin (13)  or and  Once activated, A M P K phosphorylates and  Measurement o f 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 i n 4-day diabetic hearts, an outcome that was reversed by insulin (Fig. 2 - 4 A 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 o f  diabetes, there was no difference i n 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 i n choosing substrates for energy generation. as  glucose  uptake,. glycolysis and  oxidation are  impaired, the  During diabetes, heart switches  predominantly using F A to ensure sufficient production o f A T P (25).  to  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 o f a co-activator, P P A R - a augments the expression o f genes involved i n F A uptake and oxidation (3, 24).  Thus, using "gain and loss o f  function" strategies, cardiac specific overexpression (9) or knock out (2, 6) o f P P A R - a brings about enhanced or reduced F A uptake and oxidation respectively. is known to activate P P A R - a (9).  Chronic diabetes  To investigate whether P P A R - a is activated and  contributes to high F A oxidation previously demonstrated i n 4-day acute diabetic hearts (10), we measured gene expression o f cardiac P P A R - a and its targets, CPT-1 and A C O . Interestingly, acute diabetes did not change expression o f these genes.  Using real-time  P C R , a previous study has also reported that following 1 week o f S T Z , no change in expression o f P P A R - a and CPT-1 were observed (7).  Another target o f 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 i n chronic diabetic animals (20).  In 4-day diabetic animals, no change o f 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 C D 3 6 , and plasma and cardiac F A and T G .  Acute diabetes did not change  gene and protein levels o f C D 3 6 , or plasma and cardiac lipids, and could explain the  61  absence o f 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 i n 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 o f A M P K (as evidenced by elevated A M P K and A C C phosphorylation) i n hearts, but not skeletal muscle and liver, from acutely diabetic rats. The underlying mechanism for this activation remains unclear. by both A T P dependent and independent mechanisms.  A M P K can be activated  Indeed, insulin is known to inhibit  cardiac A M P K i n an ATP-independent manner (4, 13), and level o f this hormone declines within 24 hours o f S T Z injection (21).  A s a single injection o f insulin normalized both  A M P K and A C C phosphorylation, our results suggest that following acute diabetes, reduction i n insulin may be a key regulator o f cardiac A M P K .  To examine whether  A M P K stays activated i n chronic diabetes under these conditions, A M P K and A C C phosphorylation were measured i n 6-week S T Z diabetic hearts.  Cardiac A M P K and A C C  phosphorylation remained unchanged i n these diabetic animals compared with control. The mechanism behind this lack o f activation o f cardiac A M P K i n chronic diabetes is currently unknown. Interestingly, chronic diabetic hearts demonstrated increased expression o f both C D 3 6 gene and protein, consistent with a previous study (15).  A s overexpression o f C D 3 6  increases the rate o f 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 C D 3 6 , 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 i n F A utilization (9).  In the  present study, although gene expression o f P P A R - a did not change, expression o f 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 i n 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 i n 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 . is  activated.  This  In the acute S T Z diabetic heart, A M P K , rather than P P A R - a ,  activation o f A M P K  phosphorylation and inhibition o f A C C .  likely  increases  F A oxidation through  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 C D 3 6  provides the heart with excess F A .  In this condition, P P A R - a , through its regulation o f  gene expression, contributes to high F A oxidation.  63  2.5.  Tables and figures  Table 2-1.  General characteristics o f the experimental animals  ACUTE  CHRONIC  Control  STZ  Control  STZ  Body Weight (g)  319±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) o f S T Z diabetes, animals from all groups were killed, blood collected and serum separated for measurement o f various parameters. Values are mean ± S E for 6 animals i n each group.  'Significantly different from control;  "significantly different from all other groups, P < 0.05.  64  Table 2-2.  Cardiac lipids i n 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) o f S T Z diabetes, animals from all groups were killed.  Cardiac lipids were extracted and separated using H P L C .  for 3 animals i n each group.  *Significantly different from control.  65  Values are mean ± S E  CON  STZ  CON  STZ  A  CPT-1  •  |3  Chronic  Acute  Fig. 2-1 Gene expression of PPAR-a,  CPT-1 and ACO in 4-day and 6-week STZ diabetic  Hearts from control and 4-day and 6-week STZ diabetic animals were removed and immediately snap frozen in liquid nitrogen. Gene expression of P P A R - a (A), CPT-1 (B) and A C O (C) were measured using rt-PCR. Data are means ± SE; n=4. Two-way A N O V A 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,  hearts  #  66  ACUTE 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 i n liquid nitrogen.  M C D gene expression was measured by  R T - P C R and protein determined by Western Blotting. was used to determine differences between means.  67  Data are means ± S E ; n=4. t-test  ACUTE 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. using rt-PCR. n=4.  Gene expression o f C D 3 6 was measured  Protein level was evaluated by Western Blotting.  t-test was used to determine differences between means.  from control group, P < 0.05.  68  Data are means ± S E ; Significantly different  ACUTE 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 ± S E ; n=4.  One-way A N O V A followed by the Tukey test was  used to determine differences between means. P<0.05.  69  Significantly different from other groups,  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 Endocrinol 2.  lipoprotein lipase. 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Compared to glucose, F A is the preferred substrate consumed by the heart,  contributing approximately 70% A T P when supplied at physiological levels. transported acetyl-CoA.  into mitochondria as acyl-CoA, which undergoes  F A is  P-oxidation to release  In addition to entering the T C A cycle to produce ATP, acetyl-CoA can also  be converted to malonyl-CoA under the influence o f A C C (11).  M a l o n y l - C o A is an  allosteric inhibitor o f C P T - 1 , the enzyme that manages the transport o f long-chain fatty a c y l - C o A 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, C D 3 6 and fatty acid binding protein plasma membrane (24).  Exogenous sources  o f F A include the plasma free F A fraction or F A released during hydrolysis o f T G - r i c h lipoproteins; the latter is considered to be the principal source o f 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 H S P G . Subsequently, the enzyme is translocated onto comparable H S P G binding sites on the luminal side o f 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 hindered) or exercise (when A T P expenditure is augmented),  o f A T P is  changes i n intracellular  A M P / A T P levels promote Threonine (Thrl72) phosphorylation and activation o f A M P K , an important regulator  o f both lipid 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, i n 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)  6-phosphofructo-2-kinase facilitates  (25).  F A utilization.  and  subsequent  glycolysis  through  activation  of  More importantly, through its control o f A C C , A M P K  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 m a l o n y l - C o A and minimize its inhibition o f F A oxidation (19,20). The majority o f studies examining A M P K regulation o f F A utilization have focused on F A oxidation.  M o r e recently, A M P K  has also been implicated i n F A delivery to  cardiomyocytes through its regulation o f C D 3 6 (23).  