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Regulation of pancreatic and gastric endocrine secretion by GIP and GLP-1(7-36) amide Jia, Xiaoyan 1995

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R E G U L A T I O N OF P A N C R E A T I C AND GASTRIC ENDOCRINE SECRETION B Y GIP AND GLP-l(7-36) A M I D E by  X I A O Y A N JIA M . D . , Dalian Medical College,  1983  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY  in  T H E F A C U L T Y OF G R A D U A T E STUDIES Department o f P h y s i o l o g y Faculty o f M e d i c i n e W e accept this thesis as c o n f o r m i n g to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A November, © X i a o y a n Jia,  1995 1995  In  presenting  degree freely  at  this  the  University  available  copying  of  department publication  for  this or of  thesis  this  partial  of  British  reference  thesis by  in  for  his thesis  and  or for  her  DE-6 (2/88)  study.  Columbia  of  I further  purposes  gain  the  requirements  I agree  that  agree  may  be  It  is  representatives.  financial  of  The University of British Vancouver, Canada  Columbia,  scholarly  permission.  Department  fulfilment  shall  not  that  the  Library  permission  granted  by  understood be  for  allowed  an  advanced  shall for  the that  without  make  it  extensive  head  of  my  copying  or  my  written  ABSTRACT  The gastrointestinal hormones gastric inhibitory polypeptide/glucosedependent insulinotropic polypeptide (GIP) and truncated glucagon-like peptide-1 (tGLP-1; GLP-K7-36) and GLP-K7-37)) are capable of both stimulating insulin secretion and inhibiting gastric acid secretion, these being referred to as their incretin and enterogastrone actions, respectively. It has not yet been established as to whether either peptide is predominantly responsible for these actions physiologically, or whether they act i n concert to achieve a common end point. There are, however, studies described i n the literature which suggest that tGLP-1 exhibits stronger insulinotropic activity than GIP in humans, rat islets or perfused pancreases. There are also data suggesting that tGLP-1 and GIP exert similar effects on gastric somatostatin secretion, a component of their enterogastrone activity, but opposite effects on gastrin release. These suggestions were reassessed in the studies described in this thesis. In addition, there is evidence for possible roles both for GIP and tGLP-1 i n the pathophysiology of obesity and noninsulin dependent diabetes mellitus (NIDDM) i n humans. In previous studies on GIP i n an animal model of obesity with accompanying hyperinsulinaemia, the Zucker rat, it was found that circulating GIP levels were normal i n obese animals but the glucose threshold for the insulinotropic activity of G I P was greatly reduced. This raised the possibility that there was a general defect i n the enteroinsular axis i n these animals, and that alterations i n the secretion and/or actions of tGLP-1 may exist. Responses of immunoreactive  (IR-) GLP-1 i n lean  and obese animals to an oral glucose tolerance test, and the glucose-dependence of the insulinotropic activity of GLP-l(7-36) i n their perfused pancreases, were therefore investigated.  In order to assess whether tGLP-1 and GIP exhibit different insulinotropic activities gradient perfusions of peptides were administered to an isolated perfused rat pancreas preparation. This enabled the accurate measurement of peptide thresholds for insulinotropic activity and the maximal effects of the peptides. In addition, glucose thresholds for action were determined with a similar gradient system. These studies demonstrated that insulin secretion was increased by both synthetic porcine (sp) GIP and GLP-1 (7-36) at concentrations of -16 pmol/L. Mean maximal responses to GLP-1 (7-36) i n the presence of 16.7 mmol/L glucose were slightly greater than to sp GIP or natural porcine (np) GIP, but i n the presence of 10 mmol/L glucose sp GIP and GLP-1 (7-36) exerted similar effects. In contrast, responses to synthetic human (sh) GIP were greatly reduced when compared to those to the porcine peptide. In the presence of 50 pmol/L sp GIP or G L P - 1 (7-36) the glucose thresholds were identical: 4.5 ± 0.11 mmol/L. Interestingly, increasing either peptide concentration from 50 to 300 pmol/L did not have a major effect on maximal responses to the glucose gradient plus peptide, the reason for which is obscure.  Gastric endocrine secretory responses to different preparations of GIP or tGLP-1 were studied i n an isolated perfused stomach preparation also using gradient perfusion of peptide. A l l peptides increased the secretion of somatostatinlike immunoreactivity (SLI) with a threshold concentration of -50 pmol/L. The initial rate of increase i n response to sp GIP (119 ± 39 pg/min) was greater than with other forms of GIP or with tGLP-1. Maximal increases with sp GIP and np GIP did not differ. Gastrin secretion was stimulated by similar concentrations of sp GIP and np GIP to those effective on SLI secretion. In contrast, both GLP-1 (736) and GLP-1 (7-37) suppressed gastrin secretion. Gastrin and SLI responses to sh GIP were greatly reduced compared to the porcine peptides.  Obese Zucker rats were hyperinsulinemic and demonstrated exaggerated responses to the oral glucose tolerance test compared to their lean litter mates. Basal glucose levels were slightly elevated i n the obese rats but responses to the oral load did not differ from those in the lean animals. Significant increases i n IRGLP-1 levels i n response to oral glucose were only observed i n the obese animals. Perfused pancreases from obese rats hypersecreted insulin at a l l glucose concentrations tested, and exhibited potentiated responses to GLP-1 (7-36). In the presence of 4.4 mmol/L glucose GLP-1 (7-36) increased insulin secretion i n the perfused pancreases from obese animals ~25-fold, whereas there was only a 5-fold increase i n pancreases from lean animals. Pancreases from obese rats perfused with a glucose gradient (2.8-11 mmol/L) i n the presence of GLP-1 (7-36) responded with an immediate increase i n insulin secretion at a glucose concentration as low as 2.8 mmol/L, whereas pancreases from lean animals required a minimum of 4.22 mmol/L glucose for stimulation. In the presence of high glucose (16.7 mmol/L) both obese and lean pancreases exhibited similar concentration thresholds for GLP-1 (7-36) of <50 pmol/L.  In summary, these studies provide clear evidence that GLP-1 (7-36) and porcine GIP are equally insulinotropic in the perfused rat pancreas, and share the same glucose threshold for activity, whereas sh GIP is less active. The latter result is probably due to problems i n the chemical synthesis of the peptide. It is speculated that, although both GIP and tGLP-1 are physiological incretins, at the concentrations observed postprandially GIP is likely to be more important. The results of these studies also show that GIP, GLP-1 (7-36) and GLP-1 (7-37) all stimulate S L I secretion from the perfused stomach but that, whereas G I P stimulates the release of gastrin both forms of GLP-1 inhibit secretion. The  possible significance of this to the regulation of gastric function is discussed. Finally, IR-GLP-1 responses to oral glucose are exaggerated i n the obese Zucker rat, and the glucose threshold for the insulinotropic action of GLP-1 (7-36) is aberrant, suggesting that there is a general alteration i n the hormonal component of the enteroinsular axis i n these animals which may contribute to the fasting hyperinsulinemia and exaggerated insulin responses to glucose.  ABSTRACT  ii  LIST OF T A B L E S  xv  LIST OF F I G U R E S  xvi  ACKNOWLEDGEMENTS  x*  INTRODUCTION  1  I.  ENTEROGASTRONES  1  A.  Enterogastrones Originating in the Proximal Small Intestine  3  1.  Secretin..*.  3  2.  Cholecystokinin  B.  C.  •.  4  Enterogastrones from the Distal Intestine  5  1.  Neurotensin  5  2.  Polypeptide Y Y  6  Somatostatin  6  II. T H E ENTERO-INSULAR AXIS  8  A.  The entero-insular Axis and Incretins  8  B.  Candidate Incretins  11  1.  Secretin.....  11  2.  Gastrin  12  3.  Cholecystokinin........  12  III. GASTRIC INHIBITORY POLYPEPTIDE  14  A.  Structure of GIP  14  B.  Distribution of GIP in the Gastrointestinal Tract  14  C.  Biological Activities of GIP  16  1.  Effect of GIP on Gastric Acid Secretion  16  2.  Effect of GIP on Gastrin Secretion  ..19  3.  Effect of GIP on Insulin Secretion  19  IV.  G L U C A G O N - L I K E PEPTIDE-1 A.  Preproglucagon Gene and Propeptide Processing in the Pancreas and Intestine  21  B.  Distribution of GLP-1 in the Gastrointestinal Tract  24  C.  Involvement of GIP and GLP-1 in a Proximal-Distal Intestinal Endocrine Loop  25  D.  Stimulation of GLP-1 Secretion  26  E.  Biological Activities of GLP-1  27  1.  Effect of Truncated GLP-1 on Insulin Secretion  27  2.  Effect of Truncated GLP-1 on Secretion of Pancreatic Somatostatin and Glucagon  3.  4.  29  Effect of Truncated GLP-1 on Somatostatin and Gastrin Secretion from Stomach  30  MECHANISMS INVOLVED IN THEINSULINOTROPIC ACTIONS OF GIP A N D GLP-K7-36) A M I D E  31  A.  GIP Receptors and Signal-Transduction Mechanisms  31  B.  GLP-1 Receptors and Signal-Transduction Mechanisms  34  C.  Potentiation of Glucose-induced Insulin Secretion by GIP and truncated GLP-1  VI.  28  Effect of Truncated GLP-1 and Oxyntomodulin on Gastric Acid Secretion  V.  21  35  P O S S I B L E R O L E S OF GIP A N D GLP-K7-36) A M I D E I N N O N I N S U L I N D E P E N D E N T DIABETES M E L L I T U S  37  VII. P O S S I B L E R O L E S F O R GIP A N D GLP-K7-36) A M I D E I N H U M A N OBESITY A N D I N T H E Z U C K E R RAT VIILTHESIS INVESTIGATIONS  38 .41  METHODS  43  I.  RADIOIMMUNOASSAY  43  A.  Development of a GLP-1 Assay  43  1.  43  Production of Antibodies 1.1  (KLH)  43  Immunization of Guinea Pigs  44  Preparation of I-GLP-l(7-36) amide  45  2.1  Iodination of GLP-K7-36) amide  45  2.2  High Performance Liquid Chromatography  1.2 2.  Conjugation of GLP-1 to Keyhole limpet hemocyanin  125  Purification of I-GLP-1(7-36) amide 125  3.  GLP-K7-36) amide Standards  49  4.  Procedure and Conditions of GLP-1 Assay  49  5.  Separation of Bound and Free  50  6.  Characterization of Antibody  52  6.1  Anti-GLP-1 Antiserum Titers  52  6.2  Antiserum Crossreactivity  52  6.3  Assay Sensitivity  52  Reproducibility of the Assay  53  7. B.  46  125  I-GLP-l(7-36) amide  Insulin Assay  53  1.  Antiserum  54  2.  125  I-Insulin  54  3.  Standard  55  4.  Separation  56  5.  Production of Charcoal Extracted Plasma  56  6.  Procedure  56  7.  Assessment of Assay  57  ix  C.  D.  Somatostatin Assay  58  1.  Antibody  58  2.  125  I-Somatostatin  58  3.  Standard  59  4.  Separation  59  5.  Procedure  60  6.  Assessment of Assay  60  Gastrin Assay  61  1.  Antibody....  61  2.  Iodination of Gastrin  61  3.  Standard.  62  4.  Separation  62  5.  Procedure  63  6.  Assessment of Assay  63  II. ORAL GLUCOSE TOLERANCE TEST  63  A.  Animals  63  B.  Procedure  64  III. ISOLATED PERFUSED ORGAN PREPARATIONS A.  64  Stomach Perfusion  65  1.  Surgical Procedure  65  2.  Apparatus  66  3.  Solutions and Reagents  67  3.1. Perfusate  67  3.2 Peptides  68  4.  68  Perfusion Procedure  B. Pancreas Perfusion 1.  Surgical Procedure  69 69  IV.  V.  2.  Apparatus  70  3.  Solution and Reagents  70  4.  Perfusion Procedure  70  M E A S U R E M E N T OF GLP-1 I N RAT P A N C R E A S A N D I L E U M EXTRACTS  71  A.  Tissue Extraction....  71  B.  H P L C Separation  C.  Assay  ....72 73  STATISTICAL A N A L Y S E S  73  A.  Pancreas Perfusions  73  1.  WistarRats  .73  2.  Zucker Rats  74  B.  Stomach Perfusions  75  C.  Glucose Tolerance Test  75  RESULTS I.  C O M P A R I S O N OF T H E E F F E C T S O F GASTRIC INHIBITORY P O L Y P E P T I D E A N D G L U C A G O N - L I K E PEPTIDE-1(7-36) AMIDE ON INSULIN SECRETION FROM THE ISOLATED P E R F U S E D RAT PANCREAS  77  A.  Gradient Characteristics  77  1.  Measurements of Effluent Glucose Levels  78  2.  Measurements of Effluent Glucose Levels i n the Presence of the GIP or GLP-K7-36) amide  3.  78  Measurements of Effluent GIP Concentrations from Perfusions with a 0 -1 nrnol/L Gradient of GIP i n the Presence of 10 mmol/L Glucose  78  B.  H P L C Analysis of GLP-l(7-36) Amide and Different GIP Preparations Used for Perfusion  C.  82  Comparison of 0-1 nmol/L Gradient Perfusion of GIPs and GLP-K7-36) Amide on Insulin Secretion in the Presence of 16.7 mmol/L Glucose  D.  Comparison of sp GIP and GLP-1(7-36) Amide Gradients on Insulin Secretion in the Presence of 10 mmol/L Glucose 1.  2.  85  Insulin Responses to 0-1 nmol/L Gradients of Synthetic Porcine GIP or GLP-K7-36) Amide  86  Insulin Responses to 0-50 pmol/L Gradients of Synthetic Porcine GIP or GLP-K7-36) Amide  E.  82  86  Comparison of sp GIP and GLP-l(7-36) Amide on Insulin secretion in the Presence of a 2.8-11 mmol/L Glucose Gradient 1.  89  Insulin Responses to a 2.6-11 mmol/L Glucose Gradient in the Presence of 50 pmol/L sp GIP or GLP-K7-36) amide  2.  89  Insulin Responses to a 2.8-11 mmol/L Glucose Gradient in the Presence of 300 pmol/L sp GIP or GLP-K7-36) Amide  89  C O M P A R I S O N OF T H E E F F E C T S OF D I F F E R E N T F O R M S OF GASTRIC INHIBITORY P O L Y P E P T I D E A N D G L U C A G O N L I K E PEPTIDE-1 O N SOMATOSTATIN A N D G A S T R I N R E L E A S E F R O M ISOLATED P E R F U S E D R A T S T O M A C H A.  95  Effects of GLP-1, GLP-K7-36) Amide and GLP-2 on Gastric Somatostatin Secretion  95  1.  Somatostatin Secretion in Response to 1 nmol/L GLP-1  2.  Somatostatin Secretion i n Response to 1 nmol/L GLP-K7-36) Amide  3.  96  Somatostatin Secretion i n Response to a 0-800 pmol/L Gradient of GIPs or Truncated Forms of GLP-1 1.  2.  96  Effects of np GIP, sp GIP and sh GIP on Somatostatin Secretion  100  Effects of GLP-K7-36) amide and GLP-K7-37) on Somatostatin Secretion  C.  96  Somatostatin Secretion in Response to 1 nmol/L GLP-2  B.  95  100  Gastrin Secretion in Response to a 0-800 pmol/L Gradient of GIPs or Truncated Forms of GLP-1  103  1.  Responses to np GIP, sp GIP and sh GIP  103  2.  Gastrin Responses to GLP-K7-36) amide and GLP-1 (7-37)  106  STUDIES O N GLP-1 I N T H E Z U C K E R RAT  108  A.  Development of a GLP-1 Radioimmunoassay  108  1.  Preparation of Iodinated GLP-K7-36) Amide  108  1.1  108  Separation of Iodinated GLP-K7-36) Amide  1.2. H P L C Purification of Iodinated GLP-K7-36) Amide 2.  108  Characterization of Antibody  Ill  2.1 Titers of the Antisera  Ill  2.2  Crossreactivity  Ill  2.3  Sensitivity and Reproducibility of the Assay  114  3.  H P L C Analyses of GLP-1 i n Extracts of Pancreas and Ileum  114  3.1 GLP-1 Immunoreactivity i n Pancreas Extracts  114  3.2  117  GLP-1 Immunoreactivity i n Ileum Extracts  Responses of GLP-1 to a Glucose Load in vivo and Responsiveness of the Perfused Pancreas to GLP-1(7-36) amide in vivo  117  1.  Oral Glucose Tolerance Test  119  1.1  Plasma Glucose Levels  119  1.2  Insulin Secretion  199  1.3  GLP-1 Secretion  122  2.  Response of the Isolated Perfused Pancreas to GLP-K7-36) Amide i n the Presence of 4.4 mmol/L Glucose  122  2.1 Effect of 300 pmol/L GLP-K7-36) Amide  124  2.2 Effect of 50 pmol/L GLP-l(7-36) Amide i n Obese Rats 3.  124  Response of the Isolated Perfused Pancreas to 2.811 mmol/L Glucose Gradient in the Presence of 300 pmol/L GLP-1(7-36) Amide 3.1  Insulin Secretion i n Response to 2.8-11 mmol/L Glucose Alone in Obese and Lean Zucker Rats  3.2  128  128  Insulin Secretion in Response to 2.8-11 mmol/L - Glucose Gradient and 300 pmol/L GLP-1(7-36) Amide in Zucker Rats  128  4.  Responses of the Isolated Perfused Pancreas to 0-300 pmol/L GLP-1(7-36) Amide Gradient in the Presence of 16.7 mmol/L Glucose  .131  DISCUSSION  137  REFERENCES  155  APPENDIX  193  I.  Chemical Sources....  193  II.  List of Abbreviations  195  III. Systeme InternationaleUnits  197  XV  LIST O F T A B L E S  Table  Page  1.  The major gastrointestinal hormones  2  2.  Protocol of GLP-1 radioimmunoassay  51  LIST O F F I G U R E S  Figure  1.  Page  Amino acid sequences of porcine, human, bovine, rat and hamster GIPs  15  2.  Processing of proglucagon in the pancreas  22  3.  Processing of proglucagon in the intestine  23 125  4.  Outline of the H P L C system used i n the purification of  I-  GLP-1(7-36) amide 5.  Glucose concentrations i n perfusate effluent from the pancreas perfused with a 2-10 mmol/L glucose gradient (n=6)  6.  48  79  Glucose concentrations i n the perfusate effluent of the pancreas perfused with 2.8-11 mmol/L glucose in the presence of 300 pmol/L GIP or GLP-K7-36) amide  7.  .80  Immunoreactive GIP concentrations i n fractions from pancreas perfusions with a 0-1 nmol/L gradient of synthetic porcine GIP in the presence of 10 mmol/L glucose  8.  H P L C analysis of GLP-K7-36) amide and different preparations of GIP  9.  81  83  Effect of 0-1 nmol/L gradient perfusion of GLP-K7-36) amide, natural porcine, synthetic porcine or synthetic human GIP on insulin release from the isolated perfused rat pancreas....  10.  84  Effects of 0-1 nmol/L gradient perfusions of GLP-K7-36) amide or sp GIP on insulin secretion from the perfused rat pancreas i n the presence of 10 mmol/L glucose  87  xvii  11. Effects of 0-50 pmol/L gradient perfusions of GLP-1(7-36) amide or sp GIP on insulin secretion from the perfused rat pancreas i n the presence of 10 mmol/L glucose 12.  88  Effects of 50 pmol/L GLP-K7-36) amide or sp GIP on insulin secretion stimulated by a 2.8-11 mmol/L glucose gradient i n the perfused rat pancreas  90  13. Effect of 300 pmol/L GLP-K7-36) amide or sp GIP on insulin secretion stimulated by a 2.8-11 mmol/L glucose gradient i n the perfused rat pancreas 14.  92  Comparison of the effects of 50 or 300 pmol/L sp GIP on insulin secretion stimulated by a 2.8-11 mmol/L glucose gradient in the isolated perfused rat pancreas  93  15. Effects of 50 or 300 pmol/L GLP-K7-36) amide on insulin secretion stimulated by a 2.8-11 mmol/L glucose gradient i n the isolated perfused rat pancreas 16.  Effect of 1 nmol/L GLP-1 on somatostatin release from the isolated perfused stomach  17.  98  Effect of 1 nmol/L GLP-2 on somatostatin release from the isolated perfused rat stomach  19.  97  Effect of 1 nmol/L GLP-l(7-36) amide on somatostatin release from the isolated perfused stomach  18.  94  99  Effects of 0-800 pmol/L gradient perfusions of np GIP, sp GIP or sh GIP on somatostatin secretion from the isolated perfused rat stomach  20.  101  Effect of 0-800 pmol/L gradient perfusions of GLP-1(7-36) amide or GLP-K7-37) on somatostatin secretion from the isolated perfused rat stomach  102  21.  Effect of 0-800 pmol/L gradient perfusions of GLP-l(7-36) amide or sp GIP on gastrin release from the isolated perfused rat stomach... 104  22.  Effect of 0-800 pmol/L gradient perfusions of np GIP, sp GIP or sh GIP on gastrin release from the isolated perfused rat stomach  23.  105  Effect of 0-800 pmol/L gradient perfusions of GLP-K7-36) amide or GLP-1(7-37) on gastrin release from the isolated perfused rat stomach  107  24.  Sep-Pak cartridge separation of iodinated GLP-l(7-36) amide  109  25.  Reverse phase H P L C separation of I-GLP-l(7-36) amide 125  from the Sep-Pak purification  110  26.  The titers of guinea pig anti-GLP-1 antisera  112  27.  Specificity of KMJ-03 antiserum toward the recognition of GLP-K1-36) amide, GLP-K7-36) amide, GLP-K1-37), GLP-1 (7-37), glucagon and GIP  113  28.  Standard curves of the GLP-1 radioimmunoassay  115  29.  H P L C determination of GLP-1 content i n extract of rat pancreas  116  30.  H P L C determination of GLP-1 content i n two extracts of rat ileum  31.  Effect of 1 g/kg oral glucose on circulating glucose levels in lean and obese Zucker rats  32.  121  Effect of 1 g/kg oral glucose on circulating C-terminal GLP-1 levels i n lean and obese Zucker rats  34.  120  Effect of 1 g/kg oral glucose on circulating insulin levels i n lean and obese Zucker rats  33.  118  123  Effect of 300 pmol/L GLP-K7-36) amide on insulin secretion from the isolated perfused pancreas of lean Zucker rats i n the presence of 4.4 mmol/L glucose  125  xix  35.  Effect of 300 pmol/L GLP-K7-36) amide on insulin secretion from the isolated perfused pancreas of obese Zucker i n the presence of 4.4 mmol/L glucose  36.  126  Effect of 50 pmol/L GLP-l(7-36) amide on insulin secretion from the isolated perfused pancreas of obese Zucker rats i n the presence of 4.4 mmol/L glucose  37.  Effect of 2.8-11 mmol/L glucose gradient on insulin secretion from the isolated perfused pancreas in lean and obese Zucker rats  38.  127  129  Effect of 300 pmol/L GLP-K7-36) amide on insulin release from the isolated perfused pancreas of lean Zucker rats in the presence of a 2.8-11 mmol/L glucose gradient  39.  ...130  Effect of 300 pmol/L GLP-K7-36) amide on insulin release from the isolated perfused pancreas of obese Zucker rats i n the presence of a 2.8-11 mmol/L glucose gradient  40.  132  Effect of a 0-300 pmol/L gradient perfusion of GLP-K7-36) amide on insulin secretion from the isolated perfused pancreas of lean Zucker rats in the presence of 16.7 mmol/L glucose  133  41. Effect of a 0-300 pmol/L gradient perfusion of GLP-K7-36) amide on insulin secretion from the isolated perfused pancreas of obese Zucker rats i n the presence of 16.7 mmol/L glucose 42.  134  Total insulin output stimulated by 0-300 pmol/L GLP-K7-36) amide in the presence of 16.7 mmol/L glucose i n lean and obese Zucker rats  136  Acknowledgment  I would first like to thank my supervisor, Dr. C.H.S. Mcintosh, for his expert guidance, support and precious time over the past several years. His invaluable advice, patience and constructive criticism helped me to solve the riddles and made this work possible. I also thank Dr. R. Pederson for his constant support as a supervisory committee member and graduate advisor, as well as for his help i n the insulin RIA, animal experiments and for providing Zucker rats. Thanks also to Dr. Y . N . Kwok for his help i n raising the GLP-1 antibody and i n the somatostatin RIA. I thank Dr. A. Buchan for her help in the gastrin RIA and her encouragement, and Dr. J . C. Brown for his support during my stay in the department of Physiology. I would like to express my deep appreciation to my supervisory committee members Dr. K. G. Baimbridge, Dr. S Kehl, Dr. R. Pederson and Dr. Y. N . Kwok for their invaluable time, guidance and suggestions to improve and complete this work. I am very grateful for the skillful technical and secretarial assistance from Connie Chisolm, Herminia Sy, John Sanker, Joe Tay, Debbie Yaschuk, Dave Phelan, Marie Langton, Zaira Khan and Nancy Kilpatrick. In particular, I would like to thank Leslie Checknita for excellent technical assistance in the insulin and GIP RIAs and Dr. Z. Huang for helping in tissue culture. Also I would like to extend my gratitude to other graduate students i n the Department, particularly Eric Accili, Bruce Verchere, T i m Kieffer, Carole McClean, Glenn Morrow, for their encouragement, warmth and support. Finally, I thank my family, especially my parents and my sister for their endless support, and my husband and my son for their love and encouragement.  INTRODUCTION  The first gastrointestinal (GI) hormone identified, secretin (Bayliss and Starling, 1902), was discovered as a result of studies aimed at explaining the tightly regulated events involved i n controlling the various GI and pancreatic functions. Subsequently, as a result of technological developments i n peptide purification (chromatography, electrophoresis), improvements i n bioassays, and the development of radioimmunoassays, an ever-expanding number of peptide hormones have been isolated from gut extracts.  Many of these have been  purified, and their amino acid sequences determined, including secretin itself (Jorpes et al., 1962), gastrin (Gregory and Tracy, 1964), cholecystokinin (CCK; Mutt and Jorpes, 1968), gastric inhibitory polypeptide (GIP) (Brown, 1971; Brown and Dryburgh, 1971) and motilin (Brown et al., 1972; 1973). Numerous actions have been attributed to these different peptides, but there is still considerable controversy as to which peptides are physiologically important, entericallyderived, inhibitors of gastric acid secretion (enterogastrones) and/or stimulators of insulin release from the endocrine pancreas (incretins). There is evidence both for and against such a role for a range of peptides, including those which are the topic of the current thesis, GIP and truncated (t) forms of glucagon-like peptide- 1(GLP1), GLP-K7-36) amide and GLP-K7-37). The tissue and cell localization of the GI hormones which will be discussed in this Introduction are presented i n Table I.  I. E N T E R O G A S T R O N E S  The term enterogastrone was coined by Kosaka and L i m (1930) to describe a hormone, or hormones, released from the intestine by fat and fat digestion products and which inhibited gastric acid secretion. This concept was expanded  TABLE  I  T H E M A J O R GASTROINTESTINAL H O R M O N E S  Peptide  Tissue of Origin  Cell Type  Secretin  Duodenum Jejunum  S  Cholecystokinin  Duodenum Jejunum  Gastric Inhibitory  Duodenum  Polypeptide  Jejunum  Gastrin  Reference  Chey and Escoffery., 1976; Polak et al., 1971. Buffa et al., 1976; Liddle et al., 1985.  K  Polak, et al., 1973; Buchan et al., 1978.  Antrum Duodenum  G  McGuigan, 1968.  Somatostatin  Entire Gastrointestinal Tract  D  Polak, et al., 1975a; Vale et al., 1976.  Glucagon-like  Pancreas  A  Peptide-1  Ileum Colon  L  Varndell et al., 1985; Kauth and Metz, 1987; Eissele et al., 1992.  Oxyntomodulin  Ileum Colon  Neurotensin  Ileum  N  Polak, et al., 1977.  Peptide Y Y  Ileum Colon  L  Lundberg, et al., 1982.  Buchan and Polak, 1980.  later to include all acid inhibitory hormones released from the small and large intestines i n response to acid, fat and hypertonic solutions (Gregory, 1967; Johnson and Grossman, 1968).  Several peptides have been considered as  candidate enterogastrones including secretin, C C K , somatostatin and GIP, released from the upper small intestine, and neurotensin, peptide tyrosine tyrosine (PYY), and various intestinal products of the proglucagon gene, secreted by the lower intestine.  In the current section evidence for and against the  involvement of specific peptides originating in the proximal and distal regions of the GI tract i n the enterogastrone response will be outlined. Discussion of a possible role for GIP and products of the proglucagon gene will be reviewed later (Sections III.C.l. and IV.E.3.).  A. Enterogastrones Originating in the Proximal Small Intestine  1. Secretin  Secretin has been shown to be released by intraduodenal acidification (Fahrenkrug and Schaffalitzky de Muckadell, i977; Hoist et al., 1981; Chey and Konturek, 1982). A n inhibitory effect of secretin on gastric acid secretion has been observed i n humans (Brooks and Grossman, 1970; Kamionkowski et al., 1964), dogs (Johnson and Grossman, 1968; Nakajima et al., 1969) and rats (Chey et al., 1973; Thompson and Johnson, 1969; Sandvik et al., 1987). Neutralization of endogenous secretin with polyclonal antibodies increased plasma gastrin and gastric acid secretory responses to a meal i n dogs (Chey et al., 1981; J i n et al., 1991), supporting a role for secretin as an enterogastrone. A possible involvement of endogenous prostaglandins i n the gastric acid inhibitory action of secretin was first demonstrated by MacLellan et al. (1988).  These authors showed that the inhibitors of prostaglandin synthesis, indomethacin and meclofenamate, both abolished secretin-mediated inhibition of gastric acid secretion. This finding has been supported by subsequent studies. Intravenous infusion of secretin inhibited pentagastrin-stimulated gastric acid secretion which was accompanied by an increase i n the prostaglandin E content g  of the gastric mucosa i n rats (Shiratori et al., 1993). The inhibition produced by secretin was blocked by indomethacin, and the blocking action was reversed by intravenous administration of prostaglandin E  2  (Shiratori et al., 1993; Rhee et al.,  1991).  2. Cholecystokinin  Cholecystokinin has been shown to be released from the duodenal mucosa by amino acids and fat (Himenos et al., 1983; Liddle et al., 1985) and to suppress histamine- or gastrin-stimulated (Corazziari et al., 1979) and postprandial (Konturek et al., 1993) gastric acid secretion i n dogs and humans. In the studies of Corazziari et al. (1979) concentrations of exogenous C C K required to elicit its inhibitory action were higher than circulating levels and the action of C C K as a physiological enterogastrone was therefore questioned.  Several receptor  antagonists have become available recently and used i n studies on the role of C C K i n mediating inhibition of gastric acid secretion. Two types of C C K receptors have been identified, based on their affinity for selective antagonists (Sadzot et al., 1990; H i l l and Woodruff, 1990). Type A C C K receptors are located mainly i n the gastrointestinal tract and mediate classical CCK-like effects, whereas type B receptors predominate i n the central nervous system (Wank et al., 1992), where they mediate the effects of C C K on neurons.  