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Studies on the potential utilization of glucose-dependent insulinotropic polypeptide (GIP) in type 1… Piteau, Shalea Joanne 2004

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Studies on the Potential Utilization of Glucose-Dependent Insulinotropic Polypeptide (GIP) in Type 1 and Type 2 Diabetes by Shalea Joanne Piteau B.Sc. (Hons.), Queen's University, 2000 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Physiology We accept this thesis as conforming »to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 2004 © Shalea Joanne Piteau, 2004 THE UNIVERSITY OF BRITISH COLUMBIA FACULTY OF GRADUATE STUDIES Library Authorization 0 In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Name of Author (please print) Date (dd/mm/yyyy) Title of Thesis: S~WW-ec OA ~H\e Po~W\+>'gv. I U-flli^^Ho^ &^  Degree: /HflSfer Science^ Department of The University of British Columbia Vancouver, BC Canada Year: ZlOO^ grad.ubc.ca/forms/?formlD=THS page 1 of 1 last updated: 20-Jul-04 Abstract Glucose-dependent insulinotropic polypeptide (GIP) is a peptide hormone that is released from the small i ntestine in response to a meal and acts to potentiate glucose-induced insulin secretion from the pancreatic p-cell. Recently it has been shown that GIP stimulates P-cell growth, differentiation, and survival, and inhibits apoptosis, broadening the spectrum of anti-diabetic effects of GIP and the potential application of this peptide to type 1 diabetes. In the studies described in this Thesis, the GIP analog, [DAla 2]GIP, was demonstrated to exhibit both paradoxical diabetic and anti-diabetic effects in two animal models of experimentally-induced diabetes, the multiple low-dose streptozotocin diabetic rat and the single high dose streptozotocin diabetic rat, respectively. Increased expression of the P-cell glucose transporter GLUT2 was implicated as one of the potential underlying mechanisms in [DAla2]GIP-mediated exacerbation of multiple low-dose streptozotocin (MLD-STZ)-induced diabetes. Importantly, treatment with the native hormone, GIP(l-42), significantly improved the diabetic phenotype relative to MLD-STZ controls. Anti-apoptotic effects of [DAla ]GEP offered a basis for the protective effects of t his p eptide against si ngle d ose s treptozotocin (IV S TZ)-induced P -cell d estruction. The findings in these studies exemplify the importance of the pleiotropic effects of GIP receptor signaling and shed light on the potential utilization of GEP therapy in type 1 diabetes. It has long been known that the incretin effect is reduced in patients with type 2 diabetes and this defect has been partially attributed to a loss of the GIP component of the enteroinsular axis since glucagons-like peptide-1 (GLP-1) maintains insulinotropic activity i n t hese i ndividuals. H owever, t he m echanism r esponsible for t he d iminished response to GIP has not been fully elucidated. Previous studies have shown that GIP receptor mRNA levels are downregulated in the pancreatic P-cell of an animal model of type 2 diabetes and that this is correlated with a diminished responsiveness to GIP. It has also been demonstrated that elevated glucose levels are able to significantly reduce GIP receptor expression in vivo and in vitro. Accordingly, we hypothesized that normalization of hyperglycemia in vivo would reverse the down-regulation of GIP receptor expression and GIP insensitivity in type 2 diabetes. The causal role of hyperglycemia in loss of GIP receptor expression in the ZDF rat is strongly suggested by restoration after 2 weeks treatment with phlorizin, a drug that normalizes b lood glucose without increasing plasma insulin or changing plasma free fatty acid or triglyceride levels. Furthermore, restoration of GIP receptor expression was correlated with improved pancreatic GIP sensitivity. These findings suggest that GIP receptor down-regulation in type 2 diabetes is secondary to chronic hyperglycemia and that tight glycemic control leads to restoration of GIP receptor expression and subsequently, to improved GIP sensitivity at the pancreatic islet. In summary, demonstration of the anti-diabetic effects of GIP in type 1 diabetes and recovery of biological activity of GIP in type 2 diabetes offer great potential for the use of GIP analogs in the treatment of these diseases. Abstract i i Table of Contents iv List of Figures viii List of Abbreviations • • x Acknowledgements xi i i Chapter 1 - Introduction 1 1.1 GIP Physiology 1.1.1 The Enteroinsular axis and the Incretin Concept 1 1.1.1 Discovery of GIP 2 1.1.2 GIP Amino Acid Sequence and Gene Structure 3 1.1.3 Tissue Distribution and Secretion of GIP 4 1.1.4 The GIP Receptor 7 1.1.5 GIP Receptor Signaling 9 1.1.6 Biological Actions of GIP Stomach 10 Pancreas 11 Adiopose Tissue 13 Liver 14 1.1.7 Glucagon-like Peptide-1 14 1.1.8 GIP and GLP-1 Metabolism and Dipeptidyl Peptidase IV (DPIV) 16 1.1.9 GIP Analogs 16 1.2 Diabetes Mellitus 17 1.2.1 Pathogenesis of Type 1 Diabetes 18 1.2.2 Pathogenesis of Type 2 Diabetes 19 1.2.3 The Incretin Effect in Type 2 Diabetes 21 1.2.4 Regulation of GIP Receptor Expression 22 1.2.5 Animal models of Diabetes Mellitus Stretozotocin-Induced Diabetic Rat 24 Zucker Diabetic Fatty (ZDF) Rat 26 Thesis Investigation 28 Chapter 2 - Methods 30 2.1 Materials 30 2.2 Animals 30 2.3 Cell Culture 33 2.4 Cyclic A M P Measurements .33 2.5 Rat Pancreatic Islet Isolation 34 2.6 Western Blot Analysis of GLUT2 Protein 35 2.7 Caspase Assay 37 2.8 Glucose Tolerance Tests Oral Glucose Tolerance Test 37 Intraperitoneal Glucose Tolerance Test 38 Peptide Bioassay 39 2.9 Real-time Reverse Transcription-Polymerase Chain Reaction (Real-time RT-PCR) 39 2.10 Pancreas Perfusion 40 2.11 Pancreatic Insulin Content Determination 41 2.12 Data Analysis 41 Chapter 3 - Results 42 3.1 [DAla 2]GIP Treatment in Type 1 Diabetes 42 3.1.1 Bioactivity of [DAla 2]GIP and GIP(l-42) 42 3.1.2 The Multiple Low-Dose Streptozotocin-Induced Diabetic Rat. . .46 3.1.3 Peptide Dose-Response Investigation in the MLD-STZ Diabetic Rat '. 52 3.1.4 The Effects of Concomitant GIP stimulation of the p-cell on STZ Action 56 3.1.5 Regulation of Islet GLUT2 Expression by [DAla 2]GIP In Vivo ...61 3.1.6 The Effects of [DAla 2]GIP on Susceptibility to STZ-induced P-Cell Death 64 3.1.7 Inhibition of STZ-Induced p-Cell Death In Vitro 72 3.2 GIP Receptor Expression in Type 2 Diabetes 74 3.2.1 Rat Pancreatic Islet GIP Receptor Expression in Zucker Rats .. ..74 3.2.2 Glucose Regulation of GIP Receptor mRNA Expression In Vivo 77 3.2.3 GIP Responsiveness in Phlorizin-Treated ZDF Rats 80 Chapter 4 - Discussion 84 [DAla 2] GIP Therapy in Type 1 Diabetes 84 GIP Receptor mRNA Expression in Type 2 Diabetes 96 References 107 Fig. 1 Bioactivity of [DAla 2]GIP and GJP(1 -42) in vitro 44 Fig. 2 Bioassay of [DAla 2]GIP and GIP(l-42) in conscious Wistar rats 45 Fig. 3 Responses of Wistar rats to multiple low-doses of STZ in vivo 47 Fig. 4 Effect of treatment with [DAla 2]GIP or GIP(l-42) on morning fed blood glucose levels in the multiple low-dose STZ Wistar rat model 49 Fig. 5 Effect of treatment with [DAla 2]GIP or GIP(l-42) on glucose and insulin responses in OGTTs performed on day 5 in the multiple low-dose STZ Wistar rat model 50 Fig. 6 Effect of treatment with [DAla 2]GIP or GIP(l-42) on glucose and insulin responses in OGTTs performed on day 28 in the multiple low-dose STZ Wistar rat model 51 Fig. 7 Responses of Wistar rats exposed to multiple low doses of STZ to different doses ofGLP(l-42) 54 Fig. 8 Responses of Wistar rats exposed to multiple low doses of STZ to different doses of [DAla 2]GIP 55 Fig. 9 Responses of Wistar rats to treatment with a single high dose of STZ 57 Fig. 10 Effect of treatment with GIP(l-42) on fed blood glucose levels and glucose responses in OGTTs performed in Wistar rats exposed to a single high dose of STZ 59 Fig. 11 Effect of treatment with [DAla 2]GIP on fed blood glucose levels and glucose responses in OGTTs performed in Wistar rats exposed to a single high dose of STZ 60 Fig. 12 Effect of [DAla 2]GIP treatment on islet GLUT2 mRNA expression in vivo .62 Fig. 13 Effect of [DAla ]GIP treatment on islet GLUT2 protein expression in vivo ..63 Fig. 14 Effect of treatment with [DAla 2]GIP on fed blood glucose levels and glucose responses in OGTTs performed in Wistar rats exposed to a single dose (30mg/kg)ofSTZ 66 Fig. 15 Effect of treatment with [DAla 2]GIP on fed blood glucose levels and glucose responses in OGTTs performed in Wistar rats exposed to a single dose (45mg/kg)ofSTZ :....67 Fig. 16 Effect of treatment with [DAla 2]GIP on insulin responses in OGTTs and total pancreatic insulin content in Wistar rats exposed to a single dose (45mg/kg) ofSTZ 68 Fig. 17 Effect of treatment with [DAla2]GD? on fed blood glucose levels and glucose responses in OGTTs performed in Wistar rats exposed to a single dose (60mg/kg)ofSTZ 70 Fig. 18 Effect of treatment with [DAla ]GIP on insulin responses in OGTTs and total pancreatic insulin content in Wistar rats exposed to a single dose (60mg/kg)ofSTZ 71 Fig. 19 [DAla 2]GIP and GIP(l-42) stimulation of INS-1(832/13) cells protects against STZ-induced apoptotic cell death 73 Fig. 20 Responses to oral glucose tolerance tests performed on Zucker lean, Zucker obese, and Zucker Daibetic Fatty (ZDF) rats at 12 weeks of age 75 Fig. 21 GIP receptor mRNA levels in the islets of Zucker lean, Zucker obese, and Zucker Diabetic Fatty (ZDF) rats 76 Fig. 22 Fed morning blood glucose levels in lean control, Zucker Diabetic Fatty (ZDF) control, and Zucker Diabetic Fatty (ZDF) phlorizin-treated rats ..78 Fig. 23 GIP receptor mRNA levels in the islets of lean control, Zucker Diabetic Fatty (ZDF) control, and Zucker Diabetic Fatty (ZDF) phlorizin-treated rats ..79 Fig. 24 GIP responsiveness bioassays performed on ZDF control and ZDF phlorizin-treated rats 82 Fig. 25 Insulin secretion from perfused pancreata of control and phlorizin-treated ZDF rats 83 List of Abbreviations a alpha A A Arachidonic Acid Ala Alanine ADP Adenosine 5'-Diphosphate A M C 