G i v e n the importance o f L P L in  providing hearts with F A (2,41), the objective o f the present study was to investigate i f activation o f 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 o f laboratory animals published by the U S National Institutes o f Health and the University o f British Columbia. Adult male Wistar rats (220-240 g) were obtained from the U B C A n i m a l Care Unit and supplied with a standard laboratory diet ( P M I 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 killing 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 i n the coronary arteries.  Following cannulation o f 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 ( H E P E S ) buffer containing 10 m M glucose ( p H 7.4). 02/5%) CO2.  3.2.3.  Perfusion fluid was continuously gassed with 95%  The rate o f coronary flow (7-8 ml/min) was controlled by a peristaltic pump.  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 / m l ) .  76  This concentration o f  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 o f the heart) was collected i n timed fractions (10 sec) over 5 or 10 m i n where indicated, and assayed for L P L activity by measuring the hydrolysis o f a sonicated [ H ] triolein substrate emulsion (34).  Retrograde perfusion o f  whole hearts with heparin results i n a discharge o f 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 o f L P L at coronary lumen, only peak L P L activities are illustrated. oleate released per hour per milliliter.  L P L activity is expressed as nanomoles  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 B l o t 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 liquid nitrogen and 50 m g homogenized.  After centrifugation at 5,000 g for 20 min, the protein content o f 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 i n 5% skim milk in Tris-buffered saline containing 0.1% Tween-20.  Membranes were incubated either with rabbit A M P K - a or p h o s o p h o - A M P K  77  (Thr-172) antibody, and subsequently with secondary goat anti-rabbit HRP-conjugated antibody, and visualized using an ECL®detection kit. 3.2.5.  Measurement of cardiac LPL expression  L P L gene expression was measured i n the indicated groups using R T - P C R . RNA  from  hearts  (100 mg) was extracted  using  Trizol  Briefly, total  (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). primers  (8);  c D N A was amplified using L P L ( N M O 1 2 5 9 8 ) specific  5' - A T C C A G C T G G G C C T A A C T T T - 3 '  5'-AATGGCTTCTCCAATGTTGC-3'  (right).  (left)  and  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'-ATCCTGTCAGCGATGCCTG between 15-30 cycles.  G G - 3 ' (right).  The linear range was found to be  The amplification parameters were set at: 94°C for 1 min, 58°C for  1 m i n and 72°C for 1 m i n , for a total o f 30 cycles.  The P C R products  electrophoresed on a 1.7% agarose gel containing ethidium bromide.  were  Expression levels  were represented as the ratio o f 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 i n myocytes and then translocated across the interstitial space onto H S P G binding sites on the luminal surface o f endothelial cells (38,39). using isolated cardiomyocytes.  L P L protein and activity measurement was done  Ventricular calcium-tolerant myocytes were prepared by a  78  previously described procedure (29,30). Cardiac myocytes were suspended at a final cell density o f 0.4 x 10 cells/mL, medium separated by centrifugation, and L P L protein and 6  activity assayed i n cell pellets. For  Western Blot  analysis, 25  u,g total protein was  size fractionated  SDS-polyacrylamide gel, and blotted onto a nitrocellulose membrane.  in a  After blocking  overnight at 4°C, the membrane was transferred to a solution o f 1:1000 diluted primary antibody (5D2, a monoclonal mouse anti bovine L P L generously provide by Dr. J. Brunzell, University o f Washington, Seattle, W A ) , and kept for 2 h at room temperature with gentle shaking.  After washing with T B S - 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 o f 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. and paraffin  embedding,  3  pm  sections  were  After formalin-fixation  cut on silane-coated  Immunostaining was carried out as described before (30).  glass  slides.  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. biotinylated  rabbit  Following washing with T B S , slides were incubated with  anti-chicken  IgG  (Chemicon  Corp.,  1:150  dilution)  and  Streptavidin-conjugated C y 3 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 inhibitor o f A M P K .  (Ara-A), a precursor o f Ara-ATP, is a competitive  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 o f  A M P K phosphorylation can influence cardiac L P L , isolated control hearts were perfused with perhexiline (1-10 u M ) , both i n the presence or absence o f 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 o f F A , and in preliminary experiments, i n the absence o f glucose, has been shown to activate the phosphorylation o f AMPK.  To deplete intracellular A T P and induce metabolic stress and activation of A M P K ,  oligomycin (1-5 u M ) , an inhibitor o f 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 i n  cardiomyocytes to activate A M P K (23). 3.2.9.  Serum measurements  B l o o d 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 i n 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 o f A M P K phosphorylation was observed on immediate removal o f the heart (Fig. 3-1).  Interestingly, perfusion with Krebs buffer for 1 hour further augmented  AMPK  phosphorylation, but only i n 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 i n 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 o f myocardial sections were performed to complement our observation that the augmented L P L i n fasted hearts was mainly localized at the endothelial cells.  Whereas  L P L immunofluorescence  was found throughout the control and fasted myocardium,  capillary  in  blood  vessels  the  fasted  heart  demonstrated  immunoreactivity compared with control (arrows, F i g . 3-2B).  a  more  intense L P L  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 i n L P L is a consequence o f changes in gene expression, L P L m R N A was measured. were independent  Interestingly, changes i n luminal L P L activity  o f 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 i n cardiomyocytes, the predominant c e l l t y p e 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 v i a posttranslational mechanisms. Given that A M P K phosphorylation increased further i n fasted hearts perfused for 1 hour, we evaluated whether there would be an additional increase or maintenance o f L P L over this period, i n 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 i n L P L activity (Fig. 3-2A, right panel). high heparin-releasable  A b i l i t y o f these hearts to maintain their  L P L i n vitro suggested either an increased recruitment from  myocytes or decreased displacement from the coronary lumen.  T o evaluate this further,  during the 60 m i n perfusion with heparin free buffer i n 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 o f 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 o f A M P K phosphorylation should decrease L P L activity.  A r a - A , a precursor o f Ara-ATP, is a competitive inhibitor o f A M P K (27).  Perfusion o f 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 o f glucose, perfusion o f 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 o f glucose from the perfusion buffer significantly augmented both phosphorylation and luminal L P L activity (Fig. 3-5A and B ) .  AMPK  With oligomycin, there was  a rapid and concentration dependent increase i n 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 i n 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 A T P 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 i n 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 i n 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 o f cardiac oleate oxidation do increase following fasting (45).  Given the role o f A M P K i n regulating F A oxidation, it is conceivable that it could also influence F A delivery.  