In the stomach, type B C C K  receptors mediate the effects of gastrin on acid secretion. Lloyd et al. (1992a)  demonstrated that MK-329, a selective type A C C K receptor antagonist, significantly reversed the inhibition of gastric acid output produced by fat perfusion of the intestine in dogs. A similar result was obtained i n a study using L364,718, another selective antagonist of C C K - A receptors (Konturek, et al., 1992). Administration of fat or acidified meal into the duodenum inhibited gastric acid while significantly increasing plasma C C K levels in dogs. L-364,718 reversed this decrease i n gastric acid secretion. Furthermore, intravenous infusion of exogenous C C K in a dose, which resulted in levels found postprandially, resulted in a similar inhibition of acid secretion which was completely abolished by pretreatment with L-363,718. Therefore C C K should be considered to be an important enterogastrone, at least in some species.  B. Enterogastrones from the Distal Intestine  Several peptides located in the ileum and colon have been found to inhibit gastric acid secretion and considered to be candidate enterogastrones including neurotensin, P Y Y , and two products of the proglucagon gene, truncated GLP-1 and oxyntomodulin. The latter two peptides are discussed in Section IV.E.3.  1. Neurotensin  Neurotensin has been suggested to be a hormonal regulator of gastric acid secretion since it is released by fat and other nutrients (Go and Demol, 1981; Rosell and Rokaeus, 1979; Shaw and Buchanan, 1983) and is capable of inhibiting pentagastrin- (Hammer et al., 1991; Andersson et al., 1976) and meal-stimulated (Eysselein et al., 1990) acid secretion. Furthermore, immunoneutralization with a neurotensin monoclonal antibody i n rats (Seal et al., 1988) prevented the  inhibition of gastric acid secretion produced by intra-ileal fat, indicating that it may play an important role in distal small intestine, fat-induced, inhibition of acid secretion. Results from studies on the action of neurotensin i n humans are controversial with evidence both for (Blackburn et al.,1980; Skov Olsen et al., 1983) and against (Mogard et al., 1987) a physiological effect of the peptide on meal-stimulated gastric acid secretion. Therefore further studies are needed to clarify as to whether neurotensin is an enterogastrone i n humans.  2. Polypeptide Y Y  P Y Y and GLP-1 are co-localized i n endocrine L cells of the distal intestine (Ali-Rachedi et al., 1984; Kreymann et al., 1991). P Y Y is released into the circulation i n response to luminal fat (Aponte et al., 1985; Pappas et al., 1985) or ingestion of a meal (Adrian et al., 1985). Exogenous administration of P Y Y , resulting i n physiological circulating concentrations, caused dose-dependent inhibition of meal-stimulated (Pappas et al., 1985; Lloyd et al., 1991) and pentagastrin-stimulated (Pappas et al., 1986; Guo et al., 1987) acid secretion i n dogs, as well as vagally-induced gastric acid secretion i n humans (Wettergren et al., 1994), suggesting that P Y Y is a candidate enterogastrone.  C. Somatostatin  Studies on the molecular nature of tissue somatostatin i n the GI tract have shown that there are two principal bioactive forms, somatostatin-14 (SS-14) and  somatostatin-28 (SS-28) (Polonsky et al., 1983; O'Shaughnessy et al., 1985; Ensinck et al., 1989) both of which have been suggested to act i n an endocrine manner on the GI tract and pancreas (reviewed by Mcintosh, 1985). Somatostatin i n the stomach and pancreas is present mainly as SS-14, whereas SS-28 is the major form found i n the small intestinal mucosa (Trent and Weir, 1981; Penman et al., 1983; Baldissera et al., 1985a,b). Although SS-14 is present in both stomach and pancreas, the stomach appears to be the major source of circulating SS-14, with the pancreas making only a minor contribution (Taborsky and Ensinck, 1984). Circulating SS-28 originates mainly from the intestine (Ensinck et al., 1989). Numerous investigators have reported increases i n circulating somatostatin in response to a meal (Chayvialle et al., 1980; Penman et al., 1981; Polonsky et al., 1983; Colturi et al., 1984). Fat and protein were originally considered to be the major stimuli for gastrointestinal somatostatin release (Lucey et al., 1984; Schusdziarra etal., 1979), but in vitro studies have shown that acid also regulates the secretion of gastric somatostatin (Schubert et al., 1988). Both SS-14 and SS28 increase i n the circulation after meal ingestion i n humans (Ensinck et al., 1989) and dogs (Greenberg et al., 1993), suggesting that both could exert endocrine effects.  In addition to an endocrine mechanism, somatostatin may affect  gastrointestinal function i n a paracrine manner (Short et al., 1985; Makhlouf and Schubert, 1990; Mcintosh et al., 1991) as D cells i n the stomach appear to be structured to facilitate local regulatory actions via direct contacts with both parietal and G cells (Kusumoto et al., 1979; Larsson et al., 1979) Exogenous somatostatin, infused to achieve concentrations close to those observed after a meal, was capable of inhibiting gastric acid secretion i n humans (Colturi et al., 1984) and dogs (Seal et al., 1982). In the urethane-anesthetized gastric fistula rat, monoclonal antibodies to somatostatin were shown to block the  inhibition of gastric acid secretion produced by an intraduodenal infusion of oleic acid (Seal et al., 1987), indicating a role for somatostatin i n intestinal fat-inhibited acid secretion. However, these studies do not distinguish between an endocrine effect of intestinal somatostatin somatostatin.  and enterogastrone-induced  release  of gastric  Several GI hormones have been shown to possess potent  somatostatin-releasing activities, including GIP (Mcintosh et al., 1981b), C C K (Soli et al., 1985; Bengtsson et al., 1989), neurotensin and oxyntomodulin (Hammer et al., 1992), and GLP-K7-36) amide (Eissele et al., 1990). Further studies have shown that CCK-stimulated somatostatin release in vivo was blocked by application of MK-329, a C C K - A receptor antagonist,  and  administration of a somatostatin monoclonal antibody prevented CCK-induced inhibition of gastrin or meal stimulated gastric acid secretion i n rats and dogs (Lloyd et al., 1992b; Lloyd et al., 1994). CCK-stimulated somatostatin release was also inhibited by MK-329 in cultured human antral epithelial cells (enriched for D cells) (Buchan et al., 1993). Unfortunately, there are no antagonists available to perform similar studies on the other potential enterogastrones. Evidence for and against roles for GIP and GLP-1(7-36) amide i n gastric acid secretion will be discussed later in Sections III.C.l. and IV.E.3.  II.  T H E E N T E R O - I N S U L A R AXIS  A.  The Entero-Insular Axis and Incretins  The term "incretin" was introduced by Zunz and Labarre i n 1929 to describe a humoral substance produced in the gut that enhanced the release of insulin from pancreatic islets. The insulin response to intravenous glucose was later shown to  be much smaller than to either oral glucose (Elrick et al., 1964) or intrajejunal glucose infusion (Mclntyre et al., 1964), even though there was a greater increase in blood glucose levels following intravenous administration. It was postulated that absorption of glucose in the GI tract stimulated the secretion of one or more hormones which were responsible for stimulating insulin secretion. In 1969, Unger and Eisentraut introduced the term "entero-insular axis" to describe the connection between the gut and the pancreatic islets. This axis comprised all stimuli originating i n the intestine which stimulate the islets of Langerhans. Therefore, i n contrast to "incretin" which was restricted to hormonal factors, the term "entero-insular axis" includes hormonal, neural and nutrient signals from the gut to the islet, and their interactions i n the regulation of insulin, glucagon, somatostatin and pancreatic polypeptide release (Creutzfeldt, 1979).  The  nutrient factors include absorbed glucose, amino acids and fatty acids which stimulate secretion of islet peptides. The innervation of the islet has been shown to include cholinergic, adrenergic and peptidergic neurons (Miller, 1981; Ahren et al., 1986; Rehfeld et al., 1980). Parasympathetic preganglionic nerve fibers are carried by the vagus nerve, terminating on postganglionic fibers i n pancreatic ganglia of the islets. Most preganglionic sympathetic fibres travel i n the splachnic nerve, synapse with postganglionic fibers i n the celiac ganglia, and then enter the islets. The major postganglionic (intrinsic) neurotransmitter is considered to be acetylcholine (Amenta et al., 1983).  It is now believed that both parasympathetic and  sympathetic nerves act primarily by regulating the activity of intrinsic nerves (Ahren et al., 1991). However, i n recent years evidence has accumulated that i n addition to this classical autonomic innervation, peptidergic neurons also exist i n the pancreas, either as terminals of sympathetic efferent nerves or as intrinsic neurons. Among the pancreatic neuropeptides which have been identified are  vasoactive intestinal polypeptide (VTPXBishop et al.,1980; De Giorgio et al., 1992), substance P, enkephalins (Larsson and Rehfeld, 1979), gastrin releasing peptide (GRP; mammalian bombesin)(Ghathei et al., 1984; De Giorgio et al., 1992), C C K (Rehfeld et al., 1980), neuropeptide Y (NPY)(Carlei et al., 1985), neurotensin (Reinecke, 1985), calcitonin-gene-related peptide (CGRPXSternini and Brecha, 1986), and galanin (Dunning et al., 1986). The majority of these peptides have been shown to have effects on insulin secretion. In general, VIP, C C K , and G R P seem to be stimulatory neuropeptides, whereas galanin, N P Y and C G R P are inhibitory (Ahren et al., 1986). Recently, nervous connections between the myenteric plexus and the pancreatic ganglia were found.  Exposure of the  duodenal mucosa to veratridine, a neuronal activating compound, demonstrated activation of a duodenal-islet neuronal circuit leading to activation of cells i n the islets (Kirchgessner and Gershon, 1990). It is possible that intraluminal nutrients are monitored via these nervous connections which in turn report to the endocrine pancreas directly. According to Creutzfeldt (1979), an incretin should fulfill two criteria: firstly, the hormone must be released i n response to nutrients, particularly carbohydrates, and secondly, physiological concentrations of the hormone must stimulate insulin secretion in the presence of elevated blood glucose levels. As will be discussed, these criteria exclude most of the known gastrointestinal hormones with the exception of GIP and the newest incretin candidate GLP-1(7-36) amide. They preclude insulinotropic hormones that are released i n response to intestinal stimuli other than glucose, and hormones which exert effects on the secretion of islet peptides other than insulin. It has been suggested that the incretin concept should be expanded to include circulatory peptides released by nutrients other than glucose and which stimulate islet secretion in the presence of nutrients such  as amino acids (Rushakoff et al., 1987). Under this definition, as discussed below, C C K is a potential incretin, although a neurotransmitter role is possible.  B. Candidate Incretins  Several gastrointestinal hormones have been demonstrated to possess insulinotropic activity including secretin, gastrin, C C K , GIP and tGLP-1. The latter two are discussed i n Sections III.C.3. and I V . E . l .  1. Secretin  Intravenous secretin was shown to increase circulating insulin levels by several investigators (Unger et al., 1967; Kaess et al., 1970; Fahrenkrug et al., 1978; Dupre et al., 1969). However, it is unlikely that it was a physiological effect, because it occurred only with pharmacological doses of secretin (Fahrenkrug et al., 1978; Schaffalitzky De Muckadell et al., 1976; Shima et al., 1978). Furthermore, it was demonstrated that ingestion of glucose (Bloom, 1974) or a mixed meal (Bloom et al., 1975) was without effect on secretin secretion and, although intraduodenal acid infusion released insulin, the infusion of secretin i n concentrations that mimicked plasma levels did not stimulate insulin secretion even under hyperglycemic conditions (Fahrenkrug et al., 1978). In view of the high concentrations required to influence islet secretion it is therefore unlikely that secretin plays a major role i n the intestinal stimulation of insulin secretion, and it is now generally excluded from the accepted incretin candidates.  2. Gastrin  Although gastrin has been shown to stimulate insulin secretion (Dupre etal., 1969; Creutzfeldt et al., 1970), its insulinotropic effect was weak, and the serum concentrations of exogenous gastrin necessary to exert an effect were far greater than the concentrations measured after ingestion of a meal (Rehfeld and Stadil, 1973). Furthermore, glucose was shown to be a very weak stimulant of gastrin secretion, making it a poor incretin candidate, under the classical definition (Rehfeld and Stadil, 1975). Although the products of protein digestion are the major stimuli for gastrin secretion, gastrin is also not considered to be an important factor i n amino acid-induced insulin secretion (Rehfeld et al., 1978). Therefore, gastrin has also been excluded from the list of incretin candidates.  4. Cholecystokinin  It was demonstrated by Dupre and Beck (1966) that a crude preparation of C C K possessed insulinotropic activity, but this potent insulinotropic effect was later ascribed to GIP contamination (Hedner et al., 1975; Dupre et al., 1973; Brown and Otte, 1979). Subsequent studies with synthetic C C K - 8 , however, demonstrated a stimulation of insulin release in vivo (Verspohl et al., 1992; Ahren et al., 1991) and in vitro (Muller et al., 1983; Verspohl et al., 1986; Verspohl and Amnion, 1987). Several other molecular forms of C C K , including CCK-4, CCK-33 and CCK-39, have also been shown to stimulate insulin secretion (Ahren et al., 1991; Okabayashi et al., 1989). Furthermore, C C K receptors on the B-cell were demonstrated i n autoradiographic (Sakamoto et al., 1985) and receptor binding studies (Verspohl etal., 1986).  Following the development of C C K radioimmunoassays (Schafmayer et al., 1982; Jasen and Lamers, 1983) and bioassays (Liddle et al., 1985), it has become clear that i n many of the earlier studies pharmacological, rather than physiological, concentrations  of C C K were used i n the  experiments.  Measurements of plasma C C K showed that ingestion of fat and amino acids released C C K , whereas oral glucose, the most important incretin stimulus, was found to release no detectable C C K as measured by either bioassay (Liddle et al., 1985) or radioimmunoassay (Reimers et al., 1988). In humans, infusion of exogenous CCK-8 at a rate which achieved postprandial circulating levels did not release insulin during basal or hyperglycemic conditions (Rushkakoff et al., 1987; Reimers et al., 1988). Additionally, CCK-8 did not enhance phenylalanine-induced insulin secretion under hyperglycemic clamp conditions (Reimers et al., 1988). However, a similar concentration of CCK-8 was capable of augmenting insulin release stimulated by arginine or mixed amino acids (Rushkakoff et al., 1987; Hildebrand etal., 1991). Therefore, while C C K has been shown not to contribute to the incretin effect during carbohydrate ingestion, it may act as an incretin for amino acid-stimulated insulin secretion. The development of specific C C K receptor antagonists has provided an additional tool for the study of the role of C C K i n insulin secretion. The C C K antagonist L-364,718 inhibited CCK-8-stimulated insulin secretion in vitro i n isolated rat pancreatic islets (Zawalich et al., 1988), and in vivo i n the mouse (Reagon et al., 1987). However, since C C K has been detected i n pancreatic intrinsic nerves these experiments do not establish with certainty that the physiological effect of C C K is mediated via hormonal or neuronal pathways. Nevertheless, apart from GIP and tGLP-1, C C K is the strongest candidate for an incretin.  III. GASTRIC INHIBITORY P O L Y P E P T I D E  A. Structure of GIP  Porcine GIP is a 42 amino acid polypeptide, the amino acid sequence of which was determined by Brown and Dryburgh (1971) and later amended by Jbrnvall et al. (1981).  The sequences of bovine, human, rat and hamster GIPs were  subsequently determined from the isolated peptides or from cloned cDNAs (Carlquist et al., 1984; Moody et al., 1984; Higashimoto et al., 1992). The amino acid sequences of GIPs from these different species are shown i n Figure 1. Human GIP differs at two amino acid positions from the porcine peptide with a histidine for arginine substitution at position 18 and asparagine for serine at position 34 (Moody et al., 1984; Takeda et al., 1987), and one amino acid position from the rat peptide with an isoleucine for leucine substitution at position 40 (Higashimoto et al., 1992). This high degree of conservation of GIP sequence between species supports an important biological role for this peptide i n mammals. GIP belongs to the glucagon superfamily of peptides which includes GLP-1, GLP-2, secretin, V I P , peptide histidine methionine (PHM) and growth hormone releasing factor (GRF) (Bell, 1986).  B. Distribution of GIP in the Gastrointestinal Tract  Immunohistochemical studies demonstrated that GIP was localized i n endocrine K cells i n the mucosa of the upper small intestine, i.e. duodenum and jejunum, i n humans (Polak et al., 1973; Buchan et al., 1978). The distribution of GIP-containing cells extends to the terminal ileum in the dog and rat. However, no  15 Porcine 1. Tyr 2. Ala 3. Glu 4. Gly 5. Thr 6. Phe 7. Ile 8. Ser 9. Asp 10. Tyr ll.Ser 12.11e 13. A l a 14. Met 15. Asp 16. Lys 17.11e 18. Arg 19. Gln 20. Gln 21. Asp 22. Phe 23. V a l 24. Asn 25. Trp 26. Leu 27. Leu 28. A l a 29. Gln 30. Lys 31. Gly 32. Lys 33. Lys 34.Ser 35. Asp 36. Trp 37. Lys 38. His 39. Asn 40.11e 41. Thr 42. Gln  Human  Rat/Hamster  Bovine  Asn  lie  His  Asn  Leu  Figure 1. Amino acid sequences of porcine (Jornvall et al., 1981), human (Moody et al., 1984), bovine (Carlquist et al., 1984), rat and hamster (Higashimoto et al., 1992) GIPs. Only those amino acids which differ from porcine GIP are shown for the other peptides.  immunoreactive cells were found i n the colon (Buchan et al., 1982). Studies on expression of the GIP gene did not demonstrate the presence of human preproGIP m R N A i n tissues other than the gastrointestinal mucosa (Inagaki et al., 1989).  C. Biological Activities of GIP  The two major physiological actions of GIP are considered to be the inhibition of gastric acid secretion (Pederson and Brown, 1972) and stimulation of insulin release (Dupre et al., 1973).  1. Effect of GIP on Gastric Acid Secretion  Since partially purified preparations of C C K were found to stimulate acid secretion weakly under fasting conditions, while suppressing histamine- or gastrin-stimulated acid secretion (Magee and Nakamura, 1966; Brown and Magee, 1967), Brown and Pederson (1970) suggested that these disparate effects might be due to impurities i n available preparations of the hormone.  It was  demonstrated that a side fraction from the purification of C C K possessed enterogastrone activity (Brown et al., 1969). A n active substance was purified from this side fraction and named gastric inhibitory polypeptide, or GIP (Brown et al., 1970; Brown et al, 1971a,b). Studies on the secretion of GIP demonstrated that it was released i n response to ingestion of fat (Cleator and Gourlay, 1975; Pederson et al., 1975). The model used i n the first studies on the acid inhibitory action of GIP was the vagally and sympathetically denervated pouch of the body of the dog stomach. Intravenous administration of porcine GIP inhibited pentagastrin-stimulated acid secretion i n a dose-dependent manner (Pederson and Brown, 1972) with the  highest dose producing over 80% inhibition. The inhibitory effect on histaminestimulated acid secretion i n the pouch and insulin-stimulated secretion i n the vagally innervated gastric remnant were, however, much less (about 40% inhibition). Subsequent studies challenged a role for GIP as a physiological inhibitor of gastric acid secretion. Soon-Shiong et al. (1979a) also found that GIP was a potent inhibitor of pentagastrin-stimulated acid secretion i n vagally denervated Heidenhain pouches i n the dog, but had only weak effects i n the innervated stomach. Yamagishi and Debas (1980) studied the relative inhibitory effects of duodenal infusion of fat (oleic acid) with that of infused GIP on meal-stimulated acid secretion by the innervated stomach. Fat induced a complete inhibition of acid secretion, which exogenous GIP infusion failed to reproduce, despite the much higher plasma GIP levels reached i n the latter.  They concluded that i n the  innervated stomach the enterogastrone action of fat was not mediated by GIP. Maxwell et al. (1980) observed that GIP was also a weak inhibitor of pentagastrinstimulated acid secretion i n humans.  A n infusion of GIP sufficient to raise  circulating levels far i n excess of food-stimulated levels (>7 ng/ml) showed only a weak inhibitory effect on pentagastrin-stimulated acid secretion.  Thus the  increase i n circulating GIP levels produced by feeding was considered to be inadequate to exert a significant inhibitory action on gastric acid secretion. Similar results were obtained i n vagotomized patients (Simmons et al., 1981). Studies i n humans therefore did not support a role for GIP as a fat-released enterogastrone. Further investigation indicated that the GIP effect on acid secretion was more complex than previously thought. Background infusion of the cholinergic agonist bethanechol was found to totally abolish the inhibitory action of GIP i n both the vagally denervated Heidenhain pouch and the vagally innervated gastric  remnant (Soon-Shiong et al., 1979b; 1984). It was suggested that GIP may act indirectly on the parietal cell via release of a secondary substance (Mcintosh et al., 1979; 1981a), somatostatin, and parasympathetic nervous activity inhibited release of this substance, thus diminishing the acid inhibitory activity of GIP. Concentrations of GIP as low as 1 nmol/L were shown to have a marked stimulatory effect on the release of somatostatin-like immunoreactivity (SLI) from the perfused rat stomach (Mcintosh et al., 1981a). Furthermore, the GIPstimulated S L I release was abolished by electrical stimulation of the vagus nerves. These studies therefore supported a role for somatostatin as a mediator of GIP's inhibitory action (Mcintosh et al., 1981a). Mcintosh et al. (1981a) suggested that GIP could act as a physiological inhibitor of acid secretion i f fat i n the duodenum activated sympathetic fibres at the same time as releasing GIP. Involvement of the sympathetic nervous system i n the regulation of gastric somatostatin release was suggested by the observation that catacholamines (Koop et al., 1980; Goto et al., 1981) or sympathetic  stimulation (Mcintosh  et al.,  1981b) stimulated gastric  somatostatin release i n the perfused rat stomach.  In addition, basal and  postprandial somatostatin secretion were decreased i n sympathectomized dogs (Schusdziara et al., 1980). These studies indicated an indirect pathway for the action of GIP on gastric acid secretion, via somatostatin release with neuronal modulation by the sympathetic and parasympathetic nervous system. Although further in vivo studies i n dogs have attempted to discredit the possible involvement of G I P as an enterogastrone the lack of a suitable model has precluded such attempts (Creutzfeldt et al., 1983; Konturek et al., 1986).  2. Effect of GIP on Gastrin Secretion  Studies on the effect of GIP on gastrin secretion have produced ambiguous results. Pederson et al. (1981) demonstrated a paradoxical increase of gastrin release by GIP i n the perfused rat stomach which appeared to contradict reports that G I P had inhibitory actions on gastrin release in vivo.  However such  inhibitory actions were only observed during stimulation of gastrin secretion with a meal (Villar et al., 1976), via vagal stimulation (Hoist et al., 1983), or with carbachol (Wolfe and Reel, 1986). Therefore, further studies are necessary to clarify the effect of GIP on gastrin release.  3. Effect of GIP on Insulin Secretion  GIP has been generally accepted for some years to be an important incretin. Interest i n a role for GIP i n the regulation of insulin secretion was first stimulated by the observation of Dupre et al. (1973) that an intravenous infusion of GIP i n humans together with glucose resulted in a greater increase i n insulin release and an improvement in glucose tolerance when compared to glucose infusion alone. It was established later, in vivo i n the dog (Pederson et al., 1975) and man (Elahi et al., 1979), and i n the isolated perfused rat pancreas (Pederson and Brown, 1976) that the insulinotropic action of GIP was glucose-dependent, and that there existed a threshold glucose concentration of approximately 4.4-5.5 mmol/L i n the perfused rat pancreas. This glucose-dependence was included i n an alternate explanation of the acronym GIP: Glucose-dependent Insulinotropic Polypeptide. In the same in vitro preparation, the maximum potentiating action of glucose on GIP-induced insulin secretion was observed to be at a concentration of 16 mmol/L (Brown, 1982).  A n oral glucose load was shown to result in an increase i n circulating levels of GIP i n humans whereas intravenous glucose was ineffective (Cataland et al., 1974; Anderson et al., 1978). The dependency of GIP release on absorption of glucose was suggested to be a safeguard protecting the body against potential hypoglycemia (Creutzfeldt, 1979).  In contrast to oral glucose, fat-induced  increases i n GIP were not associated with a concomitant rise i n plasma insulin because the action of GIP on insulin secretion required glucose levels to be raised above fasting (Cleator and Gourlay, 1975; Ross and Dupre, 1978). Thus, the glucose dependence of GIP action on insulin secretion is another safeguard against hypoglycemia. With the hyperglycemic clamp model, Andersen et al. (1978) and Elahi et al. (1979) established that endogenous GIP released by oral glucose contributed to the insulin response  i n humans, and Kreymann et al. (1987)  showed that circulating levels of immunoreactive GIP attained following oral glucose or a mixed meal were sufficient, when mimicked by exogenous infusion of GIP, to stimulate insulin secretion. There is uncertainty as to whether porcine and human GIPs have different insulinotropic activities. In comparative studies on insulin responses to natural porcine GIP and a recombinant human peptide i n the isolated perfused pancreas almost identical insulin responses were obtained (Chow et al., 1991). However, in vivo studies with a human peptide produced by classical peptide synthesis revealed only weak insulinotropic activity i n humans (Nauck et al., 1993c). One component of this thesis is concerned with the comparison of the insulinotropic activities of the human and porcine peptides. For several years, GIP was considered to be the most important, i f not the sole, incretin. However, immunoneutralization of GIP only reduced the incretin effect approximately 20%-50% leading Ebert et al. (1983) to suggest the existence of additional insulinotropic gut hormones which may also be released following oral  glucose. The following section discusses the peptides now thought to account for the remaining incretin activity.  IV. G L U C A G O N - L I K E PEPTIDE-1  A. Preproglucagon Gene and Propeptide Processing in the Pancreas and Intestine  Cloning of cDNAs encoding preproglucagon led to the identification of the glucagon-like peptide sequences, i n addition to glucagon, within the prohormone. The sequences of hamster, bovine and human preproglucagon genes have been determined (Bell et al., 1983a; Lopez et al., 1983) and the encoded precursor contains the sequences of a signal peptide, an NH -terminal peptide (glicentin2  related pancreatic peptide, GRPP), glucagon, and two carboxyl-terminal glucagonlike peptides (GLP-1 and GLP-2). Both GLP-1 and GLP-2 show regional homology to glucagon. The GLP-1 sequence is identical in human, bovine, hamster and rat, whereas minor differences exist between the corresponding GLP-2 sequences (Schmidt et al., 1985; Hasegawa et al., 1990). This suggested that the peptides are biologically important rather than merely "spacer" peptides i n the precursor. The processing of proglucagon differs i n the mammalian pancreas and small intestine (George et al., 1985; Mojsov et al., 1986; Orskov et al., 1994; Hoist et al., 1994; Figure 2 and 3).  