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Regulation of cardiac lipoprotein lipase during hypertension and diabetes Sambandam, Nandakumar 1999

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) REGULATION OF CARDIAC LIPOPROTEIN LIPASE DURING HYPERTENSION AND DIABETES by Nandakumar Sambandam B.Pharm., Madurai Kamaraj University, India, 1987 M.Pharm., Banaras Hindu University, India, 1990 A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies Division of Pharmacology and Toxicology Faculty of Pharmaceutical Sciences We accept this thesis as conforming to the required standard The University of British Columbia September 1999 ©Nandakumar Sambandam, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ? V W ZQAJ~%d-*-X S<-VH*MLS> The University of British Columbia Vancouver, Canada DE-6 (2/88) ii A B S T R A C T Hypertension and diabetes often co-exist in humans and the cardiac dysfunction greatly exceeds that observed with either condition alone. Induction of streptozotocin (STZ) diabetes in the spontaneously hypertensive rat (SHR) produces a more extensive cardiomyopathy relative to normotensive rats. Both diabetes and hypertension are associated with early metabolic alterations that could underlie the pathogenesis of cardiac dysfunction. In diabetes, the heart is exclusively dependent on free fatty acid (FFA) oxidation for its energy production. On the other hand, during hypertension, the heart has been shown to utilize glucose predominantly. Interestingly, when diabetes was induced, SHR hearts showed a remarkable shift towards FFA oxidation that strongly correlated with severe cardiac dysfunction observed in SHR-diabetic hearts. To date, the underlying mechanism(s) for these metabolic lesions is not understood. Lipoprotein lipase (LPL), a "gate-keeping" enzyme plays an important role in supplying free fatty acid (FFA) to the heart and therefore could play a potential role in cardiac metabolism. We hypothesized that changes in cardiac metabolism that occur during hypertension and diabetes are a consequence of alterations in cardiac L P L activity. Clinical and experimental evidence suggest that hypertension and hypertriglyceridemia co-exist, and may be causally related to each other, arguably as a result of defective L P L action. In fact, L P L activity is decreased in skeletal muscle and adipose tissues in hypertensive patients, Dahl-hypertensive rats and in stroke-prone SHR rats. We investigated the effect of hypertension on cardiac L P L in two hypertensive animal models: a) the Spontaneously hypertensive (SHR) rat that has a genetic propensity to develop hypertension, and b) the fructose hypertensive rat which is an acquired model of hypertension. Hearts from spontaneously hypertensive (SHR) rats were examined before or after the development of severe hypertension in SHR rats. Age matched Wistar Kyoto (WKY) rats were iii used as normotensive controls. With the development of hypertension in SHR rats, there was a concomitant and progressive reduction in the heparin-releasable coronary endothelial L P L activity. Neither insulin action nor cell-associated enzyme activity could explain this low L P L 2+ activity in coronary blood vessels. However, acute vasodilation with nifedipine (a Ca influx blocker) or CGS-21680 (A2-purinergic receptor agonist) increased the peak heparin-releasable L P L activity in hearts isolated from SHR rats. Fructose feeding induced hypertension, hypertriglyceridemia and hyperinsulinemia in male Wistar rats. Acute fructose treatment did not alter cardiac heparin-releasable L P L activity, whereas a significant decrease in L P L activity was seen in the chronic treated group. Withdrawal of fructose treatment normalized blood pressure and cardiac heparin-releasable L P L activity. Similar to the SHR study, acute vasodilation by in vitro perfusion of coronary vasodilators like nifedipine and CGS-21680 increased cardiac heparin-releasable L P L activity in the chronic group to control levels. These studies demonstrate that hypertension may play a significant role in regulating cardiac L P L activity and that the decrease in enzyme could be a result of poor perfusion through the cardiac vasculature. The diabetic heart has elevated levels of FFA and TG, being supplied from various sources. The relative contribution of LPL to this supply of FFA during diabetes is not clear. We previously demonstrated that moderate diabetes (induced by 55 mg/kg STZ) augments heparin releasable L P L and that this augmentation is possibly due to an increased translocation of the enzyme from its site of synthesis (i.e. cardiomyocytes). To determine the precise location of the augmented LPL, a modified Langendorff retrograde perfusion was used to isolate the enzyme at the coronary lumen from that in the interstitial effluent. In response to heparin, a 4-fold increase in L P L activity and protein mass was observed in the coronary perfusate after 2 weeks of STZ-diabetes. Release of LPL activity into the interstitial fluid of control hearts was slow but iv progressive, whereas in diabetic hearts, peak enzyme activity was observed within 1 to 2 min after heparin, followed by a gradual decline. Immunohistochemical studies of myocardial sections confirmed that the augmented L P L in diabetic hearts was mainly localized at the capillary endothelium. To study the acute effects of insulin on endothelial L P L activity, we examined rat hearts at various times after the onset of hyperglycemia. A n increased heparin-releasable L P L activity in diabetic rats was demonstrated shortly (6 to 24 hours) after STZ injection or after withdrawal from exogenous insulin. Heparin-releasable coronary L P L activity was also increased after an overnight fast. These studies indicate that the intravascular heparin-releasable fraction of cardiac LPL activity is acutely regulated by short-term changes in insulin rather than glucose. Thus, during short periods (hours) of hypoinsulinemia, increased L P L activity at the capillary endothelium can increase the delivery of FFAs to the heart. To study the effect of this enlarged LPL pool on triglyceride (TG) rich lipoproteins, we examined the metabolism of very low-density lipoprotein (VLDL) perfused through control and diabetic hearts. Diabetic rats had elevated lipoprotein TG levels compared to control. However, fasting for 16 hours abolished this difference. When the plasma lipoprotein fraction of density < 1.006 g/ml from fasted-control and -diabetic rats (and thus containing mainly V L D L ) were incubated in vitro with purified bovine or rat LPL, diabetic V L D L was hydrolyzed as efficiently as V L D L obtained from control animals. Moreover, V L D L s derived from diabetic rats were found to have a similar apolipoprotein pattern when compared to control V L D L . Post-heparin plasma lipolytic activity was comparable between control and diabetic animals. [ 3 H]VLDL obtained from control rats was metabolized at a significantly faster rate by perfused diabetic hearts than by control rat hearts. This increased V L D L - T G hydrolysis was essentially abolished by prior perfusion of the diabetic heart with heparin, implicating L P L in this process. These findings suggest that the enlarged L P L pool in the diabetic heart is present at a functionally relevant location (at the capillary lumen), and is capable of hydrolyzing V L D L . In summary, hypertension per se regulates cardiac L P L in SHR and fructose hypertensive rats. The decreased L P L at its functional location could possibly restrict the supply of FFA (derived from circulating TGs) to the cardiac tissue leading to an increased glucose oxidation. On the other hand, cardiac L P L is enhanced within hours after induction of diabetes, inducing a corresponding increase in TG breakdown. These changes complement the observed increases in FFA oxidation in the diabetic heart. Interestingly, previous studies in our lab indicate that induction of diabetes in SHR rats counteracted the hypertension-induced decrease in cardiac L P L activity and significantly increased functional L P L in the heart. This may serve to increase the delivery of FFA to the heart and the resultant metabolic changes may lead to the severe cardiomyopathy observed in the hypertensive-diabetic heart. T A B L E O F C O N T E N T S VI Title page i Abstract i i Table of Contents vi Table Legends x Figure Legends xi List of Abbreviations xvii Acknowledgments xx Dedication xxi 1. INTRODUCTION 1 1.1. Lipoprotein lipase 1 1.2. LPL Molecular Biology 2 1.2.1 L P L gene 2 1.2.2 L P L structure 3 1.3. Synthesis, Activation and Translocation 8 1.3.1 L P L synthesis and activation 8 1.3.2 L P L secretion 12 1.3.3 L P L translocation 13 1.4. LPL in the Heart 18 1.5. LPL Regulation 20 1.5.1 Transcriptional regulation 20 1.5.2 Po st-transcriptional regulation 21 1.5.3 Post-translational regulation 23 1.5.3.1 Glycosylation 23 1.5.3.2 Binding sites 24 1.5.3.3 LPL turnover by intracellular degradation/recycling 26 1.5.3.4 LPL regulation by substrates 27 1.6. LPL Regulation by Various Physiological Factors 28 1.6.1. Fasting and Feeding 28 1.6.2. Hormonal regulation of L P L 30 1.6.2.1. Growth hormone 30 1.6.2.2. Thyroid hormone 31 1.6.2.3. Catecholamines 32 1.6.2.4. Other Factors 33 1.7. LPL Regulation by Pathological Conditions 34 1.7.1 L P L in Hypertension 36 1.7.2 L P L in Diabetes 38 1.7.2.1. Diabetes Mellitus: An Overview 38 1.7.2.2. LPL regulation during diabetes 41 RATIONALE, HYPOTHESIS AND OBJECTIVES 48 2.1 Hypertension and Diabetes 48 2.2 Metabolic lesions during Hypertension and Diabetes 49 2.3 Possible role of LPL in the development of cardiac dysfunction 50 2.4 Objective 1 51 2.5 Objective 2 52 METHODS 54 3.1 Hypertension Study 54 3.1.1. Experimental animals 54 3.1.1.1. Spontaneously Hypertensive Rats 54 3.1.1.2. Fructose Hypertensive Rats 55 3.1.2. Measurement of blood pressure 55 3.1.3. Isolated whole heart perfusion 56 3.1.4. Preparation of cardiac myocytes 58 3.1.5. L P L assay 59 3.1.5.1. LPL assay in the medium 59 3.1.5.2. Cellular LPL assay 59 3.1.5.3. Post heparin plasma lipolytic activity 60 3.1.6. Triglyceride secretion rate 60 3.1.7. Triglyceride clearance rate 61 3.2. Diabetes Study 62 3.2.1. Cardiac L P L regulation during diabetes 62 3.2.1.1 Experimental animals 62 3.2.1.1.1. Induction of diabetes 62 3.2.1.1.2. Insulin reduction 63 3.2.1.2. Modified Langendorjfperfusion 64 3.2.1.3. Preparation of cardiac myocytes 65 3.2.1.4. Heart tissue homogenization 65 3.2.1.5. Enzyme-linked immunosorbant assay for LPL mass 66 3.2.1.6. Immunolocalization of LPL 67 3.2.1.7. Effect of food restriction on LPL activity 68 3.2.2. Hydrolysis of V L D L in the diabetic heart 68 3.2.2.1. Separation of lipoproteins and lipid profile 68 3.2.2.2. VLDL purification and characterization 69 3.2.2.3. Analysis of apolipoproteins of VLDL using gradient gel electrophoresis 70 3.2.2.4. In vitro lipolysis of VLDL by bLPL and rLPL 70 3.2.2.5. In vivo radiolabeling of VLDL 71 3.2.2.6. 3 H-VLDL perfusion of the isolated beating heart 72 3.3. Plasma measurements 74 3.4. Materials 74 3.5. Statistical analysis 75 RESULTS 76 4.1. Hypertension study 76 4.1.1. L P L regulation in spontaneously hypertensive rat hearts 76 4.1.1.1. General characteristics and plasma parameters 76 4.1.1.2. Coronary endothelial LPL 76 4.1.1.3. Cardiac myocyte LPL 11 4.1.1.4. Insulin and LPL activity 78 4.1.1.5. Acute effects of vasodilators 78 4.1.1.6. Secretion and clearance of TGs and plasma lipolytic activity 19 4.1.2. L P L regulation in fructose hypertensive rats 95 4.1.2.1. General characteristics 95 4.1.2.2. Coronary endothelial LPL 95 4.1.2.3. Myocyte surface and intracellular LPL activity 96 4.1.2.4. Effects of insulin and vasodilators perfusion on endothelial LPL activity 96 4.2 Diabetes Study 111 4.2.1. L P L regulation in the diabetic heart 111 4.2.1.1. General characteristics of diabetic rats 111 4.2.1.2. Modified Langendorffperfusion 111 4.2.1.3. LPL activity in whole heart homogenate 112 4.2.1.4. Immunolocalization LPL 112 4.2.2. Acute L P L regulation by hypoinsulinemia 113 4.2.2.1. Insulin depletion study 113 4.2.2.2. Insulin withdrawal study 113 4.2.2.3. Fasting 114 4.2.3. Hydrolysis o f V L D L in the diabetic heart 137 4.2.3.1. Plasma lipid profile and apolipoprotein analysis 137 4.2.3.2. Post-heparin plasma lipolytic activity 137 4.2.3.3. Lipolysis of VLDL by bovine or rat LPL 13 8 4.2.3.4. 3H- VLDL clearance by isolated rat hearts 139 5. DISCUSSION 158 5.1 Cardiac LPL regulation during hypertension 158 5.1.1. L P L regulation in spontaneously hypertensive rat heart 158 5.1.2. L P L regulation in fructose hypertensive rat hearts 161 5.2 LPL during diabetes 164 5.2.1. L P L regulation in the diabetic rat heart 164 5.2.2. Hydrolysis of V L D L in the diabetic rat heart 168 6. SUMMARY AND CONCLUSIONS 172 7. FUTURE DIRECTIONS 176 8. REFERENCES 178 TABLES Tables Page No 1 General characteristics and plasma parameters of W K Y and 80 SHR rats at different ages 2 General features of control, acute-fructose and chronic- 98 fructose treated Wistar rats. 3 Characteristics of diabetes (55 mg/Kg STZ) at 2 weeks after 115 STZ injection. 4 L P L activity, mass and specific activity in coronary perfusate 118 from hearts isolated from 2-week D55 rats and age matched controls 5 Fed and fasted plasma parameters of control and diabetic rats 141 FIGURE LEGENDS XI Figures Page No 1 A schematic representation of L P L structure and the hydrolysis of 7 triacylglycerol (TAG) by LPL. (This model is adapted and modified from Carriere etal 1998) 2 Schematic diagram of LPL synthesis, activation and secretion from 11 cardiac myocytes 3 Scheme of probable mechanism that could be involved in L P L 17 transcytosis across endothelial cells 4 Effect of hypertension on heparin releasable L P L activity in perfused 81 hearts from W K Y and SHR rats at 7-8 (A), 11-12 (B) and 15-16 (C) weeks of age. 5 Effect of hypertension on L P L activity in cardiac myocytes from SHR 83 and W K Y rats at 7-8 (A) and 15-16 (B) weeks of age. 6 Acute effect of in vitro insulin on the release of LPL from isolated 85 perfused hearts of 15-16 week old W K Y and SHR rats. 7 The effect of nifedipine in vitro and in vivo on heparin releasable L P L 87 activity in perfused hearts from W K Y and SHR rats at 15-16 weeks of age. 8 Effect of in vitro treatment with CGS-21680 on heparin releasable L P L 89 xii activity in perfused hearts from SHR rats at 15-16 weeks of age. 9 Plasma triglyceride (TG) clearance over a 60 minute post-heparin 91 period for 7-8 weeks (A), and 15-17 weeks (B) old SHR and W K Y rats is shown. 10 Post heparin plasma lipase activity of 15-17 week old SHR and W K Y 93 rats. 11 Plasma insulin (A), triglyceride (B), and blood pressure (C) of acute (2 99 weeks) and chronic (4-6 weeks) fructose treated rats. 12 Heparin releasable L P L activity in perfused hearts from rats treated 101 acutely (A) or chronically (B) with fructose. Panel C represents animals that were withdrawn from fructose treatment for 2 weeks after chronic treatment. 13 A. Cell surface L P L activity on isolated cardiac myocytes from 103 chronically fructose treated (4-6 weeks) rats. 14 Effect of increasing concentration of insulin (100, 200 and 500 ng/ml) 105 and heparin (5 U/ml) perfusion on the release of L P L from isolated hearts of control Wistar rats. 15 In vitro effects of nifedipine and CGS-21680 on heparin releasable 107 L P L activity in perfused hearts from control (A) and chronic fructose-treated (B) rats. 16 The effect of heparin (0.5 U/gm) injection on the release of L P L in 109 X l l l control and chronic fructose treated rats. 17 Effect of chronic diabetes (2 weeks) on heparin-releasable L P L activity 116 in coronary perfusate (A) and interstitial fluid (B) from C O N and D55 rat hearts 18 L P L activity in heart homogenates. Hearts from C O N and D55 were 118 frozen immediately in liquid N2, after clearing the blood of the capillaries. 19 Representative photographs showing the immunocytochemical 121 localization of L P L following chronic diabetes induced with 55 mg/kg STZ. 20 The chronological changes in plasma insulin (left panel) and glucose 123 (right panel) levels during a 24-hr period following an IV injection of 100 mg/kg STZ. 21 Peak heparin-releasable L P L activity in coronary perfusate at different 125 time points following injection of STZ. The lower panel demonstrates peak heparin-releasable L P L activity in coronary perfusate from control and diabetic rats following the development of hyperglycemia. 22 Effect of insulin treatment in STZ diabetic rats. Animals were made 127 severely diabetic with 100 mg/kg STZ (D100), and then treated subcutaneously with a long-acting Ultralente insulin (D100+I) once daily. An insulin dose of -18-20 U/kg/day was required to maintain normoglycemia for at least a 24-hour period. Treatment was continued xiv for 7 days. Body weight (A) and fluid intake (B) were measured before injecting insulin. 23 Plasma glucose (A) and insulin (B) levels of control (CON) and 129 diabetic rats following withdrawal of exogenous insulin administration. 24 Peak heparin-releasable L P L activity in coronary perfusate from 131 control (CON) and diabetic rats following withdrawal of insulin treatment. 25 L P L activity in cardiac myocytes from control and 24 hour 133 hyperglycemic rats. Myocytes were prepared as described in METHODS. 26 Effect of fasting on heparin-releasable L P L activity in coronary 135 perfusate (A) and interstitial fluid (B). 27 A. Triglyceride (TG), B. cholesterol (Choi) and protein content of 142 lipoprotein fraction (< 1.006 8) separated from the fed serum of control (CON) and diabetic (D55) rats. 28 Post-heparin plasma L P L activity from C O N and D55 rats. The L P L 144 activity was determined as a difference between total lipase (LPL + HL) activity and H L activity (after L P L inhibition). M U = nmole FFA released/ml/min. 29 In vitro lipolysis of (A) V L D L (0.3 m M TG) versus bovine L P L 146 (increasing concentrations viz., 10, 20, 50, 100 mU) and (B) 20 mU of XV bovine LPL versus increasing concentration of VLDL-TG (0.1, 0.2, 0.3, 0.5, l.OmMTG). 30 Double reciprocal plot of CON and D55 VLDL-TG concentrations 148 (1/[S]) versus rate of lipolysis by LPL (1/V). 20 mU of LPL was exposed to various concentrations of VLDL-TG obtained from 18 hour fasted CON and D55 serum. 31 The FPLC elution profile of LPL and HL from heparin sepharose 150 affinity columns. LPL elutes at higher salt concentrations (~1.4 M NaCl) and hepatic lipase (HL) at ~ 0.8 M NaCl. 32 In vitro lipolysis of CON and D55 VLDL-TGs (0.3 mM) by (A) CON 152 LPL and by (B) D55 LPL. LPL was purified from post-heparin plasma obtained from CON and D55 rats by heparin sepharose affinity chromatography. 33 Poly Acrylamide Gel Electrophoresis of VLDL fractions isolated from 154 CON and D55 serum (A). This is a representative of three experiments using VLDL samples from three animals from CON and D55 groups. 34 In vitro lipolysis of 3 H VLDL-TG (obtained from CON group) by 156 isolated-perfused hearts from CON and D55 rats. Hearts were perfused with 3H-VLDL via a re-circulating Langendorff retrograde perfusion mode for 90 minutes. 35 Schematic representation of LPL synthesis, translocation, various 175 functions and its possible pathophysiological role in the diabetic heart. L I S T O F A B B R E V I A T I O N S xvii 5' -UTR 5' -Untranslated region Ala Alanine A N O V A Analysis of variance ApoA Apolipoprotein A ApoCII Apolipoprotein CII ApoE Apolipoprotein E Arg Arginine Asn Asparagine Asp Aspartic acid B A T Brown adipose tissues bLPL Bovine L P L BP Blood pressure B S A Bovine serum albumin cAMP Cyclic adenosine mono phosphate cDNA Complementary deoxyribose nucleic acid CHO-cells Chinese hamster ovarian cells C H O L Cholesterol C H Y L Chylomicrons C O N Control Cys Cysteine D100 Diabetes induced with 100 mg/kg body weight of STZ D55 Diabetes induced with 55 mg/kg body weight of STZ E D L Extensor digitorum longus EDTA Ethylenediaminetetraacetic acid ELISA Enzyme linked immunosorbent assay endo-H Endo-p-N-acetylglucosamindase-H ER Endoplasmic reticulum FFA Free fatty acids FSE-2 Fat specific element-2 G H Growth hormone GlcNAc N-acetyl glucose Gly Glycine Gp Glycoprotein GPAGE Gradient polyacrylamide gel electrophoresis GRE Glucocorticoid responsive element H D L High density lipoprotein His Histidine H L Hepatic lipase hLPL Human L P L hrp 116 Heparin sensitive protrein HS-oligos Heparan sulfate oligosaccharides HSPG Heparan sulfate proteoglycans i.p. Intraperitoneal i.v. Intravenous IRS Insulin responsive sequence KDa Kilodalton L D L Low density lipoprotein Leu Leucine LPDS Lipoprotein deficient serum L P L Lipoprotein lipase LRP Low density lipoprotein receptor related pro Lys Lysine MEF Myocyte enhancer factor Mr Relative molecular weight mRNA Messenger ribonucleic acid NCSS Number Cruncher Statistical System N E F A Non-esterified fatty acid Nifed Nifedipine NSF N-ethylmaleimide-sensitive fusion protein Oct-1 Octamer-1 OTF Octamer transcription factor P A G E Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PL Pancreatic lipase RAP Receptor associated protein RIA Radioimmunoassay rLPL Rat LPL s.c. Sub-cutaneous SDS Sodium dodecyl sulfate Ser Serine SNAP Soluble NSF attachment protein SNARE SNAP-receptors STZ Streptozotocin TG Triglyceride TGCR Triglyceride clearance rate TGSR Triglyceride secretion rate Thr Threonine TRE Thyroid responsive element Tip Tryptophan t-SNARE target membrane-SNARE VDRE Vitamin D3 responsive element VLDL Very low density lipoprotein v-SNARE vesicular-SNARE WAT White adipose tissues A C K N O W L E D G M E N T S XX I express my sincere gratitude to my research supervisor Dr. Brian Rodrigues whose supervision and commitment were instrumental in the successful completion of my research thesis. Brian always treated me as his equal, gave me lots of independence, and spent countless number of hours in scientific discussions all of which made my graduate program very rewarding and productive. His energy and enthusiasm in academic and social activities will always be remembered. My heartfelt thanks go to Dr. John McNeill who has always been sympathetic, understanding and supportive in my academic and social life in Canada. I thank the members of my supervisory committee (Dr. Mike Allard, Dr. Roger Brownsey, Dr. Helen Burt, Dr. Jack Diamond and Dr. John McNeill) for their valuable input and guidance in my research project. I greatly appreciate my lab mates Mr. Mohammed Abrahani for his meticulous technical and friendly support and Scott Craig for his diligent assistance in my research projects. I also thank Osama Al-Atar, Edith St. Pierre, Xunsheng Chen, Fiona Lim, Esther Fok, True Phan, and other summer students who helped me in various aspects of this research project. I also sincerely acknowledge Dr. Kishor Wasan and Ms. Manisha Ramaswamy for teaching me lipoprotein separation techniques. I am indebted to Dr. David Severson for donating anti-LPL antibodies and performing ELISA for LPL protein. Dr. Severson also spared his valuable time in scientific discussions and input to this project. I owe great deal in my life to Suriya Prakash, Mrs. Narmadha, Mr. Rathinaswami & family, Mr. Mohan Manikkam & family, Maggi Li, Rajesh Krishna, Swami Subramanian and others who gave tremendous support and help when I really needed them. Finally, I sincerely acknowledge Heart and Stroke Foundation of B.C. & Yukon for supporting me with a studentship during the Ph.D. program. D E D I C A T I O N xxi To my Mom, Dad and my sister for their love and support in all walks of life To my wife Sumathi and son Santhosh for their love and understanding 1. I N T R O D U C T I O N 1.1. L i p o p r o t e i n L i p a s e Triacylglycerols (TG) constitute 90% of the dietary fat of an average adult food in the Western society (Enerback and Gimble 1993). After being absorbed, lipids are packaged and transported in the form of lipoproteins such as chylomicrons (CHYL, produced by enterocytes) and very low-density lipoproteins (VLDL, secreted by the liver). The T G component of these lipoproteins must be enzymatically hydrolyzed to release free fatty acids (FFA) which are then re-esterified and stored as intracellular TGs or utilized as an energy source (Enerback and Gimble 1993). In adult extrahepatic tissues, lipoprotein lipase (LPL) plays a primary role in the hydrolysis of circulating TGs. LPL [EC 3.1.1.34] was initially identified as a "clearing factor" by Hahn (1943) since it cleared severe alimentary lipemia that occur due to irregular eating habbits in dogs. Heparin injection to these dogs cleared milky plasma which was later found to be due to the lipolytic action of an enzyme called lipoprotein lipase (Anfinsen et al, 1952; Korn 1955). Subsequent studies have established that LPL is present in several tissues and is located close to the capillary wall (Hamosh and Hamosh 1983). Thus, LPL, present on the luminal surface of the capillary endothelium, hydrolyzes the TG component of circulating lipoproteins like C H Y L and V L D L , and releases FFA, which are subsequently utilized by tissues for various metabolic tasks. In this way, LPL plays a rate limiting role in the clearance of TG-rich lipoproteins from the circulation and in the supply of TG derived FFA to various tissues. L P L activity and mRNA have been detected in a wide variety of tissues such as white and brown adipose, adult heart, skeletal muscle, lungs, lactating mammary glands, kidney, spleen, 2 thoracic aorta, ovary, small intestine, testes, hippocampus of brain, and neonatal liver (Braun and Severson 1992). Adult liver has very little LPL activity and no LPL-mRNA. In white adipose tissue (WAT), L P L provides FFAs which are then re-esterified as TG for storage. In brown adipose tissue (BAT), LPL plays a role in the regulation of thermogenesis. LPL provides FFA as an energy source in skeletal and cardiac muscles. In lactating mammary glands, LPL facilitates milk formation. The physiological role for LPL in some tissues, like brain, is not clear (Braun and Severson 1992). 1.2. L P L - m o l e c u l a r b io logy 1.2.1. LPL-gene The LPL gene has been mapped to the short arm of chromosome 8 (8p22) and spans approximately 30 kb (Murthy et al 1996). The L P L gene has 10 exons separated by 9 introns. Exons 1 -9, which code for the protein sequence are highly conserved among various species and exon 10, the largest of all, shows the maximum species variability (Enerback and Gimble 1993). Exon 1 encodes the 5' untranslated region (UTR) and the signal peptide, exon 2 codes for N -linked glycosylation site (Asn-Xaa-Ser or Thr, where Xaa is any amino acid but proline), exon 4 for the interfacial lipid binding region, exon 5 for catalytic site (consensus sequence Gly-Xaa-Ser-Xaa-Gly), exon 6 for a putative heparin binding site, exon 8 for a second N-linked glycosylation site and exon 10 codes for 3' UTR (Braun and Severson 1992). 5'-upstream region is highly conserved up to ~210 bp in various species including humans and rats. Several cis-acting elements have been identified within the 5'-flanking region. The notable control elements include: octamer-1 (Oct-1), fat specific element (FSE-2), glucocorticoid responsive elements (GRE), thyroid responsive element (TRE), A P I , AP2, Spl , LP a/p\ and more recently, FSE-2 like, G A T A , insulin responsive sequence (IRS), vitamin D3 responsive element (VDRE), and 3 myocyte enhancer factor (MEF-2) (Bey et al 1998). It is proposed that differences in these cis-acting sites may be responsible for the differential regulation of LPL across the species. 