Provision o f F A to the heart involves: a) release from adipose  tissue and transport to the heart (22), b) breakdown o f endogenous cardiac T G stores, c) internalization o f whole lipoproteins, and d) hydrolysis o f circulating T G - r i c h lipoproteins to F A by lipoprotein lipase ( L P L ) positioned at the endothelial surface o f the coronary lumen (7), and A M P K has been implicated i n some o f these processes.  Thus, activation  of A M P K is known to augment F A uptake i n 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 i n adipocytes, A M P K is suggested to mediate the lipolytic effect o f adrenergic stimulation (44).  G i v e n that L P L  mediated hydrolysis o f lipoproteins was recently suggested to be the principal source o f F A for  cardiac utilization (2,41), we examined its relationship to A M P K  85  activity.  In  agreement with previous studies, we observed an increase o f heparin-releasable L P L activity following overnight fasting, and immunostaining revealed that most o f the L P L protein was located at the coronary lumen.  Our data suggests that A M P K phosphorylation  may play a role i n increasing cardiac functional L P L . were independent  A s changes i n luminal L P L activity  o f shifts i n 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 i n cardiac L P L activity are likely through posttranslational mechanisms that do not involve alterations i n m R N A levels, protein synthesis or specific activity o f the protein (10). Moreover, at least i n adipose tissue, down-regulation o f L P L during fasting is also post-translational, and involves a shift from active to inactive forms o f the lipase (5). To further examine this relationship, hearts from fasted animals were perfused with Krebs buffer for 1 hour (in the absence o f 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 o f albumin-bound F A and circulating lipoproteins i n the perfusate.  It should  be noted that even though A M P K phosphorylation increased with time i n 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 o f the rat heart has a finite number o f L P L binding sites and that under normal conditions only a fraction o f these binding sites are  86  occupied by L P L (29), our present data is indicative o f a model which suggests that once A M P K activation fills these sites (following overnight fasting) with the enzyme, no further increase o f L P L is possible.  Interestingly, throughout the 1-hour perfusion, fasted hearts  showed greater basal release o f L P L .  Given that this increased basal L P L release was not  followed by a decline i n heparin releasable L P L activity, our data suggest that i n 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, i n skeletal muscle, stimulation o f 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 i n the fasted heart.  M o r e importantly, i n  these hearts, insulin and A r a - A also reduced the endothelial bound L P L pool.  Our data  suggest that i n vitro, the fasted heart is able to maintain its high L P L activity through A M P K , and inhibition o f 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 o f intracellular A M P concentrations.  It should be noted that as the  effect o f A M P can be antagonized by high concentrations o f ATP, a higher A M P / A T P ratio is more efficient i n 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 o f A T P production that in turn increases intracellular generation o f A M P (12).  Perhexiline, a CPT-1 blocker that is widely used in  87  the treatment o f 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. when using perhexiline.  To test this idea, glucose was removed from our perfusion media 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 i n a dose-dependent manner.  Given  the rapid (8 minutes) augmentation o f L P L following oligomycin, and in the absence o f any change i n L P L m R N A , or protein and activity i n cardiomyocytes, our data suggest that the regulation o f L P L by A M P K is likely through posttranslational mechanisms.  One such  mechanism could include increase i n secretion o f L P L from the myocyte to the coronary lumen.  In this regard, despite the high basal release o f 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 i n myocytes from fasted animals suggesting that reduced intracellular lysosomal degradation may also be occurring concomitantly.  In adipocytes, stimulation o f L P L secretion is known to reduce enzyme  88  degradation (9,42).  Additionally, A T P depletion i n 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 o f 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 i n addition to its direct role in promoting F A oxidation, increased A M P K recruitment o f 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 o f 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 m e a n ± S E for 6 animals.  Following an overnight fast, animals were killed,  blood collected and serum separated for measurement o f various parameters. * Significantly different from control, P < 0.05.  90  Control Phospho-AMPK-a  Fasted  Control  • - ,,.„„„„.. .,,,..,-.,„„  .  Fasted m  111  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  p h o s p h o - A M P K - a were measured using Western Blotting, and either rabbit A M P K - a or p h o s p h o - A M P K (Thr-172) antibodies. group.  Data are m e a n s ± S E o f four different hearts in each  'Significantly different from control, "Significantly different from all other groups,  PO.05.  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, L P L 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 L P L 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. measured using R T - P C R ( A ) . using isolated cardiomyocytes.  L P L gene expression in the whole heart was  L P L protein (B) and activity (C) measurements were done Data are m e a n s ± S E 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 i n fasted hearts was inhibited using A r a - A (500 u M ) or insulin (5 U / m l ) 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 m e a n s ± S E o f four different  'Significantly different from control, P O . 0 5 .  94  hearts i n each  group.  +  ~  -  Glucose Perhexiline  +  Phospho-AMPK-a AMPK-a  3.0 - |  +  +  -  Glucose  -  +  +  Perhexiline  B  F i g . 3-5 Consequence  of  CPT-1  +  +  —  -f-  inhibition  —  +  on  heparin-releasable LPL activity in control hearts  AMPK  Glucose Perhexiline  phosphorylation  and  Hearts from control animals were  perfused either i n 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 ) . four different hearts i n each group.  Data are m e a n s ± S E of  'Significantly different from control, P<0.05.  95  1  Control  Control  3  1  5  3  Oligomycin (uM)  5  O l i g o m y c i n (pJ\fl)  Control  1  3  Oligomycin  Fig. 3-6 Effect  of  inhibiting  ATP  synthesis  on  5 (pM)  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  Blood vessel  Interstitial space  AMPK  j  Degradation  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. 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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 M C , Rodrigues B.  Localization of lipoprotein lipase in the diabetic heart: regulation by acute changes in insulin. 37.  Arterioscler Thromb Vase Biol 19:1526-1534, 1999.  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.  endothelial cells.  Polarized binding of lipoprotein lipase to  Implication for its physiological actions.  Arterioscler Thromb  12:1437-1446, 1992. 40.  Sugden  MC,  Greenwood  Peroxisome-proliferator-activated  GK,  receptor-alpha  103  Smith  ND,  Holness  (PPARalpha) deficiency  MJ.  leads  to  dysregulation o f hepatic lipid and carbohydrate metabolism by fatty acids and insulin. Biochem 7364:361-368, 2002.  41. LM.  Teusink B, Voshol PJ, Dahlmans V E , Rensen PC, Pijl H , Romijn JA, Havekes Contribution o f fatty acids released from lipolysis o f plasma triglycerides to total  plasma fatty acid flux and tissue-specific fatty acid uptake. 42.  Vannier C, Ailhaud G.  adipocytes.  Diabetes 52:614-620, 2003.  Biosynthesis o f lipoprotein lipase in cultured mouse  II. Processing, subunit  assembly,  and  intracellular transport.  J  Biol  Chem.264:13206-13216,1989. 43.  Witters L A , 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. 44.  Arch Biochem Biophys 308:413-419, 1994.  Yin W, Mu J, Birnbaum M J .  