In the A-cell of the pancreas, the prohormone is  predominantly processed to glicentin-related pancreatic peptide (GRPP) (1-30), glucagon (33-61), the carboxyl-terminal fragment (72-158), or major pancreatic proglucagon fragment (MPF), and small amounts of free GLP-1 and GLP-2. The fate of M P F i n the pancreas has been studied i n several species. In earler studies, 0 r s k o v et al. (1986) reported that the M P F is not further processed i n the pancreas  of pig and human (0rskov et al. 1986; 1987).  However, by gel  O)  CD CD CM  CD  CD CO CD o CO CHi O c  UJ  2 CO CM  o < DC u. z o a  0  •E | •4-*  cu ai_> .2 C  < o _l  "5)"co CD E §w  O 00 O  o cc a. cc o  •n  o  O  CM  o o cc  Q.  CO CD  CC o <  cc  o  «- S c o°E co « o SS o g o CO Dl O.CD • O * CO c  co £ E S> E o c5  CD  .  w  »- U- o Q-Q_ C D o=• c .oQ+ "O H—• CD 0) c e > CD S (fl C O CD t CO <-> CO .£ CO *  <  <  CO  £ a>  Q-T3 CO CD C O _c CO c ~ c 2 .£ o — _ CD W 5 CO ~ O O T3 2 "cbco  CO CO Q . O C CD  2-^8CM O O CC Q)^ (5 c a — CO CD I L  a a  23  Ul Q  a> LO  CM  CM  OJ  Q. _l  a. _i o  CL _l o  1  I  CD  CD > CO CP  co co UJ  CL _I CD  _l CL  co  CD  E  I f  CM  co cu  CO  a  3 o CO  CL — I  CO CO  a  CM I  CL _I CD  —  **—  ,o  -C  CD  o ^ > c 0 ^  x: o o  2a. o  e"S  <  O CO  BE  00  01  "D  _l CL  Q _l.  C _JL  CD  CL _I CD  CD  CM  CD  CO  CD _ 1^ c o. > Vca c _a) co CLI CD  l i s  O *; t co g co 3 §£ CD  iz co  oj ¥ ~ ai co ^5 .g ~ T J  COI  Id. *  CO  +=: —I CD  .E o "g a? . E £ C M co  3  a o E O  CO I  o  o  CD <  CD < O  z> _l c o co o 1  X  o  — I CD  H Z  o  — I  a  C3)T3 -—ca cz •<-  3 <°CL [fid  o  goo  ^ a.  co CD  CD <  O _l  o. a. rr  o  o  o -co .E 3 ^ " CO "O i  UJ o  H Z >X  CL CL CC CD  C  co o °? CD 3"  LU  rr  o^r  _ g P 0_l TJ  LL Q.O  permeation chromatography and H P L C analysis of extracts of the human pancreas the same authors demonstrated later that a certain amount (< 30%) of free GLP-1 also exists i n human and pig pancreas (0rskov et al., 1994; Hoist et al., 1994), which is i n agreement with results reported by George et al. (1985). Evidence has been provided for the presence of GLP-1 and both forms of truncated GLP-1 i n rat pancreatic extracts (Mojsov et al., 1986; 1990). This suggests that there may be species differences i n processing, with the rat producing small amounts of GLP-1 and tGLP-1 by the pathway described below for the intestine. The intestinal L cells produce glicentin (1-69), oxyntomodulin (33-69), GLP-1 and GLP-2 (Conlon, 1988; Fehmann and Habener, 1992). The production and subsequent processing of GLP-1 in the intestine can potentially produce at least four peptides: GLP-K1-37), GLP-K7-37), GLP-K1-36) amide and GLP-K7-36) amide (Figure 3). It is believed that GLP-1 is initially produced, C-terminally amidated and then cleaved to GLP-K7-36) amide. In extracts of human small intestine, 80% of the GLP-1 immunoreactivity corresponded to GLP-K7-36) amide and 20% to GLP-K7-37) (0rskov et al., 1994).  B. Distribution of GLP-1 in the Gastrointestinal Tract  Studies on the histochemical distribution of immunoreactive (IR) GLP-1 and GLP-2 have demonstrated localization to A-cells of the pancreas, L-cells of the lower small intestine and colon, and some neurons i n the brain (Varndell et al., 1985; K a u t h and Metz, 1987; Eissele et al., 1992).  Eissele et al. (1992)  demonstrated that i n human and pig the highest number of GLP-1 containing cells were i n the rectum, whereas i n the rat the highest cell density was found i n the ileum. A continuous increase i n GLP-1 positive cells was found from the proximal  to the distal portion of small and large intestines. Similar results were reported i n radioimmunoassay studies of tissue extracts from dog (Namba et al., 1990), human (Kreymann et al., 1987) and rat (Mojsov et al., 1986). The biological significance of these observations is not clear.  C. Involvement of GIP and GLP-1 i n a Proximal-Distal Intestinal Endocrine Loop  Increases i n secretion of GLP-K7-36) amide are observed within 15-20 minutes of food ingestion (Kreymann et al., 1987; D'Alessio et al., 1993), a time which is probably too early for a direct effect of nutrients on the L-cell, indicating that there is a proximal to distal signal possibly involving neural and hormonal pathways. Roberge and Brubaker (1991) found that administration of fat into either the duodenum or ileum stimulated an identical increment i n gut proglucagon-derived peptide (PGDP) secretion. It was suggested that duodenal fat either stimulated the enteric nervous system, extrinsic nerves, or the secretion of some factors which i n turn stimulated the release of gut PGDP, including tGLP-1. Studies on cultured fetal rat intestinal cells demonstrated that GIP stimulated P G D P secretion at physiological concentrations and i n a concentration-dependent manner (Brubaker, 1991). This led the authors to hypothesize that a proximaldistal intestinal endocrine loop may exist and tGLP-1 release may be stimulated by GIP before arrival of nutrients in the ileum. In the rat, in vivo studies showed that intravenous infusion of GIP to give concentrations similar to those observed after duodenal fat administration induced a two-fold increase i n plasma levels of intestinal P G D P which was independent of the glycemic state (Roberge and Brubaker, 1993). Infusion of another duodenal peptide, C C K , did not alter the levels of P G D P . In agreement with the finding of Kreymann et al. (1987), they also reported that the rise i n plasma GIP levels in response to a duodenal fat load  occurred slightly before the increments i n intestinal G L P - 1 levels. It was concluded that this enteroendocrine loop between the duodenal peptide GIP and the ileal P G D P may contribute to the early rises of tGLP-1 secretion observed i n response to nutrient ingestion. Recently Herrmann et al. (1993) reported that intra-arterial infusion of GIP stimulated GLP-1 release from the isolated perfused rat ileum supporting the above conclusion. However, this hypothesis was challenged by Nauck et al. (1993 a). They failed to observe an increased tGLP-1 secretion i n response to intravenous human GIP infusions which mimicked levels attained after oral glucose in human individuals.  D. Stimulation of GLP-1 Secretion  Circulating immunoreactive GLP-1 increases i n response to an oral glucose load, mixed meal or fat ingestion (Kreymann et al., 1987; Elliott et al., 1993). There is variation between the reported levels of plasma GLP-1 from different groups since antibodies of different regional specificities were used. Mean fasting levels of plasma immunoreactive GLP-1 ranged between 15 pmol/L (Kreymann et al., 1987) and 236 pmol/L (Hirota et al., 1990) in man. After oral glucose or meal ingestion circulating GLP-1 rose two to three fold (Kreymann etal., 1987; 0rskov et al., 1987, 1991). The majority of studies indicated that circulating levels of GLP-K7-36) amide do not exceed 50-90 pmol/L following a meal (Kreymann et al., 1987; 0rskov et al., 1991, 1994; Takahashi et al, 1990). Infusion of peptide to mimic this concentration resulted i n the potentiation of glucose-induced insulin secretion i n man (Kreymann et al., 1987; Nathan et al., 1992; Nauck et al., 1993 a).  27  E. Biological Activities of GLP-1  1. Effect of Truncated GLP-1 on Insulin Secretion  The amino acid sequence of G L P - 1 starting at histidine position 7 demonstrates a strong degree of similarity to both glucagon and GIP (Schmidt et al., 1985). GLP-K1-37) and GLP-K1-36) amide exerted only weak stimulatory activity on insulin release (Schmidt et al., 1985; Mojsov et al., 1987), whereas both, synthetic GLP-K7-36) amide and GLP-K7-37) were shown to stimulate insulin secretion from both cultured islets and the isolated perfused pancreas (D'Alessio et al., 1989; Hoist et al., 1987; Mojsov et al., 1987; Fehmann et al., 1992). It was thus demonstrated that histidine at position 7 of GLP-1 must exist as a free amino-terminal amino acid in order for the peptide to exert insulinotropic activity. Potent insulinotropic effects of t G L P - 1 were demonstrated  in vivo  (Kreymann et al., 1987; Hoist et al., 1987) and in vitro (Kawai et al., 1990; Mojsov et al., 1987; Weir et al., 1989). Stimulation of insulin secretion was observed at concentrations of GLP-1 as low as 10-90 pmol/L (Kreymann et al., 1987; Mojsov et al., 1987; Hoist et al., 1987; Suzuki et al, 1989; 0rskov et al., 1994). Insulin release i n response to tGLP-1 was highly dependent on the prevailing glucose concentration, and the glucose threshold for GLP-1(7-36) amide was shown to be between 2.8 and 6.6 mmol/L (Weir et al., 1989; Komatsu et al., 1989; Suzuki et al., 1990). During perfusion of the pancreas with GLP-l(7-37), the typical biphasic pattern of insulin release was maintained (Weir et al., 1989). The importance of the N-terminal histidine has been confirmed i n studies on the biological actions of different fragments of GLP-1 i n rat and dog (Suzuki etal., 1989a; Gefel et al., 1990; Ohneda et al., 1991). Gefel et al. (1990) demonstrated  that the N-terminal histidine was crucial not only for insulin secretion but also for cyclic adenosine 3', 5'-monophosphate (cAMP) formation in the |3TC-1 cell line. In contrast, GLP-2 has not been shown to augment the secretion of insulin i n several studies (Schmidt et al., 1985; Mojsov et al., 1987; Komatsu et al., 1989). Biological roles, if any, of unprocessed GLP-1 and GLP-2 remain to be identified. Although GIP and tGLP-1 are considered to be the most important incretins there is controversy as to the relative effect of these peptides i n stimulating insulin secretion. Truncated GLP-1 was reported to have a greater insulinotropic effect than GIP in some studies (Kreymann et al., 1987; Shima et al., 1988; Siegel et al., 1992), whereas similar effects were reported by other investigators (Schmid et al., 1990; Suzuki et al., 1990; Suzuki et al., 1992a). However, since infusions of single concentrations of peptides were used in these studies it was not possible to obtain accurate measurements of the concentration threshold for stimulation or the maximal responses to the peptides.  One of the objectives of the current  studies was to clarify these differences. Therefore linear gradients of peptide or glucose were applied to the perfused pancreas to enable measurement of responses to a broad concentration range of peptide or glucose.  2. Effect of Truncated G L P - 1 on Secretion of Pancreatic Somatostatin and Glucagon  In addition to stimulating the release of insulin from B cells, tGLP-1 also stimulates somatostatin release from pancreatic D cells, and suppresses glucagon secretion from A cells of the pancreas (0rskov et al., 1988; D'Alessio et al., 1989; Kawai et al., 1989; Komatsu et al., 1989; Suzuki et al., 1989; Fehmann and Habener, 1991). Since tGLP-1 receptors have been found on a somatostatinsecreting islet cell line, the stimulation of somatostatin release is probably due to  the direct action of tGLP-1 on D cells (Fehmann and Habener, 1991; Gros et al., 1992). It is not clear, however, whether the glucagon-suppressing effect of t G L P 1 is a direct result of the peptide. D'Alessio et al. (1989) demonstrated in islet cell monolayer cultures from rat pancreas that GLP-K7-36) amide increased somatostatin release but left glucagon release unaffected. Further, no receptors for tGLP-1 were found on the glucagon-producing cell line I n R l G 9 (Fehmann and Habener, 1991). The discovery that glucagon stimulated somatostatin and insulin secretion and, conversely, that insulin suppressed glucagon secretion, led to the hypothesis of a paracrine action of these peptides (Samols et al., 1986). If tGLP-1 were produced i n the rat pancreas it is possible that it acts to decrease glucagon secretion indirectly via the stimulation of the release of somatostatin or via insulin in a paracrine manner.  3. Effect of Truncated GLP-1 and Oxyntomodulin on Gastric Acid Secretion  GLP-K7-36) amide was reported to be a potent inhibitor of pentagastrininduced gastric acid secretion i n man and therfore is a potential enterogastrone. Infusion of GLP-1(7-36) amide to approximate physiological concentrations caused a 36% to 50% inhibition of pentagastrin-stimulated gastric acid secretion (Schjoldager et al., 1989; O'Halloran et al., 1990). Recently, tGLP-1 was also found to inhibit postprandial acid secretion i n man (Wettergren et al., 1993). In contrast, Nauck et al. (1992) reported that GLP-l(7-36) amide was unable to reduce pentagastrin-stimulated acid secretion i n humans. Furthermore, in vitro studies demonstrated that GLP-l(7-36) amide stimulated the production of cAMP in isolated glands from rat fundic mucosa and i n human gastric cancer cells (Hansen et al., 1989; Gespach et al., 1989) suggesting that it exerts a stimulatory, rather than an inhibitory, effect on the parietal cell. Support for this was obtained  by demonstrating parallel responses i n parietal cells: increases i n c A M P levels and increased secretion (Schmidtler et al., 1991). The reason for these apparently discrepant results is not clear, although stimulation of gastric somatostatin secretion by GLP-1(7-36) amide may be the dominant response in vivo (Eissele et al., 1990).  Nauck et al. (1992) suggested that direct stimulatory and  somatostatin-mediated indirect inhibitory effects of GLP-1(7-36) amide balance each other out under certain conditions. Oxyntomodulin was showed to be an effective inhibitor of pentagastrinstimulated acid secretion i n the anesthetized rat (Dubrasquet et al., 1982), and i n conscious rats whose vagal and splanchnic innervation were intact (Jarrousse et al., 1985), consistent with a possible role as a physiological regulator of gastric acid secretion. Oxyntomodulin was also found to be a potent inhibitor of gastric acid secretion i n man (Schjodager et al., 1988; Le Quellec et al., 1992), and the Cterminal octapeptide of oxyntomodulin was also shown to inhibit pentagastrinstimulated gastric acid secretion (Veyrac et al., 1989).  Oxyntomodulin can  therefore be included among the potential enterogastrones.  4. Effect of Truncated GLP-1 on Somatostatin and Gastrin Secretion from the Stomach  In pigs, GLP-K7-36) amide was found to have no effect on somatostatin or gastrin secretion from the antrum, or on somatostatin secretion from the nonantral stomach (0rskov et al., 1988). However, tGLP-1 was found to stimulate somatostatin and inhibit gastrin release i n the isolated perfused rat stomach (Eissele et al., 1990; Yanaihara et al., 1990), suggesting that species' differences may exist. In preliminary studies, GIP was observed to induce a transient increase i n gastrin release (Pederson et al., 1981), suggesting that GIP and tGLP-  1 had similar effects on somatostatin but opposite effects on gastrin secretion. A n objective of the current studies was to compare the actions of different types of GIP and tGLP-1 on gastric endocrine secretion.  V . M E C H A N I S M S I N V O L V E D I N T H E INSULINOTROPIC A C T I O N S O F GIP A N D GLP-K7-36) A M I D E  A. GIP Receptors and Signal-Transduction Mechanisms  GIP has been found to act on a number of responsive tissues including stomach, pancreas, fat and liver.  U n t i l recently attempts at studying the  receptors that mediate the action of GIP on normal B-cells were unsuccessful. Brown et al. (1989) suggested two possible explanations. First, G I P receptors may be critically influenced by collagenase during islet isolation. This procedure may reduce the content of GIP receptors. This possibility is supported by studies on the insulinotropic action of GIP i n isolated islets which showed that the cells only responded to pharmacological concentrations of G I P , unlike their responsiveness to other stimuli (Schafer and Schatz, 1979; Schauder et al., 1975; Szechowka et al., 1982). Second, iodination of GIP results i n a heterogeneous population of iodinated peptides which may alter its receptor binding and biological activity. Neoplastic B-cell lines have been used widely as homogeneous B-cell preparations to study direct effects of GIP on these cells. GIP binding sites were demonstrated i n membranes derived from hamster B-cell tumors (Maletti et al., 1984; Couvineau et al., 1984), i n the In III B-cell line (Amiranoff et al., 1984) and in a human insulinoma (Maletti etal., 1987). GIP binding was shown to correlate with increases i n cAMP levels and insulin release suggesting that GIP acted  directly on the pancreatic B-cell to stimulate insulin release via specific receptors and stimulation of adenylyl cyclase. The GIP receptor i n a hamster pancreatic beta cell tumor was partially characterized as a protein monomer of approximately 59000 molecular weight (Amiranoff et al., 1986). These cells, however, may differ greatly from normal B-cells in their response to physiological stimuli and may not accurately reflect the physiological distribution of the receptors i n situ. Verchere (1991) was able to demonstrate binding sites for GIP 125 on cultured rat islets using H P L C purified, biologically active  I-GIP.  Displacement of the radioligand by GIP was concentration-dependent starting at concentrations as low as 1 nmol/L. Usdin et al., (1993) have cloned and functionally expressed a receptor for GIP using a cDNA isolated from an islet tumor cell line. From the amino acid sequence of this receptor, it was placed i n the secretin-VIP receptor family of G-proteincoupled receptors (reviewed by Segre and Goldring, 1993) which includes those for glucagon (Jelinek et al., 1993), secretin (Ishihara et al., 1991), vasoactive intestinal peptide (Ishihara et al., 1992), G L P - 1 (Thorens, 1992),  growth  hormone-releasing hormone (Mayo, 1992), parathyroid hormone (Juppner et al., 1991) and calcitonin (Lin et al., 1991). Using reverse-transcription polymerase chain reaction (PCR) and i n situ hybridization histochemistry methods, Usdin and coworkers (1993) demonstrated receptor m R N A i n tissues known to respond to GIP such as the pancreas, GI tract and adipose tissue. GIP receptor m R N A was also identified in other locations such as heart, brain, lung and inner layers of the adrenal cortex (Usdin et al, 1993). However, the role of the GIP receptors i n these regions i n unknown. Recently, Wheeler et aZ.(1995) demonstrated that rat pancreatic islets express a receptor identical to that isolated from the islet tumor cell line.  The mechanism of action of GIP on insulin release from the B-cell is not fully understood. Although tumor cells may not fully reflect the physiological situation in a B-cell, they have been useful for obtaining information about the mechanism of G I P action.  Using hamster insulinoma cells, Amiranoff et al. (1984)  demonstrated that GIP produced a concentration-dependent increase i n c A M P content i n the cells which paralleled insulin release. Subsequent studies i n cultured rat islets (Siegel and Creutzfeldt, 1985) and human insulinoma tissue (Maletti et al., 1987) supported the idea that GIP acted via stimulation of a B-cell G-protein coupled adenylyl cyclase. GIP was also found to increase intracellular levels of Ca  in hamster insulin tumor (HIT) cells (Lu et al., 1993) and isolated  islets (Wahl et al., 1990), suggesting that B-cell calcium transport is an essential part of the mechanism of GIP-stimulated insulin secretion.  In H I T cells the  2+  increase i n intracellular Ca  concentration was reported to be due to the 2+  activation of voltage-dependent Ca  channels, since the L-type calcium channel  antagonist nifedipine inhibited GIP-stimulated increases i n intracellular levels of Ca  and insulin secretion (Lu et al., 1993). cDNA transfection studies with COS  and C H O - K 1 cells have confirmed that the GIP receptor can couple to both 2+  adenylyl cyclase and pathways regulating intracellular Ca  levels (Usdin et al., 2+  1993; Wheeler et al., 1995). The increase in intracellular Ca  appears to involve  2+  the mobilization of intracellular C a stores, by a mechanism not involving inositol 1, 4, 5-triphosphate (IP3) and via the activation of cell surface cation 2+  channels to allow Ca  influx (Wheeler et al., 1995).  It was found that in vivo (Ahren et al., 1983) and in isolated perfused islets (Zawalich, 1988) C C K and GIP interact in a synergistic fashion to dramatically enhance insulin secretion in the presence of a postprandial glucose level. In addition, blockade of C C K binding to its receptor with the C C K antagonist L 364, 718 abolished this synergistic effect (Zawalich, 1988). It is known that C C K  generates several second messengers including IP3 and diacylglycerol. Therefore, the synergistic action of GIP and C C K on insulin secretion is probably due to their ability to generate separate B-cell second messengers (Zawalich, 1988) and interaction between them (Fehmann et aZ.,1989; Suzuki et al.,1992b).  B. GLP-1 Receptors and Signal-Transduction Mechanisms  The molecular mechanisms underlying the effects of tGLP-1 have also been partially elucidated as a result of the recent cloning of GLP-1 receptor cDNAs (Thorens, 1992; Wheeler et al., 1993; Dillon et al., 1993). The human receptor consists of 463 amino acids and is 90% identical to the rat receptor sequence. As discussed earlier, the GLP-1 receptor belongs to the secretin subclass of G-protein coupled receptors. Studies on the signal transduction mechanisms of cloned G L P 1 receptors i n transfected COS cells showed that both rat and human G L P - 1 receptors activated adenylyl cyclase. A similar stimulation of cAMP production i n insulinoma cells was observed (Drucker et al., 1987; Goke et al., 1989). It has been concluded therefore that one intracellular pathway by which tGLP-1 acts is via increasing cAMP and activation of protein kinase A. It is not certain whether a phospholipase C pathway is also involved in GLP-1 actions. In GLP-1 receptortransfected COS cells GLP-K7-37) increased intracellular calcium and activated phosphoinositide turnover (Wheeler et al., 1993; Dillon et al., 1993). However, other investigators have been unable to demonstrate coupling of the receptor to inositol triphosphate production or mobilization of calcium from intracellular stores i n transfected COS cells (Thorens, 1992), insulinoma cell lines (Widmann et al., 1994), or cultured rat islets (Fridolf and Ahren, 1991). Thus further studies are necessary to clarify whether the GLP-1 receptor, like some peptides of the Gprotein-linked receptor family, couples to multiple signal transduction pathways.  C. Potentiation of Glucose-induced Insulin Secretion by GIP and Truncated GLP-1  The mechanisms underlying the glucose dependency of insulin responses to GIP and tGLP-1 are also not fully understood. D-glyceraldehyde, an intermediate in the glycolytic pathway, was also capable of potentiating the insulinotropic action of G I P i n the absence of glucose i n the perfused pancreas (Elahi et al., 1982).  In a similar preparation mannoheptulose, which blocks glycolysis,  abolished GIP-stimulated insulin secretion (Muller et al., 1982). These studies strongly suggested that glucose metabolism was essential for GIP to stimulate insulin release. Actions of other insulinotropic peptides such as C C K (Zawalich et al., 1987), and GLP-K7-36) amide (Weir et al., 1989) have also been demonstrated to be glucose-dependent. A common mechanism was proposed by Rasmussen et al. (1990). They suggested that moderate increases i n glucose concentration resulted i n a partial depolarization of the B-cell membrane via the closing of ATPsensitive K  +  channels, which sensitized the B-cell to glucose-dependent  secretagogues. Once in this state, GIP or other incretins may induce increases i n c A M P content which cause further memberane depolarization, and produce 2+  sufficient C a  influx for insulin release. Therefore, the postprandial change i n  plasma glucose concentration is suggested to serve primarily as a conditional modifier of insulin secretion. This dependence of B-cell secretion on interaction between glucose and cAMP signalling pathways has been demonstrated in studies on isolated pancreatic B cells. Single B cells seem not to respond to a glucose signal alone, but require the simultaneous activation of a second signaling pathway, such as stimulation of adenylyl cyclase and cAMP production (Pipeleers, 1987; Holz et al., 1993). This bidirectional cross talk between the cAMP-mediated (tGLP-1) and ATP-mediated (glucose) signalling systems has been referred to as glucose competence. Holz et  al.(1993) measured the glucose competence state of single B-cells by recording the depolarization of the plasma membrane potential that followed exposure to glucose and/or GLP-K7-37), and showed that glucose-insensitive B-cells were rendered glucose-competent (capable of responding to glucose) by pretreatment with GLP-K7-37). Conversely, GLP-l(7-37)-insensitive cells were rendered G L P l(7-37)-sensitive by prior application of glucose. Closure of ATP-dependent K  +  channels is believed to be important i n the depolarization of the B cell and 2+  consequent insulin secretion, including opening of voltage-dependent C a 2+ . . . . 2+ channels, an increase in cytosolic Ca concentration, and the initiation of C a dependent exocytosis of insulin-containing secretory vesicles. To close the A T P dependent K channel, both input of the A T P signal generated by glucose uptake +  (by glucose transporter 2) and metabolism (by glucokinase), and the cAMP signal produced by the activation of adenylyl cyclase are necessary.  The authors  hypothesized that mechanistically the binding of A T P to ATP-dependent K  +  channels as well as the phosphorylation of this channel by cAMP-dependent protein kinase A are required to effect closure of the channel. It is, however, not yet clear whether the channel itself can be phosphorylated.  A previous  electrophysiological study on RINm5F insulinoma cells failed to detect any direct effect of either increased intracellular cAMP levels or of the catalytic subunit of protein kinase A on ATP-dependent K channel activity (de Weille et al., 1989). +  As discussed earlier, Holz et aZ.(1993) also observed that GLP-l(7-37) could prime the cells to respond to subsequent exposure to glucose. Fehmann et aZ.(1991) showed a similar priming effect of B-cells by GLP-K7-36) amide and GIP using the isolated perfused rat pancreas. They first perfused the pancreas with GLP-K7-36) amide or GIP (10, 100, 1000 pmol/L) for 10 min in the presence of a substimulatory glucose concentration (2.8 mmol/L). Then the pancreas was perfused with 2.8 mmol/L glucose to wash out the peptides. Finally, 10 mmol/L  glucose was administered for 44 min. The insulin secretion response to this high glucose challenge was markedly stimulated by pre-exposure to the incretins. The first phase of insulin secretion was more affected by this priming effect than the second phase.  VI. P O S S I B L E R O L E S OF GIP A N D GLP-K7-36) A M I D E I N N O N - I N S U L I N DEPENDENT DIABETES MELLITUS  Because a major role for GIP is to augment glucose-stimulated insulin release, impairment i n the production, secretion or actions of GIP may contribute to diseases associated with insulin deficiency. There are two major forms of diabetes mellitus, type I insulin-dependent diabetes mellitus (IDDM) and maturity onset type II non-insulin-dependent diabetes mellitus (NIDDM). In well-controlled I D D M , G I P secretion is not altered.  As expected, the insulin response to  exogenous GIP infusion in IDDM patients was reduced due to the loss of functional B-cell mass in this disease (Krarup et al., 1987). It is controversial whether GIP levels are elevated, suppressed, or normal in N I D D M . Hypersecretion of GIP has been demonstrated following oral glucose or a test meal (Ross et al., 1977; Elahi et al., 1984; Jones et al., 1989). In other studies a diminished or normal response of GIP to the oral administration of nutrients was described (Service et al., 1984; Groop, 1989; Alam et al., 1992).  However, most workers agreed that the  insulinotropic action of GIP i n N I D D M patients was impaired when compared to normal subjects (Jones et al., 1987; Krarup et al., 1987; Nauck et  al.,1993a;  Meneilly et al., 1993). The potential involvement of tGLP-1 i n N I D D M has also been studied. Postprandial GLP-1 responses i n N I D D M patients were found to be higher than those of healthy volunteers (0rskov et al., 1991; Hirota et al., 1990). Thus the lost  incretin effect observed in N I D D M (Nauck et al., 1986) can not be explained by a defective release of the incretins GIP and tGLP-1, but rather could result from a decreased responsiveness  of the pancreatic B-cells to the peptides.  