Adipose Tissue Metabolism: Role Of

ADIPOSE TISSUE METABOLISM: ROLE OF CALCIUM AND CYCLIC NUCLEOTIDES IN INSULIN ACTION THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (Ph.D) IN CLINICAL BIOCHEMISTRY BY NADARAJEN VYDELINGUM BSc; MSc DEPARTMENT OF HUMAN METABOLISM ST. MARYtS HOSPITAL MEDICAL SCHOOL LONDON UNIVERSITY OF LONDON 1978 DEDICATED TO ROSEMARY ACKNOWLEDGEMENTS I would like to thank Dr, A.H. Kissebah for his guidance, inspiration and continuous encouragement throughout the course of this project. I am also very grateful for his unfailing efforts and his scientific comments during the preparation of the manuscript. My thanks to Professor V. Wynn for extending the facilities of the laboratories in the Department of Metabolic Medicine for the completion of the project. I am especially grateful to Mrs. C. Bair for the excellent typing and retyping of the countless scripts and her enthusiastic willingness to participate in the compilation of this work. Finally, I wish to thank my wife, Rosemary, for the critical evaluation of the literary style and for her invaluable support over the years. ABBREVIATIONS qtr l ATP -. Adenosine 5kphosphate ATP-ase - Adenosine triphosphatase c-AMP - Cyclic 3':5'-Adenosine Monophosphate c-GMP - Cyclic 3':5'-Guanosine Monophosphate Ca2+ - Calcium ion CPM - Counts Per Minute DBc-AMP - Dibutyryl-c-AMP DNA - Deoxyribose Nucleic Acid DPiEI - Disintegrations Per Minute -au+t;np el ehr-)~ N v EGTA - Ethylene Glycol etra-acetic Acid FFA - Free Fatty Acid La3+ - Lanthanum Leu - Leucine LPL - Lipoprotein Lipase Mol - Mole M - Molar mRNA - Messenger Ribonucleic Acid - Oxid`sed Nicotinamide Adenine Dinucleotide NAD} r NADH - Reduced Nicotinamide Adenine Dinucleotide PDE - Phosphodiesterase PEP - Phosphoenolpyruvate PPO - 2,5-Diphenyloxazole; a primary fluor TCA - Trichloroacetic Acid TEAM - Triethanolamine &ris TRIS -Ilsydroxy methyl) Amino Methane VLDL - Very Low Density Lipoprotein TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ABBREVIATIONS ii ABSTRACT 1 CHAPTER I INTRODUCTION AND REVIEW OF THE LITERATURE A. The Adipocyte as a Source of Fuel.... 3 B. Hormonal Regulation of Fat Mobilization and Storage 1. The Catecholamines 14 (a)Adrenergic Receptors 14 (b)Adrenergic Effects and c-AMP 18 2. Insulin 18 (a)Insulin Receptors 19 (b)Adipose Tissue Response to Insulin 21 C. Subcellular Mode of Insulin Action 1. The Modulation of Plasma Membrane by Insulin 23 2. Insulin Second Messenger 23 D. The Role of Calcium Ions 34 CHAPTER II MATERIALS AND METHODS Materials 39 Methods A. Preparation and Incubation Conditions for Fat Cells 41 B. Preparation and Incubation Conditions for Fat Tissue Pieces 44 C. Cyclic-AMP Assay 47 D. Cyclic-GMP Assay . 54 E. Glycerol Assay 59 F. Lipoprotein Lipase (LPL) Assay 65 G. Measurement of 3H-Leu Incorporation into Fat Cell Protein 71 H. Ester Assay 76 CHAPTER III HORMONAL REGULATION OF FAT CELL LIPOLYSIS: RELATION OF C-AMP AND CALCIU1 TO THE EFFECTS OF ADRENALINE AND INSULIN I. Background and Objectives 80 II. Experimental Procedures 84 III. Results 87 IV. Discussion . A. Relation between c-AMP and Hormonal Regulation of Lipolysis 116 B. Relation of Ca2+ to the Effects of Adrenaline and Insulin on Lipolysis 125 CHAPTER IV HORMONAL REGULATION OF LIPOPROTEIN LIPASE ACTIVITY AND PROTEIN SYNTHESIS: RELATION OF C-GMP AND CA2+ IONS TO INSULIN ACTION I. Background and Objectives 150 II. Experimental Procedures and Results 152 III. Discussion A. Insulin, Protein Synthesis and LPL Activity 186 B. Relation of c-GMP to the Effects of Insulin on Protein Synthesis and LPL Activity 194 C. Relation of Cato the Effects of Insulin on c-GMP, Protein Synthesis and LPL Activity 198 CHAPTER V General Conclusion 205 CHAPTER VI Bibliography 210 Publications from Thesis 226 Addendum - Selected Papers from the Author's Bibliography which are Relevant to the Thesis 227 ABSTRACT The hypothesis that the cellular effects of insulin may be mediated by changes in intracellular calcium concentration has been re-examined in vitro using isolated rat fat cells. The effects of alteration in extracellular and/or intracellular calcium concentration on insulin regulation of lipolysis, protein synthesis and lipoprotein lipase activity and their relation to c-AMP and c-GMP levels were studied. When fat cells were incubated with adrenaline an early and transient rise in c-AMP level was observed which was followed by a gradual increase in lipolysis. Insulin inhibited the adrenaline stimulated lipolysis but this occurred without any detectable reduction in c-AMP levels. Incuba- tion of cells in media without added calcium did not alter the antilipolytic effect of insulin whereas pretreatment of fat cells with EGTA to deplete intracellular calcium abolished the inhibitory effect of insulin on lipolysis. In addition, the calcium ionophore A23187 and lanthanum chloride, agents which facilitate an increase in cellular calcium concentration, like insulin, inhibited lipolysis in the fat cells. These results suggest that the anticatabolic effect (inhibition of