Role o f AMP-activated protein kinase i n cyclic  AMP-dependent lipolysis i n 3T3-L1 adipocytes.  45.  JBiol  Chem 278:43074-43080, 2003.  Young M E , Guthrie PH, Razeghi P, Leighton B, Abbasi S, Patil S, Youker K A ,  Taegtmeyer H.  Impaired long-chain fatty acid oxidation and contractile dysfunction i n  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% o f 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. selection. manufacture  A M P K is likely a key player i n modulating this substrate  Thus, during exercise (when A T P expenditure is augmented) or ischemia (when o f A T P is hindered), changes i n intracellular A M P / A T P levels promote  threonine ( T h r l 7 2 ) phosphorylation and activation o f A M P K (22, 25).  U p o n 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). phosphorylated A M P K  In heart and skeletal muscle,  stimulates glucose uptake (23, 55) and subsequent glycolysis  through the activation o f 6-phosphofructo-2-kinase (38). More importantly, through its control o f 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 o f the F A transporter, C D 3 6 (36). Finally, results from our laboratory have demonstrated  a strong correlation between  activation o f cardiac A M P K and increases i n coronary lumen L P L activity (2).  105  L P L is a rate-limiting enzyme for hydrolysis o f T G rich lipoproteins, thus regulating the supply o f F A to meet the metabolic demands o f 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 i n 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 o f TG.  Recently, LPL-mediated hydrolysis o f 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 o f lipoproteins (40, 56, 63). Through its role i n T G hydrolysis, L P L activity directly affects the level o f circulating lipoprotein-TG.  For example, i n transgenic rabbits with global overexpression o f L P L ,  attenuation o f hypertriglyceridemia was observed, an effect suggested to contribute toward amelioration o f insulin resistance and obesity (28).  Contrary to systemic overexpression,  tissue-specific overexpression o f L P L i n skeletal muscle and heart is associated with insulin resistance i n these tissues as well as severe myopathy, characterized by both muscle fiber degeneration and extensive proliferation o f 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 , lipid oversupply and deposition were  106  observed, along with excessive dilatation and impaired left ventricular systolic function (cardiomyopathy) (69).  In vascular smooth muscle, overexpression o f 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 o f the sympathetic nervous system, and higher levels o f circulating catecholamines (14, 17).  Although the P-adrenergic agonist isoproterenol had  no influence on myocyte cell surface or intracellular L P L ( 6 0 ) , 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 o f L P L i n cardiac and vascular pathology, the objective o f 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 i n workload andexcessive energy expenditure, rather than a direct effect.  107  4.2. M a t e r i a l s and M e t h o d s 4.2.1.  Experimental  animals  The investigation conforms to the guide for the care and use o f laboratory animals published by the U S National Institutes o f Health and the University o f British Columbia. Adult male Wistar rats (220-240 g) were obtained from the U B C A n i m a l Care Unit and supplied with a standard laboratory diet ( P M I Feeds, Richmond, V A ) , and water ad libitum. A l l o f 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. cause cardio depression.  Anesthesia is known to  Following stabilization, animals were either injected i.p. with  saline or 10 pg/kg o f the P-adrenergic agonist, Isoproterenol (ISO).  Blood pressure and  heart rate were recorded every 20 m i n 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 killing 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 i n the coronary arteries.  Following cannulation o f 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% 0 2 / 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 o f 11.5 m m H g , and an aortic afterload o f 80 m m H g , for 15 min.  Heart function (rate pressure product, R P P ) 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 o f 15 min, heart perfusion was switched from the  working mode back to the Langendorff method, and L P L released by the addition o f 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). anesthetized, and the hearts carefully excised.  Briefly, rats were  Following cannulation o f 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, I S O (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 / m l ) .  This concentration o f  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 o f the heart) was collected in timed fractions (10 sec) over 5 or 10 m i n where indicated, and assayed for L P L activity by  109  measuring the hydrolysis o f a sonicated [ H] triolein substrate emulsion (52). 3  Retrograde  perfusion o f whole hearts with heparin results i n a discharge o f 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 o f  L P L at coronary lumen, only peak L P L activities are illustrated. as nanomoles oleate released per hour per milliliter.  L P L activity is expressed  Subsequent to L P L displacement  with heparin, hearts were rapidly removed, washed with Krebs buffer, frozen i n 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 o f calcium.  Our method o f isolation yields a highly enriched  population o f calcium-tolerant myocardial cells that are rod-shaped i n the presence o f 1 m M C a , with clear cross striations. 2 +  vesiculated spheres.  Intolerant cells are intact but hypercontract into  Yield o f myocytes was determined microscopically using an  improved Neubauer hemocytometer.  Myocyte viability was assessed as the percentage o f  elongated cells with clear cross striations that excluded 0.2% trypan blue.  Cardiac  myocytes were suspended at a final cell density o f 0.4x10 cells/ml, incubated with or 6  without ISO (10 u M ) at 37°C for 1 hr, and basal L P L activity i n 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. suspension  was  removed,  medium  After incubation for 10 min, an aliquot o f cell  separated  by  centrifugation  microcentrifuge (1 min, 10,000 g), and assayed for L P L activity.  in  an  Eppendorf  The total cellular L P L  activity was measured by sonicating (Vibra Cell™ sonicator at a frequency o f 40 H z for 2x30 s) the cell pellets after resuspending them in 0.2 m l o f 50 m M N H C 1 buffer (pH 8.0) 4  containing 0.125% (v/v) Triton X - 1 0 0 .  After sonication, the volume was adjusted to 1 m l  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 p i o f the cell sonicate  and heparin (2 U / m l ) . 4.2.7.  Cardiac LPL gene expression  L P L gene expression was measured i n the indicated groups using R T - P C R (2, 44). total R N A from hearts (100 mg) was extracted  Briefly,  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). primers  (6),  c D N A was amplified using L P L (NM_012598) specific  5' - A T C C A G C T G G G C C T A A C T T T - 3 '  5'-AATGGCTTCTCCAATGTTGC-3'  (right).  The  (left)  p-actin gene ( N M 031144)  and 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). between 15-30 cycles.  The linear range was found to be  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.  111  The P C R products  were  electrophoresed on a 1.7% agarose gel containing ethidium bromide.  Expression levels  were represented as the ratio o f 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).  homogenized.  Briefly,  hearts were  ground under  liquid nitrogen, and  50 mg  After centrifugation at 5,000 g for 20 min, the protein content o f 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 m i l k i n Tris-buffered saline containing 0.1%  Tween-20.  Membranes  were  incubated  either  with  rabbit  AMPK-a,  p h o s p h o - A M P K (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 o f A M P K is a surrogate for estimation o f 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 o f triglycerides and F A was achieved using H P L C (Waters  2690 Alliance H P L C , M i l f o r d , 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 m m 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 i n the specific figure legends).  