This  hypothesis was tested by infusing physiological and supraphysiological concentrations of the peptides during hyperglycemic clamp conditions in N I D D M (Nauck et al., 1993c). GLP-l(7-36) amide was shown to be a potent insulinreleasing substance i n N I D D M , but only i f given in supraphysiological doses, whereas GIP was ineffective. Plasma glucagon levels also decreased i n response to GLP-K7-36) amide (Nauck et al., 1993; Nathan et al., 1992). The preserved incretin response of tGLP-1 i n N I D D M , coupled with its ability to suppress glucagon secretion evokes the possibility for clinical use. Wettergren et al. (1990) showed that GLP-1(7-36) amide also delayed the rate of gastric emptying, and this i n turn may delay absorption and contribute to the "anti-diabetic effect". In agreement with the glucose-dependence of insulinotropic actions of GLP-l(7-36) amide reported i n normal subjects, when N I D D M patients were hyperglycemic, insulin levels were high, with a return towards basal levels when glucose concentrations approached normal fasting levels (Nauck et al., 1993d). This should limit the risk of hypoglycemic responses unlike existing drugs used for N I D D M such as the sulphonylureas. Therefore synthetic tGLP-1 or similarly acting analogues are potential therapeutic agents for N I D D M .  VII. P O S S I B L E R O L E S F O R G I P A N D GLP-1(7-36) A M I D E I N H U M A N OBESITY A N D I N T H E Z U C K E R RAT  A role for GIP i n the hyperinsulinemia associated with obesity has been postulated (Chan et al., 1984). Obesity in humans is often associated with glucose intolerance and hyperinsulinemia (Salera and Barbara, 1987).  The factors  responsible for initiation of the hyperinsulinemia have not been established. However, since the abnormal insulin release is more pronounced after oral than after intravenous administration of glucose (Perley and Kipnis, 1967), the enteroinsular axis has been suggested to be overactive i n obesity. This could be a result either of an excessive release of insulinotropic peptides from the gut or hypersensitivity of B-cells to gut hormones i n obese subjects.  Among the  evidence for abnormalities in the endocrine component of this axis playing a role i n obesity are the demonstration that circulating levels of GIP are increased i n some groups of obese humans (Creutzfeldt et al., 1978; Ebert et al., 1979; Salera et al., 1982; Elahi et al., 1984; Mazzaferri et al., 1985), although not i n others (Amland et al., 1984a; Service et al., 1984). Unlike diabetics, however, B-cell sensitivity to GIP appears to be unaltered i n obese humans. Intravenous infusion of porcine GIP during a hyperglycemic clamp produced a similar insulin response i n both lean and obese individuals (Amland et al., 1985). Elahi et al. (1984) calculated B cell sensitivity to GIP from the insulin and GIP responses to oral glucose and found it to be normal in obese subjects. The genetically obese Zucker (fa I fa) rat has been widely used as an animal model of obesity. This strain of rat originally developed from a spontaneous mutation resulting from a Merck Stock M rat and Sherman rat breeding cross (Zucker and Zucker, 1961). The obesity is transmitted through a recessive autosomal gene. Homozygous (Fa I Fa) or heterozygous (Fa I fa) animals are lean and phenotypically normal, while those which are homozygous for the fatty gene (fa I fa) are obese. The obesity in these animals is accompanied by hyperphagia (Zucker and Zucker, 1961; Bray and York, 1972), and hyperinsulinemia (Zucker and Antomiades, 1972). In addition, the obese phenotype showed normal or mild hyperglycemia and a similar insulin resistance to the human condition (Bray, 1977; Shafrir, 1992).  40 Hyperinsulinemia i n the obese Zucker rat appears as early as just after weaning (Godbole et al., 1978). Whether hyperinsulinemia plays a causative role i n the development of obesity or is secondary to other metabolic changes is unclear. The mechanism underlying the B-cell hypersecretion i n obese Zucker rats is also obscure. It is associated with marked hypertrophy and hyperplasia of pancreatic B-cells within the islet (Shino et al., 1973, 1984). In addition to exaggerated insulin responses to glucose (Chan et al., 1984), those induced by arginine (Pederson et al., 1991; Hirose et al., 1994) and GIP (Chan et al., 1984; Chan et al., 1985) are also increased. Several theories have been put forward to explain the hyperinsulinemia (Marcello and Barbara, 1987) including a primary insulin resistance in target tissues; an abnormal regulation of insulin secretion by the central nervous system due to a hypothalamic defect; a primary defect i n the pancreas; and an overactivity of the entero-insular axis. The most popular theory evokes a primary role for increased parasympathetic activity accompanied by decreased sympathetic activity (Jeanrenaud, 1985; Jeanrenaud et al., 1985). The pancreas from preobese (17 day old) animals exhibited an exaggerated response to vagal stimulation, and atropine reversed the glucose-induced hypersecretion of insulin (Rohner-Jeanrenaud et al., 1983). Vagotomy also reduced the elevated pancreatic blood flow found in obese Zucker rats (Atef et al., 1992). However, atropine-sensitive hypersecretion of insulin to secretagogues persisted i n long term culture (21 days) of isolated islets (Hayek, 1980). It is therefore likely that additional factors apart from increased cholinergic drive are involved. Chan et al. (1984,1985) investigated the contribution of the entero-insular axis and GIP to the hyperinsulinemia of obese Zucker rats. It was found that circulating GIP levels were normal i n the obese animals when compared with their lean litter mates, but the insulin response to GIP was enhanced in the perfused pancreas of obese rats. A more interesting finding was that the glucose threshold for the  insulinotropic action of GIP was well below fasting levels i n the obese animals. The threshold glucose concentration had been shown to be 4.4-5.5 mmol/L i n both lean Zucker and Wistar rats. In the obese Zucker rats, an insulinotropic effect of GIP was observed at glucose concentrations as low as 2.8 mmol/L. Overall these studies support the proposal that there are changes associated with the enteroinsular axis i n obesity. There is little information regarding GLP-1 i n obesity. Circulating levels of GLP-1 were found to be elevated in obese patients (Fukase et al., 1993) suggesting there is a general change i n the entero-insular axis. There is no information available regarding GLP-1 i n animal models of obesity. Thus, it is not clear whether circulating levels of immunoreactive GLP-1 differ between lean and obese Zucker rats, or whether the insulinotropic activity of GLP-l(7-36) amide exhibits a similar defect i n glucose dependency in obese animals to that observed with GIP. Studies on the secretion and actions of GLP-l(7-36) amide i n the Zucker rats could provide a better understanding of the entero-insular axis in these animals.  VIII. THESIS INVESTIGATIONS  In summary, there is strong evidence that both GIP and tGLP-1 can act as enterogastrones and incretins. In addition, there is evidence for possible roles for these two peptides i n the pathophysiology of obesity and N I D D M . Additionally, GIP or tGLP-1, or analogues of these hormones, might be useful i n the treatment of diseases of disordered insulin secretion. However, at the commencement of these studies there were several uncertainties regarding their physiological actions and some of these are addressed i n the current thesis. Firstly, there was controversy as to the relative effectiveness of GIP and tGLP-1 i n stimulating insulin secretion. In addition, the glucose threshold for the insulinotropic actions of  GIP and tGLP-1 and the sensitivity of the pancreas to the two peptides required more accurate determination. This was addressed through the use of gradient perfusion of peptides i n the isolated perfused pancreas. This method allowed the investigation of a broad range of peptide or glucose concentrations to be studied. Secondly, although one report indicated that GLP-1(7-36) amide exerted a stimulatory effect on gastric somatostatin secretion and an inhibitory effect on gastrin secretion, there had been no comparative studies with GIP. In view of the preliminary studies suggesting that GIP exerted a stimulatory effect on gastrin release, it was considered important to compare the effect of the peptides on gastric endocrine secretion. Similar gradient perfusion of peptides to that for pancreatic perfusion was used i n the isolated perfused stomach. Finally, it was considered possible that there is a general change i n the entero-insular axis i n obesity since the glucose concentration threshold for the insulinotropic action of GIP was found to be aberrant in obese Zucker rats and circulating levels of both G I P and GLP-1 are elevated i n obese patients.  However, there was no  information regarding the secretion or biological actions of tGLP-1 i n the Zucker rat, including circulating levels of IR-GLP-1 and the glucose threshold for the insulinotropic activity of GLP-K7-36) amide in the lean and obese animals. In order to measure GLP-1 levels in vivo, a sensitive RIA was developed and circulating levels of IR-GLP-1 in response to a OGTT compared i n lean and obese animals. In addition the insulinotropic activity of GLP-l(7-36) in vitro was examined to determine whether it exhibited a defect in glucose dependency similar to that observed with GIP.  43  METHODS  I. R A D I O I M M U N O A S S A Y  The introduction of radioimmunoassay (RIA; Yalow and Berson, 1960) was one of the most important advances i n the biological measurement of hormone levels. It is a competitive protein binding assay used to measure an immensely wide range of peptides with high sensitivity, specificity and reproducibility. This method was used to determine concentrations of immunoreactive G L P - 1 i n plasma and tissue extracts, and insulin, somatostatin and gastrin i n perfusate samples.  The hormone concentrations were expressed i n conventional units.  The conversion factors of hormone concentration and Systeme Internationale (SI) Units are listed i n the Appendix. Since the GLP-1 assay was developed completely during the time of this research program it is described i n full. The other assays were routinely in use i n the laboratory.  A. Development of a GLP-1 Assay  1. Production of antibodies  Antibodies against C-terminally amidated GLP-1 were raised i n guinea pigs immunized with GLP-l(7-36) amide conjugated to keyhole limpet hemocyanin.  1.1 Conjugation of GLP-1 to Keyhole limpet hemocyanin (KLH)  Carbodiimide was used to couple GLP-l(7-36) amide to the carrier protein K L H . Synthetic human GLP-l(7-36) amide (1 mg; Bachem Inc., Torrance, CA),  was dissolved i n 200 (xl of distilled water and combined with 8 mg (700 ul) dialyzed K L H (Calbiochem-Behring Corp., L a Jolla,CA). Fifty milligrams of l-ethyl-3-(3 dimethylaminopropyl) carbodiimide hydrochloride (Calbiochem-Behring) were dissolved i n 1 ml distilled water and then added dropwise with constant stirring to the mixture. The reaction mixture was left mixing on a rolling rack for 2 h at room temperature. It was then dialyzed against 2 liters of distilled water overnight using dialysis membrane tubing (Spectrum Medical Industries Inc., Annex, LA) with a molecular cut off of 12,000-14,000. The final volume (2 ml) of dialyzed conjugate was stored at -20°C i n 1 ml aliquots until used for immunization. This procedure was repeated for booster immunizations.  1.2 Immunization of Guinea Pigs  Four Hartley guinea pigs weighing 300-500 g were obtained from The Animal Care Center, U.B.C. Three of these were immunized with G L P - 1 - K L H conjugates and one used as a control. For the primary injection, 1 ml of immunogen (500 itg GLP-l(7-36) amide) was mixed with 0.5 ml distilled water, and then emulsified with an equal volume (1.5 ml) of complete Freund's adjuvant (Gibco Laboratories, Grand Island, New York). This emulsion was injected subcutaneously at 4-5 sites (approximately 0.2 ml emulsion/site) in the back of the neck, each guinea pig receiving approximately 160 ug. The guinea pigs received booster injections three weeks after the primary. Incomplete Freund's adjuvant was used to emulsify GLP-l(7-36) amide for the boosts, and animals received the same dose of conjugate as for the primary. Guinea pig #3 received six booster injections in total and the antiserum from this  animal was used i n the assays. Animal #1 received four booster injections, but did not produce detectable antibody, and animal #2 died during the second bleeding. After the second, third, fourth and sixth booster, the animals were anesthetized with diethyl ether (BDH Inc.,Toronto,Ont.,Canada) and bled by cardiac puncture. After collection, the blood was allowed to clot for 2-4 hours at room temperature. The serum was then removed and centrifuged at 10,000 rpm for 20 min at 4°C. After centrifugation, serum was stored at -20°C until testing of antibody titers.  2. Preparation of I-GLP-1(7-36) amide 125  GLP-K7-36) amide was iodinated by the chloramine-T method and then purified by High Performance Liquid Chromatography (HPLC).  2.1 Iodination of GLP-l(7-36) amide  The chloramine-T method used was modified from that of Hunter and Greenwood (1962). Five to ten micrograms of synthetic human GLP-l(7-36) amide (Peninsula Laboratories, Inc., Belmont, CA) were dissolved in 10 JLLI distilled water and 10 | i l of 0.5 M phosphate buffer(pH 7.5) added. Five microliters (0.5 mCi)  of carrier-free  1  2  5  l  sodium  iodide (Du Pont  N E N Canada  Inc.,Mississauga,Ont.) and then 10 jul chloramine T (2 mg/ml i n 0.04 M phosphate buffer, p H 7.5) were added to the reaction tube. The tube contents were gently mixed for 30 s by tapping the tube and the reaction stopped by the addition of 10 uT sodium metabisulphite (5 mg/ml i n 0.04 M phosphate buffer, p H 7.5). A SepPak C-18 cartridge (octadecyl-silyl-silica reverse phase cartridge;Waters Associates Inc.,Milford,MA), was used to separate the  125  I - G L P - l ( 7 - 3 6 ) amide  46  from the iodination mixture. Prior to use, the cartridges were primed by flushing with 10 m l of H P L C grade acetonitrile (BDH) containing 0.1% H P L C grade trifluroacetic acid ( TFA, Pierce Chemical Company, Rockford, Illinois) followed by 10 m l of distilled H 0 containing 0.1% TFA. It was then dried with 10 m l of air. 2  The iodination mixture was diluted with 1 ml of distilled water containing 0.1% T F A and applied to the primed cartridge.  Samples were collected from the  cartridge following elution with acetonitrile in distilled water containing 0.1% T F A of the following volumes and concentrations: 5 ml of 10% and 20%; five x 1 m l of 30%, 40%, and 50% and 5 ml of 60%. Radioactivity i n 10 (il aliquots of each sample collected was counted for 0.1 min i n a Searle gamma spectrometer (NCS 125  Instruments, Inc.,Toronto) with approximately 69% counting efficiency. The  I-  GLP-K7-36) amide eluted with the second 40% wash. 125  2.2 High Performance Liquid Chromatography Purification of  I-GLP-K7-36)  amide Reverse phase H P L C was used for purification of peptides.  The term  reverse phase chromatography refers to the inversion of stationary and mobile phase polarity compared with conventional chromatographic methods.  The  sample molecules interact preferentially with either the polar mobile phase or the non-polar stationary phase of the column. To decrease the interaction of a sample with the non-polar stationary phase and, thus allow the sample to elute at a certain retention time, an organic solvent is added i n the form of a solvent gradient produced by two pumps. During the chromatographic run, the effect of an increase i n solvent concentration i n the mobile phase is to decrease the polarity difference between the mobile and stationary phase and decrease the  retention of samples. The basic scheme for reverse phase H P L C is shown i n Figure 4. The iodination mixture of I-GLP-1(7-36) amide, eluted during the second 125  40% wash of the Sep-Pak cartridge, was purified by reverse phase H P L C on a uBondapak C-18 column (Waters Associates Inc., Milford, MA). The solvent gradient was produced by two Beckman model H O B Solvent Delivery Module pumps (Beckman Instrument Inc., San Ramon, CA). A programmable Beckman Model 421A controller regulated the speeds of two separate pumps to maintain a constant flow rate while the solvent concentrations were varied. Before use, the solvents, H P L C grade distilled water from a M i l l i Q water filtration system (Waters) and H P L C grade acetonitrile (BDH), were degassed and filtered through 0.22 Lim and 0.45 um filters (Waters) respectively. Both solvents contained 0.1% TFA.  To separate the different iodinated forms of GLP-1(7-36) amide, an  increasing linear gradient from 38 to 42% was run over 10 min followed by washing with 60% acetonitrile. The initial and final acetonitrile concentrations were altered in order to establish these separation conditions. The flow rate was 1 ml/min. The capacity of the Beckman Model 210A injector port was 100 | i l and this volume of  125  I-GLP-1(7-36) amide was injected using a Millipore 180 needle  syringe (Waters) for each run. The column effluent was passed through a Model 170 Radioisotope Detector (Beckman) and the effluent radioactivity recorded (1 ml) on a Recordall Series 5000 Recorder (Fisher). Effluent fractions (1 ml) were collected into 13 x 100 mm glass test tubes (Fisher). The radioactivity i n aliquots (10 ul) from these tubes was measured i n a gamma spectrometer, the peak fractions transferred into Eppendorf polypropylene micro test tubes (Brinkmann Instruments Co.,Westbury,NJ), and stored at -20°C until use. To obtain a sufficient amount of purified tracer for GLP-1 RIA, several runs were required.  Gradient Controller  Filter  Filter  Injector Filter  Chart Recorder  Column  Radioisostope Detector  Waste  Fraction Collector  Figure 4. Outline of the HPLC system used in the purification of I-GLP-1 (7-36) amide. Solvent A was HPLC grade water with 0.1% TFA, while solvent B was HPLC grade acetonitrile with 0.1% TFA. Separation was performed on a reverse phase LtBondapak C18 column. 125  49 The column was equilibrated with the starting concentration of acetonitrile for at least 30 min between runs.  3. GLP-K7-36) amide Standards  Synthetic human GLP-1(7-36) amide (Peninsula; 5-10 ug) was dissolved at a concentration of 80 ng/100 ul i n a solution consisting of 0.02 M acetic acid and R  0.05% bovine serum albumin(Pentex , Miles Canada Inc., Etobicoke,Ont.). Aliquots of 50 |xl, containing 40 ng of GLP-K7-36) amide were added to 12 x 75 mm glass test tubes (Fisher). These were then lyophilized and stored at -20°C until use. On the day of assay, an aliquot was reconstituted i n 1 m l assay buffer to give a concentration of 40 ng/ml and further diluted 1:10 to achieve a concentration of 4000 pg/ml. Serial dilution of this solution i n assay buffer generated the additional standards used in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25 and 15.625 pg/ml. 4. Procedure and Conditions of GLP-1 Assay  The antiserum collected from guinea pig #3 (KMJ-03) was used to establish the assay. Different assay buffers, antisera dilutions, tracer dilution, incubation times and sample volumes were investigated to obtain the optimum binding of GLP-1(7-36) amide as well as the highest sensitivity. Assays were set up i n 12 X 75 mm polypropylene test tubes to minimize peptide adsorption. A l l procedures were carried out on a refrigerated table at approximately 4°C. Total counts, standard, zero and non-specific binding tubes were assayed i n triplicate while samples and control were assayed in duplicate.  In the protocol used routinely, 100 ul of standard, control, or sample were added to tubes containing 500 | i l assay buffer.  One hundred microliters of  antibody (1:20,000 dilution) were then added and the tubes vortexed. The assays were incubated for 24 h at 4°C prior to the addition of 100 | i l (3000 cpm) H P L C purified  125  I-GLP-l(7-36) amide and then incubated for a further 48 h at 4°C.  5. Separation of Bound and Free I-GLP-1(7-36) amide 125  Both double antibody and dextran-coated charcoal methods were tested during the development of the GLP-1 assay. Assays using the dextran-coated charcoal method were found to be more rapid, reproducible and sensitive. A suspension of charcoal was prepared by dissolving 4 g/L dextran T70 (Pharmacia A B , Uppsala, Sweden) i n assay buffer which contained 0.05 M sodium barbitone, 0.05 M ethylenediaminetetra-acetic acid tetrasodium salt (EDTA) and 2% R I A grade B S A (Sigma Chemical Co.,St. Louis,MO), p H 8. Charcoal (Fisher) was then added to the solution (80 g/L). The mixture was stirred constantly at 4°C for at least 1 h prior to use i n the assay. One hundred [il dextran-coated charcoal were added and the mixture centrifuged for 30 min (3000 rpm, 10°C). Supernatants were decanted and the pellets allowed to dry with the tubes inverted for at least 2 h. The charcoal pellets were counted for 3 min on an L K B Wallac Automatic Gamma Counter Model 1277.  The non-specific  binding(NSB), determined as the amount of I-GLP-l(7-36) amide bound i n the 125  absence of antibody, was obtained for the assay buffer and for assay samples. The protocol is outlined in Table 2.  co  ft  a  03 CO  o o io  o o  o o  o o  O O  O O  a ccj CO PQ CO  CD  S3 CD  cd CO  co  o o  lO  O O  3.  o o  o o  O  o o  o o  o  rfl  o fe  ZL  o o  CD  03  PQ  CO  o o  o o  fl fl  o  CJ  o o  13 o EH  a  T3  g  %  fl 03 T3 03  EH  fl PQ  03 CO  CD  'a  a  03 CO  O  SH  CD  u  03  EH  52  6. Characterization of Antibody  6.1 Anti-GLP-1 Antiserum Titers  * ? Titers of the different bleeds were obtained by means of antiserum dilution curves. Antisera were diluted 1:10, 100, 1000 and 10000 i n assay buffer. These 125  were then incubated with 600  JLLI  assay buffer and 100 | i l of H P L C purified  I-  GLP-K7-36) amide (assayed i n triplicate) for 72 h at 4°C. Separation was achieved by the dextran-coated charcoal method. The percentage of antibodybound G L P - 1 was plotted against antiserum dilution for each bleed.  The  estimated antiserum dilution that gave 50% of the maximal binding of the tracer was considered to be the titer of the bleed. 6.2 Antiserum Crossreactivity  To test crossreactivity, serial dilutions of different peptides related i n structure to GLP-1(7-36) amide were prepared and assayed for competition with 125  I - G L P - l ( 7 - 3 6 ) amide for binding to the KMJ-03 antiserum. The resulting  curves were then compared with the GLP-l(7-36) amide standard curve. The amounts of crossreacting peptides which yielded 50% inhibition of binding were compared with the amount of standard giving the same inhibition and the crossreactivity was expressed as a percentage of that standard.  6.3 Assay Sensitivity  The definition of assay sensitivity used is the minimal detection limit of the assay estimated as a 10% decrease i n binding from the zero standard.  53  7. Reproducibility of the Assay  Intra-assay variation was obtained from a series of determinations made on a quality-control pool within the same assay. A series of duplicate tubes (n=12) were made up from each pool as controls (250 pg/ml) and included throughout the assay. The results are expressed as a coefficient of variation:  d= the difference between duplicate estimates x= the mean of duplicate estimates N= the number of duplicate estimates  The estimation of inter-assay variation was based on measurements of aliquots from a quality-control pool i n each assay. The calculation of inter-assay coefficient of variation was the same as above. Since samples containing GLP-1 could not be reliably stored for extended periods without significant loss of immunoreactivity, the routine use of these as controls was precluded.  B. Insulin Assay  Immunoreactive insulin (IRI) was determined i n the plasma and perfusion samples by an RIA for pancreatic insulin modified from Albano et al (1972).  54  1. Antiserum  A guinea pig antiserum (GP01) raised against rat insulin was used i n all insulin radioimmunoassays. This antiserum was previously shown to detect insulin standards as low as 10-20 uJJ/ml and have no cross-reactivity with GIP, glucagon, or somatostatin at the concentrations used i n the experiments. The antiserum stock was stored in lyophilized 100 ul aliquots of 1:10 dilution at -20°C. These aliquots were then reconstituted i n insulin assay buffer to 1:5000 dilution and stored as 2 ml aliquots at -20°C until use. When required, each aliquot was thawed and diluted to 40 ml of assay buffer to give a final dilution of 1:10 i n the 125  assay. A t this dilution, the zero standard binding to porcine  I-insulin was  approximately 50%. 2. I - I n s u l i n 125  125  I - i n s u l i n was prepared by the chloramine T iodination method. The  procedure was carried out at room temperature and all solutions prepared immediately prior to the iodination. Siliconized glassware was used to prevent non-specific binding. Porcine insulin (Novo Research Institute, Bagsvaerd, Danmark) (10-50 ng) was first dissolved i n 10 ul of 0.01 M HC1 and then brought to a concentration of 5 Lig/10  ul with 0.2 M phosphate buffer (pH 7.4). One millicurie N a  125  I ( 1 0 ul) was  added to the reaction tube which contained 10 ul (5 ug) of porcine insulin solution. Ten microliters of 0.2 M phosphate buffer (pH 7.4) were then added to the tube followed by the addition of 100 jig (25 ul) chloramine T (4 mg/ml i n 0.2 M phosphate buffer) to initiate the oxidation reaction. After 10 s, the reaction was terminated by the addition of 240 ug (100 ul) sodium metabisulphite (2.4 mg/ml in  55 0.2 M phosphate buffer). After another 45 s, 50 u.1 potassium iodide (10 mg/ml i n 0.2 M phosphate buffer) were added followed by a further 1.8 m l of 0.04 M phosphate buffer. ^ ^ I - i n s u l i n was purified by a silica adsorption method. The iodination mixture (approximately 2 ml) was added to 10 mg microfine silica (QUSO G-32) i n a test tube. The solution was vortex mixed for 30 s and centrifuged at 3000 rpm for 15 min. The supernatant containing unincorporated milliliters of distilled water were added to the  125  1 2 5  I was decanted. Three  I - i n s u l i n adsorbed to the silica  pellet and the solution vortex mixed and centrifuged as before. The procedure was 125 125 repeated twice to remove remaining free  I. The  I-insulin was eluted from  the silica by vortexing i n 3 ml acid ethanol, prepared by mixing 1500 m l of 95% ethanol, 500 m l of distilled water and 30 ml of concentrated HC1. Following centrifugation, the supernatant was transferred to a vial, diluted with 1.5 m l distilled water and 2 ml acid ethanol and stored at -20°C. It could be used i n the RIA for up to approximately 4 weeks. When required,  125  I - i n s u l i n was further  diluted i n an appropriate volume of assay buffer to give approximately 2000 cpm/100 ul. 3. Standards  One hundred micrograms of lyophilized rat insulin (Novo) were dissolved in 1 ml of distilled water. It was then diluted to 200 ng/ml (4.26 U/ml) i n 0.04 M phosphate buffer(pH 7.4) containing 6 g/L NaCI, 0.24 g/L sodium merthiolate and 6% BSA(Pentex R). One milliliter aliquots were stored at -70oC as stock solutions. Each aliquot was made up to 26 ml with assay buffer to obtain a solution of 160 ftU/ml. It was then stored as 2 ml aliquots at -20°C. For use i n the RIA, an  aliquot was thawed and serially diluted i n assay buffer to a concentration of 160 uU/ml to 80, 40, 20, 10 and 5 uU/ml for the assay standard curve.  4. Separation  The dextran-coated charcoal method was used to separate bound and free 125  I - i n s u l i n . Dextran T-70 (5 g/L) was dissolved i n 0.04 M phosphate buffer (pH  7.4), 50 g/L charcoal added, stirred overnight at 4°C and stored i n the refrigerator. Before use, the charcoal suspension was stirred for at least 1 h at 4°C.  5. Production of Charcoal Extracted Plasma  Charcoal extracted plasma (CEP) was prepared from outdated human plasma (Red Cross Blood Bank, Vancouver, B.C.) and used i n the assay buffer. The plasma was centrifuged for 30 min at 10000 rpm and filtered with sharkskin (15 cm, Schleicher and Schuell Inc., Keene, N H ) to remove erythrocytes. It was mixed with 1% charcoal (1 g/100 m l plasma) and stirred at 4°C for 1 h. The charcoal was removed from the plasma by centrifugation (30 min, 10000 rpm) and filtration of the supernatant through sharkskin twice. The plasma was stored in 10 ml aliquots at -20°C until use.  6. Procedure  The assay protocol was similar to that for G L P - 1 with the following exceptions. A l l assays were set up i n 12x75 mm borosilicate glass test tubes (Fisher). To obtain an appropriate reading from the standard curves, some  57  samples were diluted i n assay buffer so that the IRI concentrations fell within the range of the steepest slope of the standard curve. The buffer used i n the IRI R I A was a 0.04 M phosphate buffer (pH 7.5) containing 5% charcoal extracted plasma. The total assay volume was 1 m l including 700 |il assay buffer (except total count tubes), 100 ill standard, control 125  or sample, 100 ul antiserum (except total count and N S B tubes) and 100 ul insulin. The assays were incubated for 24 h at 4°C without  125  I-  I - i n s u l i n and a  further 24 h i n its presence. Separation was achieved by adding 200 (il dextrancoated charcoal (except total count tubes), centrifuging for 30 min (3000 rpm, 10°C, and decanting the supernatant (containing antibody bound peptide). The charcoal pellets containing unbound peptide were counted for 3 min i n the gamma spectrometer (LKB Wallac). 