7-amino-4-methylcoumarin A N O V A Analysis of Variance Asp Aspartate ATP Adenosine 5' -Triphosphate A U C Area Under the Curve (3 beta B B Biobreeding B C A Bicinchoninic Acid bp base pairs B S A Bovine Serum Albumin C a 2 + Calcium cAMP cyclic Adenosine 3',5'-Monophosphate C C K Cholecystokinin cDNA Complementary Deoxyribonucleic Acid COS green monkey kidney cells cpm counts per minute (radioactivity) CREB cAMP Response Element Binding Protein Ct Cycle threshold C-terminal Carboxy-Terminal D M E M Delbucco's Modified Eagle Media D N A Deoxyribonucleic Acid DPIV Dipeptidyl Peptidase IV EC50 Effective Concentration where a 50% maximal response occurs E C L Enhanced Chemi-Luminescence E D T A Ethylenediaminetetraacetate E R K Extracellular Regulated Kinase F A Fatty Acid fa/fa homozygous recessive for non-functional leptin receptor FSH Follicle Stimulating Hormone G A P D H Glyceraldehyde-3-Phospate Dehydrogenase G H Growth Hormone GIIS Glucose-Induced Insulin Secretion GIP Glucose-Dependent Insulinotropic Polypeptide GIPR Glucose-Dependent Insulinotropic Polypeptide Receptor Gin Glutamine (amino acid) GLP-1 Glucagon-Like Peptide-1 GLP-2 Glucagon-Like Peptide-2 Glu Glutamate GPCR G-Protein-Coupled Receptor GRF Growth Hormone Releasing Factor HBSS Hank's Balanced Salt Solution HEPES Af-2-hydroxyethylpiperazine-AP-2-ethane sulfonic acid HIT-T15 Hamster P-cell line HPLC High Performance Liquid Chromatography I B M X 3 -Isobutyl-1 -Methylxanthine IGT Impaired Glucose Tolerance IP Intraperitoneal n>3 Inositol Triphosphate IPGTT Intraperitoneal Glucose Tolerance Test IR-GIP Immunoreactive Glucose-Dependent Insulinotropic Polypeptide IV Intravenous kDa Kilodaltons K R B H Krebs-Ringer Bicarbonate HEPES buffer M A L D - T O F Matrix-Assisted Laser-Desorption Ionization-Time of Flight M A P K Mitogen Activated Protein Kinase M L D Multiple Low-Dose mRNA Messenger Ribonucleic Acid N A D Nicotinamide Adenine Dinucleotide NGT Normal Glucose Tolerance NOD Non-Obese Diabetic N-terminal Amino-terminal ob/ob homozygous for the obese spontaneous mutation, (Lepob) OGTT Oral Glucose Tolerance Test P A C A P Pituitary Adenylate Cyclase Activating Polypeptide PARP Poly-(ADP-ribose) Polymerase PCR Polymerase Chain Reaction P K A Protein Kinase A P L A 2 Phospholipase A2 PPAR Peroxisome Proliferator Activated Receptor Pro Proline (amino acid) RIA Radioimmunoassay RTN 1046-38 Clonal rat insulinoma cell line R N A Ribonucleic Acid RT Reverse Transcription SC Subcutaneous SDS-PAGE Sodium Dodecylsulphate-polyacrylamide gel electrophoresis S E M Standard Error of Mean SGLT Sodium Glucose Cotransporter STZ Streptozotocin Taq T. Aquaticus D N A Polymerase TBST Tris-Buffered Saline with 0.5% Tween 20 T G Triglyceride Val Valine V D F Vancouver Diabetic Fatty VIP Vasoactive Intestinal Polypeptide Vmax maximal rate of transport into cell Z - D E V D - A M C benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin ZDF Zucker Diabetic Fatty I would like to first and foremost thank Chris and Ray for providing me with the opportunity to work with them and everyone else in the lab. It has been a learning experience that I will take with me for the rest of my life. Thank you for giving me the freedom to ask and pursue scientific questions and develop my own individual skills in the process. Your enthusiasm for science and knowledge were a daily inspiration to me. And to Pops, for your mentorship, thank you so much for the kindness and training you so freely gave to me. I learned a great deal from you and I really appreciate your support in my first year. Su-jin, I am forever grateful to you for your wisdom, insight, and friendship. Your curiousity and work ethic will forever inspire me, and I will never forget our late night talks! Thanks Jan, Simon, and Francis for paving the way for Kyle and I and providing us with excellent examples from which to learn. Specky, what can I say girl, it's been great! It certainly wouldn't have been quite as exciting without you, "you sure know how to squeeze 'em...". Kyle, thanks for your help and always offering insight into my project and Cuilan, thanks for always accommodating my requests. Sarah, thanks for making me laugh and Rhonda, for being such a strength to our program, it was great to travel this journey with you guys. To U l i , thank you for kindness and genorousity towards our lab, we wouldn't have half the data without you. You provided so much opportunity for us to pursue scientific endeavours, our sincerest thanks to you always! Thank you Brian R. for your ideas, friendly demeanor, and the opportunities you gave me to further explore my project, it was a joy to work with you. M y warmest thanks goes out to my housemates Monica and Erin, who always encouraged, challenged, and supported me. You guys are my second family and it has been a great journey these past two years. Thanks for being there! I would also like to thank my Athletes in Action (AIA) crew, you guys rocked my world this year! Thanks for cracking me up day in and day out, I don't think I have ever had so much fun. I also want to thank my Mom and my brother Tyron, for loving me, making me laugh at myself, and broadening my perspective on life. I loved our times on Sundays, wow they were as precious as gold, let me tell ya! Lael, Rus, and Brad, I love you guys so much, thanks for your continuous encouragement and just being you! I want to dedicate this project to my Dad, who has forever inspired me and given me someone to look up to and strive to become just like one day. You are forever in my heart and I thank you for the legacy you left us. Praise the Lord! \ Chapter 1 - Introduction 1.1 - GIP Physiology 1.1.1 - The Enteroinsular Axis and the Incretin Concept After their discovery of secretin in 1902, Bayliss and Starling proposed that signals arising from the gut after the ingestion of nutrients could influence pancreatic secretory responses that control carbohydrate disposal (1). Subsequently, in 1906, Moore et al postulated a role for gut secretions in the stimulation of internal secretions from the pancreas; however, their attempt at treating diabetes by injecting gut extracts was unsuccessful (2). It was not until the discovery of insulin in 1921 by Banting and Best, that interest in the intestinal control of "internal secretion" from the pancreas was renewed. La Barre and colleagues further pursued this gut factor and found that crude extracts of secretin could lower blood glucose levels in dogs (3,4). La Barre proposed that the secretin extract contained another factor, which they termed incretin for its ability to stimulate endocrine secretion from the pancreas (4). Despite considerable efforts, Loew, Gray, and Ivy were unable to demonstrate that an incretin isolated from the gut possessed a glucose lowering effect (5) and therefore, interest in isolating an intestinal hypoglycemic factor declined. It was not until the development of the radioimmunoassay in 1960 that the postulated effect of the gut-derived incretin on stimulating insulin-dependent reduction in hyperglycemia was established. Light was shed on the search for the unidentified incretin when it was demonstrated that the superiority of oral versus intravenous glucose tolerance was the result of a more profound insulin response (6,7). The contribution of the incretins was later estimated to account for more than 50% of nutrient-stimulated insulin secretion (8,9). In 1969, Unger and Eisentraut introduced the term "enteroinsular axis" to describe the hormonal connection between the gut and the pancreatic islets (10). Ten years later, Creutzfeldt expanded the definition to include both neural and substrate stimulants of endocrine secretion. Furthermore, Creutzfeldt defined the criteria for the hormones contributing to the enteroinsular axis: 1) they must be released by nutrients, particularly carbohydrates; and 2) at physiological levels, they must stimulate insulin secretion in the presence of elevated blood glucose levels (9). One hormone that clearly fits the requirements to be an incretin is glucose-dependent insulinotropic polypeptide (GIP). 1.1.2 - Discovery of GIP Glucose-dependent insulinotropic polypeptide (GIP) was originally isolated as an enterogastrone, which is a hormone released from the small intestinal mucosa by fat that inhibits gastric acid secretion (11). Brown and Pederson (12) isolated GIP from two different preparations of cholecystokinin-pancreozymin that were being assayed for their ability to stimulate gastric acid secretion in the vagally and sympathetically denervated canine Heidenhain pouch of the stomach. Upon discovery that the most pure preparation produced a greater s timulatory e ffect o n g astric a cid s ecretion, t hey c oncluded t hat a n inhibitor of gastric acid secretion had been removed during the purification procedure (12). GIP was then purified from extracts of the hog duodenojejunal mucosa and shown to be a potent inhibitor of gastric acid secretion' It was therefore originally named 'gastric inhibitory polypeptide in 1971 (13,14). Previous to these studies, Dupre and colleagues demonstrated that impure preparations of C C K also possessed insulinotropic activity (15) and that this activity could be removed by further preparation of the extracts (16). This observation paralleled that found by Brown and Pederson and led Dupre et al to hypothesize that GEP may also possess insulinotropic action. In 1973, Dupre and Brown demonstrated the insulinotropic activity of GIP in humans. They intravenously infused purified porcine GIP into humans with concomitant glucose and observed that GIP stimulated the secretion of greater quantities of insulin than the same dose of glucose alone (17). The GIP-stimulated insulin secretion was not demonstrated in the euglycemic state suggesting that this insulinotropic nature of GEP was glucose-dependent. Subsequent studies have further demonstrated the glucose-dependent nature of GEP-stimulated insulin secretion in dogs (18), humans (19), and the perfused rat pancreas (20). The glucose dependency of GEP-stimulated insulin secretion provides an important protection against hypoglycemia elicited by unfavorable insulin release during a low-carbohydrate m eal. This i mportant p hysiological function of GEP i nspired Brown and Pederson to rename this peptide glucose-dependent insulinotropic polypeptide (GEP) (21). 