1.2.2. LPL Structure LPL is a glycoprotein, which in its active form occurs as a non-covalent homodimer. The molecular weight (Mr) of LPL, as predicted from mRNA, is 50,394 assuming removal of the 17-27 amino acid signal peptide. Mature LPL is therefore a 448 amino acid long polypeptide (Auwerx et al 1992). Sedimentation equilibrium measurements give a Mr of 41,700 for the LPL monomer (Olivecrona et al 1982). The Mr of active dimer is determined to be -72,000 (Olivecrona et al 1985). The species difference in molecular weights was primarily attributed to variable lengths of signal peptide and degree of glycosylation. LPL has a significant homology with other lipases like hepatic lipase (HL) and pancreatic lipase (PL). Although the crystallographic structure for LPL is not yet deduced, it is proposed to be similar to that of human PL (Hide et al 1992). The LPL molecule has an amino (N)-terminal spanning amino acids 1-312, and a carboxy (C)-terminal from 313-448 (Murthy et al 1996). N-terminal region has several active sites and C-domain has lipid and heparin binding sites. LPL monomer has 8 putative functional sites: (1) a catalytic triad, (2) an oxyanion hole, (3) a 'lid' or 'flap' structure, (4) a f$-5 loop, (5) an apolipoprotein CII binding site, (6) an interfacial/lipid binding site, (7) a heparin binding site and (8) a non-covalent dimer formation site (Braun and Severson 1992, Murthy et al 1996). Human LPL has 8 highly conserved serine (Ser) residues out of which Ser 132 is the crucial residue required for enzyme catalysis (Faustinella et al 1991). Ser 132 is part of the consensus sequence Gly-Xaa-Ser-Xaa-Gly that is present in all serine proteinases and in human 4 PL. It is also proposed that these highly conserved Ser residues could play a potential role in conserving three-dimensional structure of LPL similar to PL. Recently, the catalytic triad was identified as Serl32-Aspl56-His241 and is well conserved in all three lipases of human and of all other species studied so far (Hide et al 1992). The oxyanion hole and p-5 loop along with the ' l id ' structure probably control access of the substrate to the active site. The oxyanion hole is formed by Trp55 and Leul33, which are next to Serl32 of the catalytic triad. The ' l id ' or the 'flap' structure is a mobile surface loop that covers the active site and by repositioning, it permits the substrate to have access to the catalytic site. The lid is flanked by two cysteine residues (Cys216 and Cys239) which form disulfide bridges in the LPL molecule and stabilizes the lid within the protein during activation. Mutation analysis reveals that the charge and periodicity in the proximal and distal segments of the lid is crucial for L P L activity while apical residues (up to 8 amino acids length) of the loop contribute very little to LPL activity (Henderson et al 1993). It is proposed that these differences in the open (active) conformation of the lid structures in LPL and PL may account for their different substrate specificity. The (3-5 loop, another mobile loop that occupies His53 to Trp64, is also involved in rendering the active site more accessible to the substrates by bringing the oxyanion hole into a catalytically competent position (Murthy et al 1996, Figure 1). The apolipoprotein CII (apoCII) binding site is located at Lysl47-Lysl48 in the N -, terminal domain. This site interacts with the dipeptide (Lys-Gly, Glu-Glu) present in the C-terminal region of apoCII. The heparin binding sites on LPL interacts with cell surface heparan sulfate proteoglycans (HSPG). The hypothetical consensus sequences situated in the N-terminal region are X - B - B - X - B - X and X - B - B - B - X - X - B - X , where B stands for a positively charged residue and X denotes a neutral residue. Additionally, positively charged clusters like Lysl47, Lysl48, Argl51 may also be involved in heparin binding activity. Recently, Sendak and 5 Bensadoun (1998), using mutational analysis, demonstrated that the distal carboxy-terminal domain comprising a cluster of positively charged Lys321, Arg405, Arg407, Lys409, Lys416 residues constitute the major heparin-binding domain. The disulfide bridges between Cys264-Cys275 and Cys278-Cys283 in the LPL molecule may stabilize these regions upon heparin binding (Murthy et al 1996). Therefore, it appears that each LPL molecule has several heparin binding sites. The interfacial/lipid-binding domain probably resides in the C-terminal region of LPL, particularly in the last 56 amino acids (Lookene and Bengtsson-Olivecrona 1993). In a recent study, Lookene et al (1997) expressed several mutants by replacing tryptophan (Tip) residues 390, 393 and 394 with alanine (Ala) and determined that these residues are important for the productive orientation of LPL at the lipid/water interface. In the same study, the authors proposed that Tip residues in the N-terminal region (55 and 114) might be involved in subunit interaction and dimer formation. Previato et al (1994) showed that the C-terminal domain, in addition to its lipid and heparin binding function, may also play an important role in the formation of an active L P L dimer. The mechanics of LPL-substrate interaction and the process of lipolysis is discussed in Murthy et al (1996). The LPL-lipoprotein interaction is supposed to produce some conformational changes in the LPL molecule such that the lid structure opens up and exposes the hydrophobic residues of the lid to undergo interfacial activation. This activation results in the formation of a cleft to which the F A chains (snl or 3) of T G probably bind and the glycerol backbone of the TG occupies the oxyanion hole. The catalytic triad then executes the hydrolysis of TG (Figure 1). The sn-2 fatty acyl chain probably interacts with the lipidic matrix and therefore would not be hydrolyzable (Carriere et al 1998). In addition to being a lipolytic enzyme, L P L also acts like a ligand and facilitates uptake of lipoproteins into cells. This receptor independent lipoprotein uptake requires LPL in its dimeric form. Moreover, heparin and lipid binding sites between amino acids 390-421 are necessary for LPL-mediated lipoprotein uptake (Krappetal 1995). 7 Figure 1. A schematic representation of LPL structure (above) and the hydrolysis of triacylglycerol (TAG) by LPL (below). (These models are adapted with permission from Carriere etal 1998). NH NH Oxyanion hole $5-loop 8 1.3. Synthesis , ac t iva t ion a n d t rans loca t ion 1.3.1. LPL synthesis and activation L P L biosynthesis has been extensively studied in cell cultures. 3T3-L1 adipocytes (Previato et al 1991), Ob-17 preadipocytes, and Chinese hamster ovarian (CHO) cell cultures (Ben-Zeev et al 1992) were some of the cell lines used to delineate biosynthetic pathways for LPL. LPL is synthesized as an inactive, monomeric proenzyme in the rough endoplasmic reticulum (ER) and activated somewhere between the ER and Golgi apparatus. The active enzyme is a dimer with asparagine (Asn) linked glycosylation. Glycosylation of the N-terminal domain at the Asn residue of LPL is required for its catalytic activity. There are two Asn residues to which oligosaccharide moieties are added to the LPL molecule. Asn 43 (N-terminal domain) and 359 (C-terminal domain) in guinea pigs, Asn 44 and 361 in cow and Asn 45 in chicken LPL, are glycosylated. Asn 43 in is the most important glycosylation site of human L P L (Auwerx et al 1992). In the rat, two Asn (43 and 359) glycosylation sites are conserved as in other mammals. Ben-Zeev et al (1994) used mutational techniques to systematically delete glycosylation sites of LPL and determined that N-glycosylation is essential while glycosylation at the C-terminal domain is not required for the expression of fully active LPL. This was also reinforced by Busca et al. (1995) who showed that replacing Asn 43 with Ala (Asn43->Ala43) using site directed mutagenesis caused retention of non-glycosylated human L P L (hLPL) in ER. Neither LPL activity nor protein was found in the medium of cells expressing mutant hLPL and all detectable protein was present exclusively in the ER. In addition, this accumulation of non N -glycosylated LPL also caused morphological changes in ER and prevented the transport of other proteins like B°' + amino acid transporter (rBAT) and glucose transporter (glut-4) to plasma membrane (Busca et al 1995). 9 Glycosyl moieties are added co-translationally to Asn residues on nascent LPL polypeptide by a dolichol (a lipid)-linked oligosaccharide [Glc3-Man9(GlcNAc)2] (Braun and Severson 1992). This glycoprotein with mannose rich glycosyl residues in the ER is endo-(3-N-acetylglucosaminidase H (endo-H) sensitive. Indeed, N-linked oligosaccharide undergoes a series of modifications: 1) terminal glucose residues are removed by glucosidase I and II in ER, 2) one mannose residue is then removed by a-mannosidase giving rise to high mannose peptide [Man8-(GlcNAc)2-protein] which is then transferred to cis-Golgi, a receiving center of the Golgi complex. Although the exact location of Golgi processing enzymes is not clear, three mannose residues are removed by mannosidase I in cis-Golgi. 3) As the glycoprotein moves through medial-Golgi, GlcNAc is added and two more mannose residues are removed by mannosidase II resulting in the formation of [GlcNAc-Man3(GlcNAc)2]-protein. 4) In the trans-Golgi, this protein is further modified by a series of transferases, which add GlcNAc, galactose and sialic acid residues to give rise to a endo-H resistant glycoprotein (Figure 2, Braun and Severson 1992). Vannier and Alihaud (1989) proposed that LPL exists as an inactive monomer within the ER and the activation of LPL occurs only after translocation to the cis/medial-Golgi. However, Ben-Zeev et al (1992) demonstrated that translocation of LPL from ER to cis-Golgi is not required for full expression of enzyme activity. Various approaches like brefeldin A treatment (to disassemble the cis/medial-Golgi complex), incubation at 16°C (to prevent the movement of proteins from ER to Golgi), and tagging with KDEL (a specific ER retention signal) caused retention of fully active LPL in its high mannose form within the ER, blocking secretion into the medium. Also, it was shown that mannose trimming was not necessary for the activation and secretion of LPL (Ben-Zeev et al 1992). However, a significant reduction in LPL specific activity and secretion occurred when glucosidase inhibitors like castanospermin and N-methyl-deoxynojirimycin inhibited glucose trimming of LPL oligosaccharides. Therefore, the authors 10 concluded that while glucose trimming is essential for activation and secretion of LPL, further oligosaccharide processing or translocation to the cis-Golgi is not required for full expression of lipolytic activity (Ben-Zeev et al 1992). Liu et al (1993), utilizing metabolic labeling of perfused guinea pig hearts, demonstrated that LPL is assembled into dimers in the ER and that the processing of oligosaccharide chains occurs after dimerization. Recently, Scow et al (1998), using the cld/cld mouse model showed that glycosylation and dimerization alone do not produce active L P L but transport of LPL out of the ER is essential for the secretion of active enzyme. L P L accumulates in the trans-Golgi in its fully active form with a half time of approximately one hour (Eckel 1989). C a r d i a c M y o c y t e s 11 Endoplasmic Reticulum Golgi Complex Secretory Vesicles •Protein synthesis •N-glycosylation •dimerization •glucose trimming •Mannose trimming •GlcNAc, Galactose and Sialic acid residues addition 1 •Fully matured active LPL dimer 1" Endo H sensitive Endo H resistant Lysosomal degradation Interstitial space Temporary binding to cell surface HSPGs Translocation to luminal surface A Functional LPL bound to luminal HSPG projections Vascular lumen Figure 2. Schematic diagram of L P L synthesis, activation and secretion from cardiac myocytes. 12 1.3.2. LPL secretion LPL, like other proteins, reaches the plasma membrane from the ER through a vesicular transport system. In general, vesicles are sculpted from the ER with the help of ER coat proteins like COP I/COP II (Rothman and Wieland 1996). These coat proteins are then removed to allow the fusion of the vesicle to the target membranes like Golgi and plasma membranes. The specific proteins called SNAP receptors (SNARE) bring about the docking of the vesicles to the target membrane. SNAREs (present on vesicles, v-SNAREs) bind to specific t-SNAREs (present on the target membranes) to dock the vesicle to appropriate target membranes. NSF (N-ethylmaleimide-sensitive fusion protein) and SNAP (soluble NSF attachment protein) then bind to the SNARE complex and initiate an energy dependent fusion process. NSF mediated ATP hydrolysis produces energy required in this process. The target specific vesicular transport is dictated by the intrinsic signal from the protein being transported. A protein may have multiple sorting signals, each determining the fate of that protein at successive stages and jointly controlling its journey (Rothman and Wieland 1996). In the LPL molecule, a signal peptide of 17-27 amino acids and oligosaccharides were proposed to be involved in the directional transport of the protein (Eckel 1989, Braun and Severson 1992), but the details of the transport process remain to be elucidated. L P L is secreted by the parenchymal cells as an active enzyme in its homodimeric form by constitutive and regulated mechanisms (Braun and Severson 1992). The constitutive release of L P L occurs spontaneously and the rate of release matches the rate of synthesis. The regulated release occurs in response to a secretogogue (e.g. heparin). For regulated release to occur, the enzyme has to be packaged and stored in secretory vesicles until it is released. A l l cell types that produce LPL exhibit constitutive release while some cells exhibit both constitutive and regulated 13 release (for example Ob-17, 3T3-L1 adipocytes, cardiac myocytes and mesenchymal cells). Heparin is a major secretogogue, acts by releasing LPL from its cell surface binding sites and therby stimulates secretion of additional enzyme from within the cells (Eckel 1989). Release of L P L from cells is also possibly controlled by metabolic demand (Olivecrona and Bengtsson-Olivecrona 1993). Addition of increasing concentrations of oleic acid prevented the release of L P L by cultured endothelial cells (Stins et al 1992). Recently, Anderson et al (1997) demonstrated that FFA inhibited LPL secretion by isolated cardiac myocytes suggesting a feed-back inhibition of L P L secretion by the lipolytic products. 1.3.3. LPL translocation LPL, after being secreted, binds to parenchymal cells transiently before it is translocated to the endothelial surface in the vascular lumen. Translocation of LPL from its site of synthesis (myocytes/adipocytes) to the endothelial cell surface in the vascular lumen is not clearly understood. The subendothelial basement membranes are proposed to sequester and stabilize the L P L secreted by non-endothelial cells (Chajek-Shaul et al 1990). Extracellular matrix (ECM) in the subendothelial space is composed of collagens (type I, III, and IV), fibronectin, laminin and glycosaminoglycans (GAG) like heparan sulfate-, dermatan sulfate-, chondroitin sulfate-proteoglycans of which LPL binds mainly to the heparan sulfate proteoglycans (HSPG). It was first proposed that LPL could move along bridges of HSPG molecules that connect parenchymal cells with capillary endothelial cells (Blanchette-Mackie et al 1989). In fact, binding of L P L to HSPG at the basolateral surface of the endothelium was shown to be necessary for an efficient transport to the apical surface (Saxena et al 1991). The removal of HSPGs by heparinase digestion suppressed this transport process (Saxena et al 1991). However, co-localization of HSPG and L P L within the transport vesicles was not required for the secretion of active LPL as 14 shown in HSPG deficient Chinese hamster ovarian (CHO) cells (Berryman and Bensadoun 1995). Nevertheless, L P L produced by wild type CHO cells (which express HSPG) was found to be more stable than L P L produced by the mutant cells. Stins et al (1992) further proved that the movement of LPL across the endothelial cells was polarized towards the apical (also known as luminal) surface. Transport of LPL was therefore appreciably greater from the basolateral (also known as abluminal) to the apical surface than in the opposite direction. Recently, fragments of HSPG, heparan sulfate (HS) oligosaccharides have been shown to act like extracellular chaperones for LPL and enable the transport of the enzyme in its active form across the endothelial cells (Sivaram et al 1997). Endothelial cells release a heparanase-like enzyme during the lypolytic encounter of LPL with V L D L in the co-cultures of endothelial cells and adipocytes. This heparanase-like enzyme then cleaves surface bound HSPGs to release HS-oligosaccharides (Sivaram et al 1997). HS-oligosaccharides bind non-covalently to LPL molecules and escort them to the apical surface of endothelial cells and probably prevent LPL degradation in interstitial fluid. Moreover, HS-oligosaccharides have also been shown to enhance transport of L P L across endothelial cells (Sivaram et al 1997). HS-oligosaccharides only partly saturate the heparin binding domains on the L P L molecule and, therefore do not affect further binding of L P L to cell surface HSPGs (Sivaram et al 1997). A l l of these in vitro results were complemented by the fact that feeding animals also increases the activity of LPL as well that of heparanase-like activity in adipose tissue (Sivaram et al 1997). Movement of LPL across endothelial cells has been suggested to occur via a poorly understood process called transcytosis. Endothelial transcytosis involves endocytosis of macromolecules like LPL at the basolateral side of endothelial cells, vesicular transport across the cell, and exocytosis at the luminal surface. Much of the understanding comes from basic cellular transport processes that are applicable to various macromolecules in general. Endothelial 15 cells lining the arterioles, capillaries and venules of glomeruli and peritubules of kidney and capillaries associated with intestinal mucosa and secretory glands are fenestrated. However, endothelial cells lining the microvessels of the heart are not fenestrated but continuous. Therefore, the transport of macromolecules across the endothelial barrier has to be either by fluid phase or transcytotic mechanisms (Michel 1998). Several studies suggest that caveolae could act as ferries for macromolecules between luminal and abluminal surfaces of the endothelial cells. Caveolae are endothelial vesicles involved in the receptor-mediated endocytosis or transcytosis of macromolecules. A specific inhibition of vesicular fusion protein, NSF (N-ethylmaleimide sensitive fusion protein) by N-ethylmaleimide could prevent the macromolecular permeability, supporting a vesicular transport system across the endothelial cells (Oh et al 1998). A combination of phosphatidylinositol-3 kinase (PI-3 Kinase) and tyrosine kinase regulated endocytosis, and SNARE-SNAP-NSF complex mediated exocytosis are indicated in this caveolar transport across endothelium (Niles and Malik 1999). Additionally, involvement of actin filaments has been suggested in the caveolar transport process since cytochalasin B prevented transport of these vesicles (Niles and Malik 1999). In this regard, Martinho et al (1996) demonstrated that binding of LPL to HSPG triggers the aggregation and distribution of HSPGs along the actin cytoskeleton. In the absence of such ligand binding, HSPGs appear distributed irregularly on the fibroblast cell surface, without any apparent co-localization with the actin cytoskeleton. Whether this organization of HSPG along the actin cytoskeleton is involved in the transcytosis of LPL from the abluminal to the luminal surface is still not known. Earlier studies in the heart demonstrated that antimitotic agents like colchicine and vinblastin inhibited the transport of LPL to the luminal surface of endothelial cells (Chajek et al 1975). Another antimitotic drug, cyclophosphamide, also had similar inhibitory effects on the secretion of L P L in rabbit hearts (Lespine et al 1997). Recently, Ewart et al (1999) demonstrated that the actin 16 cytoskeleton is involved in the transport of LPL from within the cardiac myocytes. Because the microvascular endothelium of the heart is known to be enriched with caveolae (Michel 1998), it would be interesting to apply the various above interventions to examine whether LPL translocation follows this pathway (Figure 3). 17 Abluminal surface «mc rafter Luminal Surface • J o HSPG LPL dimer Vesicular SNARE (v-SNARE) Target SNARE (t-SNARE) N-ethylmaleimide sensitive fusion protein (NSF) Soluble NSF attachment protein (SNAP) Figure 3. Scheme of probable mechanism of LPL transcytosis across endothelial cells. The transcytotic process has been depicted to involve four stages (1. Endocytosis, 2. Actin alignment, 3. Vesicular traffic along the actin cytoskeleton, and 4. Vesicle fusion and exocytosis). 18 1.4. L P L in the H e a r t In the heart, LPL gene expression and protein synthesis begins only after birth and reaches a maximum level within 3 weeks of age. The fetal heart does not express LPL (Hamosh and Hamosh 1983). Synthesis, processing and translocation of LPL in the heart occurs by mechanisms similar to those of adipose tissues and has been mainly studied in experimental animals like mouse, guinea pigs, rats, and rabbits. In the adult mouse heart, L P L is synthesized and processed in cardiac myocytes (Blanchette-Mackie et al 1989). Similarly, in human myocardium, in situ hybridization for LPL mRNA revealed that LPL was primarily synthesized by cardiac myocytes (O'Brien et al 1994). In the rat heart, cholera toxin induced L P L mRNA expression primarily in interstitial cells (Stein et al 1991) although cardiac myocytes also exhibited a diffuse but a definite localization of LPL mRNA. Since the LPL gene has muscle specific motifs and calcium- and cAMP-responsive elements, it is likely that under normal conditions, cardiac myocytes and possibly some vascular pericytes (which comprise a portion of interstitium in the heart) could be the primary sites of L P L synthesis (O'Brien et al 1994). Distribution of immunoreactive LPL protein was observed in different compartments of myocardium. Electron microscopic studies of immunogold-labeled sections of mouse myocardium delineated that 78% of total LPL was present in the cardiac myocytes, 3-6% in the interstitial space, and 18% in the capillary endothelium (Blanchette-Mackie et al 1989). Within the myocytes, L P L was found localized within the sarcoplasmic reticulum, Golgi complex and secretory vesicles (Blanchette-Mackie et al 1989). Outside the myocyte, LPL was associated with the plasma membrane and the basal surface of the capillary endothelium. LPL in the endothelium was seen inside the cells, within the vesicles and along the luminal surface of the endothelial cells (Blanchette-Mackie et al 1989). LPL on the luminal surface of endothelial cells 19 were also seen associated with chylomicron particles. The functional fraction of LPL in the perfused heart was immunocytochemically localized in the vascular lumen (Pedersen et al 1983). Although immunoreactivity for LPL protein was observed in endothelial cells, no mRNA expression was detected (Camps et al 1990). Further, treatment with protein synthesis inhibitors like cycloheximide, or with transport inhibitors like colchicine reduced the LPL activity in the vascular lumen (Chajek et al 1975, Liu and Olivecrona 1992). Therefore, it was concluded that endothelial cells do not synthesize LPL and the enzyme must have been synthesized in the cardiac myocytes and translocated to the endothelial surface in the vascular lumen. Pulse-chase studies demonstrated that it takes approximately 30 minutes for the newly synthesized L P L to be translocated to the luminal surface of vascular endothelium (Liu and Olivecrona 1991). When perfused with heparin via a Langendorff retrograde perfusion, isolated hearts release LPL. This heparin-releasable LPL was shown to occur in three phases over a 60 minute period in guinea pig hearts (Liu and Olivecrona 1992, Rodrigues et al 1997): 1) a rapid phase, occuring within seconds after heparin perfusion, probably from the endothelial luminal surface; 2) a shoulder of release, occuring between 2-30 minutes, probably from within and from the abluminal surface of endothelial cells; and 3) a slow steady release of LPL, probably explained by release from the site of synthesis. The rapid, heparin releasable, vascular-bound L P L fraction is also called 'functional' LPL since it is involved in the hydrolysis of circulating TGRLs. It is this heparin-releasable fraction of LPL that is more sensitive to physiological (e.g. fasting and feeding, Braun and Severson 1992), and pathological (e.g. diabetes, Tavangar et al 1992) changes and this can correspondingly affect exogenous FFA supply to the heart. 20 1.5. L P L R e g u l a t i o n LPL synthesis and activity is regulated in a tissue specific manner during developmental stages by various physiological conditions like fasting, feeding, cold exposure, lactation etc., and by various pathological conditions like diabetes, hypertension, obesity etc. For example, LPL synthesis can be observed in the fetal liver but the adult liver does not synthesize LPL. On the contrary, heart LPL activity is very low in the fetus but reaches maximum levels within three weeks after birth (Hamosh and Hamosh 1983). Several transcriptional, post-transcriptional and post-translational mechanisms have been proposed for this differential regulation of L P L . 1.5.1. Transcriptional regulation A greater understanding of transcriptional regulation of LPL has been possible due to the availability of cDNA for LPL from various species including human, mouse, rat, baboon, bovine, chicken and guinea pig (Enerback and Gimble 1993). Most of the transcriptional regulation of LPL has been studied in pre-adipocyte cell cultures. During differentiation of pre-adipocytes to mature adipocytes, several genes are activated, earliest among them being the LPL gene (Enerback and Gimble 1993). The LPL gene has several cis-acting regulatory sequences in its 5'-flanking region. Regulation of LPL gene transcription involves binding of specific trans-acting factors to these cis-acting elements. Oct-1 (octamer sequence ATTTGCAT) at nucleotide -46 (relative to the transcriptional start site) is one such site which binds to trans-acting factors like OTF-1 (octamer transcription factor). Using transient transfection studies, deletion of the Oct-1 site has been shown to decrease the transcription of LPL gene. The specificity of Oct-1 and 2 protein binding to this octamer sequence is determined by the D N A sequence adjacent to the Oct-1 site (Previato et al 1991, Currie and Eckel 1992). NF-Y, another protein which specifically binds to the C A A T motif at position -65, is also involved in the transcriptional 21 regulation of L P L (Currie and Eckel 1992). The presence of positive (-368 to -35, increases LPL expression) and negative (-724 to -565, suppresses LPL expression) regulatory sequences in the 5'flanking region have been indicated to play a role in the tissue specific regulation of L P L (Previato et al 1991). Similarly, LP-cx/p\ Ap-1 complex, and C/EBP proteins are all involved in the transcriptional regulation of LPL (Enerback and Gimble 1993). Differences that exist in these regulatory sequences and in the expression of trans-acting factors have been postulated to play a role in tissue specific regulation of L P L in various species. The involvement of the 3'untranslated region (3'UTR) region in the tissue specific regulation of L P L is increasingly evident from recent studies. L P L cDNA from various species including human, bovine and mouse exhibits two polyadenylation signals (corresponding to the human sequence at nucleotides 3155 and 3550) in its 3'-UTR and these sites are highly conserved in all species examined so far (Ranganathan et al 1995). Adipose tissue in humans expresses two LPL mRNA species (3.2 and 3.6 kb) probably due to a random choice of one of the two polyadenylation sites (Ranganathan et al 1995). However, skeletal and heart muscles express only the 3.6 kb mRNA form probably due to a non-random choice. In addition to humans, several other species express multiple mRNAs for LPL. However, in rat adipose and muscle tissues only the second polyadenylation site is utilized to express the 3.6 kb L P L mRNA (Ranganathan et al 1995). Therefore, in addition to the 5'-flanking region, the 3'UTR can be speculated to play a role in the tissue and species specific regulation of L P L expression. 1.5.2 Post-transcriptional regulation Two mRNA species have so far been identified for LPL; one is 3.2 kb and the other 3.6 kb in length. When transfected in CHO cells, the longer mRNA form more efficiently translated to L P L protein that had higher specific activity than the 3.2 kb mRNA transcript (Ranganathan et 22 al 1995). Steady state mRNA levels depend on message stability as well as gene transcription. Stability and translatability of mRNA determine the amount and rate of protein translation. The polyadenylation tail confers stability to mRNA. Although the exact mechanism by which the 3.6 kb mRNA translates more efficiently is not known, it is proposed that in general, changes in the secondary structure of mRNA may allow better interactions of certain trans-acting binding proteins. The 3'-UTR may also be involved in mRNA stability and in its translational efficiency (Ranganathan et al 1995). Hormones like insulin and epinephrine may alter the translational efficiency of mRNA by modulating mRNA-binding proteins (Yukht et al 1995). Turnover of LPL mRNA is usually very slow under normal conditions (~40 hours). This slow turnover of mRNA means a continuous production of active L P L even when there is no physiological need. Therefore, posttranslational regulation is required to control the enzyme concentration at a level that is absolutely necessary to fulfill the physiological demand (Mitchell et al 1992). The rate of LPL protein translation from its mRNA is commonly measured by 3 5 S -methionine incorporation and immunoprecipitation techniques. The increase in rate of LPL synthesis can either be dependent on or independent of mRNA levels. For example, insulin stimulated L P L synthesis in rat adipocytes is dependent on an increase in mRNA levels whereas glucose further increased LPL synthesis in these cells without altering mRNA levels (Ong and Kern 1989). In another instance, the diurnal variation (both increase and decrease) in L P L mass and activity was not accompanied by corresponding changes in L P L mRNA levels indicating a posttranslational regulation (Bergo et al 1996a). However, certain conditions like treatment with TNF-a, and cold/warm adaptations may markedly alter LPL mRNA production and degradation with a corresponding change in LPL mass and activity (Mitchell et al 1992). LPL regulation at multiple levels such as transcriptional, pretranslational and posttranslational levels was observed in Djungarian hamsters' brown adipose tissue in response to cold adaptation. In early phases of 23 cold adaptation posttranslational mechanisms are activated whereas, prolonged cold exposure involved posttranscriptional, pretranslational and posttranslational mechanisms (Klingenspor et al 1996). 1.5.3 Post-translational regulation Posttranslational regulation of LPL can occur at various sites including glycosylation, turnover of LPL protein, dimerization to an active form and also through control of LPL binding sites on the endothelium. The LPL activity can also be regulated by physiological substrates such as VLDL and CHYL and breakdown products like FFA. 1.5.3.1. Glycosylation 6-7% of the mass of each molecule of LPL is contributed by carbohydrate structures. This carbohydrate is in the form of N-glycosylated sugars on the LPL protein. Core glycosylation and glucose trimming occurs in ER and further processing takes place in the Golgi complex (section 1.3.1). Inhibition of glycosylation and glucose trimming in ER by tunicamycin (prevents N-linked oligosaccharide chain transfer) and castanospermin (glucosidase I inhibitor) led to the production of inactive monomeric LPL that had low affinity for heparin. However, treatment with 1-deoxymannojirimycin (Golgi mannosidase I inhibitor) and swainsonine (inhibits Golgi processing) produced active, dimeric LPL that had high affinity for heparin. Therefore, it was concluded that glycosylation and subsequent removal of distal glucose residues was necessary but processing by the Golgi mannosidase I was not required for the production of active and dimeric LPL that had high affinity for heparin binding (Masuno and Okuda 1994, Park etal 1995). 