lipolysis) of insulin on fat cells is not associated with a reduction in c-AMP level while changes in intracellular calcium level markedly influence this response. Insulin produced a dose-dependent stimulation of protein synthesis and lipoprotein lipase activity in fat cells and fat tissue pieces, re- spectively. Both processes were associated with an early and transient rise in c-GMP level. Omission of calcium from the medium did not alter the tissue's response to insulin while on depletion of intracellular calcium with EGTA, the effects of insulin were no longer discernible. Addition of calcium ionophore A23187 or lanthanum chloride to adipose tissue incubates, like insulin, stimulated protein synthesis and -2- lipoprotein lipase activity and caused a significant rise in c-GMP level. These findings indicate that the anabolic effects of insulin on protein synthesis and lipoprotein lipase activity are associated with a rise in adipose tissue c-GMP and that these effects are also mediated through alteration in cellular calcium level. CHAPTER I INTRODUCTION AND REVIEW OF LITERATURE -3- INTRODUCTION A. The Adipocyte as a Source of Fuel The adipose tissue consists of a variety of cells, among these it is the adipocytes which function as the storage compartment for fat. The major fraction of the cell is the single lipid droplet located in the center of the cell surrounded by a thin film of cytoplasm within which the nucleus lies (see Figure 1). The adipocytes have an organelle sys- tem which is common to most mammalian cells. Fat tissue is a highly active organ, although its relative activity depends on the species and on the anatomical origin of the tissue within a given animal. Since the adipocyte is a major target cell for many hormones such as adrenaline and insulin, the plasma membrane contains a variety of surface receptors for these hormones. It appears that the antilipolytic effects of insulin on adipose tissue are specialized evolutionary development restricted primarily to pre- datory animals which are adapted to sporadic feeding habits (Fritz 1961). Moreover, the need for an efficient storage organ as a source of energy during periods of food deprivation seems to have coincided with the development of homoiothermy. To permit prolonged survival during a time of food deprivation, it is essential that certain cells store energy in a convenient form. This energy is obtained from the food ingested during periods of caloric excess. The fuel reserve is primarily in the form of triacylglycerol, more than 98% of which is in the adipose tissue (Havel _ 1972). It is considerably more efficient to store energy as fat than as glycogen or protein. The triacylglycerol present in the lipid droplet of the adipocyte does not require the accumulation of water and ions. Fat not only occupies less volume, but weighs less per calorie of stored chem- k4 ical energy than carbohydrate or protein. Thus, glycogen yields lkcalorie ao per 800 mg whereas ljc lorie is obtained from 125 mg of triacylglycerol. 0 f ***MM. FIGURE 1 • Electron micrographs of white adipose cell. Rat tissue pieces were pre- fixed in Karnowski's solution and post-fixed in 1% osmium tetroxide made up in 0.1 M PO4 buffer. The following structures are identified: bulk lipid storage compartment, L; cytoplasmic compartment, C; mitochondria, M; plasma membrane (PM), poor- ly preserved due to fixation ārtifact; (Mag x 5Ō00). -5- Moreover, a 70 kg man has a total fuel reserve of approximately 166,000 Kcal; 141,000 as lipid; 24,000 as protein; and 1,000 as carbohydrate (Cahill 1970), yet only 10% of his total body weight is made up as fat. From the above consideration, it appears that man can withstand severe caloric deprivation without total loss of metabolic activity. The development of this highly efficient form of energy storage allows man to survive starvation for approximately 30 days provided he has a supply of water (Wertheimer 1965). During the fasting state fatty acids are the prime source of energy for metabolic activity (Gordon et al 1956). To mobil` e fuel stored in the adipose tissue for use elsewhere, the stored triacylglycerol is hydrolyzed to free fatty acid (FFA) and glycerol (Heindel et al 1975) (see Figure 2). Since the glycero-kinase (EC 2.7.1.30) activity in white adipose tissue is low, most of the glycerol formed during lipolysis cannot be re-utilized but is released to the blood (Persico et al 1975). In contrast, FFA can be released from the adipocyte or be re-esterified and re-deposited as triacylglycerol. The extent of the re-esterifica- tion depends upon the availability of glycerophosphate from glucose or gluconeogenesis (Reshef et al 1970). The rate of blood flow to the adipose tissue is probably another factor which influences the rate of re- lease of the lipolytic products (Mayerle et al 1969, Neilsen et al 1969). The rate limiting step of lipolysis appears to be the conversion of triacylglycerol to diacylglycerol and FFA (Strand et al 1964).

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