1.1.3 - GIP Amino Acid Sequence and Gene Structure The complete amino acid sequence of porcine GIP(l-42) was first described by Brown and Dryburgh (14), who reported GEP to be a 43 amino acid polypeptide. Later, the sequence was corrected after the removal of an erroneous glutamine residue at position 30, leaving GIP as a 42 amino acid polypeptide with an apparent molecular weight of approximately 5 kDa (22). Comparison of GEP sequences revealed that the peptide is highly conserved among mammalian species; the human sequence differs by only two amino acids from the porcine and rat sequences and by three amino acids from the mouse and bovine sequences. This high degree of sequence conservation across species suggests that GIP is an important regulatory hormone. Based upon gene structure homology, GIP has been classified as a member of the glucagon superfamily, which includes GLP-1, GLP-2, (GEP), secretin, pituitary adenylate cyclase activating p olypeptide (PACAP), growth hormone r eleasing f actor ( GRF), and vasoactive intestinal polypeptide (VEP), in order of degree of homology. Size differences in the GEP gene exist between species as well: the human GEP gene spans approximately 10 kb and the rat gene spans 8.2 kb (23,24). The intron-exon arrangement of the human and rat GEP genes are similar, but not identical, and both genes are derived by proteolytic processing of preprohormones consisting of 153 and 144 amino acids, respectively. Both human and rat preproGIP consist of six exons separated by five introns and the mature GEP polypeptide is encoded by exons 3 and 4 (23,24). 1.1.4 - Tissue Distribution and Secretion of GIP GEP expression is restricted to the upper small intestine (duodenum and jejunum) (25,26), the duct cells of the rat submandibular gland (27), and the stomach (28). Ultrastructural studies of GEP cells in the human intestine have shown that GEP is localized to K cells (26), which are "defined by the characteristic appearance of the intracellular secretory granules having a small electron-dense core surrounded by a concentric electron-lucent halo" (29). The development of a radioimmunoassay (RIA) for GIP (30) allowed for the investigation of the physiology and regulation of GEP secretion. However, the species differences in GEP have had consequences for the recognition of GEP by RIAs that employ antibodies with different affinities for GIP from different species. Unfortunately, antibodies have been raised against an epitope of GIP within amino acids 15-42, and this is the region in the peptide in which most species differences exist; therefore, inconsistent results have been reported when used for measuring interspecies immunoreactive-GIP (IR-GIP) levels (33). Reported values for fasting and postprandial GIP concentrations have varied widely between different laboratories (30-33,18), although the reported relative increases in GIP concentrations over basal in response to nutrients have been similar. Mean peak values of IR-GIP after ingestion of glucose have ranged from 400-1900 pg/ml, with an increment of 300-1500 pg/ml over basal levels. Despite the variability of results, general agreement exists that GIP concentrations increase 5-6 fold in response to a nutrient stimulus and remain elevated for 2-3 hours (29). In addition, GIP has recently been shown to be inactivated by the enzyme dipeptidyl peptidase I V (DPIV; Section 1.1.8); and the inactive metabolite, GIP(3-42) represents a significant proportion of circulating IR-GIP, thus further confounding interpretation of the results. For these reasons, the specific methodology utilized needs to be taken into account when interpreting in vivo GIP release experiments. GIP release can be stimulated by the carbohydrate, fat, and protein component of meals; therefore, it follows that GIP is secreted from the upper small intestine into the circulation after nutrient intake. The role of GIP as an incretin depends on luminal glucose as a stimulus of GIP release. Kieffer et al confirmed that glucose acts directly on GIP-secreting cells by demonstrating that isolated and cultured porcine GIP cells release GIP in a dose-dependent manner in response to glucose (34). GIP has been reported to increase in response to oral glucose, but not in response to intravenous glucose (35,36), suggesting a necessary role for luminal stimulation of K cells for GIP release. The absorption and metabolism of glucose, rather than its mere presence, is necessary for the release of GIP. Glucose and other monosaccharides (galactose) that are actively transported into and metabolized by the GIP cell (37), stimulate GIP secretion, whereas those monosaccharides that are not actively transported (mannose) nor metabolized (2-deoxy-glucose) do not stimulate GIP secretion (38). In addition, Sykes et al demonstrated that phlorizin, an inhibitor of sodium-dependent glucose transport, completely abolished glucose-stimulated GIP release in the perfused rat intestine (37). The most potent stimulus of GIP release is ingestion of triglycerides. The GIP response to fats is greater in magnitude and more prolonged than to glucose (20), which may be the result of the inhibitory effects of lipids on gastric acid emptying. In response to g lucose, G IP r elease reached p eak 1 evels a 14 5 m inutes a nd r eturned to b asal a 19 0 minutes in the dog. In response to triglycerides, GIP release reached peak levels (2x peak levels in response to glucose) at 60 minutes, and remained elevated after 2 hours in the dog (20). In addition, the chain length of fatty acids is related to the potency of GIP release. Long-chain triglycerides, which form chylomicrons, lead to long-lasting stimulation of GIP secretion while medium-chain triglycerides, do not form chylomicrons and do not release GIP (39). The differential stimulation of GIP release by different sized fatty acid chains is therefore thought to be associated with chylomicron formation. Interference w ith chylomicron f ormation, b ut n ot t hat of t riglyceride a bsorption i n t he gut, was reported to inhibit GIP release (38). Interestingly, fat-stimulated GIP secretion was not found to be insulinotropic in the absence of glucose, suggesting that GIP may also play a role in fat metabolism (17,18). In addition to glucose and fat stimulation, GEP release has also been shown with intraduodenal administration of protein and specific amino acids. Thomas et al reported that an amino acid mixture containing arginine, histidine, isoleucine, leucine, lysine, and threonine was able to markedly stimulate GEP release, while an amino acid mixture containing methionine, phenylalanine, tryptophan, and valine produced minimal increases in plasma GIP concentrations (40,41). In response to the intraduodenal perfusion of amino acids, GEP secretion occurred immediately (within 5 minutes), and reached peak concentrations at 30 minutes (40), indicating that amino acids elicit a more rapid GIP secretory profile than that elicited in response to glucose or fat. 1.1.5 - The GIP Receptor In 1993, Usdin et al first cloned the rat GEP receptor (GEPR) from a cerebral cortex cDNA library (42). Sequence comparisons between species indicated that the greatest sequence similarity was found with the rat glucagon (-44%) and GLP-1 (-40%) receptors, suggesting that the GEPR belongs to the secretin/VEP family of serpentine, seven transmembrane domain G-protein-coupled receptors (GPCRs). Following Usdin's initial work, there were a number of studies reporting the cloned GIPRs for human pancreatic islets (43), human insulinoma/lung cells (44), hamster insulinoma cells (45) and isolated rat islets (46). Interspecies amino acid sequence comparisons show a high degree of GEPR homology: the human GEPR is 79-81% homologous to the rat and hamster GEPR, and there is 86% identity between the rat and hamster GEPR. The genomic structure of the GEPR has been characterized for both rat and human (44,47). The intron-exon organizations o f the human and rat GEPR are identical, except for an additional exon found in the rat GIPR gene. The human gene is 13.8kb in size and contains 14 exons; splicing out the intronic sequence yields a 1389 bp open reading frame coding a 466 amino acid protein with a predicted molecular weight of ~50kDa (43,44). The rat gene is 10.2kb in size and contains 15 exons; splicing out the intronic sequence yields 1365 open reading frame coding for a 455 amino acid protein (42,46,47). GIPR mRNA has been reported to be expressed in the pancreatic P- and cc-cells, intestine, adipose tissue, lung endothelium, heart, blood vessel endothelium, pituitary, inner layers of the adrenal cortex and in several brain regions, including the cerebral cortex, hippocampus, and olfactory bulb, but it was not found in the kidney, spleen or liver (42,48). In some tissues where GIP receptor expression was detected, GIP has known biological effects; however, it has been difficult to determine which of these effects are direct, since GIP may affect the release and action of other substances. For example, although detection of GIPR expression in the stomach has confirmed that GEP does act directly on the stomach, further analysis is needed to determine which cells express the receptor for the clarification of GIP's specific role in gastric acid secretion. Additionally, while many of these tissues exhibit characterized responses to GIP, for others there are still no known effects of GIP. For example, GIP is not known to act in the brain, except for one report that GIP injected intraventricularly affected FSH and G H secretion in ovariectomized rats (49). Interestingly, mRNA for GIP could not be detected in the brain, suggesting the possibility of an alternative ligand that acts on