24 1.5.3.2. Binding Sites LPL, after secretion binds to HSPG binding sites that are present both on parenchymal and endothelial cell surfaces (Cheng et al 1981, Shimada et al 1981, Wang-Iverson and Brown 1982, Williams et al 1983, Cisar et al 1989, Chajek-Shaul et al 1989,). Recently, the active fragment of HSPG was found to be a decasaccharide (made up of the repeating structure -[IdceA(2-S04)al-4GlcNS04(6-S04)]); with a linear array of negatively charged sulfate groups. This active fragment may bind to peptide region(s) containing basic amino acid residues of LPL with high affinity (Parthasarathy et al 1994). LPL bound to HSPGs can be displaced by a competitive ionic interaction by heparin. This release of LPL by heparin is believed to stimulate L P L secretion from the parenchymal cells. The exact mechanism by which this stimulatory effect of heparin occurs is not yet known. Binding of LPL to HSPG also stabilizes L P L activity (Berryman and Bensadoun 1995). Unbound LPL present in the medium loses its activity much faster than the bound form. In vivo, LPL released into the circulation from its endothelial binding sites is subsequently taken up by the liver and degraded (Chevreuil et al 1993). Intravenous injection of heparin results in a release of large amount of functional L P L from all extrahepatic binding sites into the circulation. Heparin delays but does not abolish the hepatic clearance of L P L (Chevreuil et al 1993). In addition to heparin, LPL can also be displaced from its HSPG binding sites by physiological factors like antithrombin III, a-thrombin and its inactive derivative, DIP-a-thrombin (Chajek-Shaul et al 1990). LPL that is bound to HSPGs has a higher K m but lower V m a x than LPL in solution. In other words, LPL in solution is more efficient as a lipolytic enzyme as compared to the endothelium-bound counterpart. This is probably due to a better accessibility of the enzyme for its substrate (e.g. V L D L , CHYL) and proposed to be an additional mechanism by which HSPG binding could regulate L P L action (de Man et al 1997). 25 Glycosylphosphatidylinositol (GPI) is another binding site identified on the cell surface for LPL. GPI binding sites can be selectively cleaved by phsophatidylinositol (PI) specific phospholipase C or D (PLC or PLD). PLC is activated by insulin and therefore, incubation of 3T3-L1 adipocytes with insulin resulted in a PLC mediated release of LPL from GPI binding sites on the surface (Eckel et al 1978, Spooner et al 1979, and Chan et al 1988). In addition to insulin, agents like glimepiride (a sulfonylurea) also induced release of LPL anchored to GPI from rat adipocytes via activation of GPI specific phospholipase (GPI-PL). Glucose free medium completely abolished the stimulatory effect of insulin as well as glimepiride on GPI-PL and therefore on LPL release (Muller et al 1994). This set of data indicates that increased glucose transport rather than insulin stimulates GPI-PL and release of L P L from rat adipocytes. Bruin et al (1994) have questioned the above possibility by showing that the C-terminal domain of L P L does not have an addition signal for GPI anchoring. l4C-ethanolamine, a component of the GPI anchor, was not incorporated into LPL molecules by metabolic labeling, suggesting that L P L is not a GPI anchoring protein (Bruin et al 1994). Other binding sites for LPL include low density lipoprotein receptor related protein (LRP), a glycoprotein (gp330), a heparin releasable protein (hrp-116), and a novel 85-kDa glycoprotein (Wolle et al 1995). Physiological importance of these non-HSPG LPL binding sites is not yet known. Similarly, another putative binding of LPL with unknown physiology has been shown to occur with oc2-macroglobulin (0C2-M) in the plasma. a2-M is a broad spectrum protease inhibitor known to bind various basic proteins like nerve growth factor, basic fibroblast growth factor, cationic aspartate aminotransferase, myelin basic protein and eosinophil cationic protein. LPL, being a basic protein, also binds to CC2-M in a non-covalent manner via its heparin binding domain and thus loses its ability to bind to cell surface HSPGs. Whether this binding of L P L to 0C2-M plays any physiological role is not yet known (Vilella et al 1994, Neilsen et al 1997). 26 1.5.3.3. LPL turnover by intracellular degradation/recycling: L P L turnover by intracellular degradation is an important post-translational mechanism by which L P L activity is rapidly regulated during conditions like feeding and fasting (Lee et al 1998). About 80% of newly synthesized LPL are degraded before being secreted. This turnover by intracellular degradation in adipocytes is a very rapid process with a ti/2 of about 40 minutes. Degradation could occur either in a leupeptin inhibitable, lysosomal compartment or a tunicamycin inhibitable, ER compartment (Braun and Severson 1992). Surface bound L P L also undergoes degradation after being internalized. Adipocytes degrade only a fraction (<28%) of total surface bound L P L and this process involves internalization of the enzyme and a subsequent degradation in lysosomal compartment (Obunike et al 1996). HSPGs mediate this process of L P L turnover by facilitating internalization of LPL by various cells including adipocytes and hepatocytes (Obunike et al 1996, Hoogewerf et al 1991, and Sehayek et al 1995). Berryman and Bensadoun (1995) demonstrated that HSPG binding sites are essential for the internalization and degradation of the enzyme by cultured CHO-K1 cells. The degree of sulfation of HSPGs can alter LPL binding. Hoogewerf et al (1991) demonstrated that a decrease in sulfation of HSPGs on the adipocytes decreased the maximum binding of LPL to the cell surface. This decreased binding of LPL to HSPGs also resulted in less degradation of the enzyme as a result of decreased internalization. Similar decreases in internalization and degradation can be achieved by releasing the surface bound L P L by heparin. Release of surface bound LPL by heparin stimulates secretion of the enzyme from within cells. In adipocytes, this secretory process diverts the newly synthesized LPL away from lysosomal compartment and thereby significantly reduces intracellular degradation. In cardiac myocytes, release of surface bound LPL does not alter the amount of intracellular degradation of LPL but in compensation, intracellular degradation of L P L in cardiac myocytes is slow (Liu and Olivecrona 1992). In addition to HSPGs, receptor-27 associated protein-sensitive LRP binding sites also mediate this LPL turnover process, however, it contributes less compared to proteoglycan binding sites (Obunike et al 1996). Unlike adipocytes and cardiac myocytes, endothelial cells degrade LPL to a very small extent. Using in vitro cell cultures, endothelial cells were in fact shown to recycle the surface bound L P L in its active dimeric form (Saxena et al 1990). Involvement of HSPG binding sites is once again emphasized. The probable recycling mechanism involves internalization of HSPG-L P L complexes into an endocytotic compartment. The acidic pH in the endocytotic vesicles favors HSPG-LPL binding, thereby enabling the complex to be released back into the medium or inserted onto the cell surface. However, the following issues have yet to be clarified to substantiate the above mechanisms. Are HSPG/HS-oligosaccharides being internalized with L P L during the recycling process? If it is undegraded HSPG, then is the HSPG-LPL complex inserted back into the cell surface? 1.5.3.4. Regulation of LPL by substrates Physiological substrates like V L D L and C H Y L and lipolytic products like FFA also regulate L P L activity. Apolipoproteins present on TGRLs such as V L D L and C H Y L influence their interaction with LPL. For example, binding of apolipoprotein CII (apoCII) present on the TGRL surface to L P L enhances lipolysis while, binding of apoCIII inhibits LPL activity (Olivecrona and Bengtsson-Olivecrona 1993). Recently, apoE, another major apolipoprotein of TGRLs, has been shown to inhibit LPL activity (Jong et al 1997a). LPL activity is also regulated by the substrates via a feed-back mechanism. For example, FFA released from the TGs by the lipolytic action of LPL can bind to LPL and competitively inhibit LPL activity (Bengtsson-Olivecrona and Olivecrona 1980). However, albumin can prevent this feed-back inhibition due to its higher affinity for FFAs (Bengtsson-Olivecrona and Olivecrona 1980). Another 28 mechanism by which LPL activity can be inhibited by its substrates is by displacement of LPL from its binding sites (Saxena and Goldberg 1990). LPL bound to endothelial cells can be displaced from its binding sites by TGRLs and oleic acid. In vitro experiments demonstrate that FFA binding interferes with the LPL-heparin interaction. In addition, FFA also affects LPL binding to its activator apolipoprotein CII (Saxena and Goldberg 1990). It is proposed that when more FFA than the tissue can utilize is released by LPL, FFA would probably bind to L P L and displace it from its binding sites, thereby regulating the enzyme activity via a negative feed-back mechanism. The above results generated by cell culture experiments were questioned due to the fact that no L P L was released when FFAs were perfused through isolated beating hearts from rats (Rodrigues et al 1992). Alternatively, incubation of cardiac myocytes with oleic acid resulted in a decreased L P L activity, not due to release of the enzyme from its binding sites but due to alterations in intracellular processing and secretion of the enzyme (Anderson et al 1997). 1.6. L P L regu la t ion by va r ious phys io log ica l factors 1.6.1. Fasting and Feeding Fasting increases LPL activity in muscles (skeletal and cardiac) but decreases the enzyme activity in adipose tissues in different animal models such as rats, guinea pigs and hamsters. Feeding has opposite effects in these tissues (Robinson 1960, Salaman and Robinson 1966, Robinson and Jennings 1965, Pedersen and Schotz 1980). Regulation at post-transcriptional and post-translational levels has been suggested. Decrease in rat adipose L P L due to short term fasting was restored within 4 hours of refeeding. However, long term fasting (36 hr, 60 hr, and 3-5 days) took several days of refeeding before LPL levels were restored (Bergo et al 1996a). It was shown that short-term fasting, similar to diurnal rhythms did not affect LPL mRNA levels (Bergo 1996a) and was in fact due to an increase in the proportion of inactive form of LPL in 29 adipose tissues (Bergo 1996b). In addition, the short term fasting period was also shown to increase degradation of newly synthesized LPL and markedly decrease secretion of LPL into the medium (Lee et al 1998). Thus, the regulation during short term fasting was at a post-translational level. Prolonged fasting (>24 hours) on the other hand, resulted in a decreased level of mRNA and thus required transcriptional regulation for the restoration of LPL activity in rat adipose tissues (Bergo et al 1996a). Recent studies have further confirmed that this differential level of L P L regulation in rat adipose tissue is associated with the duration of fasting (Lee et al 1998). Fasting and feeding respectively increased and decreased heparin releasable fraction of heart LPL in rats. There was no change in LPL activity in soleus or extensor digitorum longus (EDL) in response to short term fasting or feeding. However, L P L activity and mRNA in skeletal muscle increased only after a 6-day fast. It was concluded that heart LPL is more sensitive to fasting/feeding than is the skeletal muscle (Ong et al 1994, Ladu et al 1991). The increase in HR-LPL in the heart is also attributed to increased uptake of LPL from the blood (Olivecrona et 125 al 1995). Although plasma LPL level was not measured in this study, uptake of exogenous I-LPL was shown to be increased in the heart during fasting. In the mouse myocardium, immunocytochemical studies revealed that fasting induced a 5-fold increase in LPL at the luminal projections of endothelium (Blanchette-Mackie et al 1989). When pulsed with 3 5 S -methionine (which incorporates into newly synthesized LPL molecule) and chased with non-radioactive methionine, guinea pig hearts exhibited no difference in the rate of transport of newly synthesized [ 3 5S]-LPL during fed or fasted states (Liu and Olivecrona 1992). The above studies in general support that changes in heart LPL during fasting and feeding are controlled at the posttranslational level (Ong et al 1994, Ladu et al 1991, and Doolittle et al 1990). 30 In humans, studies were mainly done in adipose tissues and skeletal muscle. Adipose LPL decreases in humans similar to animal models during fasting, but skeletal muscle exhibits either no change or increase in LPL activity depending on the duration of fasting (Enerback and Gimble 1993). LPL regulation in obese subjects is different in that LPL activity decreased both in adipose tissue as well as in skeletal muscle as a result of fasting. Weight reduction in obese individuals caused adipose LPL to remain elevated during fasting suggesting that the metabolic set point is primed for continued weight gain in these individuals (Schwartz and Brunzell 1981, Eckel and Yost 1987). These alterations in LPL activity are however not accompanied by any change in mRNA or immunoreactive mass of LPL. Thus it is proposed that fasting/feeding induced changes in LPL is due to posttranslational mechanisms. 1.6.2. Hormonal regulation of LPL Various hormones like growth hormone, thyroid hormone, insulin, glucagon, catecholamines, prolactin, and steroids regulate LPL activity in a tissue specific manner. The effects of insulin on LPL will be discussed under the "diabetes and LPL" section. 1.6.2.1. Growth hormone Growth hormone (GH) induces LPL gene transcription, increases mRNA stability and LPL activity in preadipocytes in vitro (Enerback and Gimble 1993). Overexpression of GH receptor in rat liver cells increased the ability of the cells to augment LPL expression in response to GH. GH-mediated LPL regulation in adipocytes has been shown to occur at the level of gene transcription (Enerback and Gimble 1993). A proto-oncogene c-fos is shown to play an intermediary role in LPL gene expression during GH stimulation of pre-adipocyte differentiation (Barcellini-Couget et al 1993). This c-fos mediated regulation is however inhibited by the rise in 31 intracellular C a 2 + indicating that the inability of pre-adipocytes to mobilize C a 2 + in response to G H could be a pre-requisite for the maximal expression of LPL (Barcellini-Couget et al 1994). G H increases LPL activity in the heart and skeletal muscles with a parallel increase in post-heparin plasma. These effects are not due to changes in LPL mRNA and also are not mediated by insulin or IGF-I (Oscarsson et al 1999). Hence, GH probably regulates L P L in the muscle tissues at a post-transcriptional level. 1.6.2.2. Thyroid hormone Effect of thyroid hormone on LPL activity is reciprocally regulated in human and rat adipose and muscle tissues (Ong et al 1994). Hypothyroidism stimulates L P L activity in brown and white adipose tissues and as well as in cardiac and skeletal muscles. These changes in LPL activity are not due to changes in mRNA levels and thus the regulation is at a post-transcriptional level (Enerback and Gimble 1993). It appears that an unknown translation repressor factor that binds to 3'UTR and inhibits LPL translation in normal adipocytes might be decreased under hypothyroid conditions (Kern et al 1996). This decrease in repressive trans-acting factor could then lead to a disinhibition of LPL translation in hypothyroid-adipocytes (Kern et al 1996). In a recent study, it was found that the increase in muscle LPL activity mainly occurs in heparin releasable fraction in hypothyroid rats (Ong et al 1994). The exact mechanism of this posttranslational regulation is not clear and suggested to be due to nutritional status of the animals. It is to be noted that hypothyroidism also obliterated the inverse relationship between muscle and adipose L P L regulation. 32 1.6.2.3. Catecholamines Catecholamines decrease LPL activity in white adipose tissue (WAT, stores lipids) but increase the activity in brown adipose tissues (BAT, involved in thermogenesis). The effect of cold exposure on B A T - L P L activity is in part mediated by the P-adrenergic system (Enerback and Gimble 1993). Surprisingly, sympathetic denervation did not prevent the cold induced increase of LPL activity (Klingenspor et al 1996). Although the reason for this is not clear, it is possible that circulating catecholamines in the plasma of cold-exposed hamsters could have caused the increase in BAT-LPL. In mice, cold exposure and in vivo noradrenaline treatment caused a similar increase in LPL mRNA and enzyme activity in B A T (Kuusela et al 1997a). This increase in B A T - L P L was found to be via a pV adrenergic receptor (Kuusela et al 1997b). a-Adrenergic stimulation on the other hand increased LPL activity in W A T but decreases it in B A T (Desfaits et al 1995). Selective oc-blockade with doxazosin decreased W A T but increased B A T -L P L activity. The effect of catecholamines on cardiac LPL regulation is not consistent. L P L activity is either increased or not changed by isoproterenol treatment (Stam and Hulsmann 1984, Friedman et al 1986, Deshaies et al 1993). LPL changes in response to adrenergic agents is through a cAMP second messenger system. Although, increases in cAMP (by cholera toxin) produced an increase in total L P L activity in heart tissue homogenates, isolated cardiac myocytes did not show any increase in LPL protein or activity (Carroll et al 1990). Further, non-selective P-blockers like propranolol did not decrease activity or mRNA level of L P L in rats (Gouni-Berthold et al 1997). However, Friedman et al (1992) showed that P-agonists and cAMP-analogs increased LPL mRNA and activity in cultured cardiac mesenchymal cells. A selective p 2 -adrenergic agonist like clenbuterol decreased heart LPL in rats (Belhasen and Deshaies 1992). In 33 humans, catecholamines did not have an effect on adipose LPL activity but stimulated skeletal muscle L P L activity and specific mRNA levels (Eckel et al 1996). Therefore, the inconsistencies in the literature could be partly due to non-specific activation or inhibition of the adrenergic system, differences in cell types in which L P L was measured and species differences. /. 6.2.4. Other Factors In the fed state in rats, steroids such as glucocorticoids stimulate LPL activity in muscle but decrease the activity in adipose tissues in rats (Enerback and Gimble 1993). Recently, it was shown that the glucocorticoid, dexamethasone, had no effect in isolated cardiac myocytes but stimulated both the HR-LPL and cellular LPL activity in combination with insulin (Ewart et al 1997). Therefore, the in vivo effects of glucocorticoids under fed conditions may be due to the presence of elevated levels of plasma insulin. Glucagon, a counter-regulatory hormone of insulin, elevated cardiac L P L activity in the fed state and prevented the decline in LPL activity during re-feeding (Jansen et al 1980, Borensztajn and Rubenstein 1973). This increase in cardiac L P L activity mainly occurs in the HR-LPL fraction (Stam and Hulsmann 1984) and this change in HR-LPL occurs within 3 minutes of glucagon in the perfused heart (Simpson 1979). This rapid action of glucagon is suggested to modify the enzyme activity at a posttranslational level. Adipose L P L activity was not affected by glucagon either during re-feeding or in the fed state (Borensztajn and Rubenstein 1973). It is proposed that these in vitro effects of glucagon, catecholamines and steroids could possibly contribute to the alterations of LPL in a tissue specific manner during stress, and other pathophysiological conditions. 34 Prolactin, a pituitary hormone that is responsible for lactation during pregnancy, also induces L P L enzyme activity and pre-adipocyte differentiation. In vivo, during the immediate 48 hour pre-partum, L P L enzyme activity rises 100 fold in mammary tissue and remained high throughout the suckling period (Enerback and Gimble 1993). Adipose LPL decreases during this period possibly to divert TG to the lactating breast (Eckel 1989). Although the mechanism of this prolactin mediated LPL regulation is not clearly understood, recent evidence on cultured mammary tissue from pregnant mice shows an increase in LPL-mRNA along with HR-LPL activity in response to prolactin incubation (Hang and Rillema 1997). Whether this increase in mRNA is due to increased transcription or increased stability is not known. Cytokines influence the metabolism of lipids, glucose and protein, in addition to mediating immune and inflammatory responses. One of the mechanisms by which cytokines regulate lipid metabolism is via regulating LPL activity (Memon et al 1995). Several pro-inflammatory cytokines like tumor necrosis factor (TNF-oc), interleukins 1 and 6, interferon y and, interleukin-11 or adipogenesis inhibitory factor decreased LPL activity in adipose tissues by inhibiting LPL synthesis and therefore control LPL action at translational level of the enzyme (Enerback and Gimble 1993, Oshumi et al 1994). The effect of these cytokines on the muscle tissues is variable. The above cytokines also suppressed L P L activity either additively or synergistically in murine macrophage J774.2 cell lines. This regulation of macrophage LPL activity by cytokines is believed to play a potential role in the pathogenesis of atherosclerosis (Tengku-Mohammad et al 1998). 1.7. L P L d u r i n g Pa tho log ica l condi t ions Several pathological conditions like type I hyperlipidemia, obesity, hypothyroidism, renal diseases, atherosclerosis, hypertension, and diabetes are associated with alterations in LPL 35 activity. For the purpose of this review, I will focus on LPL regulation during hypertension and diabetes while other conditions are discussed only briefly. Type 1 hypertriglyceridemia is a disease directly associated with L P L deficiency due to an autosomal recessive disorder. Some of the symptoms of this disease include recurrent abdominal pain, pancreatitis, hepatosplenomegaly, eruptive xanthomas, and lipemia retinalis and low levels of HDL. This disease is also sometimes associated with a deficiency in apoCII, the activator of LPL. Restriction of dietary fat and the resultant decrease in plasma chylomicrons tend to subsidize the clinical manifestations of type I hyperlipidemia (Eckel 1989). Hypertriglyceridemia that develops secondary to renal failure and nephrotic syndrome is correlated to an increased production of V L D L and also to a decreased LPL activity. Genetic of dietary obesity, is clearly associated with increased LPL activity in adipose tissues. This increased LPL activity in adipose tissue is responsible for increased adipose cell size, mass and resultant obesity. In obese patients, adipose LPL activity is maintained at a higher level even during fasting and responsiveness of adipose LPL to insulin or glucose is blunted. However, during a weight loss program, the responsiveness of adipose LPL to insulin and glucose increases markedly in obese patients. This probably enhances the lipid filling capacity of adipose tissues and therefore leads to a return of obesity (Eckel 1989). The role of L P L in the pathogenesis of atherosclerosis was shown to be critical by several pieces of evidence (Goldberg 1996). LPL deficiency leading to low levels of serum HDL can increase the risk of atherogenesis. In addition, recent evidence suggests that LPL could directly play a role in internalizing atherogenic lipoproteins in the blood vessels through a receptor independent mechanism. LPL on the endothelium acts as a ligand for various lipoprotein particles like L D L , V L D L - and, C H Y L -remnants and retains them on the blood vessels to be internalized later (Williams et al 1992a). The products of lipolysis of TGRLs (VLDL and CHYL) by LPL like FFAs, lysolecithins, and 36 lysophosphatidylcholine are involved in endothelial damage, one of the initial steps in the development of atherosclerotic plaques (Rutledge et al 1997). LPL present in macrophages is also shown to be up-regulated during the process of atherogenesis and this is proposed to play an important role in the formation of foam cells. Collectively, all these studies emphasize the potential role of L P L in atherosclerosis. 1.7.1. LPL in Hypertension According to one World Health Organization (WHO) report, hypertension is defined by a systolic blood pressure of > 140 mmHg and a diastolic blood pressure of >90 mmHg, after several repeated measurements (Chalmers and Zanchetti 1996). Hypertension is classified broadly into essential or primary hypertension (the etiology of which is still unknown) and secondary hypertension (secondary to various pathological conditions like diabetes). Essential hypertension is strongly correlated to defects in carbohydrate and lipid metabolism (Reaven 1991a, 1991b, Bonner 1994). The connection between hypertension and metabolic disorders appears to be primarily due to insulin resistance and hyperinsulinemia (Pollare et al 1990, Rao 1993, Sowers et al 1994, Kahn and Song 1995). Patients with essential hypertension have been observed to display a resistance to insulin-induced glucose disposal (Ferrannini et al 1987, Reaven 1990) and a resultant hyperinsulinemia (Welborn et al 1966). Another school of thought supports the fact that altered vascular reactivity of resistance vessels for insulin during insulin resistance results in vasoconstriction and therefore hypertension (Laakso et al 1990 & 1992, Lembo et al 1993 & 1995). In addition, increased activity of the sympathetic nervous system (Rowe et al 1981, Landsberg and Drieger 1989) and Na+/water retention (DeFronzo et al 1975) during the hyperinsulinemic state could also contribute to hypertension. Therefore, whether insulin resistance and hyperinsulinemia are causes or effects of hypertension is still debatable. To 37 further muddle the issue, there are several studies which deny a relationship between insulin and hypertension (Brands et al 1995, Buchanan et al 1992, Tsutsu et al 1989). It has been suggested that the coexistence of hypertension and hypertriglyceridemia is not incidental, and these two conditions may be causally related to each other (Reaven 1988, Reaven 1991a&b, Ferrannini and Natali 1991, Kotchen et al 1991, Reaven and Chang 1991). Although the mechanisms are not obvious, there are studies to support the notion that hypertriglyceridemia in these circumstances could be the result of defective clearance of VLDL and CHYL. Indeed, a decreased clearance of exogenously administered lipid emulsion has been reported in hypertensive patients and rats (Pasanisi et al 1988, Mackintosh et al 1991, Lind and Lithell 1993) possibly as a consequence of poor perfusion of capillary beds where there is an abundance of LPL. Vascular hypertrophy, and rarefaction of blood vessels as a result of increased sympathetic tone could account for the poor peripheral perfusion (Lind and Lithell 1993). Interestingly, vasodilation by vasodilators like neifedipine in hypertensive patients increased LPL activity in post-heparin plasma (Pasanisi et al 1988, Lind and Lithell 1993, Marotta et al 1995). LPL activity is also decreased in skeletal muscle and adipose tissue in hypertensive patients (Mackintosh et al 1991, Pasanisi et al 1988), Dahl salt-sensitive hypertensive rats (Mondon et al 1993, Lind and Lithell 1993) and in stroke prone SHR (Ogawa et al 1991). Of particular interest, a large body of evidence reveals a genetic correlation between LPL and hypertension. For example, in a group of Taiwanese type 2 diabetic patients, a definite linkage for systolic blood pressure was located to a region at or near the LPL locus on the short arm of chromosome 8p22 (Wu et al 1996). Familial dyslipidaemic hypertension (FDH), is a hypertensive disease that involves heterozygous LPL deficiency (Williams et al 1992b). DNA markers of lipid abnormalities or hypertension include LPL deficiency in addition to numerous other factors (Williams etal 1993). 38 Very few studies on cardiac LPL activity during hypertension have been reported. Mondon et al (1993) showed that total LPL activity in heart tissue homogenates was decreased in Dahl salt sensitive hypertensive rats as compared to normotensive controls. In SHR, LPL mRNA in the heart progressively decreased with the development of hypertrophy, and a parallel decrease in V L D L receptor levels was also observed in these hearts (Masuzaki et al 1996). Whether this decrease in LPL-mRNA level is a result of decreased LPL gene transcription or stability of the mRNA has yet to be determined. Therefore, it appears that hypertension/hypertrophy regulates L P L at transcriptional or posttranscriptional level. Since LPL activity was not measured, it is not known whether the decreased mRNA resulted in a corresponding decrease in LPL activity. Additionally, none of the above studies have measured L P L in the heparin releasable/functional compartment. 