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BIP, an INTESTINAL METABOLIC HORMONE a Thesis Submitted To BIP, AN INTESTINAL METABOLIC HORMONE A Thesis submitted to the University of Surrey for a Degree of DOCTOR OF PHILOSOPHY by PIOTR KWASOWSKI August 1986 Division of Clinical Biochemistry Department of Biochemistry University of Surrey BuiIdford BU2 5XH UK I W ' ProQuest Number: 27600360 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 27600360 Published by ProQuest LLO (2019). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLO. ProQuest LLO. 789 East Eisenhower Parkway P.Q. Box 1346 Ann Arbor, Ml 48106- 1346 SUMMARY Using an established radioimmunoassay for Immunoreactlve gastric inhibitory polypeptide (IR-GIP) the metabolic actions of gastric inhibitory polypeptide (GIP) were investigated. It was found that there was no circadian rhythm in basal plasma TR-GIP and immunoreactlve insulin (IRI) concentrations in normal human volunteers, but that there was in stimulated IR-GIP and IRI plasma concentrations following a mixed meal. It was shown that short-term adaptation of both humans and rats, with high-fat diets, could cause moderate insulin resistance as demonstrated by the removal of the feed-back inhibition of exogenous insulin on fat-stimulated IR-GIP release. It was shown that GIP via its role in the enteroinsular axis, might play a part in the development of the hyperinsulinaemia and obesity of the genetically obese hyperglycaemic (ob/ob) mouse. It was demonstrated that the pre-requisite for fat- stimulated IR-GIP release in the ob/ob mouse is the absorption of fatty acids and glycerides capable of being esterified into triglyceride. Any physical, chemical or metabolic interference with this process resulted in a decrease of the amount of IR-GIP released. It was further shown, using a combination of both exogenous and endogenous lipid and GIP, that GIP has a role in the removal of chylomicron triglyceride from the circulation of the rat. It was not possible to confirm this in humans using exogenous lipid and endogenous glucose-stimulated IR-GIP release. It was shown in the insulinoma bearing NEDH rat that exogenous porcine GIP was able to augment the insulin stimulating ability of glucose when co-administered intravenously with glucose, thus providing strong evidence for GIP's role as an incretin. II A.M.D.G, III ACKNOWLEDGEMENTS I should like to express my thanks to the following people for their help over the last few years. Firstly I must thank Profesor Vincent Marks for his constant help and encouragement in the execution and (0 completion of this thesis. I must also express my djepest gratitude to Dr Linda Morgan, who in the early days taught me all I knew about GIP, who advised and encouraged me through the initial experimental work and who, more recently, proof read the first drafts of this thesis. I must also thank Dr Shelagh Hampton for her immeasurable help and advice on radioimmunoassays. I would also like to thank Drs Peter Flatt and Clifford Bailey for their help with the ob/ob mice. Dr Kim Tan for his help with the insulinoma bearing rats, and Drs John Wright, Bob Cramb, Mike Dunne and John Wong for their help with the humans. Finally, I would like to thank my parents for encouraging me and making it possible for me to start, and my wife Christine, without whose proof reading and constant and untiring encouragement and help I would certainly not have finished. IV CONTENTS PAGE SUMMARY I DEDICATION III ACKNOWLEDGEMENTS IV CHAPTER ONE GENERAL INTRODUCTION 1.1.0 General Background 2 1.1.1 Historical 2 1.1.2 Special Aspects 3 1.1.3 General Concepts 5 1.1.4 Recent Developments 8 1.2.0 Enterogastrone Concept 15 1.2.1 Endogenous Lines of Research 15 1.2.2 Exogenous Lines of Research 16 1.3.0 Incretin Concept 19 1.3.1 Endogenous Aspects 19 1.3.2 Incretin Candidates 21 1.4.0 Gastric Inhibitory Polypeptide (GIP) 24 1.4.1 Discovery of GIP 24 1.4.2 Isolation and Purification 26 1.4.4 Localisation 27 1.4.5 Physiological Actions of Exogenous GIP 28 1.4.6 Metabolic Effects of GIP 31 V 1.4.7 stimulation of Immunoreactive BIP (IR-GIP) release 36 1.4.8 Inhibition of IR-GIP Release 42 1.5.0 Motilin 46 1.5.1 Historical Background and Discovery of Motilin 46 1.5.2 Structure of Motilin 