the GIPR in the brain. In addition, expression of the GIPR in the adrenal cortex implicates GIP in glucocorticoid metabolism and GIP has been shown to play a role in food-induced Cushing's syndrome (50), yet GIP's role has yet to be fully elucidated. GIP has also been suggested to play a role in vasculature relaxation (51) and the detection of GEPR mRNA expression in the endothelium of blood vessels corroborates this idea. Recently, GEPR expression was detected in primary human osteoblasts and in bone-derived cell lines and it was shown that GIP stimulation enhanced collagen synthesis and alkaline phosphatase activity, both of which reflect anabolic actions of osteoblasts (52). The authors proposed that GEP could serve to coordinate nutrient intake in the intestine with nutrient disposal in a variety of peripheral tissues including bone. The physiological significance of these findings remains to be clarified but they have opened up new directions to study the multifaceted roles of GEP. 1.1.6 - GIP Receptor Signaling GEP transduces its effects through specific G-protein coupled receptors on the P-cell, resulting in stimulation of adenylate cyclase and elevation of intracellular cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) levels (43,45,46,53). GEP-stimulated cAMP production facilitates glucose-dependent insulin release from pancreatic P-cells physiologically; however, cAMP production has been shown to be glucose-independent in pTC3 cells and INS-1(832/13) cells, suggesting that GEP's glucose-dependent effects on insulin release must be triggered downstream in the transduction cascade (54-56). In HIT-T15 and COS-7 cells, GEPR stimulation has been demonstrated to increase intracellular calcium via mobilization through intracellular calcium stores and influx through both L-type voltage dependent and voltage independent calcium channels (46,57). The pathway by which GIP increases intracellular calcium concentrations has not been fully elucidated. However, GEP has not been shown to have an effect on the formation of EP3, suggesting that the receptor unlikely to be coupled to phospholipase C (45). Ehses et al have postulated that GEP could stimulate calcium release from intracellular stores via activation of phospholipase A2 and consequent production of arachidonic acid (55). In addition, GEP has been implicated in pleiotropic stimulation of P K A / c A M P response element binding protein (CREB), p44/p42 mitogen-activated protein kinase (MAPK), and PI3 kinase (PI3K)/protein kinase B (PKB) signaling pathways (56,58,59). 1.1.7 - Biological Actions of GIP Stomach GEP was initially isolated for its ability to inhibit gastric acid secretion in dogs (12-14). However, the physiological relevance of GEP as an enterogastrone, a hormone released from the small intestinal mucosa by fat that stimulates gastric acid secretion, has been questioned. Pederson et al demonstrated that GEP induced a dose-dependent inhibition of pentagastrin-stimulated acid secretion in the vagally and sympathetically denervated pouch of the dog stomach (60). However, they also demonstrated that GEP only weakly inhibited gastric acid secretion in response to histamine or vagal stimulation of the innervated gastric remnant (60). In addition, weak inhibition of gastric acid secretion in humans has been shown to occur only using supraphysiological or pharmacological doses of GEP (36,61,62). Clarification of these inconsistent results came when studies reported both sympathetic and parasympathetic control of the enterogastrone properties of GEP (63,64). Evidence at the time indicating that somatostatin was capable of inhibiting gastric acid secretion (65) led to revision of the postulated role of GIP on the stomach to include mediation by somatostatin. Indeed, Mcintosh et al demonstrated that GIP stimulated the release of somatostatin from D-cells in the perfused rat stomach (66) and more recently, a somatostatin antagonist was shown to block GIP's enterogastrone activity when administered intravenously to rats (67). Currently, i t i s a ccepted t hat G IP' s i nhibitory effects o n g astric acids ecretion m ay b e mediated by somatostatin and modulated by nervous inhibitory mechanisms; however, no consensus exists as to the physiological status of GIP as an enterogastrone. In fact, recent data from Meier et al do not support a role for GIP in the regulation of gastric emptying in humans (68), further confounding the interpretation of GIP as an enterogastrone. Pancreas GIP is considered to be a major incretin that acts via the enteroinsular axis to augment glucose-induced insulin secretion from the pancreatic p-cell. The first study to indicate that GIP stimulated insulin secretion in P-cells in a glucose-dependent fashion in man w as r eported b y D upre e t a 1 i n 1 973 (17). F ollowing t his i nitial w ork, G IP w as shown to be insulinotropic in dogs (18), rats (20), isolated islets (69,70), and p-cell lines (71,72). In addition to its role as an insulinotropic hormone, GIP acts to stimulate proinsulin gene transcription and translation (73,74) and gene expression of glucose transporters and hexokinase in the P-cell (75). The glucose-dependence of GIP's insulin releasing action (17) was demonstrated to occur at a threshold of 5.5mM glucose, with maximum potentiation occurring at 16.7mM glucose in the perfused rat pancreas (20). Application of the glucose-clamp technique, with a concurrent oral glucose load (36) or GIP infusion (19) demonstrated unequivocally that GIP is a physiological incretin in humans. Further studies using immunoneutralization techniques (76,77), receptor antagonists (78,79) or receptor knockout mice (80,81) have corroborated this claim by reporting that GEP is responsible for 50-70% of the postprandial insulin response. As previously mentioned, GEP facilitates insulin release from P-cells via activation of its cognate receptor, inducing activation of adenylate cyclase leading to increases in intracellular cAMP and calcium concentrations. In addition, the incretins share a number of non-insulin mediated effects that contribute to effective glucose homeostasis. Recently, GEP has been shown to enhance P-cell growth, differentiation, proliferation, and cell survival (58,82,83), and to inhibit P-cell apoptosis (59,83) via pleiotropic activation of P K A / C R E B , M A P K , and PI3K/PKB pathways. GEP's actions in the islet are not restricted to the P-cell. GEP has been shown to stimulate the release of glucagon, somatostatin, and pancreatic polypeptide from the a, 8, and PP cells, respectively from the perfused rat and dog pancreas (84,85), albeit using supraphysiological concentrations in the rat pancreas. The effect of GIP on glucagon secretion has been shown to only occur in conditions of euglycemia in the rat, below a threshold glucose concentration of 5.5mM (85) but to occur in both basal and postprandial conditions in the dog (84). Although in previous studies it was found that GEP had no effect on serum glucagon levels in humans in conditions of euglycemia or hyperglycemia (19,86), in a recent study it was demonstrated that GEP dose-dependently stimulates glucagon secretion in healthy human subjects at euglycemia (87). Consequently, the role of GEP on glucagon secretion remains somewhat controversial. The differential effect of GEP on glucagon secretion may be species-dependent, dose-dependent, indirectly attributable to the paracrine actions of the intra-islet release of insulin and somatostatin, or dependent on the metabolic status of the individual. Clearly, more studies are needed to conclusively establish whether GIP is a physiological regulator of glucagon secretion. Adipose Tissue As stated earlier, GIPR mRNA is expressed in adipose tissue and fat represents the most potent stimulus for GIP secretion from the K-cells in the small intestine, the effect of which is not insulinotropic in the absence of glucose (17,18). These findings suggest a direct role for GIP in fat metabolism. Several pieces of evidence support this hypothesis. GIP may play a role in triglyceride uptake as postprandial plasma triglyceride levels are reduced in humans who have higher plasma GIP levels (88). In fact, G IP h as b een s hown b oth t o i ncrease t he r emoval o f p lasma t riglycerides and t o reduce plasma triglyceride increments in dogs and rats, respectively (89,90). In cultured 3T3-L1 preadipocytes (91) and in explants of rat adipose tissue (92), GIP was found to increase lipoprotein lipase activity, which may be the mechanism responsible for GIP's ability to increase triglyceride clearance. GIP may also play a role on lipid metabolism within adipocytes. GIP has been reported to stimulate fatty acid synthesis i n adipose tissue (93) and potentiate insulin-stimulated incorporation of fatty acids into triglycerides in rat epididymal fat pads (94). As well, GIP increased insulin-stimulated glucose transport and insulin receptor affinity in rat adipocytes (95,96) and several investigators have demonstrated that GIP inhibited glucagon-stimulated c A M P production and 1 ipolysis (95,97). R ecently, Miyawaki and colleagues demonstrated that GIPR-/- mice were resistant to weight gain when placed on a high-fat diet, in contrast to control mice (98). The same authors also showed that when mice with severe morbid obesity, ob/ob mice, were crossed with GEPR-/- mice, the severity of obesity in homozygous offspring was reduced by 23% (98). Taken together, these findings suggest that GEP plays an important role in fat deposition. On the contrary, Mcintosh et al demonstrated that GEP stimulated lipolysis in differentiated 3T3-L1 cells and that this effect could be inhibited by insulin via a wortmannin-sensitive pathway (99). The authors postulated that GEP may act to increase free fatty acid levels for optimization of i nsulin s ecretion b y t he P-cell. A lthough G IP h as a ntidiabetic p roperties and i s an important hormone for the regulation of glucose homeostasis, these findings suggest that inhibition of GEP signaling may present a new avenue for anti-obesity treatment. Liver Although, the GEPR does not appear to be expressed in the liver, GEP has been reported to augment insulin-stimulated suppression of hepatic glucose production and to inhibit glucagon-stimulated glycogenolysis in dogs (100) and humans (101). The mechanisms by which GEP is acting are unclear; however, GEP does not appear to exert a direct stimulatory effect on its target cells but rather a modulatory effect on the actions of insulin and glucagon. 