The level o f 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 C e l 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 L P L 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). 114  In the heart,  ACC280 is the predominant isoform.  Following 1 h o f I S O , phosphorylation o f 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 i n cardiac T G , without affecting cardiac F F A (Table 4-2). 4.3.3.  Isoproterenol  does not influence  myocytes or Langendorffperfused  LPL activity and AMPK  phosphorylation  in  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 cells), or cell sonicate (control, 2287 ± 317; ISO, 2142 ± 354; nmol/hr/10 6  cells) L P L activity.  6  To determine whether ISO influences L P L i n 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, I S O did not change H R - L P L i n  Langendorff isolated hearts (Fig. 4-3).  Overall, our data suggest that specifically in these  preparations in vitro, ISO does not have a direct effect i n regulating L P L activity.  In a  recent report, we described that recruitment o f 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 i n isolated myocytes and Langendorff perfused hearts.  115  We demonstrate that  ISO did not affect A M P K phosphorylation i n 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 o f A M P K and A C C , and coronary luminal L P L activity i n 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 o f insulin) demonstrated higher p h o s p h o - A M P K (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 HR-LPL  of substrate supply reduces AMPK  and ACC phosphorylation  and  activity in the isolated perfused working heart  Glucose entry into the heart is predominantly insulin dependent (7).  116  Compared to glucose,  F A is the preferred substrate, and when supplied at physiological levels, contributes approximately 70% o f 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 o f 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 o f 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 activity (Fig. 4-5 & 4-6).  ACC280  phosphorylation and L P L  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), iii) internalization o f whole lipoproteins (20, 61, 64), and iv) hydrolysis o f circulating T G - r i c h 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 o f F A i n lipoprotein-TG (39) and recently, LPL-mediated hydrolysis o f lipoproteins to F A was suggested to be the principal source o f F A for cardiac utilization (4).  L P L is synthesized i n cardiomyocytes  and subsequently secreted onto H S P G binding sites on endothelial cells i n the coronary lumen (13, 15, 39, 41).  Thus, even though the majority o f enzyme is located i n myocytes,  vascular endothelial-bound L P L likely determines clearance (41).  the rate o f plasma lipoprotein-TG  Previously, incubation o f cardiomyocytes with ISO for 30 minutes did not  change either H R - L P L or cellular L P L activity (60). even after incubation with ISO was prolonged for 1 hr.  We have confirmed these findings A s cardiomyocytes are quiescent,  we extended these experiments to an isolated retrograde perfused heart, and demonstrated a similar absence o f effect o f this P-adrenergic agonist on L P L . truly representative  o f a heart beating i n vivo.  The above models are not  In the only study that examined the  influence o f I S O i n the intact animal, L P L activity was measured  i n whole heart  homogenates (12), which does not distinguish between the heparin-releasable (localized on capillary endothelial cells) and cellular (that represents a storage form o f the enzyme) pools  118  of L P L .  Our results, for the first time, suggest that a single injection o f ISO, given i n 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 o f ISO.  It is interesting that despite an increase i n H R - L P L and plasma F F A ,  measurement o f intracellular lipids revealed a drop i n T G with no change i n 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 i n quiescent myocytes, an effect that was independent o f its inotropic response (30).  G i v e n that H R - L P L only increased following i n vivo administration o f ISO,  it is likely that under conditions o f 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 o f 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 i n intracellular A M P / A T P levels promote activation o f A M P K , an important regulator o f both lipid and carbohydrate metabolism (22, 25).  119  Thus, i n heart  and skeletal muscle, phosphorylated A M P K stimulates glycolysis through activation o f 6-phosphofructo-2-kinase  and facilitates F A utilization v i a 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 i n F A delivery to cardiomyocytes through its regulation o f C D 3 6 (36).  More  recently, we have also reported that A M P K can regulate cardiac L P L by facilitating movement o f this enzyme to the coronary lumen (2).  A s A M P K phosphorylation was  only observed following the administration o f I S O i n vivo, and not i n the isolated non-beating myocytes or retrograde non-working hearts, it appears that activation o f AMPK  is secondary  to  augmented  workload and energy  expenditure.  Although  intracellular A T P remains unchanged i n skeletal muscle during exercise, increased energy expenditure augments A M P , leading to a change i n the A M P / A T P ratio and subsequent activation o f A M P K (8, 62).  Similar effects on A M P K activation i n the heart were also  observed following increased heart rate and contractility during exercise (11).  As A M P K  activation phosphorylates and inhibits ACC280, and likely controls the increases i n L P L at the coronary lumen, our data imply that following ISO, with its associated increase i n 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 i n vivo.  In contrast, i n the working heart, buffer enters through the left atrium,  fills the left ventricle, and is expelled against an afterload.  120  Under these conditions, energy  expenditure i n the Langendorff heart is likely 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 likely not matched to A T P expenditure, and A M P K would be expected to increase.  Indeed, i n 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 o f A M P K phosphorylation is due to insufficient A T P production or increased AMP  accumulation is unknown.  In an  attempt to  decrease  AMPK  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 A k 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 i n energy status or the direct effects o f insulin. Under these in vitro conditions, provision o f 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, i n 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 o f A M P K .  Whether this increase i n 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 o f the vessel wall (9, 16). Indeed, when endothelial cell monolayers are incubated with increasing concentrations o f F A , the permeability o f the monolayer is enhanced allowing albumin to traverse faster across the vessel w a l l (24).  Additionally, tissue-specific overexpression o f L P L is  associated with severe myopathy (27, 34, 67), and promotion o f atherosclerosis (16). Thus, a novel mechanism by which excessive P-adrenergic agonists  could induce  cardiovascular complications is through their control o f H R - L P L .  !  122  (  4.5. Tables and figures Table 4-1.  General characteristics o f the animals  Blood Glucose (mM) Serum free fatty acid (mM) Serum triglyceride (mM) Systolic pressure (mm Hg)  Control  ISO  5.7 ± 0.2  5.4 ± 0 . 2  0.34 ± 0 . 0 4  0.49 ± 0.05 *  1.4 ± 0 . 2  1.3 ± 0 . 1  116±4  131 ± 10  Heart rate (Beats/min)  421 ± 1 6  Rate pressure product  49192 ± 3 1 2 1  Values are mean ± S E for 6 animals in each group.  •  474±18* 62039 ± 5 3 7 8 *  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.  B l o o d pressure and heart rate  were recorded every 20 m i n for 1 hr, and averages are presented. For measurement o f serum parameters, blood was collected at termination (1 h after ISO). * Significantly different from control, P < 0.05.  123  Table 4-2.  Cardiac lipids i n control and Isoproterenol treated groups  Control  Triglyceride  Free fatty acid  (p,g/mg protein)  (u,g/mg protein)  1.7 ± 0 . 2  4.6 ± 0 . 