46 1.5.3 Cellular Localisation of Motilin 47 1.5.4 Physiological Actions of Motilin 47 1.5.5 Modulation of Circulating IRM 49 1.6.0 Summary of Introduction and Aims 52 CHAPTER TWO 53 METHODS 2.1.0 The Gastric Inhibitory Polypeptide (GIP) Assay 54 lodination 54 Standards 56 Charcoal Stripped Serum 56 Assay 57 Antibody 57 2.2.0 The Motilin Assay 64 lodination 64 Antibody 65 Assay 65 2.3.0 Plasma Triglyceride Assay 73 VI 2.4.0 Plasma Glucose Assay 74 CHAPTER THREE 75 TWENTY-FOUR HOUR RHYTHMS IN THE ENTEROINSULAR AXIS 3.0.0 Introduction 76 3.1.0 GEP Hormones During a Twenty-four Hour Fast 77 3.1.1 Methods 77 3.1.2 Results 78 3.1.3 Discussion 79 3.2.0 Function of the Enteroinsular Axis at 1100 h and 0200 h 87 3.2.1 Methods 87 3.2.2 Results 88 3.2.3 Discussion 88 3.3.0 Investigation of the Insulinaemic Responses Following Intravenous Glucose at 1000 h and 0200 h 95 3.3.1 Methods 95 3.3.2 Results 96 3.3.3 Discussion 96 3.4.0 Function of the Enteroinsular Axis Throughout the Day 107 3.4.1 Methods 107 3.4.2 Results 108 3.4.3 Discussion 108 VII Summary of Results - Chapter Three 117 CHAPTER FOUR 119 EFFECT OF DIETARY FAT ON THE ENTEROINSULAR AXIS 4.0.0 Introduction 120 4.1.0 Effect of a Short-term High Fat Diet on the Enteroinsular Axis in Rats 123 4.1.1 Methods 123 4.1.2 Results 125 4.1.3 Discussion 131 4.2.0 Effect of Both High and Low Fat Diets on the Enteroinsular Axis in Man 134 4.2.1 Methods 134 4.2.2 Results 137 4.2.3 Discussion 147 Summary of Results - Chapter Four 151 CHAPTER FIVE 152 EFFECT OF GIP ON THE RAT ADIPOCYTE 5.0.0 Introduction 153 5.1.0 Role of i.p. GIP in the Clearance of i.p. Intralipid from the Plasma of the Rat 157 5.1.1 Methods 157 5.1.2 Results 158 5.1.3 Discussion 163 VIII 5.2.0 Role of Exogenous GIP in the Clearance of i.v. Intralipid in the Rat 165 5.2.1 Methods 166 5.2.2 Results 167 5.2.3 Discussion 171 5.3.0 Effect of Anti-GIP Antibodies on the Clearance of Plasma Triglyceride Following Oral Triolein in Rats 174 5.3.1 Methods 174 5.3.2 Results 175 5.3.3 Discussion 180 Summary of Results - Chapter Five 182 CHAPTER SIX THE ROLE OF GIP IN THE DIFFERENTIAL INSULIN RESPONSES TO ORAL AND INTRAVENOUS GLUCOSE IN THE TRANSPLANTABLE RAT INSULINOMA 183 6.1.0 Introduction 184 6.1.1 Methods 185 6.1.2 Results 187 6.1.3 Discussion 194 Summary of Results - Chapter Six 196 CHAPTER SEVEN 197 THE ROLE OF ENDOGENOUS CARBOHYDRATE-STIMULATED IR-GIP IN THE REMOVAL OF EXOGENOUS TRIGLYCERIDE IN MAN 7.1.0 Introduction 198 IX 7.1.1 Methods 200 7.1.2 Results 201 7.1.3 Discussion 210 Summary of Ressults — Chapter Seven 212 CHAPTER EIGHT 213 IMMUNOREACTIVE GASTRIC INHIBITORY POLYPEPTIDE (IR-GIP) IN OBESE (ob/ob) MICE 8.0.0 Introduction 214 8.1.0 Immunoreactlve Gastric Inhibitory Polypeptide (IR-GIP) in the Plasma and Small Intestine of the Obese Hyperglycaemic Mouse 216 8.1.1 Methods 216 8.1.2 Results 217 8.1.3 Discussion 220 8.2.0 Involvement of GIP and the Enteroinsular Axis in the Metabolic Abnormlities of the Obese Hyperglycaemic (ob/ob) Mouse 221 8.2.1 Methods 222 8.2.2 Results 225 8.2.3 Discussion 236 8.3.0 Effect of Chain-length and S^uration in Fatty Acid Stimulated IR-GIP Release in the Obese Hyperglycaemic (ob/bo) Mouse 239 8.3.1 Methods 239 8.3.2 Results 241 8.3.3 Discussion 246 Summary of Results - Chapter Eight 249 CHAPTER NINE 250 FINAL DISCUSSION AND FUTURE WORK 9.1.1 The Enteroinsular Axis 251 9.1.2 The Release of IR-GIP 256 9.1.3 The Enteroadipose Axis 257 9.1.4 Other Future Lines of Research 258 REFERENCES 263 PUBLICATIONS 280 XI CHAPTER ONE GENERAL INTRODUCTION paqe 1 1.1.0 General Background 1.1.1 Historical The physiology, origin, chemistry, pharmacology and clinical aspects of gastrointestinal hormones, or gut hormones as they have been more succinctly labelled, has become one of the most rapidly expanding and interesting fields in contemporary endocrinology and gastroendocrinology. One of the more eloquent pictures of the ever growing field of gut hormones was painted by G.B.J. Glass. "Tbis territory is reminiscent of a subtropical forest full of proud and established trees, jungle-like bushes inter-woven with each other, beautiful flowers not yet classified in the Linnaen code, and also frail and weak growths struggling for survival.
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