1.1.8 - Glucagon-like Peptide-1 Before the discovery of GLP-1, the existence of a second incretin hormone, in addition to GEP, was suspected because the removal of GIP activity by immunoadsorption in rats (76,77) and small bowel resection in man (102) did not completely block the increased insulin response to oral glucose relative to intravenous glucose. The cloning of cDNAs encoding proglucagons from pancreata of anglerfish allowed the detection of a peptide with a strong homology to the sequence of GIP (103), which was subsequently identified to be a second incretin, GLP-1 (104). GLP-1, a product of the pro glucagon gene, is secreted from the L cells in the intestine in response to carbohydrates, fats, proteins, and endocrine and neural signals. GLP-1 transduces its effects via a highly specific receptor that belongs to the secretin/VTP family of serpentine, seven transmembrane domain G-protein-coupled receptors (GPCRs) (105). Stimulation of the GLP-1 receptor leads to activation of signaling pathways common to GIP, including activation of adenylate cyclase and increase in cAMP, activation of the extracellular regulated kinase (ERK)l/2 cascade, and increase in intracellular calcium via intracellular stores and the opening of voltage-dependent calcium channels (105). The main physiological action of GLP-1 is believed to be augmentation of glucose-induced insulin secretion from the p -cell (106-108). In addition, GLP-1 has been shown to increase insulin production in the P-cell by upregulating insulin gene expression and promoting insulin biosynthesis (106,108). GLP-1 has also been described to have trophic effects on pancreatic P-cells, such as to promote P-cell glucose competence (109), enhance p-cell growth, differentiation, proliferation and cell survival (110-115), and inhibit P-cell apoptosis (116-118). There is also evidence for a number of e xtrapancreatic e ffects o f G LP-1. T he p eptide h as b een s hown t o i nhibit g lucagon secretion (119,120) and gastric emptying (121,122), contributing to its role i n glucose homeostasis. In addition, GLP-1 appears to be an anorexigenic hormone with the ability to promote satiety and suppress food intake by acting on the hypothalamus; however, the physiological significance of these actions is still controversial (105). 1.1.9 - GIP and GLP-1 Metabolism and Dipeptidyl Peptidase IV (DPIV) The enzyme dipeptidyl peptidase IV (DPIV) is ubiquitously distributed as a plasma membrane exopeptidase, in addition to its presence as a soluble plasma protein (123). DPIV preferentially cleaves and inactivates a variety of circulating regulatory peptides containing proline and alanine at the penultimate position from the N-terminus (123). Upon release into the circulation, GEP and GLP-1 are rapidly cleaved by DPIV, resulting in a circulating half life of ~l-2 minutes for the biologically active peptides (124-126). The N-terminally truncated products GEP(3-42) and GLP-l(9-36), lack insulinotropic activity (127) and this cleavage is thought to be the primary mechanism for incretin inactivation in vivo (125). In addition, at supraphysiological concentrations, the truncated peptides have been shown to function as receptor antagonists in vitro and in vivo (128-130). The development of specific DPEV-resistant analogs to exploit this physiological regulatory system through the enhancement of the antidiabetogenic effects of GEP and GLP-1 is serving as a promising therapeutic approach to diabetes. 1.1.10 - GIP Analogs Although both GEP and GLP-1 possess significant insulinotropic activity, some controversy exists as to their relative effectiveness in stimulating glucose-induced insulin release. Several reports have shown that both GEP and GLP-1 are equipotent in stimulating insulin secretion (131-133) whereas other reports have suggested that GLP-1 possesses greater insulinotropic activity (134,135). As a consequence, most research has focused on utilizing GLP-1 as a potential therapeutic intervention for diabetes and there have been limited reports investigating the therapeutic potential of long acting GEP analogs in diabetes. However, a number of significant features indicate that DPIV-resistant analogs of GIP possess unique potential for diabetes therapy. Firstly, evidence from studies comparing the insulinotropic activity of each incretin (136) and studies using receptor antagonists (79,137) or GIP-antiserum (76,77,138) underscore the importance of GIP as a physiological incretin. Secondly, the important finding that sulfonylureas improve P-cell responsiveness to GIP (139) and recent evidence from our laboratory and others on the ability of GIP analogs to overcome severe insulin resistance and P-cell dysfunction in a type 2 diabetic animal model (140,141) affords proof of concept that GIP analogs offer potential as future therapeutic agents for diabetes. Thirdly, GIP only acts to increase glucagon secretion at normal glucose concentrations and therefore, this effect of GIP therapy is irrelevant in treating diabetes (80,84). Fourthly, in contrast to GLP-1, GIP is well tolerated by human subjects due to its lack of inhibitory action on gastric emptying (142,143,65). The therapeutic potential of GIP and its analogs for type 1 diabetes has largely been unstudied; however, due to the unique features that GIP and its analogs possess, it is of interest to investigate their potential as a therapeutic modality in the treatment of type 1 diabetes. 1.2- Diabetes Mellitus Pancreatic p-cells have the remarkable ability to maintain glucose levels within a very n arrow h omeostatic r ange f or t he 1 ifetime o f m ost i ndividuals, b ut f ailure o f t his capacity is fundamental to the pathogenesis of diabetes. This P-cell inadequacy results from an inability to maintain sufficient P-cell mass and function to cope with the increased insulin demand. The two main forms of diabetes mellitus that will be the focus of this Thesis are type 1 and type 2 diabetes. 1.2.1 - Pathogenesis of Type 1 Diabetes Type 1 diabetes is an autoimmune disease resulting from specific destruction of the pancreatic P-cells (143). It has two distinct phases: insulitis (prediabetes), when a mixed population of leukocytes invades the islets; and diabetes, when most P-cells have been killed off, and insulin deficiency ensues, resulting in hyperglycemia (144). The initiation of pathogenesis precedes overt clinical onset by several years, suggesting a long prediabetic period (145,146). The initiating factor(s) that trigger type 1 diabetes is unknown, however it is hypothesized that physiological destruction of P-cells, via apoptosis, is a critical event at disease outset, initiating autoimmunity. O'Brien et al demonstrated that p-cell apoptosis is responsible for the development of type 1 diabetes in the nonobese diabetic (NOD) mouse (147) and the multiple low-dose streptozotocin model (MLD-STZ) (148), and that its onset precedes lymphocytic infiltration of the islets. Furthermore, Mensah-Brown et al demonstrated that down-regulation of apoptosis in pancreatic P-cells prevents multiple low-dose streptozotocin-induced autoimmune diabetes (149). Apoptosis is a mode of cell death that occurs under normal physiological conditions and it is most often found during normal cell turnover and tissue homeostasis, embryogenesis, and induction and maintenance of immune tolerance (150). A neonatal wave of P-cell apoptosis exists in normal developing rats and autoimmune animal models of diabetes, peaking at 12-14 days of age (151). Infiltration of immune cells into the islets of Langerhans is coincident with the neonatal peak of P-cell apoptosis in NOD mice, suggesting that the rate of apoptosis may be important (151). Using a mathematical model of p-cell dynamics (152), Trudeau et al demonstrated that NOD mice exhibit a 1.5-3 fold greater number of apoptotic cells compared with B A L B c mice during the neonatal period (151). Recent evidence indicates that apoptotic cells can induce immune responses (153-155). Taken together, these data suggest that an increased wave of apoptosis is a trigger for p-cell directed autoimmunity. Accordingly, it is important to determine whether apoptosis can be modulated during the prediabetic period as a means to inhibit the P-cell-directed autoimmune attack of type 1 diabetes. 