1.7.2 LPL in Diabetes 1.7.2.1. Diabetes Mellitus (An Overview): As defined by the recent expert committee on the diagnosis and classification of diabetes mellitus (Report of the expert committee 1997), "diabetes mellitus is defined as a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both." The acute clinical manifestations include polydipsia, polyuria, weight loss, polyphagia, and fatigue. Uncontrolled hyperglycemia may progress to life threatening diabetic ketoacidosis and coma. The chronic or secondary complications of diabetes include retinopathy, peripheral and autonomic neuropathy, nephropathy, and cardiovascular and cerebrovascular diseases like micro- and macro-angiopathy, atherosclerosis, cardiomyopathy, etc. Diabetes is classified based on clinical conditions and etiology into two major types: (1) type 1 diabetes, characterized by an absolute deficiency of insulin secretion and therefore an absolute 39 requirement for exogenous insulin; (2) type 2 diabetes, caused by a combination of resistance to insulin action and an insufficient compensatory insulin secretory response. Type 1 patients often exhibit serological evidence of an autoimmune destruction of pancreatic islets and genetic markers of the disease. Type 2 diabetics are usually asymptomatic and often diagnosed by fasting hyperglycemia and by oral glucose tolerance tests (Report of the expert committee 1997). Type 1 diabetes is further divided into immune-mediated diabetes and idiopathic diabetes. Several autoantibodies including islet cell autoantibodies (ICAs), insulin autoantibody (IAAs), autoantibodies to glutamic acid decarboxylase (GAD65), and tyrosine phosphatase autoantibodies (IA-2 and IA-2(3) have been identified in the serum. About 85-90% of the individuals with fasting hyperglycemia were detected with one or more of these autoantibodies (Report of the expert committee 1997). The genetic component of this type 1 diabetes is strongly associated with major histocompatibility complex (MHC) molecules which in humans are known as human leukocyte-associated antigens (HLA). Recent report suggest that genes like DQA and B with the influence of DRB are involved in type 1 diabetes. DR/DQ alleles of H L A can either predispose or protect an individual to or from diabetes (Report of the expert committee 1997). In addition to genetic predisposition, environmental factors may also play a role in the pathogenesis of type 1 diabetes. Idiopathic diabetes, on the other hand has no known etiology; evidence of autoimmunity, nor any H L A associations or strong familial inheritance is encountered with these patients. • Idiopathic diabetics often present with permanent insulinopenia, susceptibility to ketoacidosis and an absolute requirement for insulin replacement therapy (Report of the expert committee 1997). Type 2 diabetics, previously referred to as non-insulin-dependent or adult onset diabetics, have insulin resistance and usually have relative insulin deficiency (Report of the expert 40 committee 1997). The specific etiology of this disease is not known, neither autoimmune destruction nor any other causes mentioned in type 1 contribute to the pathogenesis. Obesity and /or abdominal body fat are strongly correlated to type 2 diabetes. Although the genetics of type 2 diabetes is more complex, a strong genetic predisposition is observed in type 2, more so than is the type 1 diabetes. The risk of developing type 2 diabetes increases with age, sedentary life style and obesity. The development of hyperglycemia is very gradual and slow and often with no noticeable symptoms of diabetes in the early stages. Ketoacidosis seldom occurs in type 2 and the individuals may not require insulin at least in initial stages and often throughout their lifetime. Insulin resistance is often associated with obesity and therefore can be improved by weight reduction and through pharmacological interventions. Diet and exercise is nevertheless the first line of therapy for type 2 diabetes, followed by oral anti-diabetic drugs, and insulin replacement as a last resort (Report of the expert committee 1997). Experimental models of diabetes include those induced chemically (by agents like streptozotocin (STZ), and alloxan), as well as genetic models (e.g. non-obese diabetic (NOD) mouse, Zucker diabetic fatty (ZDF) rats, BioBreeding (BB) diabetic rats, etc.). STZ is more commonly used to induce diabetes in laboratory animals due to its greater selectivity for pancreatic (3-cells, longer half-life in the body (15 min) and a lesser mortality rate of diabetic animals when compared to alloxan (Rodrigues et al 1999). STZ [2-deoxy-2-(3-methyl-3-nitrosourea) 1-D-glucopyranose] is a broad spectrum glycoside antibiotic secreted by Streptomyces achromogenes. The glucose moiety with a highly reactive nitrosourea side chain is suggested to produce selective (3-cell cytotoxicity. Although a stereospecific membrane receptor that closely resembles Glut-2 is said to be involved in STZ-|3-cell selectivity, the exact mechanism(s) is not known. At the intracellular level, STZ is believed to cause several effects: (1) N A D + depletion due to activation of poly ADP-ribose synthetase as a cell repair tool in 41 response to D N A damage by reactive methyl carbonium ions generated by STZ; (2) free radical generation and a resultant oxidative stress by STZ; and (3) nitric oxide generation from STZ, all possibly contributing to its cytotoxicity. Recently, it was hypothesized that STZ might generate peroxynitrite through the production of superoxide. The peroxynitrite would then dissociate into NO2 and hydroxy radicals which are involved in D N A damage and apoptosis of p-cells (Rodrigues et al 1999). Selective P-cell toxicity of STZ is explained by several possible mechanisms including: (1) high affinity sites for STZ on P-cell membranes, (2) unique thiol groups on the P-cell membrane that make these cells more susceptible to oxidative interactions, (3) less free-radical scavenging ability of P-cells, and (4) a low ratio of N A D + / D N A in islets compared to other tissues (Rodrigues et al 1999). However, non-selective cytotoxic effects of STZ on other cells cannot be excluded. The severity of p-cell destruction and diabetic condition is dependent on the dose of STZ used. In rats, for example, a 25 mg/kg STZ dose resulted in no change in serum glucose, insulin levels or pancreatic insulin content. At 65 mg/kg, STZ produces a moderate and a stable diabetes within 48 hours which is characterized by overt hyperglycemia, hyperlipidemia, loss of weight gain and a 50% decrease in serum insulin levels. Pancreatic insulin content drops down to less than 5% of control. High dose of STZ, in the range 100 mg/kg, produces absolute insulin deficiency due to more than 95% P-cell destruction and a severe hyperglycemia. These animals die of diabetic ketoacidosis i f not supplemented with exogenous insulin (Rodrigues et al 1999). 1.7.2.2. LPL regulation during diabetes L P L regulation during diabetes has been well documented and is known to occur in a tissue specific manner. The following sections review tissue specific L P L regulation in adipose tissue, skeletal muscle, cardiac muscle and post-heparin plasma, during diabetes. 42 Adipose LPL: Adipose LPL activity has been shown to be unequivocally decreased both at the functional level and at the site of synthesis during diabetes (O'Looney and Vahouny 1987). This decrease in adipose LPL was partly due to decreased irrrmunoreactive LPL protein and steady state mRNA levels but primarily as a result of decreased catalytic activity (Tavangar et al 1992). The decrease in LPL activity was reversed by insulin treatment. In vitro studies have demonstrated a direct effect of insulin on adipose LPL activity (Braun and Severson 1992). Insulin increased cellular LPL activity, rate of LPL synthesis, LPL mRNA levels and HR-LPL activity on the cell surface of adipocytes and thus regulates adipose L P L activity at transcriptional, post-transcriptional, and post-translational levels. These in vitro studies are further complemented by several in vivo experiments which demonstrate that increases in plasma insulin levels (as in euglycemic, hyperinsulinemic clamps) augmented adipose L P L activity (Enerback and Gimble 1993). In STZ-diabetic rats and non-diabetic controls, high caloric intake increased plasma insulin levels with a parallel increase in adipose LPL activity in both groups (Inadera et al 1992). This increase in LPL activity was accompanied by increases in LPL specific mRNA levels. The decreased adipose LPL in diabetic patients (both type 1 and type 2) was improved by insulin or oral hypoglycemic therapy (Simsolo et al 1992). This improvement in L P L activity was due to both an increased rate of L P L synthesis and a decreased degradation of the enzyme. Since there was no change in LPL-mRNA levels due to insulin treatment, the regulation was suggested to be at a post-translational level (Simsolo et al 1992). The insulin resistance that occurs in obesity blunts the adipose LPL stimulation in response to insulin (Eckel et al 1995). This response was even more diminished in type 2 diabetics (Eckel et al 1995). Weight reduction in obesity or exercise in type 2 diabetics increased insulin sensitivity with a parallel increase in the responsiveness of adipose L P L to insulin suggesting that the stimulatory effect of insulin on adipose LPL depends on insulin sensitivity. 43 The greater the insulin sensitivity, the better the response of adipose L P L and a threshold level of insulin sensitivity controls the enzyme response in adipose tissue (Eckel et al 1995). In this regard, it was also shown that the more severe the insulin resistance, the lower is the adipose LPL mRNA level (Maheux et al 1997). This was attributed to the inability of insulin either to stimulate transcription or to increase the intracellular LPL mRNA stability in adipose tissue in insulin resistant individuals (Maheux et al 1997). Contrary to all of the above evidence, insulin resistance and hyperinsulinemia induced by chronic high-fat feeding actually exaggerated the insulin response in adipose and muscle tissues (Boivin et al 1994). This discrepancy is probably due to differences in the severity and duration of high fat induced insulin resistance and hyperinsulinemia. Skeletal muscle LPL: Skeletal muscle LPL activity during type 1 diabetes was found to be either decreased, not changed, or increased, depending on type of skeletal muscle and the degree and duration of diabetes studied (O'Looney and Vahouny 1987). Under normal physiology of fasting/feeding cycle, insulin negatively regulates skeletal muscle L P L whereas during insulin resistance, as seen in obesity, skeletal muscle LPL is increased by insulin (Eckel et al 1995). When type 2 diabetes was present together with obesity, the severe insulin resistance that occurs did not increase the LPL activity in the skeletal muscle (Eckel et al 1995). Indeed, in type 2 diabetic patients infusion of insulin/glucose led to a blunted response on skeletal muscle LPL activity suggesting that neither plasma insulin nor glucose play an important role in the regulation of skeletal muscle LPL activity (Yost et al 1995). Microalbuminurea, another diabetic complication, by its damaging effects on endothelial cells decreases functional LPL in type 2 diabetics (Kashiwazaki et al 1998). Albuminuria was associated with defects in HSPGs in extra-cellular matrix (ECM) and therefore type 1 diabetic patients with albuminuria were suggested to have decreased vascular LPL activity leading to hypertriglyceridemia (Hansen et al 1997). 44 However, skeletal muscle LPL activity in diabetic patients with or without albuminuria did not differ although both groups had lower LPL activity as compared to healthy individuals. The elevated plasma TG levels were correlated to this decreased skeletal muscle. LPL activity in both the diabetic groups (Hansen et al 1997). Cardiac LPL: Cardiac LPL regulation during diabetes is mainly studied in experimental animal models. In spite of a plethora of literature on diabetes and its effect on cardiac LPL, results are inconclusive due to high variability. For example, studies on cardiac L P L regulation in alloxan/STZ-diabetes showed that both heparin-releasable, and total L P L activities (as determined in whole heart homogenate) were elevated during diabetes (Kessler 1963, Nomura et al 1984). Insulin treatment reversed the diabetic conditions as well as fractional and total LPL activities in the heart to normal levels. Tavangar et al (1992) demonstrated that the elevated levels of heart LPL in STZ-diabetic rats was accompanied by an increase in immunoreactive LPL protein but with decreased LPL-mRNA levels. Therefore, the elevated level of LPL activity was not due to enhanced translation of the protein but was probably due to the depressed rate of LPL turnover (degradation) in the diabetic heart. Contrary to these studies, isolated hearts from diabetic animals expressed a decreased functional LPL activity, which was reflected by a decreased lipolysis of perfused V L D L - T G (O'Looney et al 1983). These alterations were normalized by in vivo as well as in vitro insulin treatments. Further, the effects of insulin could be inhibited by a protein synthesis inhibitor, cycloheximide (O'Looney et al 1983). The latter set of experiments supports the idea that the decrease in functional LPL in the diabetic heart could be due to a decreased synthesis of the enzyme and that insulin can directly stimulate LPL synthesis in the heart. It is to be noted that the in vitro effects of insulin either in perfused hearts or isolated cardiac myocyte culture could not be reproduced by several subsequent studies (Braun and Severson 1991, 1992, Rodrigues et al 1992b, Ewart et al 1997, Ewart et al 1999). 45 Nevertheless, in vivo insulin administration did stimulate LPL activity in the diabetic heart, which led to a conclusion that insulin requires a second factor to regulate cardiac LPL activity. In this regard, Ewart et al (1997, 1999) using cardiac myocytes from control and diabetic rats demonstrated that incubation of cells with insulin and dexamethasone (a glucocorticoid) together stimulated LPL activity. Neither insulin nor glucocorticoid by themselves had any effect on myocyte LPL. Therefore, it appears that insulin regulates heart LPL in vivo in the presence of another factor (like glucocorticoid). Liu and Severson (1994) further confirmed that functional (heparin-releasable) L P L from perfused hearts as well as myocyte-associated LPL (non-functional) activities were depressed in diabetic rats. This decrease in cardiac L P L was neither accompanied by changes in steady state LPL mRNA levels and stability nor by a decrease in relative rates of L P L synthesis and turnover (degradation) (Carroll et al 1995). Also, the diabetic heart did not exhibit any changes in LPL-specific HSPG binding sites (Liu and Severson 1996). The decrease in LPL activity was therefore attributed to an absolute decrease in L P L synthesis and aberrations in post-translational mechanisms resulting in accumulation of inactive enzyme (Carroll etal 1995). Evidence supporting the idea that there is no change in heart LPL activity during diabetes has also been noted in the literature. LPL activity in the hearts of STZ-diabetic rats was not affected either by calorie intake or by the diabetic state per se (Inadera et al 1992). In summary, diabetes produced variable regulation of LPL in the heart. The disparity in the literature can be partly explained by differences in chemical agent used to induce diabetes (alloxan/STZ), dose of STZ, duration of diabetes, differences in animal models used (species and strain differences), and the techniques used to measure LPL activity (heart tissue homogenate versus isolated heart perfusion, acetone-ether extracts versus unextracted tissues, etc.) (O'Looney and Vahouny 1987). 46 Post-heparin plasma lipolytic activity (PHPLA): Diabetes is usually accompanied by hypertriglyceridemia in addition to elevated glucose levels. High plasma TG levels could be due either to a decreased clearance or increased secretion of TGRLs or both (Wilson et al 1987, Hirano et al 1991, Mamo et al 1992, Staprans et al 1992, Duerden and Gibbons 1993, Garg 1994, Yoshino et al 1996). The decreased clearance of TGRL from blood could partly be due to decreased LPL activity (Braun and Severson 1992). PHPLA is an index of overall L P L activity contributed by various tissues like, skeletal and cardiac muscles, adipose tissues, etc. PHPLA was determined to be low in diabetic patients (Taskinen 1987, O'Looney and Vahouny 1987) and STZ-diabetic hamsters (Ebara et al 1994). Treatment of diabetes with insulin resulted in the normalization of PHPLA and plasma triglyceride levels (Bagdade et al 1968). Insulin resistance and the compensatory hyperinsulinemia (often associated with type 2 diabetes) can also decrease plasma L P L activity and cause postprandial lipemia (Jeppesen et al 1995). Maheux et al (1997) further demonstrated that the degree of insulin resistance was directly correlated to the degree of decrease in plasma LPL (activity and mass), and to the degree of elevations in plasma TG concentrations. In fact, numerous studies have shown a genetic correlation between diabetic hyperlipidemia and abnormalities in LPL activity (Zhang et al 1997, Knudsen et al 1997). Specifically, Hindlll restriction fragment-length polymorphism genotype of LPL is associated with insulin resistance, hypertriglyceridemia and reduced levels of HDL-cholesterol (Ahn et al 1993). Pvu II polymorphism of the LPL gene is also significantly associated with type 2 diabetes and coronary artery disease (Wang et al 1996). Heterozygous familial lipoprotein lipase deficiency in association with hyperinsulinemia caused deleterious effects on plasma TG levels (Julien et al 1997). A novel compound NO-1886, by elevating LPL activity without affecting the hypoinsulinemic and hyperglycemic states of diabetes, decreased plasma TG levels and increased HDL-cholesterol levels (Tsutsumi et al 1995). This evidence at least demonstrates L P L is 47 directly responsible for the plasma TG levels in diabetic animals. On the contrary, several reports also demonstrated an increase or no change in plasma lipase activity in diabetic patients and in experimental animal models (Groop et al 1996, Ebara et al 1994, Hirano et al 1991). These inconsistencies could be partly due to differences in procedures to measure L P L activity, type of diabetes studied, basal insulin or TG levels in the diabetic conditions, and duration of insulin treatment and withdrawal in diabetic patients (O'Looney and Vahouny 1987). Decreased clearance of TGRLs from the circulation could also be due to factors other than defective LPL. A dissociation between LPL activity and plasma TG level was observed during diabetes (Chen et al 1980) suggesting that factors other than L P L could contribute towards the hypertriglyceridemia. Clinical and experimental diabetes show alterations in lipoprotein structure and composition resulting in a poorer interaction with LPL and this was implicated as one of the mechanisms involved in the development of hypertriglyceridemia (Bar-On et al 1984, O'Looney et al 1985). Major apolipoproteins that are packaged along with the lipids in TGRLs are apoBlOO, B48, apoEl-5, apoAl-4 and apoCl-3 (Breslow 1989). Among these apolipoproteins, apoCII is a co-factor necessary for the full expression of LPL activity. ApoCIII and apoE, on the other hand, inhibit LPL activity. Changes in ratios of apoCII/apoCIII and apoCs / apoEs have been shown to alter lipolysis of TGRLs by LPL (Bar-On et al 1984, Levy et al 1985, O'Looney et al 1985, Mamo et al 1992, Saheki et al 1993). 48 2. R A T I O N A L E , H Y P O T H E S I S A N D O B J E C T I V E S 2.1. H y p e r t e n s i o n a n d Diabetes Hypertension and diabetes are two major risk factors implicated in cardiovascular diseases (CVD) which are collectively the number one killer of western and Asian populations. Hypertension is an important risk factor in the development of cardiac dysfunction (Nicholls 1996). Epidemiological studies report that at least 50% of hypertensive patients die of congestive heart failure (Nicholls 1996). Development of left ventricular hypertrophy, impairment in coronary flow reserve and chronic myocardial ischemia result in eventual transformation to a dilated, failing ventricle (Nicholls 1996). Therefore, the presence of left ventricular hypertrophy proves to be a powerful prognostic implication for the development of heart failure (Nicholls 1996). In spontaneously hypertensive rats a similar development of left ventricular hypertrophy was observed in early stages of hypertension (Anversa et al 1984) which later transforms to heart failure (Bing et al 1995). Diabetes is another potent and independent risk factor for cardiovascular diseases in humans as well as in experimental animal models (Butler 1998, Dhalla et al 1998). The diabetic heart suffers a specific muscle disease called diabetic cardiomyopathy that is due to insulin deficiency but is seen in the absence of coronary artery disease, myocardial infarction, cardiac autonomic neuropathy, and micro and macroangiopathy (Fein and Sonnenblick 1985, McNeill and Tahiliani 1986). The cardiomyopathy is characterized by reduced heart rate, depressed left ventricular developed pressure, and decreased rates of contraction and relaxation. These functional changes are accompanied by several sub-cellular changes like abnormalities in 2 + sarcoplasmic reticulum and sarcolemmal membrane-bound enzyme systems (Ca -pumps, 49 Na+/Ca2+-exchanger, Na +/K +-ATPase activity), Ca 2 +-ATPase of myofibrils, myosin light chain (MLC) and MLC-kinase activity, various organelle membrane disturbances probably as a result of accumulation of F A metabolic intermediates, all resulting in altered Ca2+-handling, calcium overload and eventual dysfunction of the cardiac muscle (Dhalla et al 1998). At least 50% of type 1 and type 2 diabetics develop hypertension (Bilo and Gans 1998). The incidence of cardiovascular diseases and mortality increases, at least by two fold, when diabetes and hypertension exist together (Rodrigues and McNeill 1986, Bilo and Gans 1998). In humans as well as in experimental animals, hypertension was shown to cause extensive structural damage to the diabetic heart and a greater depression in cardiac function when compared to non-hypertensive diabetic animals (Knowler et al 1980, Factor et al 1980, Parving et al 1983, Rodrigues and McNeill 1986, Chukwuma 1992, Johnson et al 1992). 2.2. M e t a b o l i c lesions d u r i n g hyper tens ion a n d diabetes Glucose and FFA are major substrates that the heart utilizes to produce energy, FFA normally contribute at least 70 % in generating cardiac energy in the form of ATP. Evidence is emerging to suggest a correlation between metabolic lesions and the development of cardiac dysfunction during hypertension and diabetes (Christe and Rodgers 1994, 1995, Rodrigues et al 1995). In hearts from SHRs, glucose oxidation was shown to be markedly elevated compared to hearts from Sprague Dawley rats, yielding a 4-5 fold increase in the ratio of Glucose/FFA oxidation rates (Christe and Rodgers 1994). This shift in metabolism possibly supports an efficient energy production in the stable, hypertrophied heart. It was also hypothesized that FFAs are diverted to meet an increased demand on structure building during the development of left ventricular hypertrophy (Christe and Rodgers 1994). Therefore, it was proposed that changes in 50 cardiac metabolism probably favor the development of ventricular hypertrophy in the compensating heart during hypertension. In the diabetic heart, substantial evidence indicates that early metabolic derangements in the myocardium could underlie the pathogenesis of diabetic cardiomyopathy (Rodrigues and McNeill 1992c, Rodrigues et al 1995). Exclusive dependence on FFA oxidation due to restricted glucose entry and FFA mediated inhibition of glucose oxidation is probably the major metabolic lesion that occurs during diabetes (Randle et al 1963). Interestingly, when diabetes was induced in SHR, there was a remarkable shift towards FFA oxidation in the SHR-diabetic heart (Christe and Rodgers 1995). This drastic shift from an increased glucose oxidation to an increased FFA oxidation positively correlated with a much greater functional depression and cardiomyopathy in the hypertensive-diabetic heart as compared to a non-diabetic hypertensive heart (Rodrigues and McNeill 1986). To date, the underlying mechanism(s) for these metabolic lesions are not completely understood. 2.3. Possible role o f L P L i n the development o f ca rd iac dysfunct ion Although, FFA are the preferred energy substrate, the heart has a limited potential to synthesize them (van der Vusse et al 1992). Hence, FFAs are supplied to cardiac cells from several sources: through lipolysis of endogenous cardiac triglyceride (TG), or from exogenous sources in the blood (as free acid bound to albumin or as TGRL). FFA from circulating TGRL is released by the lipolytic action of endothelium-bound LPL in the coronary vasculature and taken up by the heart for numerous metabolic and structural tasks. LPL plays a rate limiting role in this hydrolytic process. Increase and decrease in LPL activity at the coronary lumen could be accompanied by parallel changes in FFA supply to the underlying myocardium. The relative contribution of LPL towards cardiac lipid metabolism is not known. Isolated heart perfusion 51 studies revealed that most of the FFA released by LPL was directly taken up by the cardiac tissue (O'Looney et al 1985). Recently, LPL over-expression specifically in skeletal and cardiac muscles resulted in premature death of mice due to skeletal and cardiac myopathy secondary to LPL-derived FFA accumulation in the muscle fibers (Levak-Frank et al 1995). In this regard, soleus muscle, similar to cardiac muscle, utilizes FFA extensively for its energy production and expresses 10 fold higher LPL when compared to extensor digitorum longus (EDL, type II fibers or fast twitch fibers). Indeed, EDL fibers are not dependent on FFA oxidation but on glycolytic and anerobic oxidation (Ong et al 1994). These observations strongly support the notion that L P L could play a potential "gate-keeping" role in supplying FFA to cardiac tissue from circulating TGs. During diabetes, FFA oxidation is increased in the heart whereas, during hypertension FFA oxidation is decreased. We believe that LPL could play a potential role in these metabolic alterations observed during hypertension and diabetes. We hypothesize that changes in cardiac LPL could be responsible for the alterations in cardiac metabolism that were observed during hypertension and diabetes. 2.4. OBJECTIVE 1 To study the regulation of cardiac LPL during hypertension L P L activity is decreased in adipose tissue and skeletal muscle in hypertensive patients (Pollare et al 1991, Marotta et al 1995) and Dahl sensitive hypertensive rats (Mondon et al 1993). In the heart, total LPL activity, as measured in tissue homogenates, was decreased in Dahl salt sensitive hypertensive rats as compared to normotensive controls. In SHR, LPL mRNA in the heart progressively decreased with the development of hypertrophy (Masuzaki et al 1996). However, in the above study LPL protein or activity was not measured. Changes in LPL synthesis or activity can occur independent of mRNA levels due to involvement of post-52 translational regulation (Ong and Kern 1989, Bergo et al 1996a). Therefore, it is physiologically relevant to determine the regulation of LPL enzyme activity. Additionally, there is no information on distribution of the LPL enzyme (functional versus non-functional fractions) in the hypertensive heart. Thus, we investigated the effect of hypertension on cardiac LPL in two hypertensive animal models: 1) spontaneously hypertensive (SHR) rats which have the genetic propensity to develop hypertension and 2) fructose hypertensive rats which acquire hypertension as a result of fructose feeding. We attempted to study the enzyme activity in functional (luminal) and non-functional (cardiomyocyte) compartments. 2.5 OBJECTIVE 2 To study the regulation of cardiac LPL during diabetes The diabetic heart has elevated levels of FFA and TG. Various sources like enhanced adipose tissue derived FFA, increased intracellular TG synthesis and lipolysis of TG stores, could contribute to this higher FFA level, ensuring fuel supply to the glucose-deprived diabetic heart (Murthy et al 1983, Kenno and Severson 1985, Chattopadhyay et al 1990). Several studies have consistently proven that adipose LPL is decreased during diabetes and that the decrease is directly related to the insulin deficient state (Robinson 1960, O'Looney and Vahouny 1987, Tavangar et al 1992, Inadera et al 1991, Simsolo et al 1992, Eckel et al 1995, Maheux et al 1997). In contrast, studies on cardiac LPL regulation are inconsistent and unclear. L P L activity and immunoreactive protein have been shown to be either unchanged, increased, or decreased in the diabetic heart (Rodrigues et al 1997). Factors such as the strain of rats, dose of STZ and duration of diabetes, and the techniques that were used to measure LPL could have partly 53 contributed to these disparate results. In fact, unlike in severe diabetes, heparin perfusion released a 2-3 fold greater amount of LPL in the perfusates of the isolated hearts from the moderately diabetic animals (Rodrigues et al 1997). In addition, we also observed that heparin perfusion by conventional Langendorff method releases LPL in 2 phases (fast-phase and delayed-phase of release) from the control heart into the medium. Since heparin can traverse the endothelial barrier (Lovich and Edelman 1995), we believe that the conventional Langendorff-perfused heart would release LPL not only from the coronary lumen (which probably constitutes the fast phase) but also from the abluminal, interstitial spaces and the myocyte surface (responsible for delayed-phase). Interestingly, although 2 or 12 weeks of hypoinsulinemia in STZ-diabetic rats elevated heparin releasable LPL, it obliterated the delayed-phase of LPL release (Rodrigues et al 1997). Therefore, we asked two questions: (1) Does the augmented LPL in the diabetic heart actually represent the functional pool of enzyme at the coronary lumen, exclusive of the abluminal, interstitial and myocyte pools? This question is of particular importance because the presence of the enzyme at this location would permit FFA supply to the diabetic heart in the absence of glucose utilization. (2) Can changes in L P L activity be acutely regulated by a short duration (hours) of hyperglycemia or hypoinsulinemia? This question carries clinical relevance since during diabetes, poor compliance with insulin treatment causes patients to be regularly exposed to brief periods of hyperglycemia. Diabetes induces structural and compositional changes in lipoproteins like V L D L and C H Y L . These changes make the lipoproteins poorer substrates for LPL. Thus, it was not clear whether increased LPL at the coronary lumen in the diabetic heart would correspondingly augment lipolysis of native V L D L - T G . Therefore, we also measured the lipolysis of native substrates like V L D L - T G in isolated diabetic hearts. 3. M E T H O D S 54 3 .1 . H y p e r t e n s i o n study 3.1.1. Experimental animals 3.1.1.1. Spontaneously Hypertensive Rats The spontaneously hypertensive rats (SHR), with a genetic propensity to develop hypertension, are inbred rats derived from the Wistar Kyoto (WKY) strain which in turn descended from the Wistar strain (Trippodo and Frohlich 1981). SHRs have been very well characterized and shown to express similarities to clinical hypertension (of primary or essential hypertension type) with respect to the degree of blood pressure, hemodynamics, vascular reactivity, neurohumoral, and renal factors (Trippodo and Frohlich 1981). Like human patients, SHRs develop left ventricular hypertrophy and heart failure (Bing et al 1995). The W K Y rat is generally used as normotensive control since it is the parent strain of SHR. A l l animals in the study were cared for in accordance with the principles promulgated by the Canadian Council on Animal Care and The University of British Columbia. Male SHR and W K Y rats were obtained at 6 weeks of age (Charles River, Montreal, Que.). The rats were maintained under a 12 h light (7:00-19:00)-dark cycle. Rats were housed two to three per cage and supplied with a standard laboratory chow diet (Lab Diet #5001, PMI Feeds, Richmond, V A ) and water ad libitum. As blood pressure is significantly higher only after 9-10 weeks of age in SHR rats, W K Y and SHR rats were killed before (7-8 weeks of age, representing a pre-hypertensive stage), after the development of severe hypertension (11-12 weeks of age), and after a prolonged period of hypertension (15-16 weeks of age), and LPL activity was measured in the heart. 