5  lhr  1.1 ± 0 . 1 *  5.0 ± 0 . 6  4hr  0.7 ± 0 . 1 *  3.4 ± 0 . 1  Isoproterenol  Values are mean ± S E for 4 animals in each group.  1 or 4 hrs following i n 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 i n each group.  'Significantly different from control, P O . 0 5 (upper panel). ISO, animals were killed and hearts frozen in liquid nitrogen. whole heart was measured using R T - P C R .  One hr following injection o f L P L gene expression in the  Data are means ± S E o f three different hearts  in each group (lower panel). 125  Control  1 hr  4 hr ISO  Control  1 hr  4 hr  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 o E 200 X c i  ns a>  D_  100 H o  -  1  ISO  Control  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 L P L , 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 <>  800  a:  400  i  ro  a.  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  »  + +  + + +  PA Insulin 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, I S O was added to buffer containing a l l o f the above  components.  ,Subsequently, A M P K - a  (total and phosphorylated) (upper panel) and  phospho-ACC 8o (lower panel) were measured using Western Blotting. 2  S E o f three different hearts i n each group.  Data are means ±  'Significantly different from control, P < 0.05.  129  Fig. 4-6 Consequence  of additional  substrate provision  in regulating  LPL in isolated working hearts, in the absence or presence of LSO  heparin-releasable  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. S E o f three different hearts i n each group.  Data are means ±  Significantly different from control, P < 0.05.  130  4.6. Bibliography 1.  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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 o f death i n diabetic patients, with coronary vessel disease and atherosclerosis being primary reasons for the increased incidence o f cardiovascular dysfunction (26, 41).  However, a number o f diabetic patients also suffer from a specific  impairment o f heart muscle (termed diabetic cardiomyopathy), a condition also evident i n rodent models o f chronic diabetes (20, 46). Several factors have been put forward to explain the development o f diabetic cardiomyopathy including an increased stiffness o f the left ventricular wall, and abnormalities o f various proteins that regulate ion flux, specifically  intracellular calcium (5, 14).  More recently, the v i e w that diabetic  cardiomyopathy could also occur as a consequence o f apoptosis, a process o f cell death that occurs subsequent to the activation o f 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 i n 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 o f second messengers like ceramide, provoke apoptosis (10, 16).  Enlargement o f the intracardiac F A pool during diabetes may similarly  overpower the tissue capacity for utilization, and the resultant accumulation o f cardiotoxic  141  lipid can be implicated i n cardiac myocyte apoptosis (45, 50). Metformin, a drug widely used i n the treatment o f Type 2 diabetes, decreases both plasma glucose and lipids.  Recently, A M P K has been implicated i n mediating the effects  of metformin (31, 49), in a L K B 1 dependent manner (38).  Knocking-out L K B 1 or  inhibition o f A M P K abolishes the clinical effects o f metformin (38, 49).  Once activated,  A M P K stimulates glucose uptake and subsequent glycolysis through translocation o f glucose transporter 4 ( G L U T - 4 ) and activation o f 6-phosphbfructo-2-kinase (PFK2) (15, 23, 29).  Recent  studies have reported  that metformin, through activation o f  AMPK,  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 o f lipoprotein lipase, C D 3 6 ,  and acetyl-CoA  carboxylase ( A C C ) , A M P K facilitates F A delivery and oxidation ( 1 , 2 1 , 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 i n the regulation o f lipid 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 o f laboratory animals published by the U S National Institutes o f 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 A n i m a l Care Unit and supplied with a standard laboratory diet ( P M I 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  o f isolation yields a  highly enriched  population o f  calcium-tolerant myocardial cells that are rod-shaped (over 80%) i n the presence o f 1 mmol/1 C a , with clear cross striations. 2 +  vesiculated spheres.  Intolerant cells are intact but hypercontract into  Y i e l d o f myocytes was determined microscopically using an  improved Neubauer hemocytometer.  Myocyte viability was assessed as the percentage o f  elongated cells with clear cross striations that excluded 0.2% trypan blue. Cardiomyocytes were plated on laminin-coated 6 w e l l culture plates to a density o f 100,000 cells/well.  Cells were maintained using Media-199, i n the presence or absence o f  1 mmol/1 albumin bound palmitic acid (1:2), and incubated at 37 °C under an atmosphere of 9 5 % 0 2 - 5 % C 0 2 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 i n the absence o f 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 o f 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 ( N A D + ) , 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 o f metformin on this process, cells were examined for morphological evidence o f apoptosis using the fluorescent DNA-binding dye, Hoechst 33342.  Cells were stained with 5 p g / m L 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 D C F D A ) was used to assess R O S levels. 2  144  C M - H D C F D A is a 2  cell-permeant indicator that is oxidized in the presence o f 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 D C F D A (5 u M for 30 m i n at 37°C). 2  Subsequently, cells  were washed and incubated for an additional 45 m i n after which green fluorescence was measured i n a fluorimeter at 485/530-nm wavelengths. 5.2.6.  Western blot analysis  Western blot was carried out as described previously (1). was ground under liquid nitrogen, and homogenized.  Briefly, 50 m g o f heart tissue  After centrifugation at 5,000 g for  20 min, the protein content o f the supernatant was quantified using a Bradford protein assay. Samples  were  diluted,  boiled with  SDS-polyacrylamide gel electrophoresis.  sample  loading dye,  and  50  pg  used  in  After transfer, membranes were blocked in 5%  skim milk i n Tris-buffered saline containing 0.1% Tween-20.  Membranes were incubated  with rabbit A M P K - a , p h o s o p h o - A M P K (Thr-172), phospho-ACC, i N O S , and long chain base biosynthesis protein 1 ( L C B 1 ) antibodies, and subsequently w i t h 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  chloroform/methanol from same number o f cells i n different groups (1:2, v/v).  145  using Samples  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 , 1 mmol/1 E G T A , 10 2  mmol/1 dithiothreitol, 1 mmol/1 ATP, [y- P]ATP, D A G kinase (15.5 munits/assay), 2.5 % 32  octyl-(3-D-glucoside, 1 mmol/l cardiolipin.  Following incubation, lipids were extracted  with 470 p i o f 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 o f 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 volume).  (10:2:3:4:1  by  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 o f protein were added to an equal volume o f  reaction buffer that contained 50 umol/1 o f the respective substrate, and incubated at room temperature for 30 min.  The enzyme-catalyzed release o f aminomethylcoumarin was  quantified i n a fluorimeter at 380/450-nm wavelengths. Bradford assay. 5.2.9.  Protein was determined using a  Caspase 3 activity is presented as activity per mg protein for each sample.  Rates of glycolysis and palmitic acid oxidation 3  3  To measure rates o f glycolysis and F A oxidation, 5- H-glucose or H-palmitic acid were  146  added separately into the incubation medium. were collected.  3  After 16 hrs incubation, medium samples  H 2 0 generated from glycolysis or F A oxidation was separated from  5- H-glucose or H-palmitic acid by previously described methods (25). 3  3  was measured by liquid scintillation counting. protein assay.  Radioactivity  Protein was quantified using a Bradford  Results were expressed as pmol/min/mg protein.  5.2.10. Lactate assay Glycolysis results in the formation o f protons and pyruvate.  