7. Assessment of Assay  Controls were prepared by pooling samples collected from perfused rat pancreas and diluted i n assay buffer to achieve a concentration of 50 u,U/ml. One milliliter aliquots of the control samples were stored at -20°C. The controls were later prepared from standards. It was confirmed that this change did not affect the accuracy of the RIA. Inter- and intra-assay variation were less than 10% and 5%, respectively. Peptides perfused (GIP and GLP-l(7-36) amide) did not cross react i n the RIA.  58  C. Somatostatin Assay  Measurement of somatostatin-like immunoreactivity (SLI) i n perfusion samples from isolated perfused stomach was as previously described (Mcintosh et al., 1978, 1987).  1. Antibody  S O M A 03, a monoclonal antibody against somatostatin (Buchan et al, 1985), was used i n the RIA. This antibody was previously characterized and shown to detect somatostatin-14 and somatostatin-28 equally, and not to cross-react with GIP, motilin, or gastrin (Mcintosh et al., 1987). Crude ascites fluid containing S O M A 03 was filtered through a sterile 0.22 um filter (Millipore, Bedford, Massachusetts), diluted with an equal volume of buffer containing 0.9% saline, 0.1% N a N and 0.5% BSA(Pentex ) and stored at -70°C. When required, a 20 ul R  Q  aliquot was thawed, added to 980 ul of the buffer and stored at 4°C as a stock antibody solution. In the assay, 13.5 ul stock antibody solution was diluted i n 100 ml of assay buffer to give a final dilution in the assay of 1:3.7 X 10 . 6  2.  125  I-Somatostatin  Synthetic Tyr—somatostatin was iodinated by the chloramine T method using identical reagent concentrations as described for the GLP-1 RIA. The iodination mixture was purified by adsorption of iodinated peptide to silica (QUSO G32), diluted i n 0.05% BSA, and stored as lyophilized aliquots (approximately 1 x 10  6  cpm) at -20°C. On the day of assay the lyophilized  125  I-somatostatin was  further purified on a column (0.9 x 10 cm) of carboxymethyl cellulose CM-52  (Whatman Chemical Separation L t d . , England).  The lyophilized  I-  somatostatin was reconstituted i n 0.002 M ammonium acetate (BDH) buffer (pH 4.6) and applied to the column which had been equilibrated with the same buffer. After washing with 30 ml of 0.002 M buffer at a flow rate of 1 ml/min,  1 2 5  I-Tyr 1  somatostatin was eluted with 0.2 M ammonium acetate (pH 4.6). Two min fractions were collected and the radioactivity of 100 ul aliquots were counted. The peak fractions were pooled, neutralized with 2 M sodium hydroxide and diluted i n assay buffer to 3000-5000 cpm/100 ul for use i n the assay.  3. Standard  Somatostatin-14 (Peninsula) was dissolved i n 0.1 M acetic acid containing 0.05% B S A (Pentex) to give a final concentration of 100 Lig/ml. Aliquots of 50 |il (5 ug) were transferred to test tubes, lyophilized and stored at -20°C. When needed, an aliquot was dissolved i n 200 ul distilled water followed by 300 ul assay buffer. It was then serially diluted to obtain concentrations of 500, 250, 125, 62.5, 31.25, 15.6, 7.8 and 3.9 pg/ml as standards.  4. Separation  125  Bound and free  I-somatostatin were separated by means of dextran-  coated charcoal. Dextran T 70 was dissolved in 0.05 M phosphate buffer p H 7.5 (2.5 g/L). After the addition of 12.5 g/L charcoal and 1 ml/L C E P , the mixture was stirred for at least 1 h at 4°C.  60  5. Procedure  Stock solution of the somatostatin R I A buffer consisted of 23.77 mmol/L sodium barbital (BDH), 3.9 mmol/L sodium acetate, 43.6 mmol/L sodium chloride and 0.247 mmol/L ethylmercurithiosalicylic acid sodium salt (merthiolate; Eastman Kodak), p H 7.5, stored at 4°C. On the day of assay, 5 g/L B S A (Pentex ) and 10 ml/L (10,000 KlU/ml) aprotinin (Trasylol , Miles) were added to R  R  the stock solution. Each assay contained 100 ul assay buffer, 100 ul standard or samples, 100 ul antibody and 100 ul I-Tyr"'"-somatostatin and allowed to incubate for 72 h at 125  4°C. In assays for S L I of perfused pancreas samples, 100 ul perfusate were added to N S B , zero and standard tubes instead of assay buffer. When necessary, 125  perfusate was used to dilute samples. Bound and free  I-somatostatin were  separated by adding 1 ml dextran-coated charcoal plus C E P . After centrifugation at 3000 rpm for 30 min, the supernatant was decanted and the charcoal pellet counted for 3 min on a gamma spectrometer. 6. Assessment of Assay  Similar to GLP-1, samples containing S L I could not be reliably stored for extended periods of time without significant loss of immunoreactivity and controls were not routinely included in assays. The inter- and intra-assay variations were less than 11% and 7%, respectively. Peptides perfused (GIP and GLP-1(7-36) amide) did not cross react in the RIA.  61  D. Gastrin Assay  1. Antibody  The antiserum (PM1) used in the gastrin assay to measure secretion from the isolated perfused stomach was raised in a rabbit against human gastrin. This Cterminally directed antiserum recognized rat gastrin-17, human gastrin-17, human gastrin-34 and cross-reacted with cholecystokinin C C K 8 . Since C C K is mainly located i n the small intestine, and none could be detected i n the rat stomach effluent (Mcintosh, unpublished), crossreactivity with C C K did not affect the measurements of gastrin.  2. Iodination of Gastrin  The iodination procedure was modified from the method of Stadil and Rehfeld (1972) as described by Verchere (1991). Five to ten micrograms of synthetic human gastrin I were dissolved i n 10 ixl of 0.4 M phosphate buffer p H 7.4. It was then incubated with 0.2 mCi N a  1 2 5  I and 10 ul of chloramine-T (0.5 mg/ml i n 0.04  M phosphate buffer) for 60 s at room temperature. The reaction was stopped by addition of 10 | i l sodium metabisulphite (0.5 mg/ml i n 0.04 M phosphate buffer) and 0.5 ml of 0.05 M imidazole buffer p H 7.5. Purification of the labelled hormone was achieved by ion-exchange chromatography using D E A E Sephadex A25 (Pharmacia). One and half grams of D E A E Sephadex A25 were swollen i n 0.05 M imidazole buffer and washed several time before packing the column. The column was equilibrated overnight with the imidazole buffer. The iodination mixture was applied to the column and the  1 2 5  I-  gastrin eluted using a linear gradient of 0 to 1 M NaCI in the imidazole buffer. The  flow rate was maintained at 2 ml/min by a peristaltic pump and 1 min fractions were collected. Ten microliter aliquots were counted. The 3 fractions following the second peak were tested i n the gastrin RIA. The fraction that showed the best displacement curve was diluted in RIA buffer, diluted into 1 ml aliquots (1,000,000 cpm/ml) and stored at -20°C.  3. Standard  Synthetic human gastrin I was stored i n 250 ul aliquots of 100 ng/ml assay buffer a t - 7 0 ° C . To prepare the standards, one aliquot was thawed and serially diluted i n assay buffer to generate standards ranging from 1600 to 6.25 pg/ml. These were stored at -70°C until use.  4. Separation  To prepare a dextran coated charcoal (CEP) solution, 2.5 g dextran T-70 and 12.5 g activated charcoal were dissolved i n 1 L 0.04 M phosphate buffer p H 6.5, stirred overnight at 4°C and stored at 4°C. On the day of separation, 7% C E P was added to the charcoal suspension, which was constantly stirred at least 1 h prior to use. To each assay tube (except the total count tubes) 200 ul of the mixture were added and the tubes centrifuged at 3000 rpm for 30 min a t 4 ° C . The supernatant containing antibody bound gastrin was decanted and the charcoal pellets containing free labeled gastrin were dried overnight and counted for 3 min on a gamma counter.  63  5. Procedure  The assay buffer consisted of 0.02 M barbital HC1, p H 8.4 containing 0.5% B S A (RIA grade, Sigma). Each assay tube contained 100 | i l of standard or 125  sample, 100 ul of antibody (except N S B and total count tubes) and 100 ul  I-  gastrin (2000 cpm). The total volume was made up to 1 m l with assay buffer. The assays were incubated for 48 h at 4°C prior to separation. 6. Assessment of Assay  Control samples of synthetic human gastrin I were stored as aliquots of 100 pg/ml at -70°C. Duplicate tubes were included i n each assay. Inter- and intraassay variations were 11% and 4%, respectively.  Peptides perfused (GIP and  GLP-K7-36) amide) did not cross react in the RIA.  II. O R A L G L U C O S E T O L E R A N C E TEST  A. Animals  Zucker rats were used to study the in vivo insulin and GLP-1 responses to an oral glucose challenge. Lean heterozygous females (Fa I fa) and obese homozygous male (fa I fa) Zucker rats were purchased from Charles River Laboratories (Quebec), and bred and maintained i n the animal care facilities of the Physiology Department of U.B.C.  The animals were housed i n polypropylene cages with bedding (2-6  rates/cage) i n a room which was temperature and light-controlled (24°C; 12 h cycle). The rats were allowed free access to laboratory food and tap water.  64  Lactating females with their litters and breeding pairs were housed i n separate cages. After weaning (approximately 21 days), same sex and phenotype rats were housed together in another room. Both male and female animals between 10 and 14 weeks of age were used in all studies.  B. Procedure  Equal numbers of lean and obese Zucker rats were fasted for 18 h prior to the experiment. Glucose was administered though a ball-tipped needle, connected to a syringe, at a dose of 1 g/kg (40% glucose solution). Blood samples were collected from the tail vein of unrestrained, conscious animals using a heparinized capillary at 0, 10, 20, 30 and 60 min following the glucose load. Samples were collected i n ice-chilled Eppendorf polypropylene micro test tubes containing 250 K I U aprotinin/0.5 ml blood (Trasylol ), centrifuged at 4°C, 10,000 rpm for 30 min. R  The  plasma was stored at -20° C u n t i l the analysis for glucose and  radioimmunoassay of GLP-1 and insulin. Plasma glucose was measured by the glucose oxidase method (Beckman glucose analyzer). Plasma samples of two rats from the same genotype were pooled together to obtain enough volume for measurement of glucose, insulin and GLP-1. Therefore, each n represents the results from two rats.  III. I S O L A T E D P E R F U S E D O R G A N P R E P A R A T I O N S  Isolated, vascularly-perfused organ systems were used to study peptide actions in vitro. Since the circulation of the isolated perfused organ preparation is isolated, the effects of peptides are not complicated by central or other organ  65  effects.  In this system, the composition of the circulating perfusate can be  precisely controlled.  A. Stomach Perfusion  The isolated vascularly perfused rat stomach was used to examine and compare the effects of tGLP-1 and GIP on gastric somatostatin and gastrin secretion.  1. Surgical Procedure  The surgical procedure for the isolated perfused stomach preparation was adapted and modified from Lefebvre and Luyckx (1977). Male Wistar rats (250350 g) were housed in a light-controlled room with free access to laboratory food and water.  The animals were fasted overnight (16-18 hours) prior to the  experiment to aid surgical dissection and limit variation i n basal hormone secretion. The rats were anesthetized with an intraperitoneal injection of 65 R  mg/kg sodium pentobarbital (Somnotol , M T C Pharmaceuticals, Cambridge, Ont.) and put on a heated pad. A midline incision from pubis to sternum and paired lateral incisions to the body wall were made to expose the abdominal viscera. The left kidney and adrenal gland blood vessels were doubly ligated and sectioned. In preparation for cannulation of the aorta, three loose ligatures were placed around the aorta immediately below the diaphragm, between the celiac and superior mesenteric arteries, and 1-2 cm rostral to the common iliac artery. Double ligatures were placed and the sigmoid colon was sectioned to allow removal of the small and large intestines. The right kidney and adrenal gland blood vessels were singly ligated and left intact. Preparation of the portal vein cannulation  66  involved the placement of one loose ligature close to the liver including the portal vein, bile duct and associated vassels and another loose ligature around the portal vein. The superior mesenteric artery was doubly ligated and sectioned. The proximal duodenum was sectioned to place a drainage cannula into the lumen of the stomach through the pylorus. The spleen and the head of the pancreas were cut away from the greater curvature of the stomach ensuring the preservation of the right and left gastroepiploic artery. The small intestine, large intestine, pancreas and spleen were then removed. A cannula (PE160) was inserted into the abdominal aorta and positioned adjacent to the celiac artery. Following tightening of the loose aortic ligature near the diaphragm the flow of perfusate was directed to the stomach via the celiac artery and 1 ml (71 units) of heparin-saline was administered to prevent clotting. The animal was sectioned at the level of the diaphragm and a second cannula was placed into the portal vein in order to collect the venous effluent. It has been demonstrated (Mcintosh et al., 1981a) that after the surgery, the small remnant of pancreas remaining attached to the portal vein gives only 1% of the insulin response of an intact isolated perfused pancreas to a stimulus of 16.7 mmol/L glucose. This suggested that the contribution of the remaining pancreas to the peptide secretion into the portal vein was negligible.  2. Apparatus  Perfusate was stirred i n flasks that were continuously gassed with a water vapor saturated mixture of 95% oxygen and 5% carbon dioxide to maintain p H of 7.4. Perfusate was delivered to the stomach using a peristaltic pump (No. 755330; Cole-Parmer Instrument Co., Chicago, IL) and heated to 38°C by passing through tubing (PE160; Clay-Adams, Parsippany, NJ) coiled around a heating  67  block such that the stomach temperature was maintained at 37 C. The perfusion pressure was monitored by means of a transducer and maintained between 50 and 70 mm Hg. A filter bubble trap was placed immediately preceding the celiac arterial cannula i n order to remove air bubbles and unwanted particles from the perfusate. The perfused stomach was kept warm by a heating source underneath and a piece of plastic wrap above.  This prevented  temperature disturbances due to moisture loss and circulating air. Peptides were administered as gradients of increasing concentration. This was accomplished by the use of a gradient apparatus consisting of two connected identical flasks of equal cross-section and the addition of the desired final concentration of the test peptide to perfusate i n the distal flask.  For the  administration of single peptide concentrations, peptides were introduced via a side arm using a Harvard infusion pump (Model 940; Harvard Apparatus Co. Inc., Millis, MA) from 10 ml syringes via PE90 tubing into a rubber bulb situated just before the arterial cannula. Peptides were diluted in perfusate and delivered at an infusion rate of 0.1 ml/min. A n automatic fraction collector (Pharmacia) with an ice-filled reservoir rack was used to collect the venous effluent from the portal vein into chilled test tubes.  3. Solutions and Reagents  3.1. Perfusate  The perfusate was a Krebs' solution containing 3% dextran (Clinical grade; Sigma), 0.2% B S A (RIA grade; Sigma) and 4.4 mmol/L glucose (Fisher). The dextran and B S A were dissolved overnight i n 0.9% saline solution. On the day of the experiments, appropriate volumes of Krebs' concentrate and sodium  68  bicarbonate solution were added to give the following final ionic composition: 4.4 mmol/L KC1, 2.5 mmol/L C a C l , 1.2 mmol/L MgS0 7 F L / ) , 1.5 mmol/L K H P 0 , 2  25 mmol/L N a H C O  4>  2  4  and 120 mmol/L NaCI. The glucose concentration was  monitored using a glucose analyzer (Beckman).  3.2. Peptides  GLP-K7-36) amide (lot #018862), GLP-K7-37) (lot #019860), GLP-K1-36) amide (lot #010448), synthetic human GIP (sh GIP)(lot #028798) and synthetic porcine GIP (sp GIP) (lot #028725) were purchased from Peninsula Laboratories, while natural porcine G I P (np GIP) was prepared i n the Department of Physiology. Peptides were freshly weighed, dissolved i n 1 ml of 0.01 M acetic acid plus 0.05% B S A (Pentex ) and diluted with perfusate to the desired concentration. The solutions were kept on ice during the experiments. Appropriate volumes of stock solution were added to the perfusate to achieve the desired final concentration with an infusion rate of 0.1 ml/min or a perfusion rate of 3 ml/min.  4. Perfusion Procedure  Following a 10-20 min equilibration period, portal vein effluent was collected at 1 min intervals into ice-cold test tubes. Aliquots (1 ml) from these samples were immediately transferred into test tubes containing 1000 K . I . U . aprotinin (Trasylol R , Miles) and stored at -20O C until assay of SLI. Total duration of perfusions including equilibration period did not exceed 60 min. During the perfusion, the pump and heating apparatus were adjusted to maintain a steady flow rate of 3 ml/min and perfusate temperature of 38°C to maintain the stomach temperature at 37°C.  69  B. Pancreas Perfusion  1. Surgical Procedure  The surgical procedure for the isolated perfused pancreas preparation was modified from the method of Penhos et al. (1969). Male Wistar rats and Zucker rats used i n the experiments were described i n section III A . 3 . and II A . respectively.  The animals were fasted overnight and then  anesthetized  intraperitoneally with 65 mg/kg sodium pentobarbital. The abdomen was exposed by a midline incision from pubis to sternum with paired lateral incisions of the body wall. The blood vessels supplying the left kidney and adrenal gland were tied and sectioned between double ligatures. Loose ligatures were placed around the aorta cephalad of the celiac artery, caudal to the superior mesenteric artery and 1-2 cm caudal to the second loose ligature i n preparation for subsequent cannulation. The vasculature supplying the right kidney and adrenal gland were disconnected by a single ligature. The duodenum was sectioned and a drainage tube inserted distal to the pancreas at the ligament of Treitz. The mesenteric arcades of the gut from this drainage tube to caudal to the cecum were ligated and the gut was removed. The esophagus, left gastric artery and vagus nerve trunks were doubly ligated and the vasculature connecting the stomach and pancreas along the greater curvature were singly ligated. The pylorus was doubly ligated and sectioned between ties and then the stomach was removed. The connective vasculature between the pancreas and the spleen was singly ligated and spleen was removed. A cannula (PE160 tubing) was inserted into the abdominal aorta to the level to the superior mesenteric artery. The loose aortic ligature cephalad to the celiac artery was tied such that perfusion of the pancreas occurred via both the celiac and superior mesenteric arteries. After injection of 1 ml heparin-saline  70 via this route, the animal was sectioned at the diaphragm. A venous cannula (PE160 tubing) was placed into the portal vein to collect effluent.  2. Apparatus  A l l the apparatus used i n the pancreas perfusion were the same as those used i n the stomach perfusions and described in section III A.2.  3. Solution and Reagents  The protocols for preparing perfusate and peptide solutions were similar to those used for the stomach perfusion with the exception of variations of glucose concentrations i n the perfusate. Glucose was added to the perfusate to obtain the desired concentrations which were monitored with a glucose analyzer. Linear gradient concentration changes of glucose were produced by the use of the gradient apparatus and the addition of high concentrations of glucose to the distal flask as described in section III A.2.  4. Perfusion Procedure  Prior to the collection of the samples, the preparation was perfused for 10-20 min to establish the basal insulin secretion. Subsequent perfusion periods were similar to those described for the stomach. Effluent from the portal vein cannula was collected i n chilled test tubes and stored at -20°C for subsequent assay for glucose or IRI by radioimmunoassay. Perfusion rate was 4 ml/min.  71  IV. M E A S U R E M E N T  OF GLP-1 IN RAT PANCREAS  AND ILEUM  EXTRACTS  Separation of peptides by H P L C combined with radioimmunoassay measurement is a sensitive method for the identification of peptides.  These  methods were used for the identification of GLP-l-like peptides i n rat pancreatic and ileal extracts using antiserum KMJ-03.  A. Tissue Extraction  Male Wistar rats (250-350 g) were fasted overnight (16-18 h) and anesthetized with sodium pentobarbital (65 mg/kg). The whole pancreas and 3-5 cm of ileum were immediately removed and rinsed with saline. The tissues were cut i n small pieces and placed into boiling 2 M acetic acid (5 ml/g tissue) for 15 min.  After cooling on ice, samples were homogenized with a homogenizer  (Tekmar, Cincinnati, Ohio). The homogenate was then centrifuged at 10000 rpm, 4°C for 30 min and the supernatant transferred into siliconized glass tubes. Samples were lyophilized and stored at -70°C. Samples of the lyophilized tissue extracts were dissolved i n distilled water containing 0.1% T F A and applied to Sep-Pak columns which were primed with 5 ml acetonitrile containing 0.1% T F A , and washed with 5 m l of distilled water containing 0.1% T F A . After application of the samples, Sep-Pak columns were washed with 5 ml of distilled water containing 0.1% T F A and the peptide eluted with 1.5 m l of 60% acetonitrile with 0.1% T F A . Acetonitrile was evaporated by placing the eluted samples under a stream of nitrogen gas. The remaining aqueous phase was lyophilized and the samples stored at -20°C.  72  B. H P L C Separation  A Waters Associates H P L C system was employed which included two Model 510 pumps, a Model 441 absorbance detector, a Model 712 WISP  sample  processor and a solvent stabilization system. The general system was similar to that described in section L A . 2.2 with the following exceptions. After filtration, the solvents were kept i n sealed bottles and were degassed and maintained under a constant flow of helium gas. A n absorbance detector set at 254 nm was used to monitor peptide outflow from the column effluent. The system was controlled by a microcomputer with a Maxima 820 chromatography software program. A set of standards, followed by H P L C grade distilled water, were run prior to tissue extract samples. Standard GLP-l(l-36) amide and GLP-l(7-36) amide were dissolved i n H P L C grade water containing 0.1% T F A to give a concentration of 5 (ig/ 10 ul. A mixture of 10 u.1 of each peptide was injected such that 5 |ig of each was applied to the column. In order to determine appropriate conditions for separation of the peptides, several runs were made while manipulating the initial or final acetonitrile concentrations. In the experiments, an increasing linear gradient from 38% to 42% acetonitrile was run over 10 min followed by 42% plateau for 3 min. It was then washed with 75% acetonitrile and returned to 38% solvent. The lyophilized tissue extracts were reconstituted with 500 ul H P L C grade distilled water and 200 | i l of each sample was injected into the column. The outflow was collected i n 0.5 min fractions for a total of 25 min. Acetonitrile was evaporated with nitrogen gas, the samples lyophilized, and stored at -20°C. To confirm that there was no cross-contamination between consecutive H P L C runs, distilled water was injected into the column between synthetic standards and tissue extracts, and subjected to the same separation conditions.  73  C. Assay  The GLP-1 radioimmunoassays were performed as described i n section L A . The lyophilized samples were reconstituted to 500 ul with 100 ul distilled water and 400 ul GLP-1 assay buffer prior to assay.  V . STATISTICAL A N A L Y S E S  A. Pancreas Perfusions  1. Wistar rats  In the pancreatic perfusion experiments, insulin secretion rates are expressed as mean microunits (uU) per minute + S E M ; n represents the number of perfusions. A method based on that of Brelje and Sorenson (1988) was used to determine glucose and peptide thresholds for stimulation of insulin secretion. For analysis of the results from glucose gradients, with a single concentration of peptide, a single-factor repeated measures analysis of variance (ANOVA), followed by the Dunnett t test, was used to determine significant increases (P< 0.05) compared with secretion during minute 3 i n four sequential fractions. The first sample of this group was considered to be the threshold. The secretion rate at the threshold was also compared with that i n control experiments with perfusion of glucose alone using a factorial A N O V A , and shown to be significantly increased i n a l l cases (P<0.05).  The mean glucose concentrations were  determined from measurements of levels i n effluent samples. The peptide threshold for insulinotropic action was determined by the same method but i n experiments i n which the glucose concentration was held constant  74  and the peptide perfused as a gradient. The 3-min delay before peptide appeared in the effluent was considered i n the calculation of threshold according to the formula:  Final concentration in the peptide gradient  X  Time Period X  perfusion time(min) - 3 min  To analyse the maximal responses to peptides from the gradient perfusion data, a statistical model was developed by Ping M a (Statistics Department, UBC). A parametric model with least-squares fit was applied to the data for each individual organ, and curve fitting performed using a computer program written i n C. The maximal effect, defined as the estimated maximum increase from basal to the peak plateau level, of each organ was obtained from each of the fitted profiles. Then the maximal effects were compared among the different peptides by a oneway A N O V A and Duncan's multiple-range test. Linearity of gradients with perfusion of glucose, glucose with GIP or GLP-1(736) amide, or GIP was analyzed by regression analysis using Statview on a Macintosh computer.  2. Zucker Rats  In the perfusion experiments, insulin secretion is expressed i n uU/min ± S E M , where n represents the number of perfusions.  Similar to the pancreas  perfusions with Wistar rats glucose and peptide thresholds for stimulation of insulin secretion were determined using a single-factor repeated measures of A N O V A , followed by the Dunnett t test. The only difference from the analysis described for Wistar rats is that comparisons were made with time period 1  75  (P<0.05).  Again the mean glucose concentration was determined from  measurements of levels i n effluent samples.  B. Stomach Perfusions  Since the basal rates of gastrin secretion from the perfused rat stomach varied between animals, secretion rates were calculated and presented as a percent change over basal rate ± S E M . Although the basal rates of SLI secretion did not differ to the same degree, data were expressed i n the same way to enable direct comparison. The calculation formula is shown below:  Percent Change i n Secretion =  Secretion rate during min X  Average secretion -  rate during min 1-3  X  100  Average secretion rate during min 1-3  Differences from basal rates were analyzed using the raw data (pg/min) and a single-factor repeated measures A N O V A , followed by the Scheffe F test at a confidence level of 0.05. Differences i n the initial rates (pg/min) of increase or decrease and maximal responses between different peptides were analyzed using a factorial A N O V A . Again, significance was taken as P< 0.05.  C. Glucose Tolerance Test  In the oral glucose tolerance test on Zucker rats, glucose levels are expressed in mmol/L ± S E M , whereas insulin and GLP-1 levels are expressed i n jiU/ml ±  S E M and pmol/L ± S E M , respectively. Significant differences i n fasting levels of glucose and the peptides and increases over fasting levels i n the glucose tolerance test were analyzed by A N O V A at a confidence level of 0.05; n represents the number of blood samples, where each sample was obtained from two lean or obese Zucker rats.  77  RESULTS  I.COMPARISON O F T H E E F F E C T S OF GASTRIC INHIBITORY P O L Y P E P T I D E A N D G L U C A G O N - L I K E PEPTIDE-K7-36) A M I D E O N I N S U L I N S E C R E T I O N F R O M T H E ISOLATED P E R F U S E D R A T PANCREAS  In previously published studies the glucose threshold for the insulinotropic actions of GIP and GLP-1(7-36) amide were determined by measuring insulin release from isolated perfused pancreases or islets i n response to single concentrations of peptide in the presence of varying glucose concentrations. With this technique, however, it is difficult to obtain an accurate measurement of the threshold. In addition, sensitivity to peptides was also determined using single concentrations. In the following studies the sensitivity of the pancreatic B-cell to glucose stimulation was determined by examining the pattern of insulin release from isolated perfused pancreases with linear glucose gradients. This method was also used to determine the sensitivity of the pancreas to GIP and GLP-l(7-36) amide, since it mimics the pattern of peptide secretion more closely, and to compare the insulinotropic activities of different peptide preparations.  