1.2.2 - Pathogenesis of Type 2 Diabetes Type 2 diabetes is characterized by two hallmark features: insulin resistance and a compromised function of the pancreatic p-cell such that insulin secretion is insufficient to match the degree of insulin resistance (156). It is believed that genetic factors combine with acquired defects in insulin secretion and action to manifest the phenotypic picture of type 2 diabetes (157). Genetically susceptible individuals risk developing type 2 diabetes when environmental factors, such as obesity and lack of exercise, influence the balance between insulin secretory capacity and tissue insulin sensitivity (157). The most striking acquired P-cell defect is loss of acute glucose-induced insulin secretion (GIIS). Interestingly, Kosaka et al demonstrated that diet, sulfonylurea treatment, or insulin administration improved insulin secretion in response to meals or oral glucose after hyperglycemia, s uggesting t hat a cquired p -cell d efects r esult from a m etabolic c hange induced by diabetes (158). This finding has led to the hypothesis that chronic hyperglycemia is the major metabolic factor eliciting p-cell dysfunction in type 2 diabetes, also known as the glucose toxicity hypothesis. Studies using the 90% pancreatectomized rat (159), chronic in vivo glucose infusions in normal rats (160), and the incomplete (60%) pancreatectomized rat fed sucrose in their water supply (161) clearly demonstrate t hat t he p resence o f h yperglycemia i s a p rerequisite f or t he P -cell glucose unresponsiveness in the animal models. It is important to note, however, that chronically e levated fatty a cid levels have a lso b een implicated in the pathogenesis of type 2 diabetes. Several studies have demonstrated that long-term exposure of pancreatic P-cells to fatty acids increases basal insulin release but inhibits glucose-induced insulin secretion (162-167); however, the emphasis of studies described in this Thesis is on the role of hyperglycemia on P-cell dysfunction. Recent studies have suggested the molecular basis for the P-cell glucose unresponsiveness seen in type 2 diabetes. Weir and colleagues have shown that chronic hyperglycemia triggers loss of pancreatic P-cell differentiation, with down-regulation of genes that optimize P-cell function and up-regulation of genes normally suppressed in the P-cell (168,169). The P-cell dedifferentiation and loss of GILS induced by hyperglycemia were reversed with phlorizin, in the absence of changes in plasma free fatty acid levels, emphasizing the specific role of hyperglycemia in p-cell dysfunction. Similarly, this concept is further supported by studies revealing that phlorizin treatment or an insulin infusion restored glucose-induced insulin secretion in 90% pancreatectomized rats (170,171) and in glucose-infused normal rats (172). 1.2.3 - The Incretin Effect in Type 2 Diabetes It has long been known that the incretin effect, the increased insulin response resulting from oral glucose in comparison with that elicited by an isoglycemic intravenous infusion (6), is reduced in patients with type 2 diabetes (173-175), however the cause of this abnormality is not known. One explanation for the defect in postprandial insulin release in type 2 diabetes is ineffective GIP action at the P-cell as both patients and animal models of type 2 diabetes have displayed a loss of responsiveness to GIP (86,132,176-180). Several mechanisms have been proposed to explain the GIP resistance in diabetes. Firstly, diabetic P-cells either may have altered expression of GIP receptors; including lack of expression, reduced expression, or expression of a defective protein. There are studies showing point mutations in the GIP receptor gene in human populations that affect GIP signaling in cell models; however, it has not been possible to provide a conclusive relationship between these mutations and type 2 diabetes (181,182). Secondly, it is possible that chronic homologous desensitization of the GIP receptor is the determinant of the lack of responsiveness to GIP in type 2 diabetes (54,183). Hinke et al has shown that GIPRs are quickly internalized in response to GIP, with a significant reduction in cell surface receptors occurring after only 10 minutes of exposure to GIP (54). However, there is no conclusive evidence to support this hypothesis as there is a lack of consensus regarding the circulating levels of GIP in type 2 diabetes; studies have demonstrated increased, decreased, and unchanged GIP levels (184-190). The confusion is due to the use of inappropriate RIAs that both measured C-terminal peptide, rather than the biologically active molecule, and that employ antibodies with different affinities for GIP. Alternatively, it is possible that receptor desensitization may govern GEPR activity in the short term rather than act as a causative factor for the chronic loss of GEP action at the 1 evel o f t he P -cell. T hirdly, t he r educed G IP-induced i nsulin s ecretion i n p atients with type 2 diabetes may be due to a generalized p-cell defect in type 2 diabetes or may be a consequence of defects in GEP's intracellular signaling pathways (191-194). However, these explanations are unlikely because GEP and GLP-1 share considerable overlap in their intracellular signaling mechanisms, except for transmission via highly specific receptors for each hormone, and GLP-1 retains potent insulinotropic activity in people with diabetes (86,176-178). Therefore, it is reasonable to speculate that the P-cell defect is at the level of the GEP receptor. Recent studies have demonstrated that there is a decrease in GEP receptor mRNA expression in the islets of the Vancouver diabetic fatty Zucker (VDF) rat, which results in a decrease in GEP-stimulated insulin release from the P-cells despite normal sensitivity to GLP-1 (195). This finding provides evidence that a loss in GEP receptor expression may account for the decreased incretin effect observed in many type 2 diabetic subjects. 1.2.4 - Regulation of GIP Receptor Expression The degree of GEPR down-regulation in the V D F rat seems to be correlated with the level of overnutrition in these animals. If there was a genetic mutation in the promoter or GEPR gene in the V D F rat, which directly altered GEPR expression, it would be likely that GEPR expression would be down-regulated throughout the lives of these animals. H owever, p rediabetic V DF r ats (4 w eeks o f a ge) a nd d iabetic V DF r ats (12 weeks of age) display a 30% and 75% reduction in GEPR expression respectively, which is predictive of a metabolic regulation of GIPR expression (unpublished results). In addition, the defective response to GIP in the P-cell was seen in five groups of diabetic patients, with completely different etiologies and phenotypes (196). This suggests that it is probably not a primary defect causing diabetes, but rather a defect that is secondary to the metabolic disturbances in diabetes. Until recently, there have been no data to suggest the mechanisms by which expression of the GIPR is down-regulated in type 2 diabetes. Supporting a role for glucose in the pathogenesis of GIPR expression in type 2 diabetes, Lynn et al demonstrated that high glucose levels induce a dramatic down-regulation of the GIP receptor in vivo and in vitro (197). The peroxisome-proliferator activated receptors (PPARs) are a family of nuclear transcription factors that are activated in vivo by fatty acids (198). P P A R a is expressed in the pancreatic P-cell and it has been demonstrated to tightly control expression of genes involved in fatty acid oxidation (198,199). Roduit et al found that high glucose stimulates down-regulation of P P A R a in INS(832/13) cells and pancreatectomized rats (200). Interestingly, Lynn et al also demonstrated that at normal glucose levels GIPR expression is stimulated through fat stimulated P P A R a activation, which is unable to reverse the GIP receptor down-regulation seen at higher glucose levels (197). The time course for down-regulation of P P A R a in conditions of high glucose reported by Roduit et al was similar to that reported for GIPR down-regulation. In keeping with this concept, hyperglycemia, not hyperlipidemia, is associated with increased islet TG content in the ZDF rat (201) and high glucose concentrations have been shown to stimulate lipid esterification and T G deposition in INS-1 cells (202). These studies lend support to the theory that hyperglycemia causes down-regulation of PPARoc, and therefore, negatively influences the expression of enzymes involved in F A oxidation and of the GEP receptor in the islet. Accordingly, GEP receptor levels may be decreased in type 2 diabetic subjects due to the prolonged elevated blood glucose levels. It is clearly important to examine whether normalizing glucose levels in vivo will reverse the down-regulation of GEP receptor expression in type 2 diabetes with the possible goal of altering disease therapy to optimize GEP's antidiabetic effects in this disease. 1.2.5 - Animal models of Diabetes Mellitus Stretozotocin-Induced Diabetic Rat Streptozotocin [2-deoxy-2-(3-methyl-3-nitrosourea)l-d-glucopyranose)] is an antibiotic that was first isolated from Streptomyces achromogenes in 1959, and its diabetogenic action results from its selective cytotoxic effect on the p-cell (203). STZ is composed of a cytotoxic moiety, methylnitrosourea, attached to carbon-2 of glucose and p-cell-specific toxicity is connected to the capacity for STZ to accumulate in these cells (203,204). In support of this concept, D-glucose and its analogs, 3-0-methylglucose and 5-thio-D-glucose, confer protection against STZ action in vitro and in vivo (205,206) and analogs of STZ whose sugar moieties are replaced by mannose, galactose, or oc-0-methylglucose are nondiabetogenic (207). Intracellular action of STZ in the P-cell appears to involve three mechanisms: D N A methylation, free radical generation, and nitric oxide formation. Once inside the cell, S TZ d ecomposes t o form a h ighly r eactive c arbonium i on, w hich a lkylates D N A (208). As well, STZ metabolism liberates nitric oxide and generates reactive oxygen species, which both contribute to STZ-induced D N A damage (209,210). In support of this c oncept, s tudies h ave s hown t hat S TZ-induced D N A cleavage i n r at i slets c an b e partially counteracted by inhibition of the inducible form of nitric oxide synthase (209), protection against STZ-induced diabetes in rats can be conferred by superoxide dismutase (211), and STZ stimulated H2O2 generation, which probably acts as a mediator of D N A fragmentation in vitro and in vivo (212). These STZ-induced D N A lesions are then removed by excision repair, which produces D N A strand breaks, leading to the overactivation of poly-(ADP-ribose) polymerase (PARP) to form poly(ADP-ribose) using nicotinamide adenine dinucleotide (NAD) as a substrate. This results in the depletion of cellular N A D levels and thus to nonphysiological concentrations of ATP and apoptotic P-cell death (208,210). The concept that activation of PARP is fundamental to the diabetogenicity of STZ has been confirmed in studies revealing that PARP inhibitors protect against (213), and PARP-deficient mice are completely resistant to, STZ-induced cytotoxicity (214,215). STZ a