B u i l d up o f these metabolites  augments lactic acid generation, a process catalyzed by L D H (35). accumulation is recognized as a marker o f acidosis.  Therefore, lactate  Lactate released into the myocyte  incubation medium was determined using a kit from Sigma.  Briefly, 100 p i o f culture  medium was incubated with 1 m l 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 P h o s p h o - A M P K - a , and A C C antibodies were obtained from C e l l Signaling (Beverly, M A ) . A n t i - i N O S antibody was obtained from Santa Cruz Biotech. Anti-LCBl  was obtained from B D Sciences.  D M E M was obtained from Invitrogen.  3 2  M e d i u m 199 was obtained from Sigma.  P , 5- H-glucose and H-palmitic acid were  147  3  3  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 l o w concentration o f 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 o f 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 o f metformin.  In the  control group, less than 50 out o f 1000 cells were scored as apoptotic. . H i g h fat significantly increased apoptosis (241/1000 cells).  Introducing l o w concentrations of  metformin into the medium significantly lowered the number o f apoptotic cells (1 mmol/1, 170/1000; 2 mmol/1, 185/1000).  With a higher concentration o f metformin (5 mmol/1), a  larger number o f cells (635/1000) underwent apoptosis. 5.3.3.  ROS generation following provision of high fat and metformin  R O S generated i n the mitochondria is known to induce oxidative stress and apoptosis.  In  the present study, incubation o f 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 o f 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 o f R O S was observed  when high concentration (5 mmol/1) o f 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 i n skeletal muscle, liver and cardiomyocytes (7, 31, 49).  In the present study, following one-hour incubation with  metformin, an approximately two-fold increase i n A M P K phosphorylation was observed (Fig.  5-3A).  N o difference i n A M P K phosphorylation was observed between groups  treated with 1, 2 and 5 mmol/1 metformin.  Increasing the incubation time o f 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 o f malonyl-CoA from acetyl-CoA.  A s malonyl-CoA decreases F A oxidation through  inhibition o f C P T - 1 , phosphorylation o f A C C relieves the inhibition o f C P T - 1 , favoring F A oxidation.  In  the  current  study,  metformin  cardiomyocytes within 1 hour (Fig. 5-4A).  increased  A C C phosphorylation in  Prolonging the incubation time to 16 hours  further increased A C C phosphorylation (Fig. 5-4B).  Coupled to this phosphorylation o f  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 o f serine palmitoyltransferase (SPT) (39).  Incubation o f cardiomyocytes with palmitic acid (1  150  mmol/1) for 16 hours increased L C B 1 protein, a subunit o f S P T (Fig. 5-5A), and intracellular ceramide (Fig. 5-5B).  Introduction o f 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 o f ceramide induces apoptosis in a caspase-3 dependent manner (43, 44). Incubation o f cardiomyocytes with palmitic acid (1 mmol/1) for 16 hours increased caspase activity (Fig. 5-5C).  Consistent with the lowering o f ceramide, introduction o f metformin  into the cardiomyocyte incubation medium also reduced the high fat mediated increases i n 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 o f G L U T - 4 and P F K - 2 respectively (15, 23, 29).  Uncoupling o f 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 o f 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) i n 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 o f 1 mmol/1 palmitic acid significantly reduced the rate o f glycolysis  in cardiomyocytes (Fig. 5-7).  Metformin dramatically increased glycolysis i n 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, i n the absence or presence o f metformin.  Absence o f glucose normalized p H (data not shown)  and prevented the metformin (5 mmol/1) induced increase i n L D H release (Fig. 5-8).  In  fact, i n this glucose free medium, the release o f L D H with metformin (5 mmol/1) was similar to control.  152  5.4. Discussion Metformin, a drug widely used i n the treatment o f Type 2 diabetes, lowers plasma glucose without causing either hypoglycemia or weight gain.  Recently, activation o f A M P K has  been implicated i n mediating this effect on glucose, either through a reduction in hepatic glucose production, or an increase i n glucose uptake and utilization (31, 49).  Through  activation o f A M P K , metformin also enhances F A uptake and oxidation (37).  Whether  metformin, as a consequence o f its modulated metabolism, influences cardiomyocyte cell death remained unknown. death.  In the present study, metformin per se has no effect on cell  A t low concentrations, this agent reduced high fat induced apoptotic cells and  L D H release, whereas a high concentration o f 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 i n 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 i n protecting cardiomyocytes against high fat induced cell death.  Surprisingly, incubation o f 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 o f palmitic acid decreases mitochondrial membrane potential, and the ability o f the mitochondria to produce R O S (17, 42).  Provision o f l o w concentration o f metformin (1 or 2 mmol/1) restored R O S to control  153  levels, likely due to the effect o f metformin i n promoting F A oxidation and electron flow through the mitochondrial electron transport chain (42).  In contrast, i n the presence o f  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 i n over 60% o f cardiomyocytes, likely leading to impaired oxidative metabolism and R O S generation.  Overall, our data suggest that metformin prevents high fat induced  apoptosis i n cardiomyocytes through a R O S independent mechanism. In the heart, when supply o f F A exceeds tissue oxidative capacity, accumulation o f lipids decreases cardiolipin synthesis (33), and induces myofibrillar degeneration (10), leading to lipotoxicity and apoptosis.  Given that exogenous d - c e r a m i d e mimicks the  deleterious effects o f F A , and that inhibition o f 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 o f caspase is one mechanism proposed to explain this effect o f 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 o f ceramide synthesis.  Ceramide levels can  increase as a consequence o f S P T mediated de novo synthesis, i n the presence o f excessive fatty acyl C o A .  G i v e n the role o f A M P K in decreasing the expression of S P T (3),  activation o f A M P K by metformin would be expected to decrease ceramide formation. Moreover, through activation o f A M P K and inhibition o f 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 o f 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 o f high fat induced L C B - 1 (a subunit o f S P T ) expression and intracellular ceramide levels.  Our data suggest that the beneficial effects o f l o w doses o f metformin i n  preventing cell death are likely through its modulation o f ceramide formation and caspase 3 activation.  It should be noted that although A M P K could mediate the protective effects o f  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 o f metformin elicited substantial apoptosis.  These data suggested that  mechanisms other than lipotoxicity could be playing a central role i n mediating these injurious effects o f metformin.  Metformin has been associated with lactic acidosis (2, 4).  In the present study, as 5 mmol/1 metformin caused the highest change i n lactate and p H measured i n the cardiomyocyte incubation medium, lactic acidosis could be the major reason i n 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 o f A M P K and inhibition o f A C C (48).  Indeed, measurement o f  glycolysis and F A oxidation demonstrated a dose dependent increase in utilization o f 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 o f glucose prevented the metformin (5 mmol/1) induced  increase in L D H release.  