55 3.1.1.2. Fructose Hypertensive Rats Fructose feeding induces hypertension, hyperinsulinemia and hypertriglyceridemia in rats (Dai and McNeill 1995, Dall'Aglio et al 1995, Zavaroni et al 1980, Hirano et al 1989). Adult male Wistar rats (180-200 g) were obtained from the University of British Columbia Animal Care Unit (Vancouver, BC). The rats were maintained under a 12 h light (7:00-19:00)-dark cycle, and supplied with a standard laboratory chow diet (PMI Feeds, Richmond, V A ) , and water ad libitum. Rats were randomly divided into control and fructose-treated groups. In the acute study, treated rats were given a 10% (w/v) fructose solution (p-D- (-)-Fructose, ICN, Aurora, Ohio) in drinking water for 2-weeks. In some rats, fructose treatment was continued for 4 to 6 weeks. As no differences in metabolic or biochemical parameters were observed between the 4 and 6 week fructose-treated animals, data from these rats were pooled, and this group was then defined as the chronic treated group. At the end of the acute or chronic treatment period, rats were killed, and the hearts were removed for measurement of heparin-releasable LPL activity. To determine i f the effects of fructose were reversible, fructose treatment was stopped after a 6-week period. These rats (fructose-withdrawn) were subsequently maintained for an additional 2 weeks, after which they were killed and LPL activity was determined. Age matched controls were used to compare the results. 3.1.2. Measurement of blood pressure Measurements of systolic blood pressure were done in conscious rats by the indirect tail cuff method without external pre-heating (Bunag 1973). Animals were placed in a rat holder in a chamber (Model 306, IITC Inc., Woodland Hills, CA) maintained at 29 °C. The tail cuff with pressure sensor was attached to the base of the tail and connected to a cuff pump (Model 20-NW, IITC Inc.). A semiautomatic BP analyzer and an analog-to-digital recorder (Model 179, IITC 56 Inc.) were connected to the tail cuff to amplify and record the signal from the pressure sensor. With this method, the photoelectric sensor detects the reappearance of pulsation (on gradual deflation of the cuff). Three readings out of five were averaged to determine BP. Animals were always preconditioned to the experimental procedure before conducting the actual measurements. Our readings were similar (within 5 mm Hg) to those obtained by direct cannulation of abdominal aorta (Bhanot and McNeill 1994). 3.1.3. Isolated whole heart perfusion Rats were anesthetized with 65 mg/kg of sodium pentobarbital i.p. (MTC Pharmaceuticals, Cambridge, Ont.), the thoracic cavities were opened and hearts were removed. Rats were not injected with heparin prior to killing, as it displaces LPL bound to HSPGs on the capillary endothelium. Therefore, it was necessary to remove and cannulate the heart as quickly as possible to avoid clotting of blood in the coronary arteries. Immediately upon excision, the beating heart was immersed in ice-cold (4°C) calcium-free Joklik minimal essential medium (pH 7.4) supplemented with 2 g N a H C 0 3 , 1.2 m M MgS04, and 1 m M L-carnitine. After cannulation of the aorta, the hearts were perfused retrogradely by the non-recirculating Langendorff technique for 5 min (or until the perfusate was clear of blood) (Rodrigues et al 1992a, 1992b). This period is necessary to remove proteases released by tissue damaged during the dissection that would reduce LPL activity subsequently measured in the hydrolysis assay. The perfusion fluid was continuously gassed with 95% 0 2 -5% C 0 2 in a double-walled water-heated chamber that was kept at 37 °C with a temperature-controlled circulating water bath. The rate of coronary flow (7-8 ml/min) was controlled by a Masterflex® pump (Cole-Parmer Instrument Co., IL USA). To measure the release of LPL activity into the medium, the perfusion solution was changed to Joklik containing 1% BSA (w/v, 0.15 mM, Fraction V, Boehringer Mannheim Germany), 1 m M 57 CaCl2 and heparin (5 U/ml, Hepalean*, Organon Teknika, Toronto, Canada) (Olivecrona et al 1977). This concentration of heparin was previously shown to maximally release cardiac LPL from its binding sites. The coronary effluent was collected for 10 seconds in timed fractions and frozen until assayed for L P L activity. Vasoconstriction has been suggested to affect LPL activity (Lind and Lithell 1993). Nifedipine, a C a 2 + influx blocker, is known to dilate blood vessels and decrease blood pressure in SHR rats (Rinaldi et al 1987, Tschudi et al 1994). Moreover, nifedipine can increase coronary blood flow, and these effects persist over 30 minutes (Ogawa et al 1981). Hence, nifedipine (3 mg/kg in 40% ethanol) was injected into the tail veins of 15-16 week old SHR rats. Thirty minutes following nifedipine injection, the rats were killed, hearts were removed and perfused with buffer containing heparin, and the perfusate was collected and analyzed for LPL activity. In another experiment, hearts from 15-16 week old SHR and W K Y rats were perfused in vitro with recirculating Joklik buffer containing nifedipine (100 nM) and 1% B S A for 10 minutes. This was followed by perfusion with heparin, and fractions of perfusate were collected and analyzed for L P L activity. CGS-21680, an A2-purinergic receptor agonist has also been shown to dilate coronary blood vessels with insignificant effects on cardiac function (Vials and Burnstock 1993). Hence hearts from hypertensive SHR (15-16 weeks) rats were similarly perfused with 10 u M CGS-21680 (RBI, M A , USA) for 10 min followed by a 10 min perfusion with buffer containing heparin to release endothelial bound LPL. During perfusion with nifedipine or CGS-21680, the coronary flow rate was kept constant at 7-8 ml/min. Similar protocols for in vitro experiments in the 4-6 weeks fructose treated group were followed to investigate the effects of coronary vasodilators like nifedipine and CGS-21680 on LPL activity. 58 3.1.4. Preparation of cardiac myocytes Perfusion of the heart with buffer containing heparin predominantly releases extracellular, endothelial-bound LPL. However, heparin non-releasable (cellular) LPL activity can still be measured in the myocytes. To measure this fraction, calcium-tolerant myocytes were made from hearts (ventricles) by a previously described procedure (Rodrigues et al 1992a, 1992b). In brief, hearts were removed from anesthetized rats and digested by perfusing Joklik buffer containing collagenase (228 U/ml), 0.5% BSA, and 50 mM CaCh retrogradely through the heart. Myocytes were made calcium-tolerant by successive exposure to increasing concentrations of calcium. Our method of isolation yields a highly enriched population of calcium-tolerant myocytes that are rod shaped with clear cross striations in the presence of 1 mM Ca 2 + . Intolerant cells are intact, but hypercontract into vesiculated spheres. Yield of myocytes (cell number) was determined microscopically using a Neubauer haemocytometer. Myocyte viability (generally between 75-85%) was assessed as the percentage of elongated cells with clear cross striations that excluded 0.2% trypan blue. Cardiac myocytes from WKY and SHR rats were suspended in Joklik-minimum essential medium to a cell density of 0.4 x 106 cells/ml and incubated at 37°C under an atmosphere of 95% 02-5% CO2. To release surface-bound LPL activity, heparin (5 U/ml) was added to the myocyte culture. Aliquots of cell suspension (1 ml) were removed (cell preparation was shaken before sampling to obtain a homogenously suspended sample) at specified intervals, and the medium was separated from cells by centrifugation (3,000 x g for 10 sec) in an Eppendorf microcentrifuge. The supernatant and corresponding cell pellets were stored in -70°C until assayed for LPL activity. 59 3.1.5. LPL assay 3.1.5.1. LPL assay in the medium LPL catalytic activity in coronary perfusates and incubation medium of cardiac myocytes was determined by measuring the in vitro hydrolysis of a sonicated [3H]triolein substrate emulsion. The standard assay conditions included 0.6 m M glycerol tri[9,10-3H]oleate (1 mCi/mmol), 25 m M PIPES (pH 7.5), 0.05% (w/v) albumin, 50 m M M g C l 2 , 10 % (v/v) heat-inactivated chicken serum (containing the LPL activator, apolipoprotein CII), and 100 pl of either myocyte medium or heart perfusate in a total volume of 400 pl. The release of [3H]oleate was measured after incubation for 30 min at 30°C. The released [3H]oleate in the reaction mixture was determined by adding 3 ml of fatty acid extraction solution (methanol:chloroform:heptane; 1.41:1.25:1.0 and 100 pl of oleic acid) and 100 pl of 0.1 M NaOH (Ramirez et al 1985). After vortex mixing and centrifugation (TY JS-4.2 rotor, 2,500 x g for 30 minutes) using a Beckman J-6B centrifuge, the radioactive sodium [3H]oleate in a sample (0.5 ml) of the upper phase was determined by liquid scintillation counting. A l l LPL assays were performed in duplicate where the reaction rate was linear with respect to time and volume of medium assayed. The validity of the L P L assay has been previously established (Ramirez et al 1985). Results were expressed as nanomoles of oleate released per hour per ml (coronary perfusate) or 106 cells (myocyte medium or cells). 3.1.5.2. Cellular LPL assay Heparin non-releasable cellular LPL activity was measured by sonicating (2x30 sec with a 5 second interval, at 4°C) the cell pellets (0.4 x 106cells) after resuspending them in 0.2 ml of 50 m M ammonia buffer (pH 8.0) containing 0.125% (v/v) Triton X-100. After sonication, the 60 volume was adjusted to 1 ml using sucrose buffer [0.25 M sucrose, 1 m M EDTA, 1 m M dithiothreitol, 10 m M HEPES, [pH 7.4] (Carroll et al 1995). The assay for cell sonicate LPL activity was done essentially as described above except that 20 ul of cell sonicate was used and heparin (2 U/ml) was added to release membrane bound LPL. 3.1.5.3. Post heparin plasma lipolytic activity (PHPLA) Plasma LPL activity in the basal (non-fasted) state and in response to a heparin injection was determined in control rats and those fed fructose for 6 weeks. Heparin (0.5 U/g) was injected into the tail vein of lightly anaesthetized (20 mg/kg sodium pentobarbital i.p.) rats and blood samples were collected at 5 minutes. Plasma was separated and stored at -70 °C until assayed for LPL activity (Galan et al 1994). Plasma lipase activity was determined by first measuring total lipase (hepatic + LPL) activity in 5 pl of plasma sample. Hepatic lipase activity was measured by incubating plasma with 1 M NaCl (at room temperature for 10 minutes before exposing to substrate) and conducting the assay in the absence of apolipoprotein CII, to suppress L P L activity (Galan et al 1994). Plasma LPL activity was calculated as the difference between total and hepatic lipase activity and is expressed in milliunits (mU), which corresponds to 1 nmol of fatty acid released per minute. 3.1.6. Triglyceride Secretion Rate (TGSR) TGSR is a measure of rate of secretion of V L D L - T G from the liver. TGSR was determined in SHR and W K Y rats by a previously described method (Kazumi et al 1986). Triton WR1339, when injected intravenously, inhibits T G lipolysis in the circulation. Therefore, newly synthesized TGs accumulate in the plasma and the rate of accumulation is an indirect measure of TGSR. We used 15-16 week old SHR and W K Y rats to determine the TGSR. Rats were fasted 61 for 5 hours (900-1400 hrs) to minimize chylomicron contribution in the TGSR determination. After light anesthesia, rats were injected (i.v.) with Triton WR1339 (25% w/v solution in normal saline togive a dose of 600 mg/kg body weight). Blood samples were collected at 0, 20, 40, and 60 minutes after the injection. Serum was separated and the TG concentration was measured using a Boehringer Mannheim diagnostic kit . When TG concentration is plotted against time, slope of the linear line that is generated gives the TGSR of the animals. 3.1.7. Triglyceride clearance rate (TGCR) Clearance of TG from plasma over a period of time following an intravenous injection of heparin gives the measure of plasma lipolytic activity in vivo. When heparin is injected i.v., it displaces L P L and H L from all tissue-binding sites and increases the plasma lipolytic activity several fold. This increase in plasma lipolytic activity is accompanied by an accelerated clearance of endogenous TG from the circulation. Anesthetized SHR and W K Y rats (15-16 weeks of age) were injected with heparin (i.v., 0.5 U/g body weight), blood samples were collected at 0, 1, 2, 5, 10, 30, 60 minutes. TG concentration was measured in the plasma separated from the above blood samples. An aliquot of 0 and 5 minute plasma samples were also measured for post heparin plasma lipolytic activity (for PHPLA measurement, refer section 3.1.5.3.). 62 3.2. Diabetes s tudy 3.2.1. LPL regulation in the diabetic heart 3.2.1.1. Experimental Animals Adult male Wistar (280-290 g) rats were obtained from University British Columbia animal care unit (Vancouver, BC). The rats were maintained and cared for as mentioned earlier. 3.2.1.1.1. Induction of Diabetes Selective P-cell death and the ensuing diabetic state can be produced after a single intravenous dose of STZ (Rakieten et al 1963, Junod et al 1969). A dose-dependent increase in severity of diabetes is produced by 25-100 mg/kg STZ (Rodrigues et al 1999). After an i.v., injection of 55 mg/kg STZ, stable hyperglycemia develops within 24-48 hrs and remains 2-3 times higher than normal in concert with an =50% reduction in plasma insulin levels. Although these animals are insulin deficient, they do not require insulin supplementation for survival and do not develop ketoacidosis. On the other hand, a 100 mg/kg dose of STZ causes intense p-cell necrosis, remarkable elevation of serum glucose within 24 hrs, reduced plasma insulin to ~2%-5% of control, and a 98% loss of pancreatic insulin stores. Without administration of exogenous insulin, death in most of these animals occurs within 7-10 days. Rats were randomly divided into nondiabetic (CON) and diabetic (D55 and D100) groups. Halothane-anesthetized rats were injected with STZ (55 [D55] or 100 [D100] mg/kg IV, Sigma Chemical Co.) or an equivalent volume (1 mL/kg) of saline. Glycosuria was determined 24 hours after STZ injection, and hyperglycemia was tested at 48 hours with a glucometer. A l l STZ-treated rats displayed both glycosuria (>4+) and hyperglycemia (>13 mmol/L). 63 3.2.1.1.2. Insulin Reduction To evaluate the effect of a chronic reduction in plasma insulin on cardiac LPL, D55 rats were kept for 2 weeks after the STZ injection, at which time they were killed and the hearts removed. To investigate the short-term effects of a decrease in insulin on cardiac LPL, 2 protocols were followed. In the first protocol, rats were injected with 100 mg/kg STZ. In preliminary experiments, we determined that after this dose of STZ, there is a triphasic pattern of changes in blood glucose and insulin levels in the 24-hour period after injection. An initial brief hyperglycemia is followed by a period of hypoglycemia that is brought about by massive P-cell degranulation, and an enormous release of insulin (Junod et al 1969). Blood glucose then rises to a hyperglycemic value of 13 mmol/L within 12-16 hours. Rats were monitored individually, and at the point of hyperglycemia, or 3 or 6 hours after hyperglycemia, animals were killed for the determination of cardiac LPL activity. One potential drawback with this approach is the varied metabolic changes that occur before progression to stable hyperglycemia. Hence, with our second protocol, rats were made severely diabetic with 100 mg/kg STZ. One day after diabetes induction, the animals were treated subcutaneously with intermediate-acting insulin (D100+1, Iletin NPH, Beef and Pork; Lilly) once daily. The insulin injection was given at 1000 A M with the dose adjusted daily to achieve normoglycemia. Treatment was continued for 7 days. This time was necessary to ascertain the optimal insulin dose (-18-20 U/kg) required to maintain euglycemia for 24 hours. After the animals' diabetes had been well controlled, insulin injection was stopped, and plasma glucose was monitored. After the last insulin injection, plasma glucose levels increased after 24 hours. A plasma glucose concentration of 13 mmol/L was considered a hyperglycemic value, and when this level was reached, diabetic animals were kept for either 6 or 24 hours before they were killed. Using this method, we were able to achieve a fixed duration of hypoinsulinemia and hyperglycemia. 64 3.2.1.2. Modified Langendorff Perfusion To localize and quantify the augmented LPL in diabetic rat hearts, a modified Langendorff retrograde perfusion technique was used to separate the coronary from the interstitial effluent (DeDeckere and Hoor 1977, Jansen et al 1980, de Lannoy et al 1997). Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and the thoracic cavity was opened. The left anterior vena cava was ligated below the azygous vein followed by ligation of the right anterior vena cava. The hearts were then carefully excised with the aorta, inferior vena cava, and lungs still attached. Rats were not injected with heparin before being killed because heparin displaces LPL bound to HSPGs on the capillary endothelium. Consequently, it was necessary to cannulate the heart quickly to avoid clotting in the coronary arteries. Immediately on excision, the beating heart was immersed in cold (4°C) Krebs-Ringer-HEPES buffer (pH 7.4). The concentrations of solutes in the buffer were (in mmol/L) 1 CaCl 2 , 118 NaCl, 4.96 KC1, 1.19 K H 2 P 0 4 , 1.19 M g S 0 4 7H 2 0, 24 HEPES, and 10 glucose. After the aorta was cannulated and tied below the innominate artery, the hearts were perfused by the retrograde noncirculating Langendorff technique. The perfusion fluid was continuously gassed with 95% 0 2 /5% C 0 2 in a double-walled water-heated chamber maintained at 37°C with a temperature-controlled circulating water bath. A peristaltic pump controlled the rate of coronary flow (7-8 mL/min). The right and left branches of the pulmonary artery were cut before they entered the lungs, and the 2 branches were then trimmed off at their junction. Afterward, the inferior vena cava and branches of the right and left pulmonary veins were ligated, the lungs were removed, and the pulmonary artery was cannulated and tied. At this time, most of the perfusate (=98-99%) starts flowing through the pulmonary cannula whereas a small amount of fluid (=1-2%) drips down to the apex of the heart. The pulmonary effluent represents the coronary perfusate whereas the fluid collected at the apex represents interstitial transudate (DeDeckere and Hoor 1977, Jansen et al 65 1980, de Lannoy et al 1997). To measure the release of LPL activity or protein into the medium, the perfusion solution was changed to Krebs-Ringer HEPES buffer containing 1% BSA and heparin (5 U/ml). This concentration of heparin can maximally release cardiac L P L from its binding sites, an action mediated by the interaction of negative charges on heparin with positively charged amino residues on the enzyme (Olivecrona 1993). The coronary and interstitial effluents were collected separately in timed fractions, and frozen until assayed for L P L activity. Insulin treatment: In some experiments, 2-week D55 rats were treated with a rapid-acting insulin (Iletin, 15 U , i.v.) 90 minutes before the rats were euthanized, and cardiac heparin-releasable coronary and interstitial L P L activity measured. 3.2.1.3. Preparation of cardiac myocytes Calcium tolerant cardiac myocytes were isolated from C O N and D100 rats by the previously described method (see section 3.1.4.). Cardiac myocytes were isolated to measure whether acute insulin reduction after withdrawal of insulin treatment in D100 + I group could also modulate enzyme activity. Insulin treatment reversed a number of effects of STZ-diabetes, including hyperglycemia, food and fluid intake and body weight gain, etc, to control levels. When insulin was withdrawn, the rats became hyperglycemic within 24 hours. The animals were kept for a further 6 hours following hyperglycemia and then killed. 3.2.1.4. Heart tissue homogenization Hearts from C O N and D55 rats were removed and cleared of blood by flushing them through the aorta with 10 ml of Krebs-Ringer-HEPES buffer (pH 7.4, 37°C). Atria and other tissues were removed and the ventricles were freeze-clamped in liquid nitrogen. The tissues were stored in -70°C until LPL activity was measured. For the LPL measurement, frozen tissues 66 were weighed and ground using a mortar and pestle that were pre-cooled in liquid nitrogen. To the powdered tissue, 20 ml of 50 mM ammonia buffer (pH 8.0) containing 0.125% (v/v) Triton X-100 and 10 U/mL of heparin was added and homogenized further using a Polytron (PT MR 3100, Kinematica, AG, Switzerland) homogenizer (2x30 seconds with a 10 seconds gap in between) under ice-cold conditions. After sonication, the volume was adjusted to give 25 mg/ml of protein concentration using sucrose buffer (0.25 M sucrose, 1 mM EDTA, 1 mM dithiothreitol, 10 mM HEPES, 10 U/ml of heparin, pH 7.4). The homogenate was centrifuged at 3000xg and the supernatant was separated. A 25 pl aliqout of the supernatant was exposed to sonicated [3H]-triolein emulsion and the LPL assay was performed in triplicate as before (for details see section 3.1.5.1.). 3.2.1.5. Enzyme-linked immunosorbent assay for LPL protein concentration Changes in the amount of LPL activity do not always represent changes in quantity of immunoassayed LPL protein (Olivecrona et al 1987). To measure LPL mass, coronary perfusate (=24 ml) from CON and D55 hearts was collected between 1 and 3 minutes (flow rate at 7-8 ml/min) after heparin perfusion. LPL protein was measured by a previously described sandwich ELISA method (Anderson et al 1997). Briefly, samples were lyophilized and resuspended in 0.2 ml H2O. Affinity-purified polyclonal antibodies against LPL purified from bovine milk were obtained from egg-laying hens (Goers et al 1987). Polystyrene microtiter plate wells (Immulon 1) were coated with 100 pl of anti-LPL antibody (15 pg/ml in 0.05 M carbonate buffer, pH 9.6) overnight at 4°C. After washing, 100 pl aliquots of samples diluted in phosphate-buffered saline (PBS) containing 0.05% (wt/vol) Tween-20, 1 mg/ml heparin, 0.4% (wt/vol) BSA, 1 mmol/L phenylmethanesulfonylfluoride, 10 Ug/ml leupeptin, and 1 pg/ml pepstatin A were added to the wells. The plates were then sealed and incubated overnight at 4°C. Purified bovine milk LPL 67 was used as standard for the ELISA. After extensive washing, affinity-purified biotin-labeled anti-LPL antibody was applied to the wells followed by a second overnight incubation at 4°C. The wells were then incubated with peroxidase-labeled streptavidin in PBS, 1% (wt/vol) fatty acid-free BSA for 2 hours at 25°C. The plates were washed thoroughly and incubated with o-phenylenediamine (0.8 mg/ml in 0.15 M citrate buffer, pH 5), and color development was measured at 495 nm using a Bio-Rad microplate reader. L P L concentration in coronary fluid was used to calculate LPL specific activity as mU/ng of LPL protein, where 1 mU is defined as the amount of enzyme catalyzing the release of 1 nmol oleate per minute. 3.2.1.6. Immunolocalization of LPL Immediately upon excision, CON and D55 rat hearts were perfused by the retrograde non-circulating Langendorff technique with Krebs-Ringer-HEPES buffer for 3 minutes to clear the heart of blood. Perfusion buffer was then changed to fixative (neutral phosphate-buffered 10% formalin solution, room temperature) for 2 minutes. After perfusion, hearts were stored in 10% formalin for 24 h followed by paraffin processing through graded ethanol and xylene. The blocks were then embedded in Paraplast, sectioned at 3 um and mounted on positively charged glass slides. For immunostaining, sections were deparaffinized, rehydrated, and treated with 5% (vol/vol) heat inactivated rabbit serum in Tris-buffered saline (TBS, 0.15 moles/1 NaCl pH 7.4) to block any nonspecific background. Sections were incubated with the affinity-purified polyclonal antibody against LPL (1:100 dilution in TBS containing 1% (w/v) BSA) overnight at room temperature in a humid chamber. The primary antibody was then washed in TBS and further incubated for 1 hr at room temperature with the secondary biotinylated rabbit anti-chicken IgG (1:150 dilution), followed by incubation for 1 hr. with streptavidin-biotin-peroxidase complex (ABC Kit, Vector Inc). After being rinsed, sections were stained with 0.3% (w/v) 3,3' 68 diaminobenzidine hydrochloride/H202 followed by staining with 0.1% (w/v) Nuclear Fast Red in 5% (w/v) aqueous aluminum sulfate. After a final rinse in running tap water, sections were dehydrated in ethanol, cleared in xylene, and mounted in a resinous mounting medium and photographed. Sections from CON and D55 hearts were treated in an identical manner during all incubation and washing steps. As previously demonstrated (Blanchette Mackie et al 1989b), an absence of staining was observed when the primary antibody was omitted or replaced by preimmune chicken serum. 3.2.1.8. Effect of food restriction on LPL activity To ascertain whether hypoinsulinemia in the absence of hyperglycemia could alter LPL activity, C O N rats were fasted for 16-hrs (6 PM-10 A M ) and L P L activity was measured in coronary and interstitial compartments. During fasting, food was withdrawn from the animals but they had free access to water. 3.2.2. Hydrolysis of VLDL in the diabetic heart 3.2.2.1. Separation of lipoproteins and lipid profile Rats were deeply anesthetized with sodium pentobarbital (65 mg/kg, i.p.), the chest cavity was opened, and blood was withdrawn from the inferior vena cava using a 10 ml syringe with a 20 gauge needle. The blood was transferred to glass test tubes and centrifuged at 3000x g for 20 minutes in J6B-Beckman centrifuge at 4°C to separate clear serum. A pooled serum sample was then used for the isolation of major lipoproteins. Lipoproteins like V L D L , L D L , and H D L were separated from the serum by a one-step density gradient ultracentrifugation method (Redgrave et al 1975, Chapman et al 1981). To a 3 ml aliquot of serum in Ultraclear® centrifuge tubes (Beckmann), 1.02 g of NaBr (0.34 g/ml) was added to achieve a density of about 1.25 g/mL 69 (Hatch and Lees 1968, Wasan et al. 1999) and kept at 4° C for 2 hours for pre-cooling. 2.8 ml of pre-cooled buffers of different densities (1.21, 1.063, or 1.006 g/ml) were layered carefully in the order of decreasing densities. Tubes were kept immersed in ice until they were placed into Beckman SW 41 titanium rotor buckets. Tubes with buckets were balanced and sealed with screw caps. Ultracentrifugation at 288,000 x g at 15° C for 18 hours was carried out in L8-80 Beckman Ultracentrifuge. Tubes were removed from the centrifuge with care so as not to disturb the gradient layers. V L D L / C H Y L float on top (< 1.006 g/ml), while L D L separates at 1.019 g/ml, HDL at 1.21 g/ml and lipoprotein deficient serum (LPDS) stays at the bottom. The lipoprotein layers were removed using a glass pasteur pipette and the volume of each layer was measured. Different fractions were then measured for TG, cholesterol and protein contents. T G and cholesterol were measured using respective diagnostic kits, and protein was measured by the Bradford method. 3.2.2.2. VLDL-Purification and characterization Isolation of V L D L with less amount of C H Y L was achieved by using the serum obtained from fasted animals. Animals were fasted for about 18 hours (1930 to next day 1330). As a result of fasting for this duration, rats produce more V L D L and less C H Y L (Rajaram et al. 1980). EDTA was not added to blood samples that were used for isolation of V L D L for in vitro lipolysis or for whole heart perfusion experiments since EDTA in these V L D L preparations sequesters C a Z T that is necessary for L P L activity and for heart function. However, EDTA (4.0 mM, pH 7.6) was added to blood samples that were used for V L D L to analyze apolipoproteins. To 4.0 ml pre-cooled serum samples in Beckman Ultraclear® centrifuge tubes, 7.5 ml of 1.006 g/ml buffer (11.4 g NaCl, 0.2 g disodium EDTA, 1 m M NaOH in 1003 ml double distilled water) was added. The tubes were secured in SW-41 titanium rotor buckets and ultracentrifuged at 288,000 x g for 70 18 hours and at 15°C. V L D L that floats on top of the 1.006 g/ml solution was separated carefully using a glass pasteur pipette to avoid dilution. From each tube approximately 1.0-1.5 ml of V L D L fraction was collected and pooled. Using a similar procedure, the lipoprotein fraction (<1.006 g/ml) was isolated from plasma of fed animals. In the fed state the plasma is known to contain a mixture of C H Y L - and VLDL-TGs, collectively called TGRLs. 3.2.2.3. Analysis of apolipoproteins of VLDL using gradient gel electrophoresis V L D L obtained by the above method was characterized for TG, cholesterol and protein contents as mentioned earlier. A gradient polyacrylamide gel (Ready gel, 4-20%) electrophoresis was performed to identify major apolipoproteins -ApoB (Mr 220,000), ApoAI (Mr 46,000), ApoE (Mr 31,000-35,000), and ApoCII (Mr 14,000). V L D L samples (non-delipidized) from C O N and D55 rats were solublized and reduced in SDS sample buffer (5% SDS, 20% glycerol, 1% (3-mercaptoethanol, 0.5% bromophenol blue in 0.5M Tris, pH 6.8) and boiled for 4 min at 95 °C. A broad range protein molecular weight standard (BioRad) was diluted 1:20 with sample dilution buffer, digested for 4 minutes at 95°C and 10 pL of this digested molecular weight standard was loaded into the wells. 25 pL of V L D L sample (adjusted for protein concentration to 3.2 mg/mL) was loaded and electrophoresis was run at constant current of 40 mAmps/gel. The gels were stained with Coomassie Brilliant blue R250 for 90 minutes and destained overnight. Apolipoproteins were identified based on their molecular weights by comparing to molecular weight standards. 