A. Gradient Characteristics  Linear gradients were produced over a 40 min perfusion period. Several studies were first performed to determine the reproducibility and the delay between gradient production and sample collection due to the presence of the pancreas and tubing.  78  1. Measurements of Effluent Glucose Levels  Glucose levels were measured i n the effluent perfusate from pancreases perfused with a 2-10 mmol/L glucose gradient (Figure 5). The first elevation of glucose levels occurred during minute 4. The glucose concentrations measured i n the pancreatic venous effluent samples were found to be linear over the 40 min 2  perfusion period, with a regression coefficient (R ) of 0.947 (P<0.0001).  2. Measurements of Effluent Glucose Levels i n the Presence of GIP or G L P - K 7 36) amide  To determine whether peptide administration influenced the linearity of glucose gradients, GIP or GLP-K7-36) amide (300 pmol/L) were applied during perfusion of a 2.8-11 mmol/L glucose gradient (Figure 6).  There were no  significant differences i n glucose concentrations between perfusions with glucose alone, glucose with GIP, or glucose with GLP-K7-36) amide (R =0.987 and 0.979, 2  respectively; P<0.0001) Again the first elevation of glucose levels occurred i n minute 4, and this figure was taken into account in the calculation of thresholds.  3. Measurements of Effluent GIP Concentrations from Perfusions with a 0 - 1 nmol/L Gradient of GIP in the Presence of 10 mmol/L Glucose  Immunoreactive G I P concentrations were measured i n the effluent perfusate from perfusions with a 0-1 nmol/L gradient of synthetic porcine GIP i n the presence of 10 mmol/L glucose (Figure 7). Assays were performed by L . Checknita as described in Verchere (1991). Since there was some variability i n  0  10  20  30  40  TIME (min)  FIGURE 5. Glucose concentrations in perfusate effluent from the pancreas perfused with a 2-10 mmol/L glucose gradient (n=6). The gradient developed was linear over the 40 min perfusion (R =0.947; P<0.0001). 2  FIGURE 6. Glucose concentrations in the perfusate effluent of the pancreas perfused with 2.8-11 mmol/L glucose in the presence of 300 pmol/L GIP or GLP-1(7-36) amide. The gradient was not affected by the peptides (R =0.987 for glucose with GIP; R =0.979 for glucose with GLP-1(7-36) amide; P<0.0001). 2  2  81  TIME (min)  FIGURE 7. Immunoreactive GIP concentrations in fractions from pancreas perfusions with a 0-1 nmol/L gradient of synthetic porcine GIP in the presence of 10 mmol/L glucose. Data were plotted as percentages of final GIP concentrations due to variability in the final concentration reached at 40 min. Gradients were linear (R =0.979; P<0.0001) over the 40 minute perfusion period (n=5). 2  the final concentrations of GIP at 40 min, data were calculated and plotted as percentages of the final GIP concentrations. Gradients were linear (R =0.979; P<0.0001) over the 40 minute perfusion period. Similar experiments were not performed with GLP-1(7-36) amide due to the relative insensitivity of the radioimmunoassay.  B. H P L C Analysis of GLP-K7-36) Amide and Different GIP Preparations Used for Perfusion  In order to quantify the amounts of GLP-K7-36) amide, np GIP, sp G I P and sh G I P i n the different preparations used for perfusion, the peptides (5 (ig) were analyzed using H P L C . Samples were run with a 0-70% gradient of acetonitrile containing 0.1% trifluoracetic acid. As shown in Figure 8, there were no significant differences i n peak height between the peptide profiles. Differences i n peak areas, calculated by the Maxima program, were less than 3% between the different peptide preparations. Natural porcine GIP contains 10-20% G I P elutes with G I P  0  3 4 2  which co-  under the above acetonitrile/water gradient conditions  (Jornvall et al., 1981).  C. Comparison of 0-1 nmol/L Gradient Perfusion of GIPs and GLP-1(7-36) Amide on Insulin Secretion in the Presence of 16.7 mmol/L Glucose  Since maximal insulin secretion was previously observed with perfusions of GIP i n the presence of 16.7 mmol/L glucose (Pederson and Brown, 1976) this hyperglycemic concentration was used to compare the maximal effects of the peptides. Insulin secretion in response to GLP-l(7-36) amide, np GIP, sp GIP and sh GIP are shown in Figure 9. Insulin secretion in the presence of all peptides was  nPGIP  sPGIP  sHGIP  G L P - 1 (7-36)  1 -i  0  0.2  Time (min)  FIGURE 8. HPLC analysis of GLP-1(7-36) amide and different preparations of GIP. Peptides were applied to the HPLC and eluted with a 0-70% gradient of acetonitrile containing 0.1% TFA on a iiBondapak C-18 column. There are no significant differences in peak height or peak areas.  84  14000-,  Peptide Gradient (0-1 nmol/L)  1200010000E  ID CO  sp GIP  800060004000  -O  GLP-1 (7-36)  -A  np GIP  -*  sh GIP  -O  CONTROL  2000 H 0  1  0  10  20  —r~ 30  40  TIME (min)  FIGURE 9. Effect of 0-1 nmol/L gradient perfusion of GLP-1(7-36) amide, natural porcine, synthetic porcine or synthetic human GIP on insulin release from the isolated perfused rat pancreas. The perfusate contained 16.7 mmol/L glucose. Insulin secretion increased significantly over the basal levels at minute 4 (P<0.05) for GLP-1(7-36) amide and porcine GIPs. Significant increase in insulin secretion in response to sh GIP started from minute 5. The estimated maximal effect evoked by sh GIP was much less than that for GLP1(7-36) amide or porcine GIPs (P<0.01). Control=glucose alone. N=5 for all of the peptides; n=6 for glucose control.  significantly greater (P<0.05) than with glucose alone. Perfusion of glucose alone increased insulin secretion from 549 ± 60 uU/min at 3 min to 1123 ± 127 uU/min at 40 min. Perfusion with GLP-K7-36) amide, np G I P or sp GIP, significantly increased (P<0.05) insulin release over basal levels at minute 4 at which time the peptide concentration was approximately 22 pmol/L. The mean maximal effects of the peptides were estimated using the curve fitting method and the parametric logistic model as described i n Methods.  The maximal  insulinotropic effect of GLP-1(7-36) amide (11133 ± 1420 uU/min) was slightly, but significantly greater (P<0.05) than those of either np G I P (8030 ± 1757 uU/min) or sp GIP (9032 ± 1523 uU/min). These maximal effects were achieved with peptide concentrations of between 600 and 700 pmol/L. Although sh GIP evoked greater insulin responses compared to glucose alone, between minute 18 and the end of the perfusion, the 0-1 nmol/L sh GIP gradient exhibited a much lower maximal effect (2482 ± 1345 uU/min) than with equivalent molar gradients of GLP-l(7-36) amide or either form of porcine GIP. These differences i n response to sh GIP compared with the other three peptides were significant (P<0.01). Furthermore, significant increases i n insulin secretion i n response to sh GIP started i n minute 5, which was 1 min delayed compared with the other peptides. This equates to an increase in peptide concentration of approximately 44 pmol/L.  D. Comparison of sp GIP and GLP-l(7-36) Amide Gradients on Insulin Secretion in the Presence of 10 mmol/L Glucose  To approximate the postprandial level of glycemia i n the rat, 10 mmol/L glucose was used i n these studies. Since the effect of sh GIP on insulin release was much lower than that of porcine GIPs, and sp and np GIP exhibited similar insulinotropic effects, only sp GIP was used in the following studies.  1. Insulin Responses to 0-1 nmol/L Gradients of synthetic Porcine GIP or G L P 1(7-36) Amide  Similar initial rates of increase i n insulin secretion and maximal secretory rates were observed i n perfusions of sp GIP and GLP-K7-36) amide (Figure 10). Significant increases i n insulin release occurred at minute 4 i n both groups (P<0.05) and the relative increases above basal did not differ significantly between the two peptides.  Insulin secretion reached a plateau after 12 m i n for both  peptides, a time at which the peptide concentration was estimated to be approximately 290 pmol/L. The maximal effects calculated by the curve fitting method for sp GIP and GLP-1(7-36) amide were 2470 + 319 |iU/min and 2480 + 155 LiU/min, respectively and the difference was not significant. Insulin secretion rates with equivalent peptide gradients were significantly greater (P<0.001) i n perfusions in the presence of 16.7 mmol/L glucose than those i n the presence of 10 mmol/L glucose for both sp GIP and GLP-1(7-36) amide perfusions (Figures 9 and 10).  2. Insulin Responses to 0-50 pmol/L Gradients of Synthetic Porcine GIP or G L P 1(7-36) Amide  Insulin secretion i n response to 0-50 pmol/L gradients of GIP or GLP-K7-36) amide was studied to obtain a more accurate assessment of the peptide thresholds (Figure 11). When compared with both the basal levels and rates for glucose alone significant increases i n insulin secretion did not occur until minute 13 for either peptide (p<0.05). The estimated peptide concentration at this time was approximately 16 pmol/L. Rates of insulin secretion for sp GIP (294 ± 36 (xU/min) and GLP-K7-36) amide (325 ± 29 LiU/min), and the relative increases  87  TIME (min)  FIGURE 10. Effects of 0-1 nmol/L gradient perfusions of GLP-1(7-36) amide or sp GIP on insulin secretion from the perfused rat pancreas in the presence of 10 mmol/L glucose. Significant increases of insulin release over basal levels and glucose alone occurred at minute 4 (arrow) for both peptides (P<0.05). There were no significant differences in maximal effects between the two peptides. N=7 for both peptides.  FIGURE 11. Effects of 0-50 pmol/L gradient perfusions of GLP-1(7-36) amide or sp GIP on insulin secretion from the perfused rat pancreas in the presence of 10 mmol/L glucose. Significant increases in insulin secretion over basal levels and glucose alone occurred at minute 13 for both peptides (indicated by arrow; P<0.05). Control=glucose alone. N=7 for all of the studies.  over the initial levels did not differ significantly between the two peptides. The differences between calculated maximal effects of sp GIP (2157 + 545 uU/min) and GLP-1(7-36) amide (2294 ± 365 uU/min) were also not significant. In the glucose control group, only a small increase in insulin secretion was observed, with an initial rate of 139 ± 17 uU/min and peak rate (at 40 min) of 213 + 38 uU/min.  E . Comparison of sp GIP and GLP-K7-36) Amide on Insulin secretion i n the Presence of a 2.8-11 mmol/L Glucose Gradient  In order to determine the glucose threshold for stimulation, the pancreases were perfused with a 2.8-11 mmol/L linear gradient of glucose i n the presence of a fixed concentration of sp GIP or GLP-K7-36) amide.  1. Insulin Responses to a 2.6-11 mmol/L Glucose Gradient i n the Presence of 50 pmol/L sp GIP or GLP-K7-36) amide  The pattern and increment in insulin secretion were similar for sp GIP and GLP-K7-36) amide. Insulin secretion in pancreases stimulated with either sp GIP or GLP-1(7-36) amide was increased significantly at 13 min compared with those perfused with glucose alone (P<0.05). Insulin release i n the presence of sp GIP or GLP-l(7-36) amide was also increased significantly at 13 min compared with minute 3 (P<0.05). The measured glucose concentrations i n this period were 4.5 ± 0.11 mmol/L (Figure 12).  2. Insulin Responses to a 2.8-11 mmol/L Glucose Gradient i n the Presence of 300 pmol/L sp GIP or GLP-K7-36) Amide  90  FIGURE 12. Effects of 50 pmol/L GLP-1(7-36) amide or sp GIP on insulin secretion stimulated by a 2.8-11 mmol/L glucose gradient in the perfused rat pancreas. The patterns of insulin secretion were similar and the rates of insulin secretion did not differ significantly between the two peptides. Compared with glucose alone (open triangles), a significant increase in insulin secretion occurred at minute 13 (indicated by the arrow; P<0.05). The measured glucose concentrations in this period were 4.5 ± 0.11 mmol/L. Control=glucose alone. N=7 for GLP-1(7-36) amide and 6 for sp GIP. Closed triangles: glucose concentrations in perfusate.  As shown i n Figure 13, the patterns of insulin secretion were again similar for the two peptides. There were no significant differences between the levels of stimulation of insulin secretion perfused with sp GIP and GLP-K7-36) amide. Secretion rates i n the presence of sp GIP or GLP-K7-36) were significantly increased at 13 min compared with those in the glucose alone group (P<0.05), and when compared to secretion during minute 3 (P<0.05). When insulin secretion rates i n the presence of the two different concentrations of sp GIP (Figure 14) or GLP-K7-36) amide (Figure 15) were compared, it was observed that within the same peptide treated group, the insulin responses to peptide concentrations of 50 pmol/L and 300 pmol/L were not significantly different.  92  sp GIP or GLP-1 (7-36) (300 pmol/L) Glucose Gradient (2.8-11 mmol/L)  2000-1  1500 H c  ~  spGIP GLP-1 (7-36) CONTROL  1000i  co  500 H  TIME (min)  FIGURE 13. Effects of 300 pmol/L GLP-1(7-36) amide or sp GIP on insulin secretion stimulated by a 2.8-11 mmol/L glucose gradient in the perfused rat pancreas. A significant increase of insulin secretion over the basal level (minute 3) occurred at 13 min for both peptides. Compared with the glucose control, insulin levels increased significantly at min 20 in the presence of sp GIP or GLP-1(7-36) amide. Control=glucose gradient alone. N=6 for both sp GIP and GLP-1(7-36) amide.  93  FIGURE 14. Comparison of the effects of 50 or 300 pmol/L sp GIP on insulin secretion stimulated by a 2.8-11 mmol/L glucose gradient in the isolated perfused rat pancreas. Data from Figure 12 and 13 were replotted. Although the insulin secretion rate during min 12-16 showed marginal differences, there was no significant difference in insulin secretion between the two concentrations of the peptide.  94  GLP-1 (7-36) (50 or 300 pmol/L) Glucose Gradient (2.8-11 mmol/L)  2000n  GLP-1 (7-36) (50 pmol/L) GLP-1 (7-36) (300 pmol/L)  1000H zz CO  c  0 0  10  20  30  40  TIME (min)  FIGURE 15. Effects of 50 or 300 pmol/L GLP-1(7-36) amide on insulin secretion stimulated by a 2.8-11 mmol/L glucose gradient in the isolated perfused rat pancreas. Again data from Figure 18 and 19 were replotted. There was no significant difference in insulin secretion between the two concentrations of the peptide.  II. C O M P A R I S O N O F T H E E F F E C T S OF D I F F E R E N T F O R M S O F GASTRIC INHIBITORY  POLYPEPTIDE A N D GLUCAGON-LIKE PEPTIDE-1 O N  SOMATOSTATIN  A N D GASTRIN RELEASE FROM T H E ISOLATED  P E R F U S E D RAT S T O M A C H  Previous studies demonstrated that both G I P and GLP-1(7-36) amide stimulated gastric somatostatin secretion. In contrast, whereas gastrin secretion was reported to be inhibited by GLP-l(7-36) amide (Eissele et al., 1990), a paradoxical increase i n gastrin secretion i n response to G I P was observed (Pederson et al., 1981). There have been no comparative studies on the effects of these peptides on gastric endocrine secretion. Therefore i n the following studies the actions of np GIP, sp GIP, human GIP, GLP-K7-36) amide, and GLP-K7-37) on somatostatin and gastrin secretion were compared using single concentration or gradient perfusions of peptide. In addition, since the effects of GLP-K1-36) amide (GLP-1) and GLP-2 on gastric somatostatin secretion had not been established, their effects were also studied.  A. Effects of GLP-1, GLP-1(7-36) Amide and GLP-2 on Gastric Somatostatin Secretion  1. Somatostatin Secretion i n Response to 1 nmol/L GLP-1  In order to determine the effect of GLP-1 on somatostatin release, it was infused between 10-30 min of a 40 min perfusion. During the 1 nmol/L GLP-1 perfusion, SLI levels i n the effluent did not change significantly compared to basal (Figure 16). Mean SLI concentrations at minutes 10, 20, 30 and 40 were 245 ± 41, 189 ± 17, 190 ± 18 and 184 ± 18 pg/ml respectively.  96  2. Somatostatin Secretion i n Response to 1 nmol/L GLP-l(7-36) Amide  GLP-K7-36) amide (1 nmol/L) was infused between 10 and 30 min of the perfusion. A biphasic increase i n mean SLI secretion rates occurred (Figure 17). GLP-1(7-36) amide enhanced the release of SLI rapidly from a baseline value of 500 + 110 pg/min at 10 min to 904 ± 152 pg/ml at 11 min (Figure 17). A peak increment of 1789 ± 388 pg/ml was achieved at 15 min and the S L I levels remained elevated throughout the perfusion of peptide.  3. Somatostatin Secretion i n Response to 1 nmol/L GLP-2  Perfusion with 1 nmol/L GLP-2 did not alter gastric SLI secretion (Figure 18). During GLP-2 infusion between 10-30 min, SLI concentrations remained at basal levels. Concentrations of SLI at minutes 10, 20, 30 and 40 were 127 ± 27, 102 + 23, 119 ± 34 and 99 + 15 pg/ml respectively.  B . Somatostatin Secretion i n Response to a 0-800 pmol/L Gradient of GIPs or Truncated Forms of GLP-1  In order to compare the initial response to, and maximal effects of, np GIP, sp GIP, sh GIP, GLP-K7-36) amide and GLP-K7-37) on S L I secretion, peptides were administered to the isolated perfused stomach as 0-800 pmol/L linear gradients. Both SLI and gastrin secretion data are presented as percent increase over the basal rate to facilitate comparison between perfusions, as described i n Methods.  97  FIGURE 16. Effect of 1 nmol/L GLP-1 on somatostatin release from the isolated perfused stomach. The peptide was infused between 10-30 min of the perfusion. Levels of SLI remained low even during the infusion of GLP-1 (n=6).  98  3000 i  GLP-1 (7-36) amide (1 nmol/L)  2000 H CO Q.  co  1000  0  10  20  —i—  —i—  30  40  TIME (min)  FIGURE 17. Effect of 1 nmol/L GLP-1(7-36) amide on somatostatin release from the isolated perfused stomach. GLP-1(7-36) amide was infused between minute 10-30 of the perfusion. Secretion of SLI increased rapidly in response to the peptide and remained elevated throughout the infusion of the peptide (n=6).  f  99  FIGURE 18. Effect of 1 nmol/L GLP-2 on somatostatin release from the isolated perfused rat stomach. The peptide was infused between 10-30 min of the perfusion. Levels of SLI remained low even during the infusion of GLP-2 (n=6).  100  1. Effects of np GIP, sp GIP and sh GIP on Somatostatin Secretion  A l l three peptides stimulated SLI release from the perfused stomach (Figure 19). Significant increases over the basal secretion rate occurred at minute 5 of the perfusion (P<0.05) when the peptide concentration was estimated to be approximately 50 pmol/L. Although the initial rate of increase i n SLI stimulated by sp GIP (119 + 39 pg/min) was greater (P<0.05) than that by np GIP (32 + 12 pg/min), the maximal increase i n response to sp GIP (1258 + 33%) was not different from that obtained with the native peptide (1091 ± 146%). With sh GIP, the initial rate of increase and the maximal increase were 16 ± 2 pg/min and 776 ± 52%, respectively. These responses were significantly less than those i n the presence of either form of porcine GIP. In a control group (perfusate alone) basal secretion decreased slightly during the perfusion.  2. Effects of GLP-K7-36) amide and GLP-K7-37) on Somatostatin Secretion  As shown i n Figure 20, the effects of GLP-K7-36) amide and GLP-K7-37) on SLI release were similar. The mean initial rates of increase with GLP-K7-36) amide and GLP-K7-37) did not differ significantly from each other or from that observed with sp GIP (Figure 19). The maximal increases in response to G L P - K 7 36) amide (732 ± 195%) and GLP-K7-37) (640 ± 34%) were not significantly different. These levels were, however, significantly lower than those elicited with either form of porcine GIP (P<0.05; Figure 19).  101  FIGURE 19. Effects of 0-800 pmol/L gradient perfusions of np GIP, sp GIP or sh GIP on somatostatin secretion from the isolated perfused rat stomach. Data are presented as percent increases over the basal rate. Although all three peptides stimulated SLI release, the initial rate of increase and the maximal increase in response to sh GIP were significantly less than those in the presence of either form of porcine GIP (n=5-7).  102  FIGURE 20. Effects of 0-800 pmol/L gradient perfusions of GLP-1(7-36) amide or GLP-1(7-37) on somatostatin secretion from the isolated perfused rat stomach. Data are presented as.percent increases over basal rate. The patterns of SLI release stimulated by either form of the truncated GLP-1 were similar. The rate of stimulation of somatostatin secretion with GLP-1(736) amide did not differ significantly from that with GLP-1(7-37) (n=5-7). Control=perfusate alone.  103  C. Gastrin Secretion i n Response to a 0-800 pmol/L Gradient of GIPs or Truncated Forms of GLP-1  As described i n the Methods Section, since the basal rates of gastrin secretion varied between batches of rats, data were presented as the percentage change over basal rate. In preliminary studies it was found that synthetic porcine GIP and GLP-K7-36) exerted opposite effects on gastrin secretion from the isolated perfused stomach (Figure 21), and the complete data are presented i n separate graphs to facilitate comparison.  1. Responses to np GIP, sp GIP and sh GIP  A l l peptides enhanced gastrin release from the perfused stomach (Figure 22). As observed with somatostatin secretion, significant increases over basal rate occurred 5 min after starting the perfusion with either form of porcine GIP (P<0.05). This equates to an estimated peptide concentration of approximately 50 pmol/L for the two peptides. The increases i n secretion rate reached a plateau at approximately 9 min (242 ± 53%) for sp GIP perfusion. Responses to gradients of np GIP appeared to be biphasic, with a second phase starting at minute 25 (216 ± 27%).  There were no significant differences between the maximal  responses to sp GIP or np GIP during minutes 25-35. Although the rate of sh GIP-induced gastrin secretion was similar to that with np GIP during the first 25 min, secretion rates remained low afterward, and the maximal increases during minute 25-35 were significantly lower than those obtained with either form of porcine GIP (P<0.05).  104  FIGURE 21. Effects of 0-800 pmol/L gradient perfusions of GLP-1(7-36) amide or sp GIP on gastrin release from the isolated perfused rat stomach. Data are presented as percent change over basal rate. GLP-1(7-36) amide inhibited gastrin release, whereas sp GIP stimulated gastrin release from the stomach (n=5-7).  105  400  P E P T I D E GRADIENT (0-800 pmol/L)  300 LU  CD O z 2  F < LU DC  O  LU CO  ^  O  CD  <  CO O CC  <  200 H  LU  CD  -o—  npGIP  —  spGIP  -•—  sh GIP  -o—  CONTROL  100 H  111  0  -100  10  —I— 20  30  TIME (min)  FIGURE 22. Effects of 0-800 pmol/L gradient perfusions of np GIP, sp GIP or sh GIP on gastrin release from the isolated perfused rat stomach. Data are presented as percent change over basal rate. All three peptides stimulated gastrin release from the stomach. The maximal increase of secretion during minutes 25-35 was significantly lower with sh GIP (P<0.05) than with either form of porcine GIP (n=5-7). Control=perfusate alone.  2. Gastrin Responses to GLP-K7-36) amide and GLP-K7-37)  Unlike GIP, both GLP-K7-36) amide and GLP-K7-37) inhibited gastrin secretion from the perfused stomach i n a similar manner (Figure 23). A slight, but non-significant increase i n mean gastrin secretion was observed at minute 4 for both peptides. The initial rate of decrease in response to GLP-l(7-36) amide (13 ± 16 pg/min) did not differ from that to GLP-1(7-37) (14 ± 6 pg/min). Significant decreases from the basal rate of gastrin release were observed between 9 min and the end of the perfusion for both peptides (P<0.05). The maximal inhibition obtained with GLP-K7-36) amide (-71 ± 10%) and GLP-K7-37) (-75 + 7%) were not significantly different.  107  FIGURE 23. Effects of 0-800 pmol/L gradient perfusions of GLP-1(7-36) amide or GLP-1(7-37) on gastrin release from the isolated perfused rat stomach. Data are presented as percent decrease over basal rate. Both peptides inhibited gastrin release from the stomach with a similar pattern. A significant suppression of secretion occurred between minute 9 and the end of the perfusion (P<0.05) for both peptides. Control=perfusate alone. The rate of decrease and the maximal inhibition were not significantly different with the two peptides (n=5-7).  108  III. STUDIES O N GLP-1 I N T H E Z U C K E R RAT  A. Development of a GLP-1 Radioimmunoassay  In order to measure plasma levels of C-terminally amidated GLP-1 i n lean and obese Zucker rats, a radioimmunoassay was developed as described i n the Methods.  1. Preparation of Iodinated GLP-1(7-36) Amide  1.1 Separation of Iodinated GLP-K7-36) Amide  Sep-Pak cartridges were used to separate the products of the chloramine T iodination of GLP-1(7-36) amide. Iodinated GLP-1(7-36) amide was applied to the cartridge and washed with 10-60% acetonitrile containing 0.1% T F A . A typical profile for elution of iodinated GLP-l(7-36) amide from a Sep-Pak cartridge is shown i n Figure 24. The majority of the  125  I-GLP-l(7-36) amide eluted with 40%  acetonitrile and this peak fraction was further purified via H P L C .  1.2 H P L C Purification of Iodinated GLP-K7-36) Amide  The collected peak from Sep-Pak cartridges was purified by reverse phase H P L C on a uBondapak C-18 column, with an acetonitrile gradient (38-42%) run over 10 min. Several peaks were obtained (Figure 25). Binding of the different peaks of  125  I - G L P - l ( 7 - 3 6 ) amide to C-terminal antibodies (KMJ-03) was  examined. The major peak (Figure 25) demonstrated the highest binding (50%) and was used for the radioimmunoassay.  109  300  i  200  H  x E Q. O  100  H  I—I w-JJb  0 10 20  Art  30  40  50  60  Percentage of Acetonitrile(%)  FIGURE 24. Sep-Pak cartridge separation of iodinated GLP-1(7-36) amide. Iodinated GLP-1(7-36) amide was applied to the cartridge and washed with 10, 20, 30, 40, 50 and 60% acetonitrile containing 0.1% TFA. The majority of  12*5  I-GLP-1(7-36) amide eluted with the second wash of 40% acetonitrile.  110  FIGURE 25. Reverse phase HPLC separation of l-GLP-1(7-36) amide from the Sep-Pak purification. Iodinated GLP-1(7-36) amide eluted from the Sep-Pak cartridge was purified on a LtBondapak C-18 column with a 38-42% acetonitrile gradient over 10 min. The major peak (A) showed the highest binding to the antibody and was used for the radioimmunoassay of GLP-1. iz;,  Ill 2. Characterization of Antibody  2.1 Titers of the Antisera  Titers of antisera after the second, third, fourth and sixth boosters were determined using radioimmunoassay. As shown i n Figure 26, the titers of guinea pig #3 antiserum started to rise 91 days after the first immunization and increased until day 142 . Guinea pig #2 died during the experiment. Guinea pig #3 was sacrificed on day 142 and the antiserum (KMJ-03) collected for radioimmunoassay.  Guinea pig #1 only demonstrated low titers even after  several boosters. The final dilution of KMJ-03 for use i n the radioimmunoassays (resulting i n 38-48% binding of the labelled peptide) was 1:20,000.  2.2 Crossreactivity  The crossreactivity of antiserum (KMJ-03) with GLP-K7-36) amide, G L P 1(7-36), GLP-l(l-37), GIP and glucagon was examined and is shown i n Figure 27. Between peptide concentrations of 15 pg/ml and 4000 pg/ml, GLP-1(1-36) amide 125 produced a displacement of amide.  I-GLP-K7-36) amide comparable to GLP-K7-36)  However, neither GLP-l(7-37) nor G L P - l ( l - 3 7 ) showed significant  displacement even at high concentrations (between 2 ng/ml and 1 |ig/ml). Thus the antiserum used i n the assay for immunoreactive GLP-1 was C-terminally directed and detected amidated forms of G L P - 1 , with no significant crossreactivity with non-amidated forms of GLP-1. The antibody did not show any crossreactivity with GIP or glucagon with peptide concentrations between 2 ng/ml and 1 |ig/ml.  112  1: 30000n Guinea Pig #1 Guinea Pig #3 6  Days After Immunization  FIGURE 26. The titers of guinea pig anti-GLP-1 antisera. Guinea pig #1 showed low titers even after several boosts. In contrast, antiserum titers of guinea pig #3 rose dramatically at the fourth booster. Numbers=booster time.  113  P E P T I D E S (PG/ML)  FIGURE 27. Specificity of KMJ-03 antiserum toward the recognition of GLP1(1-36) amide, GLP-l(7-36) amide, GLP-1Q-37), GLP-l(7-37), glucagon and GIP. KMJ-03 recognizes amidated forms of GLP-1 and does not crossreact with non-amidated forms of GLP-1, glucagon or GIP even at high concentrations (1 Ltg/ml).  