In fact, i n this glucose free medium, the release o f 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 likely masked by its ability to augment proton production. A limitation o f the present study was the use o f 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, i n 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 o f metformin is yet to be determined. In summary, our study demonstrates that low doses o f metformin reduce high fat induced cardiac cell death, likely through its effects i n decreasing ceramide formation and caspase 3 activity (Fig. 5-9).  However, through its role i n increasing proton accumulation  and lactic acidosis, metformin can induce cardiomyocyte cell damage i n 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, i n the presence or absence o f metformin (1-5 mmol/1). was measured ( A ) .  Following 16 hours, L D H released into the incubation medium  Cells were also examined for morphological evidence o f apoptosis  using the fluorescent D N A - b i n d i n g dye Hoechst 33342 (B & C ) . n=3-8 myocyte preparations from different animals.  Tukey test was used to determine differences between means. from control, P < 0.05.  Data are means ± S E ;  One-way A N O V A followed by the Significantly different  "Significantly different from the high fat treated group, P < 0.05.  ®Significantly different from all other groups, P < 0.05.  158  F i g . 5-2 ROS levels following  incubation with high fat and metformin  Cardiomyocytes  were incubated with 1 mmol/1 palmitic acid, i n the presence or absence o f metformin (1-5 mmol/1).  Following 16 hours, R O S levels were measured.  myocyte preparations from different animals.  test was used to determine differences between means. control, P < 0.05.  Data are means ± S E ; n=4  One-way A N O V A followed by the Tukey Significantly different from  "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  CON  1  2  1  5  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 b y the Tukey test was used to determine differences between means. 'Significantly different from control, P < 0.05. groups, P < 0.05.  160  @  Significantly different from a l l other  B  A MET (mmol/l|  MET (mmol/1)  CON  CON  P-ACC I p M H  8 < **9  5  TT  o.«  P-ACC «—__,  1  immm* t  t  E 2 1 hr  -  4  X  II2 o •= is  2 tf) O  m MET (mmol/1)  150  MET (mmol/1)  -\  I  120  100  MET (mmol/l)  F i g . 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). myocyte preparations from different animals.  One-way A N O V A followed by the Tukey  test was used to determine differences between means. groups, P < 0.05.  Data are means ± S E ; n=4  Significantly different from other  ®Significantly different from all the other groups, P < 0.05.  161  MET (mmol/1) CON  PA  1  2  5  *  I  1  I  2  CON  5  MET (mmol/l) MET (mmol/l) CON P-Ceramide  -f_  PA  1  2  ~ ~ , „ ». ——  6 '""*••"  l.'i.j.  *  I  1  ICON  2  5  MET (mmol/l)  c  1  2  5  MET (mmol/l)  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.  Fig. 5-5 Apoptotic mediators in isolated cardiomyocytes  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 differentfromall 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 o f metformin (1-5 mmol/1). measured.  Following 16 hrs, L D H was  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.  high fat treated group, P < 0.05.  165  #  Significantly different from the  M  FA  Glucose  ^=  MET/AMPK  =L  Glucose  FA  I  ACC  Glycolysis  Ceramide f H +Pyruvate +  I - t  t Cell Damage  \  LCB1 I I  Met/AMPK  Caspase 3 V  FA Oxidation  Pyruvate Oxidation  —  Apoptosis  Mitochondria Cardiomyocyte  F i g . 5-9 Proposed mechanism of how metformin influences cardiomyocyte cell death Metformin, likely through activation o f 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 o f glycolysis from glucose  oxidation, leading to lactic acidosis, and caspase 3 independent cell death.  166  5.6. Bibliography 1.  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Diabetes 51: 159-167, 2002. 20.  Jackson CV, McGrath G M , Tahiliani AG, Vadlamudi RV and McNeill J H A  functional  and  ultrastructural  analysis  o f experimental  diabetic  rat  myocardium.  Manifestation o f a cardiomyopathy. Diabetes 34: 876-883, 1985. 21.  Kudo N, Barr AJ, Barr R L , Desai S and Lopaschuk GD H i g h rates o f fatty acid  oxidation during reperfusion o f ischemic < hearts are associated with a decrease in malonyl-CoA levels due to an increase in 5'-AMP-activated protein kinase inhibition o f 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 o f AMP-activated protein kinase reduces hyperglycemia-induced mitochondrial reactive oxygen species  169  production and promotes mitochondrial biogenesis i n human umbilical vein endothelial cells. Diabetes 55: 120-127, 2006. 23.  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Diabetes 52: 1627-1634, 2003. 28.  Luna V, Casauban L , Sajan MP, Gomez-Daspet J , Powe J L , Miura A, Rivas J,  Standaert M L and Farese RV Metformin improves atypical protein kinase C activation by  insulin  Diabetologia 29.  and phosphatidylinositol-3,4,5-(P04)3  i n muscle  o f diabetic  subjects.  49: 375-382, 2006.  Marsin AS, Bertrand L , Rider M H , Deprez J , Beauloye C, Vincent MF, Van den  170  Berghe G, Carling D and Hue L Phosphorylation and activation o f heart P F K - 2 by A M P K has a role i n the stimulation o f 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 o f phosphatidate, diacylglycerol and ceramide i n 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 J M , Ljunqvist O, Efendic S, Moller DE, Thorell A and Goodyear L J Metformin increases AMP-activated protein kinase activity i n skeletal muscle o f subjects with type 2 diabetes. Diabetes 51: 2074-2081, 2002: 32.  Obeid L M and Hannun YA Ceramide: a stress signal and mediator o f growth  suppression and apoptosis. J Cell Biochem 58: 191-198, 1995. 33.  Ostrander DB, Sparagna G C , Amoscato A A , 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 E A The glucose fatty-acid  cycle. Its role i n insulin sensitivity and the metabolic disturbances o f diabetes mellitus. Lancet I: 785-789, 1963. 35.  Robergs RA, Ghiasvand F and Parker D Biochemistry o f exercise-induced  metabolic acidosis. 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Zhou YT, Grayburn P, Karim A, Shimabukuro M , Higa M , Baetens D, Orci L  and Unger R H Lipotoxic heart disease i n 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 i n the acute S T Z diabetic heart. unknown.  The mechanisms that mediate this activation in A M P K are currently  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 o f diabetes, lack o f insulin could be one potential mechanism the leads to the activation of cardiac A M P K .  This activation of A M P K  phosphorylation and inhibition o f A C C .  likely increases  F A oxidation through  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 C D 3 6  provides the heart with excess F A .  Associated with increased plasma T G and intracellular  F F A and T G , cardiac A M P K and A C C phosphorylation were normalized by mechanisms that have yet to be resolved.  Interestingly, long-chain a c y l - C o A esters are known to  inhibit phosphorylation o f A M P K by its upstream kinase L K B 1 .  Moreover, augmented  expression o f protein phosphatase 2 C has been observed i n hearts o f Z D F rats, associated with a high rate o f F A supply, which reduces A M P K  phosphorylation.  Thus,  accumulation o f F A or its derivatives i n 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, likely contributes to high F A oxidation. Using fasting and modulators o f 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 F A supply. mediate this process are currently unclear.  The mechanisms that  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. acidosis.  In clinical studies, over dose o f metformin has been associated with lactic  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. M y 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.  extrapolating these results to a clinical setting.  Hence, caution should be used when  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 i n acute, but not chronic S T Z diabetic hearts, the mechanisms for this dual effect o f 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 o f 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 i n intact animals is unknown, and needs to be determined.  177  

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