3.2.2.4. In vitro lipolysis of VLDL by bovine LPL (bLPL) and rat LPL (rLPL) In vitro lipolysis of V L D L - T G was carried out based on previously described methods (Saheki et al. 1993, Fisher et al. 1995, de Man et al. 1997). V L D L was isolated from C O N and 71 D55 rat sera by the methods described earlier. TG concentrations in CON- and D55-VLDL were adjusted to 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, or 0.35, m M and exposed to bLPL (0.2 pg protein, equivalent to 20 mU of activity against sonicated 3H-triolein) for 30 minutes at 37°C. Both V L D L and bLPL stock solutions were diluted with Tris buffer (pH 8.1) containing 0.5% essentially fatty acid free B S A (Saheki et al. 1993, Fisher et al. 1995, and de Man et al. 1997). At the end of a 30 minute incubation, the reaction was stopped by adding pre-cooled 0.3 M Na2P04 buffer (pH 6.9) and immersing the tubes in ice. From this reaction mixture, a 50 uL aliquot was pipetted in triplicate to measure FFA release due to lipolysis, using an N E F A - C kit. The results were corrected for the background using an unreacted blank with corresponding concentrations of VLDL-TGs and an equivalent volume of BSA containing Tris buffer instead of bLPL (Fisher et al. 1995). The release of FFA by bLPL was found to be linear up to 30 minutes under the conditions specified. Some of the assays were performed with rLPL purified from C O N and D55 post-heparin plasma. The purification of rLPL was carried out by heparin sepharose affinity chromatography (Iverius and Ostlund-Lindqvist 1976) using a HiTrap® column and a fast protein liquid chromatography (FPLC; Pharmacia, Uppsala, Sweden). The purified rLPL was then adjusted to 20 mU/ml in both C O N and D55 samples and exposed to 0.3 m M of CON- and D55 V L D L under the above mentioned conditions. Some in vitro lipolysis experiments with TGRLs obtained from plasma of fed rats against bLPL were also performed. 3.2.2.5. In vivo radiolabelling of VLDL V L D L was labeled with ^H-oleic acid based on a previously described method (Rajaram et al. 1980). 3H-oleic acid was complexed to BSA as follows: 20 ul (=100 uCi) of 3H-oleic acid was mixed with 200 uL of 10% NaHC03 until the solution cleared, 25% B S A in distilled water was then added to produce albumin-fatty acid complex, and heated for 3 minutes at 60°C (Nestel 72 and Barter 1971). ^pj-oleic acid-BSA complex was then injected intravenously to deeply anesthetized non-diabetic Wistar rats (CON) that were fasted for 18 hours (between 1930 and 1330). Forty minutes after injection of the ^H-oleic acid, rats were euthanized and blood was withdrawn from the inferior vena cava as described previously. Serum was then subjected to ultracentrifugation to isolate ^ H - V L D L as described earlier. Incorporation of the radiolabel was confirmed by thin layer chromatographic analysis on chloroform/methanol extract (2:1 v/v) using silica gel plates (K5F, Whatman) and hexane/diethyl ether/methanol/acetic acid (90:20:3:2 v/v) solvent system. The spot corresponding to standard triolein was scraped off and counted for the radioactivity and compared to equivalent volume (5 pl) of chloroform/methanol extract of V L D L . More than 97% of the VLDL-lipoprotein radioactivity was present in the TG moiety. 3.2.2.6. ^H-VLDL perfusion of the isolated beating heart Rate of V L D L - T G clearance in C O N and D55 hearts was studied by perfusing the hearts with 3H-VLDL by a recirculating Langendorff retrograde perfusion method (O'Looney et al. 1983). Wistar rats used in this study were induced with diabetes using STZ (55 mg/kg, i.v.) and kept for 2 weeks before sacrifice. The perfusion buffer contained 0.3 m M of V L D L - T G , 0.5% BSA, 1.0 m M CaCl2 in 20 ml of Krebs-Henseleit buffer (pH 7.4). The perfusion buffer was continuously oxygenated with 02:C02 (95:5%). The perfusion apparatus was a specially designed, closed recirculating system with a double jacketed reservoir, heart chamber (to keep the heart warm at 37°C), coil condenser to warm the buffer to 37°C, a bubble trap and an injection port to collect the sample. The total perfusate volume of the apparatus (including the tubing) was less than 30 mL. This miniature apparatus has enabled us to use small perfusion volumes (15-20 ml) and diminished the loss of V L D L due to sticking to the surfaces (average loss was 20% as determined by circulating the buffer containing V L D L through the apparatus 73 without the heart for 90 minutes). After the heart was secured through the aorta, Krebs-Ringer-HEPES buffer with no BSA and no V L D L - T G was perfused for 3 minutes in a non-recirculating mode to clear the blood from capillaries. The perfusion was then switched to recirculating mode and buffer containing V L D L - T G (0.3 mmoles/1) and BSA (0.5% w/v), and perfused for 90 minutes at the rate of 5.0 to 5.5 ml /min. The flow rate was kept constant with help of a perfusion pump (Masterflex®, 7519-00, Cole-Parmer Instruments Co., IL, USA). The heart rate was counted mannually at frequent intervals throughout the perfusion period. Samples were collected through the injection port at 0 (just before the perfusate reaches the heart), 15, 30, 45, 60, and 90 minutes, placed on the ice and immediately extracted for TG. T G extraction was performed according to a rapid extraction procedure described by Bligh and Dyer (1959). To a 400 pl aliquot of sample, 500 pl of chloroform, 1.0 ml of methanol followed by another 500 ul of chloroform and 400 pl of distilled water was added with vortexing after each addition. Thus the proportion of chloroform : methanol : aqueous phase was kept at 1:2:0.8. Centrifugation at 3300xg for 30 minutes of the extraction mixture resulted in separation of two layers: chloroform layer at the bottom and an aqueous alcoholic layer at the top with an insoluble protein interface. A n aliquot of 200 ul (in duplicate) of the chloroform layer which contains the TG moiety of V L D L was added to ACS scintillation fluid and was counted for radioactivity using a liquid Scintillation counter (LS 6000 TA, Beckman, Palo Alto, USA). In some of the perfusion studies, hearts were perfused with heparin (5 U/ml) containing Krebs-Ringer-HEPES buffer (pH 7.4, 37°C) for 5 minutes to deplete functional LPL prior to V L D L perfusion to determine whether there is an LPL independent mechanism involved in the V L D L - T G clearance by the perfused heart. After heparin, hearts were perfused with buffer (containing no B S A and V L D L ) to clear the heparin before commencing the perfusion with V L D L . 74 3.3. P l a s m a measurements After a 5 hour fasting period (0900-1400), blood samples from the tail vein were collected in heparinized glass capillary tubes. Blood samples were immediately centrifuged (3300 x g for 10 min at 4 °C) and plasma was collected and stored at -20 °C until assayed. Plasma glucose, triglyceride, cholesterol and FFA levels were measured with kits (Boehringer Mannheim Canada, Laval, Que.). Plasma insulin was measured using a rat insulin RIA kit. 3.4. M a t e r i a l s Joklik minimal essential medium was obtained from Gibco Canada (Burlington, ON, Canada), Iletin; Regular Beef and Pork insulin, Eli Lilly Canada (Toronto, Ont., Canada), [3H]triolein was purchased from Amersham Canada (Oakville, ON, Canada), [3H]-oleic acid from NEN Life Sciences Products (Boston, MA, USA), heparin sodium injection (Hapalean; 1000 U.S.P. U/ml) from Organon Teknika (Toronto, ON, Canada). CGS-21680, Research Biochemicals International (RBI-Sigma-Aldrich, Ont., Canada), and Collegenase (class-2, 325 U/mg) was obtained from Worthington Biochemical Corporation (Freehold, NJ, USA). Affinity-purified chicken polyclonal antibodies against LPL purified from bovine milk were a generous gift from Dr. D.L. Severson (University of Calgary, AB, Canada), rabbit anti-chicken IgG; Chemicon Corp, TG, Choi diagnostic kits from Boehringer Mannheim, Que., Canada, Bradford protein assay kit from BioRad Laboratories, ON, Canada. Ready gel (4-20%); BioRad Laboratories, CA, USA), NEFA-C kit (WAKO chemicals GmbH, Neuss, Germany), HiTrap® column (Pharmacia, Sweden), K5F-TLC plates (Whatman Inc., NJ, USA), triolein standard (Sigma Chemicals Co, St. Louis, MO, USA), Insulin RIA kit (Linco Research Inc., MO, USA). 75 3.5. S ta t i s t ica l analysis A l l data are reported as mean ± standard error of mean (SEM) unless otherwise stated. Unpaired Student's t test was used to determine differences between group means. Changes in whole heart LPL activity over time in response to heparin was analyzed by multivariate A N O V A followed by the Newman-Keul's test using the Number Cruncher Statistical System (NCSS®, Kaysville, Utah). The level of statistical significance was set at p < 0.05. 76 4. R E S U L T S 4.1. H y p e r t e n s i o n study 4.1.1. LPL regulation in spontaneously hypertensive rat hearts 4.1.1.1. General characteristics and plasma parameters Table 1 shows the chronological changes in general characteristics and plasma parameters of SHR and W K Y rats. There were no marked differences between groups in body weight until week 15, after which body weight in W K Y rats became significantly greater than SHR. At 7-8 weeks, the blood pressure of SHR rats was not significantly different from W K Y controls. However, SHR blood pressure gradually increased over time reaching -200 mmHg at 11-12 weeks of age. Therefore, SHR represent a chronically hypertensive group by 15-16 weeks of age. The blood pressure in 11-12 and 15-16 weeks of age groups was 74 mmHg higher in SHR than in W K Y rats. We did not observe any difference in the heart weight/body weight ratio between W K Y and SHR rats at 15-16 weeks of age ( W K Y 3.6±0.2; SHR 3.7±0.2, mg/g). A l l plasma parameters were measured after a 5 hour fast (0900-1400). There was no significant differences in plasma glucose or insulin levels between W K Y and SHR rats at any age studied. Plasma TG and total cholesterol were significantly lower in SHR rats when compared to W K Y animals at 7-8 and 15-16 weeks. However, plasma FFA concentrations in 15-16 week old SHR rats were elevated when compared to age matched W K Y controls. 4.1.1.2. Coronary endothelial LPL Retrograde perfusion of the isolated whole heart with heparin resulted in a release of LPL into the coronary perfusate (Figure 4). The heparin-induced LPL discharge was rapid, and peak 77 activity that appeared within 2 minutes was suggested to represent LPL located at or near the endothelial cell surface. On continuous perfusion with heparin, there was a progressive decline in L P L activity to values close to baseline. At 7-8 weeks, heparin-releasable L P L activity in SHR rat hearts was comparable to values obtained from W K Y animals (Figure 4A, inset). However, with increasing age and blood pressure, a decline in peak and total heparin-releasable LPL activity was observed in SHR rats relative to W K Y animals (Figure 4B and 4C, insets). 4.1.1.3. Cardiac myocyte LPL LPL is synthesized in cardiac myocytes, and then translocated to the coronary endothelium where it hydrolyzes circulating lipoprotein-TG. To determine whether the decline in cardiac heparin-releasable LPL activity in SHR rats is a consequence of alterations at the site of synthesis, cardiac myocytes from SHR and W K Y rats were isolated at different ages, and surface bound and intracellular LPL activities were measured. There was no difference in myocyte viability (% live cells) between the W K Y and SHR rats at 7-8 (WKY 79±3; SHR 81±1, n=9 in each group) or 15-16 ( W K Y 83+1, n=7; SHR 81±2, n=8) weeks of age. Similarly, myocyte yield (total number of cells x lO 6 ) at 7-8 (WKY 1L+1.5; SHR 9.210.6) or 15-16 ( W K Y 9.3±1; SHR 8.8±0.9) weeks of age was similar in W K Y and SHR rats. No difference in total cellular L P L activity was observed in SHR and W K Y rats at 7-8 weeks of age (Figure 5A, left panel). However, at 15-16 weeks, myocytes from SHR rats had higher total cellular L P L activity than cells from W K Y rats (Figure 5B, left panel). The increase in cellular L P L activity was not accompanied by a parallel rise in the secretion of enzyme. At both 7-8 (Figure 5A, right panel) and 15-16 weeks (Figure 5B, right panel) of age, there was no difference in heparin-induced release of L P L from cardiac myocytes isolated from SHR and W K Y rats. 78 4.1.1.4. Insulin and LPL activity Based on reports that: a) insulin can release LPL from its binding sites on the surface of cultured 3T3-L1 adipocyte cells (Chan et al 1988), and b) the presence of hyperinsulinemia in SHR rats (Reaven and Chang 1991, Verma et al 1994), we hypothesized that the decrease in endothelial LPL activity in SHR rats could be a result of elevated insulin levels in these animals. However, our current results indicate that 5-hour fasted plasma insulin levels in SHR and W K Y rats were similar at all time points measured (Table 1). Moreover, retrograde perfusion of isolated rat hearts from 15-16 week old W K Y and SHR rats with insulin failed to release LPL activity into the coronary perfusate (Figure 6). 4.1.1.5. Acute effects of vasodilators Nifedipine treatment in vivo effectively reduced the blood pressure of 15-16 week old SHR rats (108±6 mm Hg, n=5) as compared to pre-treatment values (199±9 mm Hg, n=5) within 30 minutes. This effect lasted over 2 hours, at which point blood pressure returned to pre-treatment values. Nifedipine had no effect on the blood pressure of 15-16 week old W K Y rats. Figures 7A and 7B demonstrate the effects of nifedipine on heparin-releasable LPL activity in 15-16 week old W K Y and SHR rats. Perfusion of isolated hearts from W K Y rats with nifedipine did not change heparin-releasable LPL activity (Figure 7A). However, nifedipine administered either in vitro for 10 min or in vivo for 30 min prior to removal of the heart increased peak heparin-releasable L P L activity in SHR rat hearts (Figure 7B). A similar increase in heparin-releasable LPL activity was observed in 15-16 week old SHR rat hearts on prior perfusion for 10 min with CGS-21680, a coronary vasodilator, in vitro (Figure 8). In vivo treatment of SHR rats with CGS-21680 could not be performed due to its extremely short duration of action. 79 Nifedipine and CGS-21680 by themselves had no effect in releasing cardiac endothelial LPL (data not shown). 4.1.1.6. Secretion and clearance of circulating TGs and plasma lipolytic activity In this study, SHR of all age groups were found to have lower plasma TG levels when compared to age matched W K Y controls (Table 1). Therefore, we measured TG clearance rates (TGCR) in 7-8 week and 15-17 week age groups. As shown in Figure 9, at 7-8 weeks the TGCR of SHR is not different from W K Y . At 15-17 weeks, the TGCR of SHR appear to be less when compared to W K Y . However, post-heparin plasma L P L activity in 15-17 week SHR rats was not different from W K Y rats (Figure 10). Preliminary experiments suggest that T G secretion rate in SHR is less compared to W K Y at 15-17 weeks of age ( W K Y = 1.43 mg/ml/min, SHR = 1.09 mg/ml/min, values are averages of two experiments). o oo o o vo IT) c« wo « •i-i c a* t« t2 w S3 <U -4-1 £ "a Tj C o u « u 73 >* o x/i CO 00 I to vo +1 00 co co * +1 uo oo +1 CO Ov CN CN w VO +1 CO oo CN 00 +1 VO 00 o co CO +1 VO 00 60 43 '5 O PQ VO O CM —< vo * OV +1 Ov Ov CO —< 00 vo +1 OV o\ * +1 IT) +1 oo CN •<* +1 ON CN 00 X £ £, PQ i n CN © +1 vd CO o +1 vd CM © +1 Ov VO CO d +1 CO CN © +1 1(0 —< >o w CN d +1 oo vd 0) CO O O a cd co cd vo w IT) +1 IT) CO CO CO +1 Ov IT) +1 CN i n vo +1 oo I/O CO +1 vo VO CO +1 =s CO e HH cd £ co cd T-H T-H OV © © © d d © +1 +1 +1 I/O VO © vq d - < © VO * * * CN © © © d d d +1 +1 +1 CN Ov Ov oo © © © CO IT) VO CN CO © — < © © © © +1 +1 +1 —H CN T-H 00 d - < d vo *—» VO 00 * * " — ' CO CO © o © d d d +1 +1 +1 CO Ov 00 © d d a H cd a CO cd O 4=1 U cd s CO cd P h cd CO cd V OH CO e 3 U cd o o <u t-l co <U "cd > o cu CO <U CO c u OH CO cd < 4 H O 1 <u ^ H w 00 +1 co c cd c3 3 oo <D 1-0) 4=1 H •5 o > I 13 81 Figure 4. Effect of hypertension on heparin releasable LPL activity in perfused hearts from W K Y and SHR rats at 7-8 (A), 11-12 (B) and 15-16 (C) weeks of age. Rats were perfused with Joklik as described in the methods. At the time indicated by the arrow, heparin (5 U/ml) was added to the buffer, and L P L activity measured in the coronary perfusate which was collected for 10 seconds, at the indicated times. The insets represents area under the curve for heparin-releasable LPL activity in perfused hearts from W K Y and SHR rats. Results are the mean + S E M from the number of experiments indicated in parentheses. Changes in LPL activity in response to heparin, over time, were analyzed by multivariate analysis of variance followed by the Newman-Kern's test; *,p < 0.05 relative to W K Y . A. 7-8 weeks 700 -600 -500 -400 -300 -200 -100 -0 -B. 11-12 weeks 700 - i 600 -500 -400 -300 -200 -100 -0 -0 2 I 4 0 - • - SHR(n=6) - 0 - WKY(n=6) T 8 10 - • - SHR(n-5) - 0 - WKY(n=5) " I 1 1 1 4 6 8 10 2700 - i 5^ 1800 O < 900 H 2700 ^1800 H O < 900 H Mil C. 15-16 weeks 700 - . 600 -500 -400 -300 -200 -100 -0 -4 - • - SHR(n=6) - 0 - WKY(n=3) T " 6 0 2 Perfusion time (min) 8 ~ l 10 2700 - i * T ^ 1800 -O < 900 - • o - • \ Figure 4 83 Figure 5. Effect of hypertension on LPL activity in cardiac myocytes from SHR and W K Y rats at 7-8 (A) and 15-16 (B) weeks of age. Myocytes were prepared as described in the methods. L P L activity in cell homogenates was determined at time zero by removing a sample of cell suspension followed by centrifugation, homogenizing of the cell pellet and determination of cellular LPL activity (left panel). Heparin (5 U/ml) was then added to the incubation medium of the homogenized cells at the time indicated by the arrow and the release of LPL activity into the incubation medium determined at the indicated times of incubation (right panel). Results are the mean ± S E M from the number of experiments indicated in parentheses. Probability values were calculated by a Student's t-test; *,p < 0.05 relative to W K Y for cellular activity at 15-16 weeks. A. 7-8 weeks 3000 H 2500 2000 1500 -1000 -500 -0 C E L L S M E D I U M h 600 W K Y S H R 0 B. 15-16 weeks h 400 200 SHR(n=9) G - WKY(n=9) 0 10 20 30 40 50 60 0 3000 -2500 -2000 -1500 -1000 -500 0 C E L L S M E D I U M h 600 a W K Y S H R 0 400 h 200 SHR (n=8) WKY (n=7) 10 20 30 Incubat ion t ime (min) 50 0 60 o o o CO 0 S Figure 85 Figure 6. Lack of an acute effect of in vitro insulin on the release of L P L from isolated perfused hearts of 15-16 week old W K Y and SHR rats. Various concentrations of insulin were added to the perfusion fluid, and samples of coronary perfusate collected for 10 seconds, at the indicated times. Results depicted show a representative experiment out of three. 86 700 - i 600 S H R W K Y 500 H 2 400 H Ins 100 ng/ml Ins 200 ng/ml Ins 500 ng/ml 300 H 200 H 100 H o H " T 0 j - e 10 ~ T ~ 20 30 Time (min) Figure 6 87 Figure 7. The effect of nifedipine in vitro and in vivo on heparin releasable LPL activity in perfused hearts from A) W K Y and B) SHR rats at 15-16 weeks of age. For the in vitro experiments, hearts from 15-16 week old W K Y and SHR rats were removed and perfused with recirculating Joklik buffer containing nifedipine (100 nM) for 10 minutes. This was followed by perfusion with heparin, with fractions of perfusate collected and analyzed for LPL activity as described previously. For the in vivo study, nifedipine (3 mg/kg) was injected into the tail vein. 30 minutes following nifedipine injection, the rats were killed, the hearts removed and perfused with heparin and the perfusate collected and analyzed for LPL activity. Results are the mean ± SEM from the number of experiments indicated in parentheses; groups are compared by using multivariate A N O V A followed by Newman-Keul's test, p < 0.05 relative to untreated SHR. A. WKY Q5-16 WEEKS') Figure 7 89 Figure 8. Effect of in vitro treatment with CGS-21680 on heparin releasable LPL activity in perfused hearts from SHR rats at 15-16 weeks of age. Hearts were perfused for 10 minutes with 10 pM CGS-21680 in Joklik buffer followed by heparin perfusion. The fractions were collected at the indicated times and LPL activity measured. Results are the mean ± SEM; multivariate ANOVA followed by Newman-Keul's test was used to determine the probablity value, *,p < 0.05 relative to untreated SHR. S H R (15-16 weeks) Figure 91 Figure 9. Plasma triglyceride (TG) clearance over a 60 minute following heparin injection (see Methods section for details) for 7-8 weeks (A), and 15-17 weeks (B) 15-16 weeks old SHR and W K Y and SHR nifedipine (SHRN) treated rats are shown. Heparin (0.5 U/g body weight) was injected into the tail vein of pentobarbitol-anesthetized rats and the blood samples were collected at 0, 1, 2, 5, 10, 30, and 60 minutes, plasma was separated, and TG concentration was measured in each sample. The error bars represent standard error of means. Multivariate A N O V A followed by Newman-Keul's test was used to determine the probablity value, * Significantly different from W K Y rats (p<0.05) and + Significantly different from untreated SHR (p<0.05). 92 Figure 9 93 Figure 10. Post heparin plasma lipase activity of 15-17 week old WKY and SHR rats. Heparin (0.5U/g body weight) was injected into the tail vein of pentobarbitol-anesthetized rats and the blood samples were collected at 0 and 20 minutes, plasma was separated, and LPL activity was measured as a difference between total lipase and hepatic lipase activities (See methods for details). The error bars represent standard error of means. Figure 10 95 4.1.2. LPL regulation in fructose hypertensive rat hearts 4.1.2.1. General Characteristics Feeding fructose to Wistar rats, as a 10% solution in drinking water reduced food intake, with a corresponding augmentation in fluid intake (Table 2). Weight gain over the acute and chronic treatment periods was the same in both groups such that control and fructose-treated rats had similar body weights at the time of death (Table 2). Whereas fructose feeding did not affect plasma glucose concentrations (Table 2), plasma insulin (Figure 11A), and triglycerides (Figure 1 IB) were elevated in the acute and chronically treated rats. Blood pressure in fructose-treated rats increased progressively, and was significantly different from the control group after 2 weeks of treatment (Figure 11C). Prolonged fructose treatment for up to 6 weeks did not produce an additional increase in blood pressure (Figure 11C). Moreover, withdrawal from fructose after a 6 week treatment period restored all of the above parameters to control values within 2 weeks (Figure 11 A , B, & C). 4.1.2.2. Coronary endothelial LPL Retrograde perfusion of isolated whole hearts from control and fructose-treated rats with heparin resulted in a rapid release of LPL into the coronary perfusate (Figure 12). At 2-weeks after fructose treatment, there was no difference in heparin-releasable LPL activity between the control and treated groups (Figure 12A). However, with prolongation of fructose treatment, a significant reduction in heparin-induced release of LPL activity was observed (Figure 12B). This decrease in LPL activity in chronically-treated rats could be reversed by a 2-week withdrawal from fructose treatment (Figure 12C). 96 4.1.2.3. Myocyte surface and intracellular LPL activity We measured LPL activity in the isolated cardiac myocytes from chronically-treated rats to verify whether the decrease in functional LPL was due to a decreased synthesis at cardiac myocytes. It appears neither myocyte surface LPL released by heparin over a 1-hour period, nor intracellular LPL activity (measured in cell sonicate) was decreased in fructose treated rats (Figure 13 A & B). This suggests that the decrease in functional LPL at coronary endothelium is probably due to factors other than defective synthesis at cardiac myocytes. 4.1.2.4. Effects of in vitro insulin and vasodilators on endothelial LPL activity Fructose treatment induces hyperinsulinemia within 2 weeks in Wistar rats (Figure 11A). Since in vitro insulin incubation was shown to release LPL from 3T3-L1 adipocytes (Chan et al 1988), we hypothesized that the decrease in endothelial LPL activity could be a result of greater release of LPL by elevated insulin levels in these animals. However, our current results indicate that retrograde perfusion of isolated whole hearts from control rats with insulin failed to release L P L activity into coronary perfusate (Figure 14). Figure 15 demonstrates the in vitro effects of nifedipine, and CGS on heparin-releasable L P L activity in control and chronic fructose-treated rats. Perfusion of isolated hearts from control rats with nifedipine or CGS did not change the amount of heparin-releasable LPL activity (Figure 15A). However, nifedipine administered for 10 min increased the basal, and heparin-releasable L P L activity in fructose-treated rats (Figure 15B). A similar increase in heparin-releasable LPL activity was observed in chronic fructose-treated rat hearts on prior perfusion of these hearts with CGS-21680 (Figure 15B). 97 As L P L is the rate-limiting enzyme in TG clearance, a decrease or dysfunction in LPL activity often parallels a reduced catabolism of TG-rich lipoproteins that results in hypertriglyceridemia. To test whether the reduction in LPL activity that we observed in the heart is a generalized phenomena and a potential contributing factor towards the elevated plasma triglycerides, pre- and post-heparin plasma LPL activity was determined from control and chronic fructose-treated rats. Figure 16 shows that both basal and post-heparin plasma LPL activity were reduced in the fructose treated rats. 98 Table 2. General features of acute and chronic fructose treated rats. Acute Fructose Tx a Chronic Fructose Tx a Control Fructose Control Fructose Body weight (g) 329±9 322±14 483±15 489±13 (6) (12) (17) (21) Food intake (g/day) 31±1 21±1* 30±1 15±1* (6) (12) (5) (15) Fluid intake (ml/day) 56±1 104±3* 64±1 116±4* (6) (6) (6) (12) Plasma glucose (mM) 6.1±0.2 6.9±0.1 7.6±0.6 6.9±0.2 (6) (6) (6) (12) a) Fructose Tx = Fructose treated. The results are the mean ± S E M for the number of animals shown in parenthesis. Values are those obtained before death. The acute treatment was for 2 weeks whereas the chronic treatment was for a duration of 4-6 weeks. 99 Figure 11. Plasma insulin (A), triglyceride (B), and blood pressure (C) of acute (2 weeks) and chronic (4-6 weeks) fructose treated rats. As no differences in metabolic or biochemical parameters were observed between the 4 and 6 week fructose-treated animals, data from these rats were pooled, and this group was hence defined as the chronic treated group. To determine if the effects of fructose are reversible, treatment of 6 week fructose-treated rats was terminated. These rats (fructose-withdrawn) were subsequently maintained for an additional 2 weeks, after which they were killed. Plasma samples were separated from tail vein blood collected from 5-hour fasted animals. Results are expressed as mean ± SEM. Probability values were calculated by ANOVA. * Significantly different from control, p< 0.05. 100 101 Figure 12. Heparin releasable LPL activity in perfused hearts from rats treated acutely (A) or chronically (B) with fructose (Fructose Tx). Panel C represents animals that were withdrawn from fructose treatment for 2 weeks after chronic treatment. Rats were perfused with Joklik MEM as described in the methods. At the time indicated by the arrow, heparin (5 U/ml) was added to the buffer, and LPL activity measured in the coronary perfusate which was collected at the indicated times. Results are the mean ± SEM from the number of experiments indicated in parentheses. Changes in LPL activity in response to heparin, over time, were analyzed by multivariate ANOVA followed by the Newman-Keul's test. *,p < 0.05 relative to control. ~~I A. Acute treatment (2 weeks) 102 600 H 400 H 200 H 0 g 800 o E s a u 600 H 400 H 200 H - J PH -2-0 0-2 2-4 4-6 6-10 B. Chronic treatment (4-6 weeks) 0 800 - i 600 400 H 200 0 -2-0 0-2 2-4 4-6 6-10 C. Fructose-withdrawn -2-0 0-2 2-4 4-6 6-10 Time (min) Control (n=4) Fructose (n=5) Control (n=7) Fructose (n=10) Control (n=7) Fructose (n=6) Figure 12 103 Figure 13. A. Cell surface LPL activity on isolated cardiac myocytes from chronically fructose treated (4-6 weeks) rats. To the cell suspension (0.4 x 106 cells), heparin (5U/ml) was added to release surface bound LPL. 1.0 ml fractions were removed from the homogenously suspended incubation medium at indicated time points after the addition of heparin. Medium was separated by centrifugation and the cell pellets were saved for intracellular LPL measurements. B. Intracellular LPL activity measured in cell sonicates of the cell pellets collected above at 0 and 10 minutes after heparin incubation. A . C e l l Surface LPL 104 Figure 13 105 Figure 14. Effect of increasing concentration of insulin (100, 200 and 500 ng/ml) and heparin (5 U/ml) perfusion on the release of LPL from isolated hearts of control Wistar rats. Retrograde perfusion was carried out as described in the methods. Results correspond to a representative experiment out of 3. 106 Time (min) Figure 14 107 Figure 15. In vitro effects of nifedipine and CGS-21680 on heparin releasable LPL activity in perfused hearts from control (A) and chronic fructose-treated (B) rats. Hearts were removed and perfused with recirculating Joklik buffer containing nifedipine (100 nM) or CGS-21680 (10 pM) for 10 minutes. This was followed by perfusion with heparin, with fractions of perfusate collected and analyzed for LPL activity as described previously. Results are the mean ± SEM from the number of experiments indicated in parentheses. Changes in LPL activity in response to heparin, over time, were analyzed by multivariate ANOVA followed by the Newman-Keul's test. , p < 0.05 relative to untreated fructose group. 108 9 0 0 1 A. Control 08 u 600 e 300 H S "o s e 0 Untreated (7) + Nifedipine (5) + CGS (5) -900 - i B. Fructose 600 H 300 0 Untreated (7) + Nifedipine (6) + CGS (4) Time (min) Figure 15 109 Figure 16. The effect of heparin (0.5 U/g) injection on the release of LPL in control and chronic fructose treated rats. After collection of a blood sample for basal values, heparin was injected into the tail vein and blood samples collected after 5 minutes. Blood was centrifuged and plasma separated and used for the measurement of post heparin LPL. Results are the mean ± SEM from the number of experiments indicated in parentheses. Probability values were calculated by a Student's t-test; *, P < 0.05 relative to control. 