1  2.3 Sensitivity and Reproducibility of the Assay  The data from 6 different assays were expressed as mean B % ± S E M (Figure 28). The non-specific binding (percent of total) was less than 4%. The intra-assay variation of the radioimmunoassay assay was less than 6% and the inter-assay variation was less than 11% at 250 pg/ml. The detection limit, defined as the concentration of peptide resulting i n 10% decrease i n binding from the zero standard, was 78 pg/ml with GLP-1.  3. H P L C Analyses of GLP-1 in Extracts of Pancreas and Ileum  Tissues were extracted as described i n Methods. The separation of G L P - K 7 36) amide and GLP-K1-36) amide was accomplished by H P L C on a C-18 reverse phase column with a 38-42% gradient of acetonitrile. As a control, a mixture of synthetic human GLP-l(l-36) amide and GLP-l(7-36) amide was applied to the column before the tissue extracts. Immunoreactive C-terminally amidated G L P 1 was monitored with the radioimmunoassay using KMJ-03 antiserum. A ± 1 min variation i n the elution positions of immunoreactive peaks was observed due to variations i n the timing of the start of the fraction collector. However, within a given set of H P L C runs the relative elution positions of synthetic standards and GLP-1 immunoreactive peaks in tissue extracts were identical.  3.1 GLP-1 Immunoreactivity i n Pancreatic Extracts  One rat pancreatic extract was examined. Two major peaks eluting at the same positions as synthetic GLP-l(l-36) amide and GLP-l(7-36) amide were observed (Figure 29). The ratio of IR-GLP-1(1-36) amide:(7-36) amide was 3:1. A  o co  10H  I  10  "I " T  I  T I I I |  100  I  I  1 1  I  I I I |  1000  I  I  I  I  1 1  I I |  10000  GLP-1 Concentration (pg/ml)  FIGURE 28. Standard curves of the GLP-1 radioimmunoassay. The data from 6 different assays were expressed as mean B% + SEM. The detection limit was 78 pg/ml GLP-1. Intra- and inter-assay variations were 6% and 11%, respectively.  116  GLP-(1-36) amide GLP-1 (7-36) amide 2000  CO Q.  Pancreas extract 1000 H  Q.  _1  o  •  0  10  i. 20  • *•* i 30  i 40  i 50  FIGURE 29. HPLC determination of GLP-1 content in extract of rat pancreas. The peptides was analyzed by HPLC followed by radioimmunoassay. Arrows indicate elution positions of synthetic GLP-1(736) and GLP-1Q-36) amide. Both GLP-l(l-36) amide and GLP-1 (7-36) amide were detected. Fractions = 0.5 min samples (approximately 0.5 ml/each).  minor peak was also detected, the identity of which is unknown. The integrated amounts of GLP-l(l-36) amide and GLP-l(7-36) amide were 4.6 ng/g tissue and 1.5 ng/g tissue, respectively.  3.2 GLP-1 Immunoreactivity in Ileum Extracts  Extracts from two rat ilea demonstrated similar H P L C elution profiles, with the majority of the immunoreactivity eluting at the same position as GLP-K7-36) amide (Figure 30, A and B). The integrated amounts of GLP-1(7-36) amide were 32 ng/g tissue (A) and 24 ng/g tissue (B) in the ileal extracts.  B. Responses of GLP-1 to a Glucose Load in vivo and Responsiveness of the Perfused Pancreas to GLP-1(7-36) amide in vivo  In studies on the enteroinsular axis i n obese Zucker rats, it was found previously that GIP levels i n the obese animals were similar to their lean littermates, but the normal glucose threshold for the action of GIP was reduced (Chan et al., 1984; Chan et al., 1985; Pederson et al., 1991). Circulating levels of GLP-1 were found to be elevated i n obese patients (Fukase et al., 1993), but there have been no reports on the secretion or actions of GLP-K7-36) amide i n Zucker rats.  Therefore the following studies were carried out.  Firstly, i n vivo  immunoreactive (IR) GLP-1 levels were determined during fasting and after an oral glucose load. Secondly, the effect of GLP-l(7-36) amide on insulin secretion from the perfused pancreas i n the presence of glucose was studied and the glucose threshold for the peptide determined. Thirdly, insulin secretion from the perfused pancreas i n response to a gradient of GLP-1(7-36) amide i n lean and obese rats was compared.  118  30000 -i  GLP-1 (1-36) amideGLP-1(7-36) amide  20000 Q.  Ileum Extract 10000  1  0  15000  10  20  30  40  1  50  GLP-1 (1-36) amide GLP-1 (7-36) amide  Ileum Extract  10  20  30  40  50  Fraction Number  FIGURE 30. HPLC determination of GLP-1 content in two extracts of rat ileum. The extract was separated by HPLC and then the fractions from the HPLC were assayed by GLP-1 radioimmunoassay. Arrows indicate elution positions of synthetic peptides. Two peaks of immunoreactivity were detected corresponding to GLP-l(l-36) amide and GLP-l(7-36) amide. Fractions = 0.5 min samples (approximately 0.5 ml/each).  119  1. Oral Glucose Tolerance Test  1.1 Plasma Glucose Levels  Glucose levels were measured before and after a glucose load (1 g/kg) administered as a 40% solution (Figure 31). After overnight fasting, the plasma glucose levels of obese and lean Zucker rats were 8.1 ± 0.56 mmol/L and 6.7 ± 0.56 mmol/L, respectively. The values for the obese rats were significantly higher (P<0.05) than those of the lean animals. However, the circulating glucose levels resulting from the oral glucose challenge were not significantly different between obese and lean animals. A t 20 min after glucose ingestion, the peak glucose occurred, with plasma levels reaching 15.4 ± 1.2 mmol/L in obese and 14.5 ± 0.7 mmol/L in lean rats. Glucose levels at 60 min were not different between obese (11.89 ± 1.23 mmol/L) arid lean (10.22 + 0.67 mmol/L) rats.  1.2 Insulin Secretion  As shown i n Figure 32, obese rats exhibited severe fasting and glucosestimulated hyperinsulinemia when compared to lean littermates. Fasting plasma insulin levels in obese and lean rats were 184 ± 17 uU/ml and 7 ± 1 uU/ml, respectively. The insulin responses to the oral glucose load reached a peak at 10 min and the peak increases from the fasting levels were greater in the obese than in the lean animals (+68 ± 17 aU/ml versus +28 ± 3 uU/ml).  120  0H 0  1  1  10  1  T  20  1  1  1  30  1  40  '  1  50  '—~~i  60  TIME (min)  FIGURE 31. Effect of 1 g/kg oral glucose on circulating glucose levels in lean and obese Zucker rats. Basal glucose levels were significantly higher in the obese rats than in lean animals (P<0.05). The glucose levels after the oral glucose challenge did not differ between lean and obese. N=7 for both rats.  121  300 1  ZD  LEAN OBESE  200  ZD  CO  TIME (min)  FIGURE 32. Effect of 1 g/kg oral glucose on circulating insulin levels in lean and obese Zucker rats. Basal and glucose-stimulated insulin release were significantly greater in obese rats than in lean animals (P<0.05). N=7 for both lean and obese rats.  122  1.3 GLP-1 Secretion  As shown i n Figure 33, mean fasting GLP-1 levels were slightly, though not significantly, higher in the obese (59 ± 12 pmol/L) compared to the lean animals (49 ± 9 pmol/L). However, peak values at 10 min following the oral glucose load were significantly greater (P<0.05) i n the obese (96 ± 10 pmol/L) when compared with the lean rats (56 ± 6 pmol/L). The increments of GLP-1 secretion at 10 and 20 min over fasting levels were significant i n the obese rat (P<0.05), whereas increases i n mean levels of the lean animals were not.  2. Response of the Isolated Perfused Pancreas to GLP-l(7-36) Amide i n the Presence of 4.4 mmol/L Glucose  Isolated pancreases of obese and lean Zucker rats were perfused with a glucose concentration of 4.4 mmol/L.  In previous studies, this glucose  concentration was shown to be below the threshold for G I P action on insulin release from lean rats (Chan et al., 1984). Between 10 and 30 min of the total 40 min perfusion period, 300 or 50 pmol/L GLP-K7-36) amide was infused via a sidearm. The peptide concentration of 300 pmol/L was used because it produced maximal insulin release from Wistar rats under hyperglycemic (10 mmol/L) conditions (Figure 10) and it exceeds the highest postprandial plasma GLP-1 level reported by Hirota et al. (1990). The lower concentration, 50 pmol/L, was within the normal range of postprandial GLP1 concentrations reported by several investigators (Kreymann, et al., 1987; 0rskov et al., 1991; 1994; Takahashi et al., 1990).  123  FIGURE 33. Effect of 1 g/kg oral glucose on circulating C-terminal GLP-1 levels in lean and obese Zucker rats. Peak levels of GLP-1 at 10 min following the glucose challenge were significantly higher in obese than in lean animals (P<0.05). N=7 for both lean and obese animals.  124  2.1 Effect of 300 pmol/L GLP-K7-36) Amide  In the presence of 4.4 mmol/L glucose, there was only a transient increase i n the insulin response to G L P - K 7 - 3 6 ) amide i n pancreases from lean animals (Figure 34). The insulin levels immediately returned to basal and remained low throughout the peptide perfusion. The mean insulin concentrations during the first 10 min of perfusion were 21 ± 2 (iU/min, while the peak values were 95 ± 27 LiU/min. This is approximately 4-5 times above the basal levels. The infusion of 300 pmol/L GLP-1(7-36) amide evoked a biphasic insulin secretion i n obese animals to peak levels of 6165 ± 1223 LiU/min (Figure 35), and the second phase was sustained during the perfusion of the peptide. The mean insulin secretion rate during the first 10 min of perfusion in pancreases from obese rats (246 + 13 LiU/min) was approximately ten times that from lean animals (Figure 34). The relative increases above basal i n the obese rat pancreases (approximately 25 times) were also greater than those from lean animals.  2.2 Effect of 50 pmol/L GLP-K7-36) Amide in Obese Rats  Even i n the presence of a much lower concentration of the peptide (50 pmol/L), obese rats exhibited an obvious biphasic insulin secretion (Figure 36). The insulin peak levels were approximately 7.5 times higher than the basal values (936 + 142.66 LiU/min versus 125 ± 29.5 LiU/min). Elevated levels of insulin secretion were maintained throughout the period of perfusion of the peptide.  GLP-1 (7-36)amide  ~  0  I  10  ~  20  l  30  l  40  Time (min)  FIGURE 34. Effect of 300 pmol/L GLP-1(7-36) amide on insulin secretion from the isolated perfused pancreas of lean Zucker rats in the presence of 4.4 mmol/L glucose. The peptide was infused between 11-30 min. Insulin secretion increased transiently during minutes 12 and 13 (P<0.05). N=7.  126  8000-,  c E  6000 4 Obese 4000 4  CO  2000 4  Time (min)  FIGURE 35. Effect of 300 pmol/L GLP-1(7-36) amide on insulin secretion from the isolated perfused pancreas of obese Zucker in the presence of 4.4 mmol/L glucose. The peptide was infused between 11-30 min. Stimulated insulin secretion was biphasic and was increased significantly during minutes 11-32 (P<0.05). N=7.  127  GLP-1 (7-36) amide  1 0  1  10  1  1  20  30  1  40  TIME (min)  FIGURE 36. Effect of 50 pmol/L GLP-1(7-36) amide on insulin secretion from the isolated perfused pancreas of obese Zucker rats in the presence of 4.4 mmol/L glucose. The peptide was infused between 11-30 min. Insulin secretion was increased above basal during minutes 12-31 (P<0.05). N=7.  128  3. Response of the Isolated Perfused Pancreas to 2.8-11 mmol/L Glucose Gradient in the Presence of 300 pmol/L GLP-K7-36) Amide  3.1 Insulin Secretion i n Response to 2.8-11 mmol/L Glucose Alone i n Obese and Lean Zucker Rats  Perfused pancreases from obese rats showed hypersecretion of insulin (335 ± 53 fxU/min) even at 2.8 mmol/L glucose when compared to lean animals (15 + 3 |iU/min; Figure 37). Elevated levels were maintained throughout a gradient perfusion of glucose, with maximal secretion rates i n obese and lean rats of 917 ± 177 jxU/min and 110 ± 23 jiU/min, respectively. However, the relative increase above basal i n lean rats (7.3 times) was higher than that i n the obese animals (2.7 times).  3.2 Insulin Secretion i n Response to 2.8-11 mmol/L Glucose Gradient and 300 pmol/L GLP-K7-36) Amide in Zucker Rats  GLP-1(7-36) amide stimulated insulin secretion i n a glucose-dependent manner i n lean animals (Figure 38).  Insulin secretion did not increase  significantly above basal (30 ± 5 uU/min) between minute 1 and minute 12 (66 + 17 |iU/min;P<0.05). Glucose levels were 4.22 ± 0.11 pmol/L at this time point. This glucose threshold was close to, but slightly lower than, that found i n Wistar rats using a similar glucose gradient and peptide concentration (Figure 12). The maximal insulin output, occurring i n minute 38 (1346 + 206 ^U/min), reflected a 41-fold increase over levels at the start of the perfusion. In obese animals, insulin secretion from the perfused pancreas increased i n response to peptide as soon as it was applied i n the presence of 2.8 mmol/L  129  FIGURE 37. Effect of 2.8-11 mmol/L glucose gradient on insulin secretion from the isolated perfused pancreas in lean and obese Zucker rats. Insulin secretion was significantly greater throughout the perfusion in the obese animals than in lean ones (P<0.05). N=7 for both lean and obese rats.  130  FIGURE 38. Effect of 300 pmol/L GLP-1(7-36) amide on insulin release from the isolated perfused pancreas of lean Zucker rats in the presence of a 2.8-11 mmol/L glucose gradient. Insulin secretion in response to GLP-1(736) amide increased significantly at minute 12 with glucose levels of 4.22 ± 0.11 mmol/L (P<0.05). N=7.  glucose (Figure 39). In minute 3 of the perfusion insulin levels increased to 1973 ± 255 p,U/min from the first min basal levels of 448 ± 73 LiU/min. Secretion increased further to a maximal of 14178 ± 1605 liU/min i n minute 37. Although the peak insulin concentrations reached i n pancreases from the lean animals (1346 ± 206 |iU/min) were only 9.5% of those from the obese animals, the increases over those i n period 1 were greater (P<0.05): 44.9-fold (± 0.9) compared with 34.4-fold (±0.6) increases with pancreases from obese animals.  4. Responses of the Isolated Perfused Pancreas to 0-300 pmol/L GLP-K7-36) Amide Gradient in the Presence of 16.7 mmol/L Glucose  In order to compare insulin release under hyperglycemic conditions i n lean and obese animals, pancreases were perfused with a 0-300 pmol/L GLP-l(7-36) amide gradient in the presence of 16.7 mmol/L. In the presence of 16.7 mmol/L glucose, basal insulin secretion i n lean Zucker rats i n minute 1 and the maximal insulin secretion rate at minute 37 were 383 ± 21 uU/min and 5114 ± 707 uU/min respectively (Figure 40). This peak value was approximately 4 times that produced i n perfusion with 300 pmol/L GLP-1(7-36) amide i n the presence of 11 mmol/L glucose (Figure 38). In the presence of G L P 1(7-36) amide, insulin levels were elevated significantly i n period 5 (1042 ± 166; P<0.05) over both those i n period 1 and those measured i n perfusions with glucose alone. Hypersecretion of basal insulin was again demonstrated (884 ± 52 jiU/min) compared with (383 ± 2 1 )U.U/min) i n the lean pancreases.  In pancreases from  obese rats the secretion remained stable during the first 5 m i n (Figure 41). Significant increases (P<0.05) over basal levels occurred at minute 6. Therefore the threshold for GLP-l(7-36) amide in obese rats was similar to that i n lean  132  F I G U R E 39. Effect of 300 pmol/L GLP-1(7-36) amide on insulin release from the isolated perfused pancreas of obese Zucker rats in the presence of a 2.8-11 mmol/L glucose gradient. Insulin secretion increased significantly over that in minute 1 and glucose alone from minute 3 to the end of the perfusion (P<0.05). N=6.  133  Lean  -•  + GLP-1 (7-36) GLP-1 (7-36)  -•  + Glucose  c JE  .r-300  14-200 CD CO  MOO  CO  I  CD  0  T I M E (min)  FIGURE 40. Effect of a 0-300 pmol/L gradient perfusion of GLP-1(7-36) amide on insulin secretion from the isolated perfused pancreas of lean Zucker rats in the presence of 16.7 mmol/L glucose. Insulin secretion increased significantly from period 5 when compared with basal levels and with glucose alone from minute 4 (P<0.05). N=7.  134  Obese  20000  -•  + GLP-1 (7-36)  -o——  + Glucose  /  R  300  GLP-1 (7-36)  15000H TTTT#U i - 2 0 0  o  E  Q_ 10000 4  CO CO  5000 H  Q. _l (3  ID CO  §««rfoc«>ooooooooc^^ 0  I  0  10  I  20  0  I  30  40  TIME (min)  FIGURE 41. Effect of a 0-300 pmol/L gradient perfusion of GLP-l(7-36) amide on insulin secretion from the isolated perfused pancreas of obese Zucker rats in the presence of 16.7 mmol/L glucose. Insulin secretion increased significantly over basal levels in the perfusions with GLP-1(7-36) amide and perfusions with glucose alone between minute 6 and 40. N=7.  animals (Figure 40). This threshold was calculated to be less than 50 pmol/L i n both lean and obese animals. The peak insulin concentrations i n perfusate from obese pancreases (14980 ± 2009 uU/min) were similar to those obtained with 300 pmol/L G L P - K 7 - 3 6 ) amide i n the glucose gradient perfusions (Figure 39), suggesting that this is close to the maximal insulin output in obese rats. As shown i n Figure 42, the total integrated insulin output from the pancreases of lean rats i n response to 16.7 mmol/L glucose alone (14137 ± 3128 uU) was approximately 30% that i n the obese (40865 ± 19025 uU). The total increment i n insulin secretion i n the obese rats stimulated with 0 - 300 pmol/L GLP-K7-36) amide i n the presence of 16.7 mmol/L glucose were 323524 ± 88762 [i\J compared with 123007 ± 43893 uU i n the lean animals. However, the relative increases i n the insulin secretory response to GLP-l(7-36) amide over perfusions with glucose alone were approximately 8 times i n both lean and obese animals, suggesting similar sensitivity to the peptide in both lean and obese animals.  136  500  _  3  400 H  •  Lean Control  0  Lean with GLP-1 (7-36)  H  Obese Control  0  Obese with GLP-1 (7-36)  E  H CL  300 H  o  CO  O  200 i  100 H  FIGURE 42. Total insulin output stimulated by 0-300 pmol/L GLP-1(7-36) amide in the presence of 16.7 mmol/L glucose in lean and obese Zucker rats. Although the total insulin secretion is greater in obese than in lean rats, the relative increases over the perfusions with glucose alone were similar in both groups. N=7 for lean and obese rats.  137  DISCUSSION  The objectives of the studies described i n this thesis were to compare the actions of truncated GLP-1 and different forms of GIP on somatostatin and gastrin secretion from the isolated perfused rat stomach, and insulin secretion from the isolated perfused rat pancreas. In addition, the secretion of C-terminally amidated G L P - 1 , and the insulinotropic action of GLP-1(7-36) amide on the pancreas were investigated i n Zucker rats. GIP and GLP-1 are both considered to be established incretins. The strong insulinotropic action of GIP i n the presence of elevated glucose has been demonstrated i n humans (Dupre et al., 1973, Brown et al., 1975) and dogs (Pederson et al., 1975) in vivo, and the isolated rat pancreas in vitro (Pederson and Brown, 1976). The physiological significance of this action is indicated by the fact that such responses were obtained when GIP was infused to achieve circulating levels found postprandially (200-600 pmol/L) (Amland et al., 1984b; Pederson et al., 1975). The threshold glucose concentration above which GIP exerted an insulinotropic effect was reported to be between 4.4 and 5.5 mmol/L i n rat and human pancreas (Pederson et al., 1976; Mc Cullough et al., 1983). However, because single concentrations of glucose were used i n these studies it was not possible to obtain accurate measurements of the threshold. Both GLP-1(7-36) amide and GLP-1(7-37) were shown later to also stimulate insulin secretion i n humans in vivo (Kreymann et al., 1987), pig (Hoist et al., 1987) and rats in vitro (Mojsov et al., 1987; Fehmann et al., 1989), also under hyperglycemic conditions. Postprandial levels of immunoreactive GLP-1 have been shown to range between 40 and 90 pmol/L i n different studies (Kreymann et al., 1987; 0rskov et al., 1991; 1994; Takahashi et al., 1990), and infusion of either form of tGLP-1 i n humans at a dose that achieved these circulating levels resulted i n potentiation of glucose-  induced insulin secretion (Nauck et al., 1993a; Kreymann et al., 1987), establishing a physiological role for these peptides. The glucose threshold for GLP1(7-36) amide was not determined with certainty, but appeared to lie between 2.8 and 6.6 mmol/L in vitro (Weir et al., 1989; Suzuki et al., 1990) or in vivo (Nathan et al., 1992). Several in vitro studies directed at comparing the effects of the two hormones led to the suggestion that GLP-1(7-36) amide is considerably more powerful than GIP i n stimulating insulin release i n the isolated rat pancreas (Shima et al., 1988) or islets (Siegel et al., 1992).  However, other studies  demonstrated an equivalent insulinotropic activity i n the rat pancreas in vitro (Schmid et al., 1990; Suzuki et al., 1990; 1992a). It is important to establish the relative potencies of these two peptides, since there is growing interest i n the potential use of incretins for controlling the hyperglycemia i n patients with N I D D M (Habener, 1993; Nathan et al., 1992; Nauck et al., et al, 1993c; 1993d). Because i n previous studies only single peptide and glucose concentrations were used, and conditions differed between experiments, it is difficult to assess the relative effectiveness of the peptides from their results. Therefore, i n the current studies a gradient of peptide or glucose was administered to the isolated perfused rat pancreas to facilitate comparison under a broad range of peptide or glucose concentrations.  The aim of the studies was to clarify whether there are  differences i n the concentration threshold at which truncated GLP-1 and GIP increase insulin secretion, the maximal effects exerted by these peptides, and whether their glucose thresholds for stimulation of insulin secretion differ. The insulinotropic activities of sp GIP, np GIP, sh GIP and GLP-K7-36) amide were first compared with a 0-1 nmol/L peptide gradient i n the presence of a level of glycemia (16.7 mmol/L) that has previously been shown to approach a maximal stimulation of the B-cell i n the perfused rat pancreas (Pederson and  Brown, 1976). The mean maximal effects obtained were similar for sp GIP, np GIP and GLP-K7-36) amide. Although slightly greater responses were obtained with GLP-K7-36) amide, the concentration required (>700 pmol/L) greatly exceeds the physiological levels of the peptide and is unlikely to be of physiological importance. The slightly lower insulinotropic activity with np GIP is probably a result of the presence of GIP (3-42) i n preparations of np GIP (Jornvall et al., 1981), a fragment with very low insulinotropic activity (Brown et al., 1981). Insulin responses to the 0-1 nmol/L gradient of sh GIP i n these experiments were much lower than those to either form of porcine GIP or GLP-1(7-36) amide, although greater than those obtained with glucose alone. It was considered possible that sequence differences were responsible for the reduced response to shGIP, since human GIP differs at two amino acid positions from the porcine peptide with a histidine for arginine substitution at position 18 and asparagine for serine at position 34 (Moody et al., 1984; Takeda et al., 1987), and from the rat peptide with a similar substitution at position 18 and an isoleucine for leucine at position 40 (Higashimoto et al., 1992). However, Chow et al. (1990) demonstrated that the insulinotropic activity of a recombinant form of human G I P i n the isolated perfused rat pancreas did not differ from that of np GIP, and an alternative possibility considered was that the reduction i n biological activity resulted from the peptide synthesis.  This is supported by the following  observations. Although the insulinotropic activity of sh GIP was found to be much less than that of GLP-l(7-36) amide i n a glucose-clamp study i n humans by Nauck et al. (1993c), the results are not consistent with those from an earlier study (Nauck et al., 1989) using a similar experimental protocol. In the former study, the peak values of insulin secretion in response to the same concentrations of shGIP were approximately 1000 pmol/L (Nauck et al., 1989), whereas they approximated only 350 pmol/L i n the latter (Nauck et al., 1993c). The insulin  levels obtained i n the early study are similar to those obtained with GLP-K7-36) amide (Nauck et al., 1993c). Furthermore, whereas i n the earlier study sh GIP exerted an effect on insulin release similar to oral glucose, i n the latter study it was significantly less. The results from the present study suggest that some of the ambiguity i n the literature may be explained by large variability i n biological activity between different batches of synthetic peptides. This has recently been confirmed by Wheeler et al. (1995), where it was shown that more recent batches of sh GIP obtained from Peninsula, and a batch from Bachem exhibited identical insulinotropic activity to the porcine peptide. Because the effect of np GIP was similar to that of sp GIP, and the effect of sh GIP was greatly reduced, only sp GIP was used for comparison with GLP-1(736) amide i n subsequent experiments. When the glucose level i n the perfusate was reduced to a level which approximately mimicked the level found after an oral glucose load i n the rat (10 mmol/L), a 0-1 nmol/L gradient of sp GIP or GLP-K736) amide produced similar insulin responses and maximal effects. As expected, the maximal effects were lower than those obtained with 16.7 mmol/L glucose. The concentrations of either peptide required for maximal increases i n insulin release i n the current study were comparable to those reported by Schmid et al. (1990) and Shima et al. (1988), i.e. between 100 and 1000 pmol/L. A further interesting finding was that the peptide concentration producing a maximal effect was dependent on the glucose concentration. For example, with a glucose level of 16.7 mmol/L, insulin secretion began to plateau when the peptide concentration exceeded 700 pmol/L. However, with 10 mmol/L glucose maximal insulin secretion was observed at peptide concentrations of approximately 290 pmol/L.  This  appears to be the first report of a glucose concentration dependency for the effect of these two peptides on insulin secretion.  Although it is difficult to estimate the threshold concentration of peptides which produced significant increases i n insulin release i n the studies using 0-1 nmol/L peptide gradients, the rapid onset of action (within min) of either peptide suggested they were effective at 22 pmol/L or less. To obtain a more accurate assessment of this threshold for stimulation of insulin secretion, sp GIP and G L P 1(7-36) amide were applied as 0 to 50 pmol/L gradients i n the presence of 10 mmol/L glucose. In agreement with the above conclusion, the sensitivity of the rat pancreas to the two peptides was identical, and increases i n concentrations of 16 pmol/L or less were effective. The minimal concentration of peptide required to exert an insulinotropic effect found i n this study is comparable to the range of 1050 pmol/L for GIP previously reported (Schmid et al, 1990; Pederson et al., 1982). The results from the current studies, however, are not i n agreement with those reported by Siegel et al. (1992) or Shima et al. (1988) that supraphysiological concentrations (>400 pmol/L) of GIP are needed to stimulate insulin release. The threshold for stimulation observed in the current studies is similar to those (10-25 pmol/L) reported for GLP-K7-36) amide (Weir et al., 1989; Komatsu et al., 1989; D'Alessio et al., 1989; Schmid et al., 1990). A n even lower (1 pmol/L) threshold for GLP-K7-36) amide was found i n islet cell monolayers i n the presence of both glucose and amino acids (D'Alessio et al., 1989). In contrast, several studies have reported thresholds of 100 pmol/L or greater for GLP-K7-36) amide (0rskov et al., 1988; Hoist et al., 1987; Shima et al, 1988; Siegel et al, 1992). The differences i n the minimal effective concentrations of GIP and GLP-K7-36) amide i n different studies may be due to the use of different glucose concentrations (Siegel et al, 1992; Weir et al, 1989), the use of islets (Siegel et al,  1992), or perfused  pancreases from a different species (0rskov et al, 1988). In the studies in which a glucose gradient was applied i n the presence of 50 pmol/L peptide, an identical glucose threshold of 4.5 ± 0.11 mmol/L was  demonstrated for both spGIP and GLP-K7-36) amide. This is consistent with earlier studies which reported a threshold of between 4.4 and 5.5 mmol/L for GIP (Pederson and Brown, 1976) and 2.8 to 6.6 mmol/L for GLP-K7-36) amide (Weir et al., 1989; Suzuki et al., 1990; Nathan et al., 1992). These results therefore confirmed that GIP and GLP-1(7-36) amide were equally potent i n stimulating insulin secretion on a molar basis and exhibited identical glucose thresholds. Supraphysiological concentrations of GLP-K7-36) amide were found to have insulinotropic activity even at glucose levels as low as 2.8 mmol/L (Fridolf and Ahren, 1991; Komatsu et al., 1989), and a similar result was reported for GIP (Pederson et al., 1975). Responses to both peptides were sigmoidal, and this may partially reflect the kinetics exhibited by glucokinase (Niemeyer et al., 1975). Although the mechanism of the sigmoidal kinetic function of glucokinase is not clear, this kinetic character has been considered to be an adaptive feature aimed at increasing the efficiency of the enzyme when operating i n the range of changes of physiological glucose concentrations (Niemeyer et al., 1975). Surprisingly, increasing either peptide concentration from 50 to 300 pmol/L, did not have a major effect on the responses to the glucose gradient. In both cases secretion reached a plateau at a glucose concentration of approximately 9 mmol/L, which is within the range of circulating levels after an oral glucose load, and the maximal effects were identical. The mechanisms involved i n this phenomenon are not clear. The overall response of the pancreatic B-cell to secretagogues is dependent upon both its previous exposure to glucose and the nature of the secretagogues themselves. Glucose, acting as a competence factor, is now considered to be a prime determinant of subsequent islet responses. Different investigators have demonstrated that prior exposure to glucose sensitizes the endocrine pancreas to subsequent stimulation by glucose itself or other secretagogues (Cerasi, 1975; Efendic et a., 1979; Grill and Rundfeldt, 1979;  Grill, 1981). This is referred to as a priming effect. Recently, Holz et al. (1993) measured the glucose competence state of single B-cells by recording the plasma membrane potential. The authors found that glucose pre-exposure rendered B cells responsive to subsequent exposure to tGLP-1. The intracellular mechanism responsible for glucose priming has not been established. B-cell insulin secretion 2+  is C a  -dependent, and depolarizing concentrations of glucose increase 2+  intracellular C a 1992).  