dministration m ay b e g iven a s a s ingle h igh d ose o r m ultiple 1 ow d oses, either intravenously or intraperitoneally, to induce type 1 diabetes. As well, the dose of STZ administered can be modulated for the induction of a diabetic state of graded severity (216). After the intravenous administration of a single high-dose of STZ, there is a characteristic triphasic response in blood glucose levels. Transient hyperglycemia exists by the first two hours, followed by a second phase of hypoglycemia at approximately 6 hours, which is primarily due to degranulation of the p -cells arid the sudden release of insulin. The third phase represents a phase of permanent hyperglycemia that begins at about 12 hours and plateaus at 24 hours to remain relatively stable thereafter (217). At this stage, very few viable p-cells exist and plasma insulin and total pancreatic insulin levels fall to -50% and - 5 % of the initial value, respectively (216). The multiple low-dose streptozotocin rat model was developed because it mimics, in some basic respects, type 1 diabetes in human patients. Injection of multiple equal low doses of STZ induces a slow progressive hyperglycemia, accompanied by lymphocytic infiltration of the pancreatic islets in mice and rats (218-220). These findings led to the concept that multiple subdiabetogenic doses o f STZ p artially damage i slets, triggering insulitis that causes further loss of p-cells and an insulin-dependent state of diabetes. Zucker Diabetic Fatty (ZDF) Rat Zucker fatty rats are homozygous recessive for a mutation in the extracellular domain of the leptin receptor gene, fa, which results in a Gln269Pro amino acid substitution and decreased expression of the leptin receptor on the cell surface, decreased leptin binding, and diminished signal transduction (221). Rats carrying one normal Fa allele are phenotypically lean and display normal glucose tolerance (222). The Zucker fatty (fa/fa) rat is a widely used animal model of human obesity that has many features in common with type 2 diabetes including hyperinsulinemia, insulin resistance, glucose intolerance, and hyperlipidemia, islet hyperplasia, but not fasting hyperglycemia. They also exhibit a left shift in glucose concentration response curves, such that increased sensitivity to low glucose results in fasting hyperinsulinemia (222). The Zucker diabetic fatty (ZDF) rats arose from the inbreeding of a substrain of Zucker obese rats that exhibited fasting hyperglycemia, and therefore display all the metabolic abnormalities of the Zucker obese model in addition to fasting hyperglycemia, making them a useful model of type 2 diabetes. A l l male ZDF rats develop overt type 2 diabetes between 7 and 10 weeks of age with average glucose levels in excess of 22mM (222). To determine whether down-regulation of GIPR mRNA expression is secondary to the chronically elevated glucose levels seen in type 2 diabetes, it is necessary to lower the plasma glucose concentration without altering circulating substrate levels (other than glucose) and without administering any agent that has a direct effect on cellular metabolism. Phlorizin is one agent that fulfills these requirements. Phlorizin is a potent inhibitor of renal tubular glucose transport and blocks proximal tubular glucose reabsorption when plasma glucose concentration is increased above the basal level. Renal reabsorption of glucose is mediated by Na+-glucose cotransporters (SGLTs) (223), three of which have been cloned and reported to be expressed in renal epithelial cells (SGLT1,2,3) (224-226). As such, phlorizin treatment leads to normalization of plasma glucose without causing hypoglycemia or altering plasma insulin, amino acid, free-fatty acid, or other substrate/hormone concentrations (170). Thesis Investigation The number of patients that are suffering from both type 1 and type 2 diabetes mellitus is rapidly increasing. The major goal in treatment of diabetic patients is to optimize blood glucose control in order to reduce the incidence and severity of diabetic complications; however, most patients do not achieve glycemic goals. Thus, the aims of the studies described in this Thesis were two-fold. The first major goal of this project was to shed light on the potential utilization of the hormone, glucose-dependent insulinotropic polypeptide (GIP), in type 1 diabetes. The therapeutic potential of GIP in this disease has largely been unstudied. Recent studies showing that GIP stimulates 0-cell growth, differentiation, and survival, and inhibits apoptosis, suggest the potential for preventative and/or therapeutic application of this peptide in type 1 diabetes. Secondly, the lack of GIP responsiveness in type 2 diabetic patients has limited investigation into the efficacy of GIP as a clinically useful anti-diabetic therapeutic. However, recent studies showing that GIP receptor mRNA is down-regulated in the pancreatic P-cell of an animal model of type 2 diabetes, and that elevated glucose levels are able to significantly reduce GIPR expression in vivo and in vitro, have provided a molecular basis for the reduced sensitivity of GIP in this disease. Accordingly, the second major goal of this project was to determine whether the loss of GIP responsiveness at the level of the P-cell is an acquired defect secondary to the chronic hyperglycemia in an animal model of type 2 diabetes by investigating whether this defect is reversible with tight metabolic control. These studies provide new information on the action of GIP analogs in animal models of type 1 diabetes and indicate that such analogs could have therapeutic potential in patients prior to the development of chronic hyperglycemia in type 2 diabetics and following restoration of GIP responsiveness by reductions in hyperglycemia. Chapter 2 - Methods 2.1 Materials A l l chemicals, which were of reagent or molecular biology grade, were from the following Canadian distributors: Sigma-Aldrich (Oakville, ON), Fisher Scientific (Napean, ON), or Lnvitrogen (Burlington, ON). A l l tissue culture disposables were from B D Falcon (San Jose, C A , USA). Specific sources for chemicals are indicated in brackets in the following methodology sections. Synthetic human GEP(l-42) and [DAla2]GEP(l-30a) were a generous gift from Dr. Hans-Ulrich Demuth (Probiodrug, Halle, G ermany). T hese p eptides w ere s ynthesized b y D r. S usanne M anhart u sing an automated peptide synthesizer (Rainin Symphony) according to published protocols (227). Peptides were purified by HPLC and subjected to mass spectrometry (MALDI-TOF) and analytical HPLC to confirm identity and purity. GLP(l-42) and [DAla2]GEP were reconstituted in 0.1M CH3OH and 0.1M NH4OH, respectively. Peptides were aliquoted into siliconized tubes and freeze dried in vacuo using a Speed-Vac. Aliquots were then covered in parafilm and stored at -20°C until use. Anti-rat GLUT2 antibody and rat GLUT2 cDNA were a generous gift from Dr. Bernard Thorens (Institute of Pharmacology and Toxicology, University of Lausanne, Switzerland). Rabbit anti-rat GLUT2 was raised against a synthetic carboxy-terminal peptide consisting of amino acids 513-522 conjugated to keyhole limpet hemocyanin (228). 2.2 Animals Several different strains of rat were used in the studies outlined in this report. Male Wistar rats were obtained from the University of British Columbia Animal Care Facility (Vancouver, Canada); and male Zucker Diabetic Fatty (ZDF), male Zucker obese, and male Zucker lean rats were obtained from Charles River (Ontario, Canada). Animals were housed in conditions that included a 12 hour light/dark cycle (lights on at 6:00am) and free access to standard rat chow (Purina 5012) and water. Animal procedures conformed to the guidelines declared by the University of British Columbia Committee on Animal Care and the Canadian Council on Animal Care. Male Wistar rats (275-325g) were used in experiments employing streptozotocin to induce diabetes. In the multiple low-dose streptozotocin studies, the animals were pretreated with either GIP(l-42) (8nmol/kg), [DAla 2]GIP (8nmol/kg), or 0.9% saline (as a control for peptide treatment) by intraperitoneal injection twice daily (0900 and 1700) for 1 week prior to the administration of MLD-STZ, and thereafter until the animals were sacrificed. STZ was dissolved in a lOmM citrate buffer (pH 4.0) immediately prior to injection. On days 0-4, all groups, save the sham (as a control for STZ administration) and [DAla 2]GIP control groups, were administered intraperitoneal injections of STZ (25mg/kg i.p.), and the sham group was administered citrate buffer alone. On days 5 and 28 (day 0 represents the first injection of STZ) an oral glucose tolerance test (OGTT; lg/kg) was performed after a 16 hour fast to analyze glucose tolerance and blood glucose and plasma insulin responses were determined. In the single high dose streptozotocin study, the animals were pretreated with either GIP(l-42) (lOnmol/kg), [DAla 2]GIP (8nmol/kg), or 0.9% saline (as a control) by intraperitoneal injection twice daily (0900 and 1700) for 1 week prior to the administration of an intraperitoneal injection of STZ (75mg/kg i.p.). On day 5 (day 0 represents the first injection of STZ) an oral glucose tolerance test (OGTT; lg/kg) was performed on the treated and control groups after a 16 hour fast and blood glucose and plasma insulin responses were determined. In the intravenous streptozotocin study, male Wistar rats (~250g) were pretreated with [DAla ]GIP (8nmol/kg) or 0.9% saline (as a control) by intraperitoneal injection twice daily (0900 and 1700) for 1 week prior to the administration of an intravenous (tail vein) injection of STZ (45mg/kg i.v.) while under halothane anesthesia, and thereafter until the animals were sacrificed. On day 2 (day 0 represents the first injection of STZ) an oral glucose tolerance test (OGTT; lg/kg) was performed on the treated and control groups after a 16 hour fast and blood glucose and plasma insulin responses were determined. Pancreatic islet GEP receptor mRNA expression was measured in three different strains of male Zucker rats (12-14 weeks): Zucker lean, Zucker obese, and Zucker diabetic fatty (ZDF) rats. For the phlorizin studies, male Z D F rats were administered 0.4g/kg phlorizin (Sigma) (in 60% propylene glycol) by intraperitoneal injection every 12 hours (0830 and 2030) and the control animals received concurrent doses of 60% propylene glycol for 2 weeks (1.0ml syringe; 23 gauge needles). Aliquots of phlorizin were made up fresh daily by redissolving phlorizin at a concentration of 0.2g/ml in 60% propylene glycol and incubating in a 60°C water bath until completely dissolved; the drug was stored at room temperature until use. Phlorizin is a potent inhibitor of glucose reabsorption by the renal tubular cells; and therefore, selectively lowers blood glucose without affecting plasma free fatty acid and lipids levels (170). As such, we monitored regulation of GEP receptor mRNA expression by glucose in vivo. Following treatment, animals were anesthetized and islets isolated as described in Section 2.5, and total R N A was extracted for the quantification of pancreatic islet GIP receptor mRNA levels as described in Section 2.9. 