110 300 250 -A 200 H 150 H Control (n=4) Fructose (n=5) 20 -I 15 10 5 H 0 X 0 min 5 min Figure 16 I l l 4.2. Diabetes s tudy 4.2.1. LPL regulation in the diabetic rat hearts 4.2.1.1. General Characteristics of diabetic rats Induction of diabetes with 55 mg/kg STZ resulted in glycosuria (>4+). Body weight gain over 2 weeks was reduced in D55 animals relative to controls, but there was no significant difference in heart weight between control and diabetic rats (Table 3). The STZ injection caused a reduction in plasma insulin levels that was accompanied by hyperglycemia. Both plasma TG and cholesterol levels were elevated in D55 rats (Table 3). 4.2.1.2. Modified Langendorff Perfusion Retrograde perfusion of hearts from CON and 2-week D55 rats with heparin resulted in release of LPL activity into the coronary perfusate that was collected via the cannulated pulmonary artery (Figure 17A). This heparin-induced LPL discharge was rapid, and peak activity in both groups was observed within 1.5 min. On continuous perfusion of these hearts with heparin, LPL activity returned to near basal levels. In D55 hearts, peak LPL activity in coronary perfusate was 3-4 fold higher than CON. To confirm that the elevated lipase activity was specific to LPL, the assay was performed in the presence of 1M NaCl and in the absence of apoCII, and under these conditions, peak activity from CON and D55 was inhibited to 10-15% of the total lipase activity measured above (CON = 253 [+LPL]; 29 [-LPL], D55 = 889 [+LPL]; 154 [-LPL] nmol/ml/h). The increase in LPL activity in the coronary perfusate of D55 rats was not due to an increase in specific activity of the protein but was a consequence of a 4-fold increase in LPL protein concentration as measured by ELISA (Table 4). Following heparin, the release of LPL activity into the interstitial fluid was clearly different from that observed in coronary 1 112 perfusate (Figure 17B). Initially, the enzyme released from CON hearts was low, but gradually increased over time. In D55 hearts, a peak release of enzyme was observed within 1-2 min followed by gradual decline such that after 10 min, it was lower than CON. Acute treatment of D55 rats with a rapid-acting insulin reduced hyperglycemia within 90 minutes (0 min = 18.1+1.2, 90 min = 5.6±0.6 mM glucose). In these insulin-treated rats, heparin-releasable LPL activity in the coronary perfusate was reduced, but remained higher than control (Figure 17A). Although insulin had no effect on the prolonged release of LPL into the interstitial fluid, the initial peak release observed in untreated diabetic rats was blunted by insulin treatment (Figure 17B). 4.2.1.3. LPL Activity in whole heart homogenate LPL regulation in the heart during diabetes was inconsistently reported in the literature. The variability could be partly due to the experimental approach that did not distinguish various LPL pools i.e., endothelial, interstitial, and myocyte-associated LPL pools. Therefore, we determined total LPL activity in heart tissue homogenates. Indeed, we have shown that LPL activity in whole heart homogenates was not different between CON and D55 groups (Figure 18) despite a 4-fold increase at coronary endothelium (Figure 17A). 4.2.1.4. Immunolocalization of LPL Immunohistochemical studies of myocardial sections were performed to complement our observation that the augmented LPL in diabetic hearts was mainly localized at the endothelial cells. Although the results are difficult to quantify, this technique provides information regarding cellular localization of the LPL protein within the cardiac tissue. Whereas LPL immunoreactivity was found throughout the control and diabetic myocardium (brown stain), 113 capillary blood vessels in the diabetic (Figure 19B) demonstrated a more intense LPL immunoreactivity compared to control (Figure 19A). Staining was not seen when the primary antibody was omitted or replaced by preirnmune chicken serum (data not shown). 4.2.2. Acute L P L regulation by hypoinsulinemia 4.2.2.1. Insulin Depletion Study Induction of diabetes with 100 mg/kg STZ produced the characteristic triphasic pattern of changes in blood glucose and insulin in the 24-hour period immediately following injection (Figure 20). An initial brief hyperglycemia was followed by a period of hypoglycemia that is brought about by a release of insulin from damaged (3-cells. Blood glucose rose gradually with a corresponding decline in plasma insulin, and hyperglycemia (>13 mmol/L) was usually attained within 12-16 hours. Interestingly, even at this early stage of diabetes, peak (Figure 21) and total (calculated as area under the curve over 10 min, Figure 21-inset) heparin-releasable LPL activity in the coronary perfusate was increased when compared to control rats. Prolonging the hyperglycemia for a further 3 or 6 hours (after glucose levels reached >20 mmol/L) maintained the elevated enzyme activity at the coronary lumen (Figure 21). 4.2.2.2. Insulin Withdrawal Study Treatment of D100 rats for 1 week with a long-acting insulin resulted in an increase in body weight and a normalization of fluid intake (Figure 22), plasma glucose and insulin (Figure 23) levels. Subsequent withdrawal of insulin produced an increase in plasma glucose from 24 hrs following the last injection. On reaching a glucose concentration of 13 mmol/L, diabetic animals were kept for a further 6 (D100-6h) or 24 (D100-24h) hours before they were killed. At termination, both the D100-6h ahd D100-24h groups were hyperglycemic and hypoinsulinemic 114 (Figure 23). As observed with the insulin depletion study, even a brief duration of 6 hours of hyperglycemia produced an increase in peak heparin-releasable LPL activity in the coronary perfusate that remained elevated after 24 hours of hyperglycemia (Figure 24). Total LPL activity was similarly high at these time points (Figure 24 inset). To examine whether the enhanced coronary LPL activity in D100-24h rats was accompanied by a parallel increase in myocyte LPL, isolated myocytes were incubated in the presence of heparin to measure both surface-bound and secreted LPL. There was a significant reduction in heparin-releasable LPL from cardiac myocytes (Figure 25) and a decrease in LPL activity in cell sonicates (Figure 25-inset) from D100-24h hyperglycemic rats compared with CON. 4.2.2.4. Fasting Fasting is known to increase cardiac heparin releasable LPL activity without affecting mRNA levels or protein synthesis. In the present study, overnight fasting for 16 hours reduced plasma insulin to diabetic levels (CON 2.1±0.1, FAST 0.5±0.0 ng/ml), with no effect on plasma glucose (CON 7.0±0.1, FAST 6.0±0.1 mmol/1). Fasting caused a two-fold increase in heparin-releasable LPL activity at the coronary lumen (Figure 26A). However, unlike in acute diabetes, overnight fasting had no effect on the release of enzyme into the interstitial fluid (Figure 26B). 115 Table 3. Characteristics of Diabetes (55 mg/kg STZ) at 2 Weeks after STZ Injection Control STZ-Injected Body Weight (g) 373+6 345±6* Heart Weight (g) Plasma Insulin (ng/ml) 1.1510.02 2.1710.25 0.9910.02 0.8410.07* Plasma Glucose (mM) 7.010.1 15.810.4* Plasma Triglyceride (mM) 1.7910.11 3.6810.41* Plasma Cholesterol (mM) 1.5410.05 2.1110.10* Data are means 1 SEM for the 6-8 control and diabetic rats. Values are those obtained before death. Plasma parameters were from fed rats. Blood was collected from the tail vein in heparinized tubes that were centrifuged for the separation of plasma. Insulin was measured using rat insulin standards. Probability values were calculated by a Student's t-test; * Significantly different from control, PO.05. 116 Figure 17. Effect of chronic diabetes (2 weeks) on heparin-releasable L P L activity in coronary perfusate (A) and interstitial fluid (B) from CON and D55 rat hearts. Hearts were perfused (7-8 ml/min) with heparin (5 U/ml) in Krebs-Ringer Hepes buffer using a modified Langendorff retrograde perfusion technique that separates the coronary perfusate from interstitial fluid. The coronary perfusate samples (for 10 seconds) were collected at the indicated time points. As interstitial fluid represents only 1-2% of the heart perfusate, samples were collected over 60 seconds, at the indicated time points. Coronary and interstitial LPL activities were also measured in insulin treated (90 minutes before killing) D55 rats. Results are the mean ± S E M for 6-8 rats in each group. Probability was calculated using multivariate A N O V A followed by Newman-Keul's test, *Significantly different from control and insulin treated D55 rats, P<0.05. A 8 0 0 - i 117 6 0 0 4 0 0 2 0 0 0 r 0 2 B 8 0 0 6 0 0 4 0 0 H 2 0 0 H 0 H n I i 8 1 0 1 2 A O CON (n=8) D55 (n=6) D55 +1 (n=6) n i i 1 1 1 •1-0 1-2 3-4 5-6 7-8 9 - 1 0 Perfusion time (min) Figure 17 118 Table 4. LPL Activity, Mass and Specific Activity in Coronary Perfusate From Hearts Isolated From 2 Week Control and D55 Rats. Rats Activity (mU) Concentration (ng/ml) Specific Activity (mU/ng) CON 4.97±0.54 5.7±1.0 0.87+0.15 D55 21.24±1.99* 22.3+5.3* 0'.95±0.05 Measurement of LPL catalytic activity (by in vitro hydrolysis of [3H]triolein) and mass (by enzyme-linked immunosorbent assay) were done in pooled coronary perfusate samples (24 ml) collected between 1-3 min after heparin perfusion. For the ELISA, coronary perfusate samples were lyophilized and resuspended in 0.2 ml H2O. 100 pl aliquots of the samples were then diluted in PBS and added to the polystyrene microtiter plate wells coated with 100 pl of anti-LPL antibody. 1 mU is defined as the amount of enzyme catalyzing the release of 1 nmol oleate per min. Results are the mean ± SEM of 6-8 control and diabetic rats. Probability values were calculated by a Student's t-test; * Significantly different from control, P<0.05. 119 Figure 18. LPL activity in heart homogenates. Hearts from CON and D55 were frozen immediately in liquid N2, after clearing the blood of the capillaries. Tissues were homogenized in the presence of heparin and the clear supernatant was separated from the tissue debris by centrifugation (see Methods section for details). Protein concentration was measured in the supernatant by Bradford-protein assay. LPL activity was normalized for gram protein present in the samples and expressed as mU/g, where mU = nmoles FFA released/ml/min. Numbers in parantheses indicate the number of hearts used in each group. 120 Figure 18 121 Figure 19. Representative photograph showing the immunocytochemical localization of LPL following chronic diabetes induced with 55 mg/kg STZ. Cross sections of control (A) and diabetic (B) ventricles were fixed and then immunolabeled using an antibody specific for LPL. Low magnification image shows immunostaining over the entire control and diabetic sections. However, in diabetic hearts, numerous capillaries were heavily stained for LPL (arrow, B) in contrast to the observations in control heart sections (A). A. CONTROL 12 Figure 123 Figure 20. The chronological changes in plasma insulin (left panel) and glucose (right panel) levels during a 24-hr period following an IV injection of 100 mg/kg STZ. The results from three individual animals are shown. 124 0 4 8 12 16 2 0 2 4 0 4 8 12 16 2 0 2 4 T i m e after S T Z inject ion (hr) Figure 20 125 Figure 21. Peak heparin-releasable LPL activity in coronary perfusate at different time points following injection of STZ. The lower panel demonstrates peak heparin-releasable LPL activity in coronary perfusate from control and diabetic rats following the development of hyperglycemia. Following STZ, rats usually developed hyperglycemia (13 mmol/L) within 12-16 hours. Some animals were killed at this time (13mM+0h) or kept for a further three (13mM+3h) or six (13mM+6h) hours before being used for the measurement of LPL activity. Hearts were perfused with Krebs buffer containing heparin using the modified Langendorff procedure. The inset shows the AUC of LPL released over 10 minute period. Results are the mean ± SEM for 6-8 rats in each group. Probability values were calculated by ANOVA, * Significantly different from control, P<0.05. 126 900 -i A U C L P L mU/10 min Control 13.6 ± 1.6 13 mM-0h 19.8 ±0.8* 13 mM-3h 21.0 ± 1.3* 13 mM-6h 29.4 ± 1.9* 13 m M - O h 13 m M - 3 h 13 m M - 6 h Duration (h) of hyperglycemia (13 mM) following injection of STZ Figure 21 127 Figure 22. Effect of insulin treatment in STZ diabetic rats. Animals were made severely diabetic with 100 mg/kg STZ (D100), and then treated subcutaneously with a long-acting Ultralente insulin (D100+I) once daily. An insulin dose of-18-20 U/kg/day was required to maintain normoglycemia for at least a 24-hour period. Treatment was continued for 7 days. Body weight (A) and fluid intake (B) were measured before injecting insulin. Results are the mean ± SEM for 6-8 rats in each group. Figure 22 129 Figure 23. Plasma glucose (A) and insulin (B) levels of control (CON) and diabetic rats following withdrawal of exogenous insulin administration. After the diabetic animals had been well-controlled (D100+I), insulin treatment was stopped, and plasma glucose monitored. Following the last insulin injection, plasma glucose usually started to increase after 24 hours. A plasma glucose concentration of 13 mmol/L was considered a hyperglycemic value, and when this level was reached, diabetic animals were kept for a further 6 (D100-6h)or 24 (D100-24h) hours before they were killed. Results are the mean ± S E M for 6-8 rats in each group. Probability was calculated using A N O V A , * Significantly different from control and insulin-treated diabetic groups, PO.05 . 130 2 0 - , 4 -i 3 CO C 1 H o CON X D100+I D100-6h D100-24h Duration of hyperglycemia following withdrawal from insulin (h) Figure 23 131 Figure 24. Peak heparin-releasable LPL activity in coronary perfusate from control (CON) and diabetic rats following withdrawal of insulin treatment. Enzyme activity in diabetic (100 mg/kg STZ) rats was measured after the animals had been well controlled with insulin for 7 days (D100+I) or following 6 (D100-6h) or 24 (D100-24h) hours of hyperglycemia after insulin injection was stopped. Hearts were perfused with Krebs-Ringer-HEPES buffer containing heparin using the modified Langendorff procedure. Results are the mean ± SEM for 6-8 rats in each group. Probability values were calculated by ANOVA; * Significantly different from control and insulin-treated diabetic groups, P<0.05. 132 AUC of LPL (mU/10 mini CON D100 +1 1)100-6 h D100-24h 13.6 +/-1.6 7.2 +/- 1.2 23.3 +/- 3.4* # 23.7 +/- 4.5* # 9 0 0 - i 6 0 0 H © £ s </> i-a I 3 0 0 S3 -J PH -J -J: 83 P* 0 CON D100+I D100 CON D100+I D100-6h D100-24h Duration of hyperglycemia following withdrawal from insulin (h) Figure 24 133 Figure 25. LPL activity in cardiac myocytes from control (CON) and 24 hour hyperglycemic rats following insulin withdrawal (D100-24h) are shown. Myocytes were prepared as described in METHODS. Heparin (5 U/ml) was added to the incubation medium at the time indicated by the arrow and the release of surface-bound LPL activity into the medium determined at the indicated times of incubation. Results are the mean ± SEM for 6-8 rats in each group. Probability values were calculated by multivariate ANOVA. Inset shows intracellular LPL activity from CON and D100-24h groups. Probability values were calculated by Student's t-test. *Significantly different from control, PO.05. 134 Figure 25 135 Figure 26. Effect of fasting on heparin-releasable LPL activity in coronary perfusate (A) and interstitial fluid (B). Control rats were fasted for 16-hrs (1800-1000) during which food was withdrawn from the animals, but they had free access to water. Hearts were perfused (8 mL/min) with heparin (5 U/mL) in Krebs-Ringer Hepes buffer using a modified Langendorff retrograde perfusion technique that separates the coronary perfusate from interstitial fluid. The coronary perfusate samples (over 10 seconds.) were collected at the indicated time points. As interstitial fluid represents only 1-2% of the heart perfusate, samples were collected over 60 seconds at the indicated time points. Results are the mean ± SEM for 6-8 rats in each group. +Significantly different from control, PO.05. 136 Figure 26 137 4.2.3. H y d r o l y s i s o f V L D L i n the diabet ic heart 4.2.3.1. Plasma lipid profile and apolipoprotein analysis In fed control rats, 75% of total TG was present in TGRL (VLDL + CHYL), and the remaining 25% was present in LDL and HDL fractions (Figure 27A,B&C). VLDL contributed the least to the total cholesterol pool which was present predominantly in the HDL (61% of total cholesterol) and LDL (30%) fractions. Diabetic rats had higher levels of TG and cholesterol compared to control animals. When compared to CON, D55 showed higher concentrations of TG as well as cholesterol in their TGRL fraction. In both LDL and HDL, there was no difference in cholesterol and TG concentrations between CON and D55 groups indicating that the increase in TG levels in D55 is mainly due to an increase in TGRL. However, fasting for 16 hours eliminated this increase in TG (Table 5) suggesting that diabetic hypertriglyceridemia is largely a result of an increase in the concentration of chylomicrons. Apolipoproteins in VLDL obtained from fed CON and D55 rats were analyzed by gel electrophoresis, and a representative gel from a CON and a D55 rat is shown in Figure 33. Smaller ApoB (>200 kDa), ApoE (~ 35 kDa), ApoA-I (-28 kDa) and ApoCs (-8-9 kDa) were identified with the help of molecular weight standards (6.5 kDa to 220 kDa) that were electrophoresed in parallel lanes 1, 5, and 6 (Figure 33). A sample comparison of CON and D55-VLDL (Figure 33) exhibits no apparent difference in apolipoprotein distribution. 4.2.3.2. Post-heparin plasma lipolytic activity As LPL is the rate-limiting enzyme in TG clearance, a decrease or dysfunction in LPL activity often parallels a reduced catabolism of TG-rich lipoproteins that result in hypertriglyceridemia. To test whether the elevated plasma triglycerides in diabetic rats result 138 from a decrease in LPL activity, basal (before heparin injection) and post-heparin plasma was obtained from CON and D55 rats at 2 weeks after diabetes induction. Neither basal nor post-heparin plasma LPL activity was reduced relative to control at 2 weeks after diabetes induction (Fig. 28). 4.2.3.3. In vitro lipolysis of VLDL by bLPL or rLPL In preliminary experiments, we determined that the optimum enzyme concentration was 20 mU of bLPL and the saturable substrate (VLDL-TG) concentration was 0.3 mM when incubated for 30 minutes at 37°C (Figure 29A&B). Incubations for 2 hours indicate that VLDL-TG hydrolysis by bLPL was not different between CON and D55. However, D55-TGRL appeared to have a lower lipolysis towards the end of 2 hour incubation (data not shown). A double reciprocal plot of 1/[V] versus 1/[S] is shown in Figure 30A. The intercept on the Y axis gives V m a x whereas the X-axis intercept gives K m values for this lipolytic reaction. The apparent K m values for CON and D55-VLDL were 0.15 mM, and 0.26 mM, respectively, whereas V m a x values were 20.4 mU and 31.3 mU FFA released for CON and D55 VLDL, suggesting that diabetic VLDL was hydrolyzed as effectively as VLDL obtained from control animals. Similarly, K m and V m a x for TGRL hydrolysis by bLPL, were the same using lipids from CON and D55 rats (Figure 30B). As shown by Mamo et al (1992), in the present study, our preliminary results indicate that VLDL obtained from CON and D55 rats may not be different from each other. Given the possibility that the interaction between VLDL and rat LPL could be different from that of bovine LPL, we purified LPL from CON and D55 post-heparin plasma. Figure 31 is a representative elution profile of LPL and hepatic lipase (another major lipase released into plasma in response to intravenous heparin injection) indicating that hepatic lipase elutes at 139 approximately 0.8 M NaCl while LPL elutes at 1.4 M NaCl. Fractions enriched with CON and D55 LPL (>90%) were adjusted to have equivalent activities and subsequently used for in vitro lipolysis of VLDL-TG obtained from CON and D55 rats. Our preliminary results indicate that the lipolysis of diabetic VLDL-TG was not decreased (Figure 32), suggesting that at this duration of diabetes, either LPL or VLDL characteristics with respect to their enzyme-substrate interaction may not be different from each other. 4.2.3.4. 3H- VLDL clearance by isolated CON and D55 hearts We next attempted to assess whether the D55 heart could clear [3H]VLDL faster than CON hearts. Having established that control and diabetic VLDL were essentially similar, CON and D55 rats were only perfused with control [3H]VLDL. Figure 34 compares the rate of [3H]VLDL-TG clearance from re-circulating buffer containing [3H]VLDL-TG when perfused through CON and D55 hearts by Langendorff method. Metabolism of [3H]VLDL by the isolated CON heart was slow (net loss 17%; rate 0.50±0.17 nmol/min, n=4), but comparable to previously reported values (O'Looney et al 1983). In contrast to CON rat hearts, the rate of disappearance of VLDL-TG (over a perfusion period of 90 minutes) was more rapid from D55 rat hearts (net loss 44%; rate 0.93±0.23 nmol/min, n=4). It has been suggested that LPL can detach from the endothelium during lipolysis (Zambon et al 1997). To determine whether LPL can be released by recirculating VLDL, enzyme activity was also determined in the buffer chamber after 30 minutes of perfusion of unlabeled VLDL. Perfusion of control and diabetic hearts with unlabeled VLDL did not result in detachment of LPL from its binding sites (n=l from CON and D55 group, data not shown). The increased VLDL-TG hydrolysis was essentially abolished by prior perfusion of the diabetic heart with heparin, implicating LPL in this process (Figure 35, inset). In all of the 140 above groups, heart rate remained unchanged (above 150 beats per minute) throughout the perfusion period. 141 Table 5. Fed and fasted plasma parameters of control and diabetic rats Fed Fasted Control Diabetic Control Diabetic Glucose (mM) 8.2±0.17 27.96±0.83* 6.00±0.18 6.21±0.48 Triglyceride (mM) 0.95±0.20 1.63+0.21 0.37±0.04 0.35±0.01** Cholesterol (mM) 1.61±0.05 1.73±0.07 1.62±0.09 1.45±0.08 FFA (mM) 0.54±0.06 0.94±0.20 1.37±0.04** 1.20±0.08 Pl glu= plasma glucose, Pl TG = plasma triglyceride, Pl Choi = plasma cholesterol, Pl FFA = plasma free fatty acid. Results are average of 6 animals from each group. Probability values were calculated by A N O V A , * Significantly different from control P<0.05. ** Significantly different from fed groups PO.05. 142 Figure 27. TG and cholesterol concentration in A. TGRL, B. LDL and C. HDL fractions separated from the serum of fed control (CON) and diabetic (D55) rats. TG and cholesterol in different fractions were measured using appropriate diagnostic kits. Results are an average of six animals from each group, error bars indicate SEM. * significantly different from CON (P < 0.05) as determined by Student's t-test. A . T G R L 143 Figure 27 144 Figure 28. Post-heparin plasma LPL activity from CON and D55 rats. The LPL activity was determined as a difference between total lipase (LPL + HL) activity and HL activity (after LPL inhibition). mU = nmole FFA released/ml/min. 145 Figure 28 146 Figure 29. In vitro lipolysis of (A) V L D L (0.3 m M TG) versus bovine L P L (increasing concentrations of 10, 20, 50, or 100 mU) and (B) 20 mU of bovine L P L versus increasing concentration of V L D L - T G (0.1, 0.2, 0.3, 0.5, or 1.0 m M TG). Incubations were carried out for 30 minutes at 37°C and pH 8.2. (For details please see METHODS section). Results are average of n=2. 147 0.0 0.2 0.4 0.6 0.8 1.0 1.2 V L D L (mmol/1) Figure 29 148 Figure 30. Double reciprocal plot of CON and D55 (A) VLDL-TG, (B) TGRL concentrations (1/[S]) versus rate of lipolysis by LPL (1/V). 20 mU of LPL was exposed to various concentrations of VLDL-TG (fasted serum) and TGRL (fed serum) obtained from CON and D55 serum. Incubations were done at 37°C and at pH 8.2. Each data point is an average of triplicate values of one experiment. K m is the substrate (VLDL-TG) concentration at half maximal velocity, and V m a x is the maximum velocity of LPL activity. A . V L D L 149 -20 -15 -10 -5 0 5 10 15 20 25 B . T G R L -20 -15 -10 -5 0 5 10 15 20 25 1 / [ S ] ( m M 1 ) Figure 30 150 Figure 31. A representative elution profile of LPL and HL from heparin sepharose affinity columns. LPL elutes at higher salt concentrations (-1.4 M NaCl) and hepatic lipase (HL) at - 0.8 M NaCl. LPL activity in each fraction was determined as a difference between total lipase and H L activities. H L activity was measured by inhibiting LPL activity in the presence of 1.0 M NaCl and in the absence of apoCII. [1 mU = nmol/ml/min] 151 Figure 31 152 Figure 32. In vitro lipolysis of CON and D55 VLDL-TGs (0.3 mM) by rat LPL: (A) CON-LPL and by (B) D55-LPL. FPLC fractions enriched with LPL (>90%) was adjusted to 20 mU in CON and D55 samples. Other experimental conditions are as mentioned earlier. Results are average of two separate experiments carried out in triplicate. 153 A. CONTROL LPL 80 - i 60 4 0 H 20 H 0 CON D55 VLDL PH B. DIABETIC LPL 80 60 H 40 H 20 H 0 CON D55 VLDL Figure 32 154 Figure 33. (A) SDS-gradient polyacrylamide gel electrophoresis comparing apolipoproteins of CON and D55-TGRL (obtained from fed rats). Lanes 1, 5, and 6 are molecular weight standards. Lanes 2, 3, and 4 are D55-TGRL (triplicate) and lanes 7, 8, and 9 are CON-TGRL (triplicate). TGRL samples from C O N and D55 were adjusted for their protein content (3.2 mg/ml) and equal volumes were loaded into the wells. SDS-PAGE was performed three times using different samples. 155 SDS-Gradient gel electrophoresis of VLDL Diabe t i c C o n t r o l 1 2 3 4 5 6 7 8 9 Figure 33 156 Figure 34. In vitro lipolysis of 3 H V L D L - T G (obtained from C O N group) by isolated-perfused hearts from C O N and D55 rats. Hearts were perfused with Krebs-Ringer-HEPES buffer containing 3 H - V L D L via a recirculating Langendorff retrograde perfusion mode for 90 minutes. Perfusate was collected at 0, 15, 30, 45, 60 and 90 minutes, extracted for T G and the radioactivity was counted. Results are the average of 4 animals in each group. Error bars represent SEM. * Significantly different from C O N (P < 0.05) as determined by multivariate A N O V A . Inset shows absence of lipolysis of V L D L - T G in diabetic heart that was pre-perfused with heparin for 5 minutes (n=l). Figure 34 5. D I S C U S S I O N 158 5.1. C a r d i a c L P L regula t ion d u r i n g hyper tens ion 5.1.1. LPL regulation in the spontaneously hypertensive rat heart During hypertension, LPL activity is decreased in skeletal muscle and adipose tissue in human patients (Pollare et al 1991, Marotta et al 1995) and Dahl salt-sensitive hypertensive rats (Mondon et al 1993). We questioned whether a similar reduction in LPL activity exists in the hypertensive SHR rat heart. To this end, we measured the rapid, heparin-releasable L P L fraction in whole hearts obtained from W K Y and SHR rats. This rapidly releasable fraction is more sensitive to altered physiological (e.g. feeding, fasting) and pathological (e.g. diabetes, hyperthyroidism) states than total tissue activity. More importantly, the ability of the heart to hydrolyze lipoprotein-TG is closely linked to changes in the rapidly releasable LPL, but not to the remaining cellular fraction (Eckel 1989). Hence, any change in cardiac L P L activity during hypertension may have significant implication in altering the supply of FFA to the heart. In this report, we show for the first time that coronary heparin-releasable L P L activity is decreased in 15-16 week SHR rat hearts when compared to age-matched W K Y animals, and propose that this phenomenon could be intrinsic to the hypertensive state per se. Insulin has been cited as an important factor affecting LPL activity in vivo. For example, in normal weight subjects, hyperinsulinemia combined with insulin resistance was associated with a reduced L P L activity in skeletal muscle (Pollare et al 1991) and post-heparin plasma (Knudsen et al 1995). In addition, Marotta et al. (Marotta et al 1995) recently observed a strong correlation between serum insulin and post-heparin plasma LPL activity in mild hypertensive patients. Since SHR rats have been characterized as hyperinsulinemic (Reaven and Chang 1991, 159 Verma et al 1994), we considered the possibility that the reduced heparin-releasable LPL activity in SHR rat hearts was secondary to high insulin levels. However, we found that insulin levels in the 5 hour fasted SHR rats were not significantly elevated when compared to W K Y rats of the same age. Similarly, other studies have reported no evidence of hyperinsulinemia in SHR rats relative to W K Y animals (Davidoff et al 1990, Buchanan et al 1992, Dai.et al 1994). The contradictory findings in the literature with regard to the state of insulinemia in SHR animals could be explained by differences such as the age group studied and procurement of animals ( W K Y rats may be endocrinologically heterogenous depending on the commercial source, Christe and Rodgers 1995). Insulin was previously reported to directly release L P L from its binding sites on 3T3-L1 adipocytes (Chan et al 1988), an effect suggested to involve the liberation of LPL via the phospholipase C-catalyzed hydrolysis of a glycosylphosphatidylinositol membrane anchor. However, in this study, insulin did not displace endothelial-bound L P L into the coronary perfusates. Similarly, LPL was not released by insulin from isolated control and diabetic myocytes (Braun and Severson 1992) or by phosphatidylinositol-specific phospholipase C from bovine aortic endothelial cells (Saxena et al 1991). Moreover, it was recently reported that LPL did not possess a glycosylphosphatidylinositol membrane anchor (Bruin et al 1994). Thus, it appears that neither a direct effect of insulin nor the presence of a hyperinsulinemic state could account for the lowered heparin-releasable cardiac L P L activity in SHR rats. L P L gene expression is located exclusively in cardiomyocytes but not in endothelial cells (Camps et al 1990). Therefore, we postulated that the reduced heparin-releasable enzyme activity may be due to decreased cellular LPL content or secretion. However, both cellular and secreted LPL activities in this study do not indicate a diminished L P L synthesis in SHR cardiac myocytes. A recent study observed that there was a duration dependent decrease in LPL mRNA levels in SHR rat hearts (Masuzaki et al 1996). However, in this study, cardiac LPL activity and 160 protein mass were not measured. Since a dissociation between LPL-mRNA levels and enzyme activity was observed previously (Bergo et al 1996a), it is not known whether the decrease in LPL-mRNA level was actually accompanied by a decrease in LPL synthesis and activity. Moreover, the above study was performed in whole heart homogenate that does not distinguish between the various pools of LPL in the heart. As reduction in enzyme activity paralleled the progressive development of hypertension, the lower heparin-releasable LPL activity in SHR rat hearts could be related to the hypertensive state per se. A positive correlation between plasma LPL activity and aortic flow velocity has been reported in mild, uncomplicated hypertensive patients (Marotta et al 1995). Factors such as poor peripheral blood flow, vascular hypertrophy and rarefaction of blood vessels have been proposed to affect LPL action during hypertension by reducing vascular surface area for enzyme binding sites and/or impeding the delivery of substrate (Lind and Lithell 1993). We examined whether the increased perfusion of coronary vasculature could enhance the heparin-releasable LPL activity in hypertensive animals. Interestingly, both nifedipine and CGS-21680, vasodilators with divergent mechanisms of action, normalized heparin-induced LPL release, suggesting that flow through coronary blood vessels may regulate LPL activity. In support of these findings, the reduced clearance of a circulating chylomicron-like emulsion in SHR rats was improved with doxazosin (an a.