concentrations (Grapengiesser et al., 1988; Hellman et al.,  Both tGLP-1 and GIP stimulate the production of c A M P i n B-cells  (Drucker et al., 1987; Thorens, 1992; Amiranoff et al., 1989; Maletti et al., 1987). Since the production of cAMP by incretins is also glucose dependent, it is possible that the B-cell adenylyl cyclase is regulated by calcium and calmodulin (Valverde et al., 1979). Thus both the pattern of insulin secretion and the maximal response may primarily depend on glucose exposure.  It is possible that both the  concentration and duration of exposure influence the degree of priming. On the other hand, Holz et al. (1993) also found that tGLP-1 could prime the cells to respond to subsequent exposure to glucose. A similar priming effect by tGLP-1 and GIP was described earlier by Fehmann et al. (1991). These hormones may thus be of crucial importance for maintaining B-cells in a glucose competent state in the basal situation to allow B-cells to respond rapidly to glucose stimulation. However, the intracellular mode of cross-talk between the c A M P and glucose signalling systems remains to be established. Although the above can explain the interdependence of the insulin response to glucose and incretins it does not explain why identical responses were obtained with 50 pmol/L and 300 pmol/L peptides.  One possible reason is that the  prolonged exposure to either peptide induced receptor desensitization prior to glucose reaching threshold, and the remaining receptor number could be maximally stimulated by 50 pmol/L peptide. Although desensitization of GLP-1  (7-36) receptors on insulinoma cells was reported by Fehmann and Habener (1991) at a concentration of 100 nmol/L or higher, the concentrations of peptide inducing receptor desensitization on tumor cells may be different from that required i n the isolated perfused rat pancreas, and conditions i n the experiments are also different.  Further studies are clearly necessary to reveal the  mechanisms responsible.  The second component of the research described i n this thesis was directed at comparative studies on the effect of GIP and tGLP-1 on gastric endocrine secretion. These studies demonstrated that both GIP and tGLP-1 stimulated somatostatin secretion from the isolated perfused rat stomach. The D-cells exhibited a similar sensitivity to synthetic and native porcine GIP, GLP-1(7-36) amide and GLP-K7-37), with significant stimulation at a concentration of 50 pmol/L or less. The results are in good agreement with previous observations that these peptides were potent stimulators of gastric somatostatin secretion (Mcintosh et al., 1979; 1981a; Eissele et al., 1990). The threshold for stimulation obtained with either form of GLP-1 was similar to that reported by Eissele et al. (1990) using single concentration perfusions. In contrast, i n the pig stomach GLP-1 (7-36) amide failed to increase somatostatin secretion (0rskov et al., 1988) suggesting that species differences may exist. In agreement with Eissele et al. (1990), GLP-1 (1-36) amide was found to have no effect on somatostatin release, and GLP-2 was also inactive. The data i n the present studies also showed that the maximum stimulatory effect of either form of porcine GIP was greater than that of GLP-K7-36) amide or GLP-K7-37). On the other hand responses to sh GIP were reduced with respect to both the initial rate of increases and the maximal increases obtained. The possible reason for this, as well as the reduced  insulinotropic activity of sh GIP i n the perfused rat pancreas, were discussed earlier. This may also explain the reported lack of effect of a synthetic human preparation of GIP on acid secretion in humans (Nauk et al., 1992). The studies on the effect of GIP or tGLP-1 on gastrin secretion demonstrated that these two peptides had opposite effects on gastrin release, unlike their similar effects on somatostatin secretion. The basal secretion and responses of gastrin were more variable than those of somatostatin. Although the origin of this greater variability is not clear, it is possible that the antrum experienced more mechanical handling during surgery which may affect gastrin secretion. In contrast, the somatostatin measured is from all regions of the stomach, and this may mask fluctuations caused by mechanical stimulation. Maximum effects on gastrin secretion were obtained earlier following the onset of peptide perfusion (9 10 min for GIP and 14 - 15 min for GLP-l(7-36) amide or GLP-K7-37)) than those on somatostatin secretion (> 20 min) suggesting that the G-cell is more sensitive to these peptides than the D-cell. The current studies demonstrated that sp GIP, np GIP and sh G I P all stimulated gastrin secretion, although the maximal stimulatory effect of sh GIP was again lower than either form of porcine GIP. This stimulatory effect of GIP on gastrin secretion was also shown earlier by Pederson et al. (1981) using the natural peptide purified from intestinal extracts. Only single concentrations of peptide were tested i n these studies, and responses were relatively small and transient.  It was therefore considered that this may have resulted from a  contaminant i n the natural product since small amounts of C C K were reported to be present. This is unlikely to be true, since the current studies showed that the synthetic peptides also stimulate gastrin secretion. This stimulatory effect of GIP is i n apparent contradiction to reports that GIP had inhibitory actions on gastrin release.  However, this inhibitory effect was only observed during  stimulation of gastrin secretion with a meal (Villar et al., 1976), v i a vagal stimulation (Hoist et al., 1983), or with carbachol (Wolfe and Reel, 1986). In an immunoneutralization study Wolfe and Reel (1986) showed that the inhibitory effect of GIP on carbachol-stimulated gastrin release from cultured antral mucosa was partly mediated through somatostatin. However, i n such studies a direct effect of GIP as a stimulator of gastrin release can not be excluded and build-up of somatostatin i n the medium i n static incubations could account for the observed responses.  To clarify this situation further studies are necessary using a  perifusion system with isolated G-cells. In contrast to GIP, GLP-K7-36) amide and GLP-K7-37) demonstrated inhibitory effects on gastrin secretion in the rat stomach. Both forms of tGLP-1 exerted similar inhibitory effects which parallels the finding that they were equally potent i n stimulating insulin secretion from the pancreas (0rskov et al., 1993b). The results of the present study are compatible with those of Eissele et al. (1990) using a similar rat stomach perfusion model, but differ from experiments performed i n the pig stomach, where GLP-1(7-36) amide had no effect on gastrin release (0rskov et al., 1988). Therefore species differences may again exist. The roles of GIP and truncated forms of GLP-1 as enterogastrones remains to be established. Arguments both for and against a role for G I P have been presented (Brown et al., 1989; Konturek, 1989). Porcine GIP was found to be a potent inhibitor of pentagastrin-stimulated acid secretion i n vagally and sympathetically denervated pouches of the dog stomach (Pederson and Brown, 1972). However, i n the innervated dog stomach (Soon-Shiong et al., 1979; SoonShi ong et al., 1984) and human (Maxwell et al., 1980) GIP showed only a weak inhibitory effect on gastric acid secretion. Mcintosh et al. (1981b) found that vagal stimulation abolished the stimulatory effect of GIP on the release of gastric somatostatin suggesting that the acid inhibitory effect of GIP may be mediated  by somatostatin, which is under inhibitory control of the parasympathetic innervation. They also suggested that during feeding sympathetic fibres are activated at the same time as GIP secretion is stimulated, and this sympathetic input to the stomach may antagonize the parasympathetic inhibition of D-cell activity (Mcintosh et al., 1981a). Unfortunately, appropriate experiments to prove, or disprove, this proposal have not been performed. The physiological relevance of a stimulatory effect of GIP on gastric gastrin secretion is not understood. The possible role of GLP-K7-36) amide and GLP-K7-37) as enterogastrones is also unclear, although the increase of somatostatin secretion and decrease of gastrin release i n response to GLP-1(7-36) amide suggests such a role. However in isolated rat parietal cell preparations GLP-K7-36) amide was found to increase, rather than decrease, cAMP production (Hansen et al., 1988; Schmidtler et al., 1991; Schepp et al., 1992; Schmidtler et al., 1994) and this would be expected to increase acid secretion, whereas i n anesthetized rats it exerted no effect on basal or pentagastrin-stimulated acid secretion in vivo (Schmidtler et al., 1991). Studies on the effect of GLP-1(7-36) amide i n humans have also resulted i n conflicting data.  Several studies demonstrated that the peptide inhibited gastric acid  secretion i n man (Schjoldager et al., 1989; D'Halloran et al., 1990; Wettergren et al., 1993), whereas Nauk et al. (1992) reported that GLP-l(7-36) amide had no effect on acid secretion. Therefore, further studies are needed to clarify the effects of this peptide on gastric acid secretion.  The third objective of the investigation described i n this thesis was to determine whether circulating levels of immunoreactive GLP-1 differed between lean and obese Zucker rats, and whether the insulinotropic activity of G L P - K 7 -  36) amide exhibited a defect i n glucose dependency similar to that observed with GIP. A sensitive radioimmunoassay specific for amidated forms of GLP-1 was developed to measure circulating levels of immunoreactive (IR) GLP-1 i n the Zucker rat. GLP-1 (7-36) amide was used for immunization of guinea pigs. The antiserum obtained was directed against the C-terminal region of the molecule, since it bound both to GLP-1 (7-36) amide and GLP-1 (1-36) amide but not to either GLP-1 (7-37) or GLP-1 (1-37). The antiserum did not cross-react with any of the structurally related peptides examined, such as G I P and glucagon, suggesting that it was specific for the amidated GLP-1 peptides. In order to establish that the radioimmunoassay was capable of detecting endogenous GLP-1 produced by the rat small intestine and pancreas reverse phase H P L C separations were performed on tissue extracts from Wistar rats followed by radioimmunoassay of the fractions. Rat ileum contained both IR-GLP-1 (1^36) amide and GLP-1 (7-36) amide with the majority existing as GLP-K7-36) amide. Both molecular forms of GLP-1 were detected i n rat pancreatic extracts also, although the GLP-1(7-36) amide levels were much lower. These results suggest that the rat pancreas contains some processed GLP-1, while intestine is the major source of tGLP-1. These findings are i n agreement with those obtained by Mojsov et al. (1986; 1990) i n the rat. It was demonstrated that a certain amount of unprocessed GLP-1 also exists i n human (0rskov et al., 1994; George et al.,1985) and pig (Hoist et al., 1994) pancreas. Circulating immunoreactive GLP-1 concentrations were measured for the first time i n the Zucker rat. The fasting levels of IR-GLP-1 were i n the same range as those reported for Sprague-Dawley rats (approximately 100 pmol/L) (Hendrick et al., 1993) and humans (10-88 pmol/L) (Kreymann et al., 1987; Takahashi et al., 1991; Elliott et al., 1993; 0rskov et al., 1994). After an oral glucose challenge mean levels of IR-GLP-1 increased slightly i n the lean rats, but  this increase did not reach significance. However, i n the obese animals circulating levels of the peptide were significantly elevated at 10 and 20 min after oral glucose load, and the peak level at 10 min was greater than i n the lean animals. This relatively weak IR-GLP-1 response to glucose i n lean animals is consistent with that observed i n normal-weight human subjects (0rskov et al., 1991), and the greater response i n the obese animals parallels that of obese humans (Fukase et al., 1993). The GLP-1 assay developed may not precisely reflect the concentrations of biologically active GLP-1 i n Zucker rat plasma since other forms of GLP-1 are secreted in vivo. The development of other antisera with specificities for the N terminally truncated forms of GLP-1 such as GLP-1 (7-36) amide and GLP-1 (737) is needed to determine the circulating levels of these active peptides. Although circulating levels of tGLP-1 have not been studied previously i n Zucker rats, another major product of the proglucagon gene, glucagon, has and conflicting reports have appeared. Glucagon secretion i n response to arginine has been reported to be increased (Nishikawa et al., 1981; Rohner-Jeanrenaud and Jeanrenaud,  1988) and isolated perfused pancreas (Hirose et al., 1994),  unchanged i n the isolated perfused pancreas or islets (Seino et al., 1981; Hayek and Woodside, 1979), and even decreased i n a study in vivo (Eaton et al., 1976). The variability i n glucagon secretion reported could be due to the detection of glucagon-like-immunoreactivity with some antisera. . The possible sources of increased circulating levels of immunoreactive GLP-1 are increased secretion or/and production i n the intestine and pancreas, and a decreased metabolism of the peptide i n the obese Zucker rats. The possibility of decreased  metabolism of GLP-1 i n the obese animals has not been studied.  However, an increased number of L-cells or/and A-cells and increased responses of these  cells could  be responsible  for the  increased  peptide  levels.  Immunocytochemical studies of the pancreas of obese Zucker rats demonstrated the presence of hyperplastic, hypertrophic islets with increased B-cells and unchanged A-cells (Shino et al., 1973; Larsson et al., 1977; Chan et al., 1984). However the secretion rate of the A-cells and the relative contribution of pancreatic GLP-1 to the elevated glucose-induced plasma GLP-1 is unknown. Using  immunocytochemistry, C h a n et al. (1987) demonstrated  that  enteroglucagon cell number was reduced in the jejunum of obese compared to lean Zucker rat and the ileal enteroglucagon cell numbers were similar.  The  enteroglucagon cell number i n the distal gut including the lower ileum and colon, the major source of GLP-1, i n obese Zucker rats is not known. However, it is possible that these obese rats would parallel the situation in ob I ob mice i n which a greater proportion of enteroglucagon cells were found compared to lean mice (Polak et al., 1975b), and the hypertrophied intestine produced excessive amounts of glucagon-like immunoreactive peptide (Flatt et al., 1983). It has been hypothesized that an abnormal stimulation of the islets by the gut, v i a overactivity of the entero-insular axis, could contribute to the hyperinsulinemia of obesity (Jeanrenaud, 1979; Frerichs et al., 1980), although this suggestion is controversial (Selara and Barbara, 1987). Support for the theory includes the finding of elevated circulating GIP levels in some obese human subjects (Creutzfeldt et al., 1978), the obese hyperglycemic (ob/ob) mouse (Flatt and Bailey, 1982), and the corpulent (cp I cp) rat (Pederson et al., 1991). Circulating GIP levels i n obese Zucker rats were not, however, found to differ from those i n the lean animals, although the glucose dependence for the insulinotropic action of this peptide was altered (Chan et al., 1984). The change i n the glucose threshold for GIP action may contribute to the fasting hyperinsulinemia seen i n these obese rats, since even under fasting conditions the B cell may be subject to continual stimulation by GIP. The current studies demonstrate that the  insulinotropic activity of GLP-1 (7-36) amide, another important incretin, exhibited a defect in glucose dependency similar to that observed with GIP. Obese Zucker rats showed a hyperinsulinemia when fasting and after oral glucose challenge. The isolated perfused pancreas from obese animals also showed a marked basal hyperinsulinemia and hypersecreted insulin at a l l glucose concentrations used for perfusion.  In the presence of 4.4 mmol/L glucose,  pancreases from both lean and obese animals responded to GLP-1 (7-36) amide with an immediate increase i n insulin secretion. However, insulin secretion from the obese rat pancreases was biphasic and sustained with an approximately 25fold increase, whereas that from the lean rat pancreases was transient with only an approximately 5-fold increase. When a gradient of GLP-1 (7-36) amide was perfused at a fixed level of glucose the pancreases from the lean and obese animals exhibited a similar peptide threshold for stimulation, of 50 pmol/L or less. In addition, the relative increases i n total insulin secretion stimulated by GLP-1 (7-36) amide were approximately 8-fold for both lean and obese rats.  These  results suggest that the sensitivity to GLP-1(7-36) amide of pancreases from animals of both phenotypes was similar. However, when the pancreases were perfused with a glucose gradient (2.8-11 mmol/L) i n the presence of a constant concentration of GLP-1 (7-36) amide (300 pmol/L), insulin secretion increased immediately at a glucose concentration as low as 2.8 mmol/L i n the obese animals, whereas a minimum of 4.22 mmol/L glucose was required for stimulation of pancreases from lean rats. This lack of glucose-dependency of the action of GLP-1 (7-36) amide parallels that observed with GIP i n earlier studies on the obese Zucker rat (Chan et al., 1984). Therefore, i n the obese animals, both GLP-1 (7-36) amide and G I P may contribute to fasting and nutrient-stimulated hyperinsulinemia.  The mechanisms involved i n the altered responsiveness of B cells from obese Zucker rats to incretins are unclear. Recently, Milburn et al. (1995) demonstrated that basal hypersecretion of insulin at normoglycemic concentrations i n obese rats may be partly due to an increase i n low K  m  glucose metabolism i n B-cells  and B-cell hyperplasia, which can be induced i n normal islets by high concentrations of long chain fatty acids such as those occurring i n obesity. It is also possible that there are alterations i n receptors and/or signal transduction systems mediating the actions of GIP and GLP-1 (7-36) amide i n obese Zucker islets. There is evidence for altered G-protein function i n tissues from obese rats. Studies on hepatocyte membranes of obese animals revealed that functional G i is not present, and the function of G is altered (Houslay et al., 1989). Levels of G i s  and Gs were also found to be decreased in adipose tissue of obese rats (Vannucci et al., 1989). To date, the only study on changes in G proteins i n isolated islets from obese Zucker rats has shown normal functional Gi-proteins (Cawthorn and Chan, 1991). However since abnormal regulation of cyclic A M P generation was detected in isolated islets and adipose tissue of obese (ob/ob) mice (Black et al., 1986; 1988) further studies are needed to clarify whether similar changes occur i n obese rats. More distal components of signal transduction pathways have also been found to be altered i n obese rats. The activation of protein kinase C (PKC) was found to be defective i n the heart and liver of obese {fa I fa) rats (Van De Werve et al., 1987) and levels of P K C were decreased i n the skeletal muscle of these rats (Cooper et al., 1993). Similar measurements have not been performed on obese rat islets. Studies on calcium uptake i n pancreatic islets from Zucker rats demonstrated that over a glucose concentration range of 0-27.7 mmol/L, calcium accumulation was 2-fold greater i n islets from the obese rats than from the lean animals (Pansini and Tolman, 1981). This increased calcium uptake i n obese rat  islets i n the presence of glucose concentrations below the physiological range suggested a defective glucose-sensing mechanism may be the primary defect i n these animals. Roe et al. (1994) found that glucose stimulation of islets isolated from the dbldb mouse, a single gene mutation animal model of N I D D M , caused a biphasic increase i n intracellular C a  2 +  concentration instead of the triphasic  pattern of intracellular C a  2 +  observed i n control islets. This lack of initial  reduction of intracellular C a  2 +  concentration after glucose administration was a  direct result of loss of endoplasmic reticulum C a - A T P a s e activity (Roe et al., 2+  1994). Although there is no study on the pattern of glucose-induced changes i n intracellular C a  2 +  concentrations i n the Zucker rat, it is possible that the obese  animals may exhibit a similar defect. A further potential site of altered function is at the level of the islet "glucosesensor".  This includes GLUT-2, the glucose transporter specific to liver and  pancreatic B-cells (Johnson et al., 1990a), and glucokinase, the first rate-limiting enzyme i n glycolysis of B-cells (Matschinsky, 1990). The K  m  of glucokinase for  glucose is 8 mmol/L which allows large changes in enzyme activity i n response to small increments i n glucose concentration over the physiological range (4-9 mmol/L) (Meglasson and Matschinsky, 1986). GLUT-2 also has a high K  m  for  glucose that ensures the uptake of glucose is proportional to the physiological extracellular glucose concentrations (Johnson et al., 1990a). Therefore, this glucosensor mechanism is essential for B-cell responses to physiological changes in circulating glucose levels. Newgard et al. (1990; 1994) have suggested that there is a physical coupling of GLUT-2 and glucokinase i n the B-cell, which is important for glucose sensing, and a defect i n these components could result i n altered insulin secretion. Unger and coworkers reported that GLUT-2 levels were reduced i n obese Zucker rats (Johnson et al., 1990 b; Orci et al., 1990; Unger, 1991). However, down-regulation of GLUT-2 was only observed i n the Zucker rat  with marked hyperglycemia and severe diabetes and islet GLUT-2 levels were normal i n a strain similar to that studied i n this thesis (Johnson et al., 1990 b; Orci et al., 1990). Thus, it is unlikely that changes i n GLUT-2 are responsible for the altered B-cell responses to GIP and GLP-1 (7-36) amide. Glucokinase is an alternative site for altered glucose sensing. Newgard (1994) demonstrated that i n islets overexpressing hexokinase 1, insulin output increased at basal glucose concentrations (3 mmol/L). This finding suggests that the enzyme could be involved i n the aberrant glucose threshold of the B-cells of obese Zucker rats, and Chan and coworkers (Chan, 1993a; Chan et al., 1993b) reported that B-cell glucokinase function was defective i n obese Zucker rats. The authors found that the K m of glucokinase for glucose was lower i n obese animals and glucokinase was not inhibited by mannoheptulose, a glucokinase inhibitor. Although studies by Liang et al. (1994) showed marginally increased glucokinase activity a reduction of glucokinase K m could contribute to insulin secretion at low glucose levels. Although these various sites of altered function could all contribute to the change i n glucose sesitivity, further study of the signal transduction pathways involved i n the actions of GIP and GLP-1 (7-36) amide and the glucose response pathway are needed to establish which are the most important. These insights into the molecular pathophysiology of B-cell failure may open the way to a greater understanding of the etiology of diabetes in humans.  155  Adrian, T.E., Ferri, G.L., Bacarese-Hamilton, A . J . , Fuessl, H.S., Polak, J . M . and Bloom, S.R. (1985) Human distribution and release of a putative new gut hormone, peptide Y Y . Gastroenterology 89: 1070-1077. Ahren, B., Hedner, P. and Lundquist, I. (1983) Interaction of gastric inhibitory polypeptide (GIP) and cholecystokinin (CCK-8) with basal and stimulated insulin secretion i n mice. Acta Endocrinol 102: 96-102. 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Arch Internat Physiol 31: 20-44.  A P P E N D I X I: Chemical Sources  Acetic acid  British Drug Houses (BHD), Toronto, Ont., Canada  Acetonitrile  BDH  Ammonium acetate  BDH  Aprotinin (Trasylol-^)  Miles Canada Inc. (Miles), Etobicoke, Ont.  Bovine serum albumin (RIA Grade)  Sigma Chemical Co. (Sigma), St. Louis, MO.; Miles  Bovine serum albumin  PentexR, Miles Canada Inc. (Pentex), Etobicoke, Ont.  Carboxymethyl cellulose CM-52  Whatman Chemical Separation Ltd. (Whatman), England  Chloramine T  Sigma  Dextran (Clinical,Industrial Grade)  Sigma  Dextran T70  Pharmacia A B (Pharmacia), Uppsala, Sweden  Diethanolamine  BDH  Diethyl ether  BDH  Ethylmercurithiosalicylic acid sodium salt (Merthiolate)  Eastman Kodak Company (Eastman), Rochester, N Y  1 2 5  I sodium  Du Pont N E N Cadana Inc. (Du Pont Nen), Mississauga, Ont.  Glucose  Fisher Scientific Company (Fisher), Fairlawn, N J .  194  Heparin  Fisher  Insulin  Novo Research Institute (Novo), Bagsvaerd, Danmark  KLH  Calbiochem-Behring Corp. (Calbiochem-behring), L a Jolla, C A  Potassium chloride  Fisher  QUSO (microfine silica, G-32)  Philadelphia Quartz  Sep-Pak C-18 cartridge  Waters Associates, Inc. (Waters), Milford, M A  Sodium barbital  BDH  Sodium chloride  Fisher  Sodium merthiolate  Eastman Kodak  Sodium metabisulphite  Fisher  Sodium pentobarbital  M T C Pharmaceuticals (MTC), Cambridge, Ont.  Somatostatin-14  Peninsula Laboratories, Inc. (Peninsula), Belmont, C A  Synthetic human GLP-1(7-36) amide  Peninsula  Synthetic human GLP-l(7-36) amide  Bachem Inc. (Bachem), (for raising the antibody) Torrance, C A  Trifluroacetic acid  Pierce Chemical Company (Pierce), Rockford, Illinois  uBondapak C-18 column  Waters  A P P E N D I X II: List of Abbreviations  ANOVA  Analysis of variance  BSA  Bovine serum albumin  CCK  Cholecystokinin  CEP  Charcoal extracted plasma  CGRP  Calcitonin gene-related peptide  EDTA  Disodium ethylenediaminetetraacetate  ELISA  Enzyme-linked immunosorbent assay  GI  Gastrointestinal  GIP  Gastric inhibitory polypeptide  spGIP  Synthetic porcine GIP  shGIP  Synthetic human GIP  npGIP  Natural porcine GIP  GLP-1  Glucagon-like peptide-1  GLP-2  Glucagon-like peptide-2  GLP-K7-36) amide  Glucagon-like peptide-l(7-36) amide  GRF  Growth hormone releasing factor  GRP  Gastrin-releasing peptide  GRPP  Glicentin-related pancreatic peptide  HPLC  High Performance liquid chromatograghy  1251  125  IP  3  Iodine  Inositol 1, 4, 5-triphosphate  IDDM  Type I insulin-dependent diabetes mellitus  IRI  Immunoreactive insulin  KLH  Keyhole limpet hemocyanin  196  MPF  Major pancreatic proglucagon fragment  NIDDM  Type II non-insulin-dependent diabetes mellitus  NPY  Neuropeptide Y  NSB  Non-specific binding  PCR  polymerase chain reaction  PHM  Peptide histidine methionine  PYY  Polypeptide tyrosine tyrosine  RIA  Radioimmunoassay  SEM  Standard error of the mean  SLI  Somatostatin-like immunoreactivity  SS-14  Somatostatin-14  SS-28  Somatostatin-28  tGLP-1  Trauncated glucagon-like peptide-1  VIP  Vasoactive intestinal polypeptide  A P P E N D I X III: Systeme Internationale (SI) Unit  Measurement  SI Unit  Common Unit  Conversion (Common to SI)  Glucose GLP-K7-36) amide Insulin Somatostatin Gastrin  mmol/L pmol/L pmol/L pmol/L pmol/L  mg/dl pg/ml LiU/ml pg/ml pg/ml  18 3.3 7 1.64 (SS-14) 2.1 (G-17)  

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