\ blocker) (Mackintosh et al 1991), whereas a 4-fold increase in post-heparin plasma LPL activity was observed in obese Zucker rats following treatment with the K+-channel opener AL0671 (Matzno et al 1994), a potent vasodilator. SHR rats have previously been reported to be hypertriglyceridemic (Reaven and Chang 1991, Reaven and Ho 1991) a finding clearly opposite to that obtained in the present study. The discrepancy in plasma TG between this and previous studies occurred despite identical 161 experimental parameters, i.e., the age of animals, and duration and time of fasting. Reduced levels of serum TG and cholesterol in SHR rats relative to normotensive Wistar or W K Y rats have similarly been reported (Iritani et al 1971, Tonooka et al 1985). The reasons for hypotriglyceridemia in SHR rats are presently less clear. Plasma T G levels generally represent a balance between production (from the liver and gut) and removal (lipolysis of TG-rich lipoproteins). In our preliminary studies, we observed a decreased TGSR as well as TGCR coupled with a low PHPLA in older SHR rats. The decreased TGCR could be as a result of vasoconstriction restricting L P L action in SHR rats (Ogawa et al 1991). Although, low TGSR appeared to have greater influence on plasma TG levels in SHR rats, more studies need to be performed to confirm this mechanism. Although plasma FFA levels are significantly higher in SHR rats, the approximate contribution of different sources of FFA (from the circulation or intracellular sources) towards (3-oxidation in the SHR rat heart is not known. We speculate that the reduced cardiac L P L action coupled with low circulating TG levels, could significantly diminish FFA supply to the heart via the exogenous pathway. In support of our hypothesis, it was demonstrated that the SHR heart utilizes more glucose than FFA, a biochemical adaptation suggested to promote energetic economy amidst adverse hemodynamic conditions (Christe and Rodgers 1994). Further studies are required to determine the impact of a reduced heparin-releasable L P L activity on FFA supply and utilization in the hypertensive rat heart. 5.1.2. LPL regulation in fructose hypertensive rat hearts In SHR study, we demonstrated that with the development and maintenance of hypertension, there is a concomitant and progressive decrease in coronary endothelial L P L activity released by heparin (Sambandam et al 1997). We questioned whether a similar 162 derangement in cardiac LPL activity existed in a non-genetic, acquired hypertensive animal model like the fructose-hypertensive rat. As reported earlier, fructose feeding through drinking water induced hyperinsulinemia, hypertriglyceridemia and hypertension. Several mechanisms were proposed in the development of hypertension during fructose feeding. Endothelial dysfunction, altered vascular responsiveness, metabolic intermediates of fructose (e.g., aldehydes), activation of the renin-angiotensin system, etc., were shown to be some of the factors responsible for elevated blood pressure in response to fructose feeding (Iyer et al 1996, Verma et al 1997, Berger et al 1998, Katakam et al 1998, Vasdev et al 1998). To our knowledge this is the first report which demonstrates that coronary functional LPL release in response to heparin perfusion is decreased in chronic fructose-hypertensive animals. Contrary to our chronic studies, short term fructose feeding for example, 4 days fructose treatment did not affect LPL activity from the perfused heart or total cellular and surface-bound LPL in isolated cardiac myocytes (Liu and Severson 1995). Similarly, acute fructose loading did not alter the fasting induced increase in cardiac LPL activity (Pedersen and Schotz 1980). Results from the present study confirm that in rats treated for 2 weeks with fructose, there was no change in cardiac heparin-releasable LPL activity. Therefore, it appears that the decreased cardiac heparin releasable LPL activity that was observed during chronic fructose feeding in our study may not be a direct effect of fructose but probably due to metabolic changes like hyperinsulinemia and hypertriglyceridemia or due to a increased blood pressure observed in these animals. Since hyperinsulinemia combined with insulin resistance has been implicated in LPL deficiency, we considered the possibility that the lower cardiac LPL in fructose-fed rats was secondary to hyperinsulinemia in these animals. However, our results indicate that 2 week 163 fructose treated rats do not demonstrate any change in LPL activity, despite being hyperinsulinemia Further, similar to the SHR study, isolated whole hearts from Wistar rats did not release LPL when perfused with insulin. Therefore, hyperinsulinemia may not have contributed to the decreased heparin-releasable cardiac LPL activity in fructose-fed rats. As in SHR rats, the reduction in cardiac heparin-releasable LPL activity in fructose-treated rats paralleled the duration of the hypertensive state. This decrease in functional LPL activity was not due to a decrease in LPL synthesis at the cardiac myocyte. Additionally, as withdrawal of fructose for 2 weeks from chronically treated rats reversed the fructose-induced increase in blood pressure and decrease in cardiac LPL activity, an association between cardiac LPL and hypertension can be suggested. Therefore, we performed in vitro treatment with coronary vasodilators in fructose treated rats to demonstrate whether improvement in perfusion of coronary vasculature could increase heparin-releasable LPL activity in fructose hypertensive animals. Indeed, in vitro perfusion of fructose hypertensive rat hearts with nifedipine or CGS-21680 increased heparin-releasable LPL activity. These results confirm our previous findings in the SHR rat heart. Together, these results suggest that poor perfusion through coronary blood vessels may be a factor in regulating LPL activity in chronically hypertensive fructose-treated rat hearts. Hence, it would appear that the hemodynamic state may be important in the regulation, not only of the amount of LPL available for its physiologic function, but also for the delivery of the substrate to it (Marotta et al 1995). Hypertriglyceridemia in fructose treated rats has been proposed to be due either to an increased hepatic secretion of VLDL-TG (Herzberg and Rogerson 1988) or a decreased removal of TG rich lipoproteins from the circulation (Hirano et al 1988). Impaired TG clearance could result from structural changes in VLDL (Mamo et al 1991) or defective LPL activity. In a 164 previous study, post-heparin plasma lipolytic activity in fructose-fed animals was reported to be similar to that of controls (Iwata et al 1990). However, Spirulina platensis (an agent which significantly increases LPL activity in post heparin plasma) decreased fructose induced hypertriglyceridemia suggesting that LPL could contribute to changes in plasma TG levels in fructose-fed animals. Our results clearly show that fructose treatment for 6 weeks reduced basal and post heparin plasma lipolytic activity. Hence, impaired TG clearance after chronic fructose treatment could be a combined effect of structural changes in VLDL together with altered post heparin plasma LPL activity leading to hypertriglyceridemia in these animals. In summary, our results demonstrate that chronic fructose treatment reduces cardiac heparin-releasable LPL activity in rats. Moreover, this effect is not due to a decreased synthesis of the enzyme in the myocytes but probably due to the development of hypertension per se. The impact of a reduced heparin-releasable LPL activity on FFA supply and utilization in the fructose hypertensive rat heart is unclear at present. 5.2. L P L d u r i n g diabetes 5.2.1. LPL regulation in the diabetic rat heart Recently, using the conventional Langendorff perfused heart, we determined that peak heparin-releasable LPL activity was augmented in the diabetic rat (Rodrigues et al 1997). This rapidly releasable LPL pool was believed to represent the endothelium-bound enzyme fraction. However, heparin can diffuse through the arterial wall (Lovich and Edelman 1995). Thus, the conventional heparin-perfused Langendorff heart may release LPL not only bound to the luminal side of the endothelium but also the enzyme present within the endothelial cell, and at the subendothelial, interstitial and myocyte cell surfaces. Indeed, using a modified Langendorff heart 165 perfusion that separates coronary from interstitial fluid, heparin was found to release L P L from both compartments (Jansen et al 1980). In the present study, we used the modified Langendorff heart to establish that in diabetes, the increase in LPL activity or protein originates mainly from the luminal surface of capillary blood vessels. Immunohistochemical studies of L P L confirmed this finding and verified that despite widespread labeling of the enzyme within control and diabetic myocytes, there was a convincing increase in LPL immunoreactivity in the capillary blood vessels of the diabetic heart. In vivo administration of insulin to the diabetic rats 90 minutes before the experiment, reversed the coronary LPL activity to levels closer to that of control. This short duration of insulin treatment however, did not increase the depressed interstitial LPL but further obliterated the initial peak-release that was observed in untreated diabetic rats. This reversal by insulin supports the fact that the altered LPL activity in the diabetic heart is not due to a non-specific cytotoxic effect of STZ but due to the diabetic state per se. In the heart, capillary endothelial LPL is largely derived from cardiac myocytes that synthesize and continuously secrete LPL (O'Brien et al 1994, Anderson et al 1997). Although we had hypothesized that the enhanced heparin-releasable LPL activity in diabetic rat hearts could be due to an increased synthesis of the enzyme, myocyte L P L in these animals was found to be dramatically reduced (Rodrigues et al 1997). In the present study, on continuous perfusion of control hearts with heparin, there was a progressive increase in the discharge of L P L into the interstitial fluid indicating that heparin could conceivably cross the capillary wall and release the enzyme from the extracellular space and myocyte surface. Interestingly, in diabetic hearts, there was a peak release of LPL into the interstitial fluid within 1 -2 min following heparin perfusion, implying an accumulation of the enzyme at or near the endothelial cell, on the abluminal side. Moreover, release of LPL into the interstitial fluid of the diabetic hearts was depressed following 166 prolonged heparin perfusion, an observation that is congruent with the previously reported reduction in myocyte L P L activity (Rodrigues et al 1997). Insulin regulates LPL, but its effects vary depending on the tissue being investigated. Thus, elevated levels of insulin in vivo (either postprandial or following an euglycemic clamp) (Eckel 1987, Sadur and Eckel 1982) or in vitro (Ong et al 1988) increases L P L activity in adipose tissue. In the heart, administration of insulin in vivo to control rats increases heparin-releasable LPL activity in isolated cardiac myocytes within one hour. However, incubation of control myocytes with insulin in vitro has no effect on L P L activity indicating that in the heart, additional metabolic factors must be required for the regulation of LPL (Braun and Severson 1991 & 1992). Given these observations, a predicted outcome of insulin deficiency would be an attenuated L P L activity in cardiac cells. Indeed, in this study, a brief duration of hyperglycemia (24 hours) reduced both heparin-releasable and intracellular LPL activity in cardiac myocytes. However, as demonstrated previously in 2-week diabetic rats (Rodrigues et al 1997), even short-term diabetes (within hours) increased capillary luminal LPL stores. These results indicate that in the heart, an acute reduction in insulin has distinct and immediate regulatory effects on LPL at two levels: a decreased synthesis of LPL at the myocyte and an augmented association of L P L with the luminal surface of capillary endothelial cells. Hypoinsulinemia can also be induced by fasting which enhances cardiac and skeletal muscle LPL activity but reduces LPL in adipose tissues (Rodrigues et al 1992, Semb and Olivecrona 1986). The fasting-induced changes in cardiac L P L activity were suggested to be posttranslational and did not involve altered mRNA levels, protein synthesis or specific activity of the protein (Doolittle et al 1990). As previously reported using the modified Langendorff heart (Jansen et al 1980), the fasting-induced increase in cardiac heparin-releasable L P L activity 167 occurred mainly at the coronary lumen. Using immunogold staining, other studies have also reported that the primary effect of fasting on distribution of LPL occurred at the surface of endothelial luminal processes (Blanchette-Mackie et al 1989a & 1989b). Interestingly, enzyme activity in the interstitial fluid of fasted rats remained unchanged and is consistent with the finding that fasting does not alter LPL synthesis in the myocyte (Doolittle et al 1990). As the degree of hypoinsulinemia was comparable between fasted and diabetic rats, it appears that although hypoinsulinemia alone can enhance endothelial LPL activity, it may not entirely influence the synthesis of LPL. Hence, other short-term diabetes-related factors may be necessary for a reduction in myocyte LPL production (Anderson et al 1997). At present, the mechanism(s) by which insulin regulates LPL at the vascular endothelial cell is not known. Endothelial LPL is regulated by detachment from its HSPG binding sites into the circulation, followed by hepatic degradation (Olivecrona and Bengtsson-Olivecrona 1993). HSPGs associate with endothelial cells via their core proteins or a glycosylphosphatidylinositol (GPI) linkage (Yanagishita and Hascall 1992) and cleavage of the GPI anchor by insulin-sensitive phospholipases could release HSPGs, and hence LPL (Eckel et al 1978, Spooner et al 1979, Chan et al 1988). Provided this mechanism operates in vivo in the heart, hypoinsulinemia would enable the enzyme to remain attached to its endothelial binding site. In perfused guinea pig hearts, LPL moves from its site of synthesis in the parenchymal cells to the endothelial surface within 30 min (Blanchette-Mackie et al 1989a, Liu and Olivecrona 1991). Thus, the enhanced capillary LPL pool in diabetic rats could involve an accelerated translocation of LPL from myocytes to the lumen (Rodrigues et al 1997, Stins et al 1992). It should be noted that the augmented endothelial LPL could conceivably be derived from the circulation (Wallinder et al 1984, Olivecrona and Bengtsson-Olivecrona 1989, Olivecrona et al 1995). However, we have reported that when the heparin-releasable LPL pool was allowed to recover for 1 hr after removal 168 of the enzyme, diabetic hearts continued to demonstrate a higher peak LPL activity after a second heparin perfusion (Rodrigues et al 1997). Finally, the amount of luminal LPL can be regulated by the endothelial cell. This process involves the internalization and recycling of LPL to the cell surface, thereby allowing endothelial cells to maintain an additional pool of the enzyme at the vascular endothelium (Saxena et al 1990). Alterations in pH can bring about dissociation of LPL from its binding site, with less detachment of cell surface bound LPL at pH 5.5 compared with pH 7.4 and 8.5 (Saxena et al 1990). Hence, the assumption is that inside the endothelial cell, an acidic pH would permit LPL to remain bound to proteoglycans, and thus promote recycling of internalized LPL (Saxena et al 1990). As diabetes results in an altered ability to regulate pH (Feuvray and Lopaschuk 1997), it is possible that changes in pH in endothelial cells may augment this auxiliary pool of LPL. 5.2.2. Hydrolysis of VLDL in the diabetic heart Hypertriglyceridemia is a common metabolic disorder associated with STZ-diabetes (Rodrigues et al 1986). In an insulin deficient state, excessive circulating TGs could be a consequence of increased production of lipoproteins or a reduced clearance of TG from the blood (due to a depleted post-heparin plasma lipolytic pool of LPL). However, TG secretion rates in control and diabetic rats have been reported to be comparable (Chen et al 1979, Hirano et al 1991) and post heparin plasma lipolytic activity in this and other (Chen et al 1980) studies was not significantly reduced in diabetic rats. The possibility remained that changes in structure of lipoproteins could explain this hypertriglyceridemia. Several in vitro and in vivo studies in clinical and experimental diabetes have shown that VLDL and chylomicrons undergo changes in structure and composition such that they may become poorer substrates for LPL (Bar-On et al 1984, Hirano et al 1991, Levy et al 1985, Mamo et al 1992, O'Looney et al 1985). If VLDLs and chylomicrons are altered during diabetes, another question of overriding importance is whether 169 or not the diabetic heart (with its augmented LPL activity) is in fact capable of metabolizing these TG rich lipoproteins. In the present study, we were unable to detect any difference in the lipolysis rates or in the characteristics of LPL substrate kinetics (Km and Vm a x) was observed between control and diabeic rats. Thus, VLDL prepared from diabetic rats appears to be a normal substrate for LPL and impaired lipolysis of these particles may not be a causal factor towards the development of hypertriglyceridemia. As the diabetic rats in the present study were hypertriglyceridemic in the fed but not in the fasted state, it is likely that excessive accumulation of triglycerides occurs as a result of changes in the characteristics of chylomicrons, the other major triglyceride containing lipoprotein. Mamo et al. (1992) have demonstrated that lipoproteins (plasma lipoprotein fraction of density < 1.006 gm/ml that includes chylomicrons and VLDL) of diabetic origin are hydrolysed by LPL at a significantly slower rate than lipoproteins from normal rats. However, as fasting for 16 hours prior to recovery of lipoproteins eliminated this difference in rates of lipolysis, the authors concluded that during diabetes, the lipolytic defect might be specific for chylomicrons. Interestingly, [14C]chylomicrons obtained from STZ diabetic rats when intravenously injected into control animals disappeared from the circulation at a significantly slower rate than similarly prepared chylomicrons from control animals (Levy et al 1985). The question of whether hearts from diabetic animals in our study are able to clear chylomicrons efficiently remains to be answered. However, an earlier study has reported that despite poor peripheral clearance of chylomicrons, cardiac tissue from diabetic rats exhibit a 3-fold higher uptake of this lipoprotein when compared to non-diabetics (Levy et al 1985). When radiolabeled VLDL-TG were perfused through the isolated heart and the decline in triglyceride radioactivity monitored with time, diabetic hearts metabolized VLDL at a faster rate than that observed in control rat hearts. This increased VLDL-TG clearance from the diabetic 170 heart is in agreement with the enhanced LPL activity and mass that was reported previously (Rodrigues et al 1997, Sambandam et al 1999). As prior removal of LPL by perfusion with heparin prevented this increased clearance of V L D L , this argued against a receptor-mediated removal of this lipoprotein by the diabetic heart (Wyne et al 1996). Moreover, the increased clearance of V L D L from the diabetic heart was not a result of an increased lipolysis in the buffer chamber due to detachment of LPL. We (Rodrigues et al 1992) and others (DeMan et al 1997) have demonstrated that V L D L does not result in detachment of L P L from its HSPG complex. In conclusion, our results demonstrate that during diabetes, rapid removal of V L D L from the diabetic heart could be one mechanism for the provision of FFAs for energy production, to compensate for the diminished contribution of glucose as an energy source. It should be noted that in an insulin-deficient state, adipose tissue lipolysis is enhanced, resulting in elevated circulating FFAs (Rodrigues et al 1992). In addition, an increased activity of myocardial enzymes that catalyze the synthesis of TG promotes the accumulation of intracellular TG stores during diabetes (Murthy et al 1983). Subsequent hydrolysis of this augmented TG store could also lead to high tissue FFA levels (Kenno and Severson 1985). In view of these mechanisms that enhance cardiac FFA levels, the relative significance of cardiac LPL activity in delivering FFA to the diabetic heart is unknown. Interestingly, when the uptake pattern of albumin-bound versus V L D L - T G derived FFA were compared in human placental cells (macrophages and trophoblasts), the uptake of albumin-bound FFA after 24 hours was 4 to 6% of the initial amount contained in the incubation medium. In contrast, when these cells were incubated with labeled V L D L - T G , cellular TG-FFA content was many folds higher (Bonet et al 1992). Thus, at least in some cell types, TG-rich lipoproteins may be a more efficient means to deliver FFA than are albumin bound FFA and emphasizes the importance of LPL in the supply of FFA to the heart (Bonet et al 1992, Levak-Frank et al 1995). A caveat is that this abnormally high capillary 171 endothelial LPL activity could provide excess FFA to the diabetic heart leading to a number of metabolic, morphological and mechanical changes, and eventually to cardiac disease (Rodrigues and McNeill 1992, Rodrigues et al 1995). 6. S U M M A R Y A N D C O N C L U S I O N S 172 Hypertension and diabetes are two important risk factors of cardiovascular disease, a leading cause of deaths in Western population. Cardiac dysfunction is one of the cardiovascular diseases, which develops secondary to hypertension and diabetes. Although the underlying mechanisms are not clear, it is proposed that early metabolic derangement could be causally related to cardiac dysfunction. In this regard, we investigated LPL regulation, a key enzyme in FFA supply to the heart, during hypertension and diabetes. Our results show that cardiac LPL activity decreases at the capillaries (functional LPL pool) with the increase in blood pressure and duration of hypertension in both SHR and fructose hypertensive rats. This decrease in functional LPL activity was not due to a defective synthesis of the enzyme but probably due to poor perfusion of coronary blood vessels of hypertensive hearts. Coronary vasodilators with divergent mechanisms of action reversed the decreased LPL to normotensive levels by improving the coronary perfusion. Therefore, it appears that poor capillary perfusion during hypertension per se regulates cardiac LPL at the functional level. Based on the literature evidence that hypertensive hearts utilize less FFA and more glucose for their energy transduction (Christe and Rodgers 1994 & 1995), we speculate that the decrease in LPL at the functional location could be linked to this altered metabolism in the hypertensive hearts. Capillary-bound and myocyte LPL are distinctly regulated during diabetes. Acute hypoinsulinemia augments cardiac capillary LPL, presumably within or at the luminal and abluminal surface of the endothelial cell (Figure 36). In contrast, the level of LPL in isolated cardiac myocytes is low. So far, the mechanism for this selective augmentation of the enzyme at the capillary is not known. We have identified that this enhanced recruitment of LPL is an intrinsic ability of the diabetic heart. Various posttranslational mechanisms could play a role in 173 this regulatory process. Increased dimerization of LPL to the active form during its transit from myocytes to capillary endothelium, a faster translocation of the enzyme from its site of synthesis, and endothelial recycling, could be some of the mechanisms involved. Regulation at this location is essential as it permits a rapid response to an acute demand for FFA in the absence of glucose utilization. We have also demonstrated that the diabetic heart hydrolyzes significantly higher amounts of VLDL-TG at a higher rate as compared to the non-diabetic heart. In fact, the diabetic heart has elevated levels of FFAs and TGs. Hyperlipidemia combined with higher functional LPL in the diabetic heart could possibly contribute to the accelerated supply of FFA to the heart. Although various other sources like enhanced adipose tissue derived FFA, increased intracellular TG synthesis and lipolysis of TG stores could contribute to these higher FFA levels (Murthy et al 1983, Kenno and Severson 1985, Chattopadhyay et al 1990 ), a role of LPL in the intracellular fat accumulation should not be underestimated. For example, overexpressing human LPL in skeletal and cardiac muscles in transgenic mice leads to elevated FFA uptake and severe myopathy, characterized by muscle fiber degeneration, and extensive mitochondrial and peroxisomal proliferation (Levak-Frank et al 1995). Therefore, we believe that the augmented LPL in the diabetic heart could enhance FFA supply thereby playing a potential pathogenic role in the development of diabetic cardiomyopathy. However, further studies are needed to examine the fate of hydrolytic products of VLDL-TG when perfused through normal and diabetic hearts, and the role of chylomicrons in normal and diabetic hearts. Also, future studies to prove that increased LPL in the diabetic heart is causally related to the development of cardiomyopathy is of great clinical significance. This awaits the discovery of a specific pharmacological agent that inhibits LPL activity in vivo. In a previous report from our laboratory, we have demonstrated that hypertensive-diabetic rats have an augmented capillary LPL (Shepherd et al 1998). Thus the diabetes-induced 174 augmentation of capillary LPL counteracts the reduction in enzyme activity associated with hypertension. We believe that these changes in cardiac LPL activity in the hypertensive-diabetic heart, especially at the functional location, could play a key role in altering cardiac metabolism and thus initiating the development of cardiac failure. 175 Blood vessel Interstitial space Myocardial cell Figure 35. Schematic representation of L P L synthesis, translocation, various functions, and its possible pathophysiological role in the diabetic heart. 7. F U T U R E D I R E C T I O N S 176 Future studies would be pursued to investigate the contribution of TGRL derived FFA to cardiac lipid metabolism in the diabetic heart in the absence or presence of NEFA. The question of whether the FFAs are oxidized or re-esterified in the cardiac tissue subsequent to their release from perfused TGRLs should also be determined. Changes in insulin levels acutely regulate cardiac LPL during diabetes. The acute changes in functional LPL appear to be an intrinsic ability of the heart to recruit more enzyme in response to changes in insulin levels. Although our preliminary studies indicate there could be a faster translocation of the enzyme from myocytes, more studies using S-methionine to measure the rate of translocation of newly synthesized LPL will be carried out in the future. In addition, the signaling events that bring about the faster translocation of LPL from its site of synthesis (cardiac myocytes) to the functional site (coronary lumen) have yet to be uncovered (Figure 35). Experiments are underway to determine whether a cAMP mediated mechanism could play a role in this enhanced secretory process. Transcytosis of macromolecules across the endothelium occurs via a caveolar transport process, which involves PI3-kinase, tyrosine kinase, actin cytoskeleton rearrangement, and SNARE-SNAP-NSF fusion processes. Whether this mechanism plays a role in LPL transport, and if so, whether there is any augmentation of this process in the diabetic heart, has yet to be discovered. Another mechanism that could play a role in the acute regulation of coronary LPL is endothelial recycling (Figure 35). Previous studies have shown that LPL attached to the HSPG binding sites on the luminal side of the endothelial surface are constantly internalized and 177 recycled back. Therefore, it is possible that augmentation of this endothelial recycling could play an additional role in the enhancement of capillary LPL of the diabetic heart. Endothelial recycling is currently being investigated. Vascular LPL plays a central role in atherosclerosis, both by releasing deleterious lipolytic products and internalizing proatherogenic lipoproteins like LDL in the vasculature (Figure 35). Diabetes is associated with an increased incidence of atherosclerosis and coronary heart disease. Since we observed an increased coronary LPL activity in the diabetic heart, it would be of great clinical relevance to investigate whether this altered LPL activity could play a role in atherosclerosis. Since rats do not develop atherosclerosis, experiments should be performed in other animal models of diabetes such as rabbits and mice. 8. R E F E R E N C E S 178 Ann YI, Ferrell RE, Hamman RF, and Kamboh MI. 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