2.3 Cell Culture Two cell types were used in the experiments outlined in this Thesis: PTC-3 cells, a clonal mouse p-cell line derived from transgenic mice expressing a hybrid insulin/oncogene (229); and INS-1(832/13) cells, an x-ray-induced rat insulinoma cell line obtained as a generous gift from Dr. CB Newgard (Duke University, Durham, NC). PTC-3 cells (passages 8-15) were cultured in low glucose (5.5mM) D M E M (pH 7.4) with 2mM glutamine and 110 mg/L pyruvate, 100 U/ml penicillin G, 100 ng/ml streptomycin, 12.5% horse serum, and 2.5% fetal calf serum (Cansera, Rexdale, ON). INS-1(832/13) cells (passages 5 0-65) were cultured i n R P M I 1640 supplemented with 1 0% fetal calf serum, l l m M glucose, 100 U/ml penicillin, 100 ng/ml streptomycin, 10 m M HEPES (pH 7.4), 2mM glutamine, 1 m M pyruvate, and 50 |aM P-mercaptoethanol. Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C. Cells were fed every three days, until 80-90% confluence was attained, and then harvested using 0.25% trypsin/0.3% EDTA solution (w/v) made up in Ca 2 + - and Mg2 +-free Hank's balanced salt solution. 2.4 Cyclic AMP Measurements To compare the effectiveness of GIP(l-42) and [DAla 2] GIP in vitro, cAMP studies were performed in a P-cell model (PTC-3 cells). Cells were cultured in standard growth media and seeded at 5 x 105 cells per well into 24-well plates 48 hours prior to experimentation. Before all experiments, PTC-3 cells were cultured for 18h in l.OmM glucose D M E M with serum and antibiotics. PTC-3 cells used in these experiments were of passages 8-15. Cells were washed twice with 37°C Kreb's-Ringer bicarbonate HEPES (KRBH) buffer (0 or l l m M glucose) and allowed to preincubate for 1 h at 37°C. A 30-min stimulation of cells followed in the same buffer (0 or l l m M glucose), additionally supplemented with 0.5 mmol/1 isobutylmethyl xanthine (Research Biochemicals International, Natick, M A ) and 1% Trasylol (aprotinin; Bayer, Etobicoke, Ont., Canada), at the peptide concentrations indicated in the figures. Cells were then lysed in ice-cold ethanol (70%), cellular debris was removed by centrifugation, and cell contents were dried in vacuo (Speed Vac; Savant, Farmingdale, NY) . Cyclic A M P levels were determined using a radioimmunoassay kit (Biomedical Technologies, Stoughton, M A ) and expressed in terms of % maximal peptide-stimulated cAMP production. The cAMP stimulation protocol is based upon previous research techniques (230). 2.5 Rat Pancreatic Islet Isolation Rat pancreatic islets were isolated as described by Van der Vliet et al, 1988 (231). Briefly, rats were anaesthetized (sodium pentobarbital 65 mg/kg), the common bile duct was cannulated, and the pancreas was distended with collagenase (32mg/100ml, Type XI) in Hank's Balanced Salt Solution supplemented with 10 raM HEPES, 2mM L-glutamine and 0.2% bovine serum albumin (BSA; Fraction V , RIA grade) (supplemented HBSS). The pancreas was then excised and cut into 2mm 2 pieces and digested in a total volume of 25 ml collagenase (in supplemented HBSS) in a shaking 37°C water bath for 10 minutes. The islets were then firmly shaken and i f the solution appeared cloudy, they were washed using supplemented HBSS. A second digestion was carried out for 1-5 minutes, depending on the degree that the pancreas was digested (very cloudy, minimal pancreatic clumps signified sufficient digestion). Following digestion, the islets were washed and then filtered through a 1 mm nylon mesh and purified from exocrine tissue via dextrose gradient centrifugation (l,500g/4°C) for 20 minutes. To prepare the gradient, tissue was resuspended in 10ml of 27% dextran solution (using supplemented HBSS without BSA), then 6ml of 27% dextran solution was gently layered below and 10ml of a 23% solution was layered above the tissue, followed by a final layer of 10ml of 14% dextran solution above. Islets were retrieved from the 23%-14% dextrose interface, the 27%-14% dextrose interface, or the pellet and then rewashed and hand-picked under a dissecting microscope for further experimentation. It is important to note that islets isolated from the pellet were generally contaminated with exocrine tissue and therefore, the preparation had to be further diluted in supplemented HBSS in order to purify the preparation before hand-picking the islets. 2.6 Western Blot Analysis of GLUT2 Protein Regulation of GLUT2 protein by [DAla ]GIP was measured in vivo. Male Wistar rats were treated with [DAla 2]GIP (8nmol/kg) twice daily (0900 and 1700) by intraperitoneal i njection for 1 week t o d et ermine t he e ffect o f [ DAla ]GIP o n G LUT2 protein expression. Following treatment, islets were isolated, lysed in ice-cold RIPA buffer (150mM NaCl, 20mM Tris-Cl pH 7.5, 1 m M EDTA, 1% Nonidet P-40, 1% deoxycholate, 0.1% SDS, 5mM NaF, ImM phenylmethylsulfonyl fluoride, 1 m M DTT, 10 fig/ml leupeptin, 10 ug/ml pepstatin A , 10 ug/ml bestatin and 1% Trasylol (Bayer Pharmaceuticals, Etobicoke, ON) (100 ul/100 islets), subjected to 3 freeze-thaw cycles and centrifuged (12,000rpm for 30 min). To examine GLUT2 protein expression, Western blot analysis was performed. Briefly, protein content in the samples was quantified using the B C A reagent (Pierce) to ensure equal loading of gels for subsequent Western blotting. Protein samples (50ug protein/well) were denatured under reducing conditions (100 m M dithiothreitol (DTT)) at room temperature for 15 minutes, separated on a 13% sodium dodecyl sulfate (SDS)/polyacrylamide gel, and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, M ississauga, O ntario, C anada). M embranes were t hen b locked w ith 5 % skim milk (in tris-buffered saline with 0.5% Tween 20 (TBST)) and then incubated for 2 hours with an anti-rat GLUT2 antibody (dilution 1:1,000). Membranes were then washed 6 times (lOmin/wash) in TBST and incubated for 1 hour with a horseradish peroxidase-coupled donkey anti-rabbit immunoglobulin antibody (Jaskon Immmunoresearch Laboratories, West Grove, PA, USA) (dilution 1:2,000). Following a further 5 washings, the immunoreactive bands were visualized using enhanced chemi-luminescence (ECL) (Amersham-Pharmacia). For quantification of band density, films were analyzed using densitometric software (Image J; National Institutes of Health). 2.7 Caspase Assay Caspase-3 is a key effector in the apoptosis pathway, amplifying the signal from initiator caspases (such as caspase-8) and signifying full commitment to cellular disassembly; therefore, measurement of caspase 3 activity was used as an indicator of apoptosis. INS-1 cells were seeded into 6 well plates (2 x 106 cells/well) in l l m M glucose RPMI one day prior to experimentation. After establishing metabolic quiescence in the absence of serum overnight (3.3mM glucose RPMI with 0.1% BSA), the cells were stimulated with either [DAla 2] GIP or GIP(l-42) 10 minutes prior to STZ exposure (2mM; 30 minutes). After subjection to STZ treatment, the medium was replaced and the cells allowed t o r ecover for 2 4 h ours. C aspase 3 a ctivity w as d etermined a ccording t o t he manufacturer's protocol [standard: 7-amino-4-methylcoumarin (AMC), substrate: Z-D E V D - A M C ; Molecular Probes]. Caspase activity/well was assessed using a microplate fluorescence reader (Bio-tek FL600, excitation/emission at 360/460nM) and corrected for total protein content using the B C A protein assay (Pierce, Roxford, IL). 2.8 Glucose Tolerance Tests Oral Glucose Tolerance Test. Oral Glucose Tolerance Tests (OGTTs) were performed after a 16-18 hour fast and peptides were not administered the morning of the OGTT to ensure complete peptide washout (16h from last peptide administration; 1.5h clearance half life). D-glucose (lg/kg; Abbott Laboratories, QU) was administered by oral gavage at t=0 and then blood glucose was measured and blood samples were collected into heparinized tubes via the tail vein 5 minutes prior to (basal) and 10, 20, 30, 60, and 120 minutes following glucose ingestion. Blood samples were centrifuged at 14000g and p lasma w as s eparated and s tored at- 20°C. Blood glucose was m easured using a SureStep Glucose analyzer (Lifescan Canada, Burnaby, B C , Canada) plasma insulin levels were measured using a rat-specific sensitive insulin kit (no dilutions; detection range of 0.02-1.0 ng/ml; 100% cross-reactivity with rat insulin I&II, and human, porcine, sheep, hamster, and mouse insulin; Linco Research Inc.) or an in-house radioimmunoassay (RIA; detection range of 0.125-4.0 ng/ml). Using the inrhouse RIA, samples were diluted (ZDF rats 1:3, STZ rats no dilution) in insulin RIA buffer (0.5% charcoal extracted human donor plasma, in 40mM phosphate buffer, pH 7.5) and incubated with insulin antisera (GP01; 1:1,000,000 final dilution) on day 1. Rat insulin (Linco Research Inc.) was used as a standard. After 24 hours (day 2), 2000 cpm of chloramine-T iodinated porcine insulin (Sigma) was added to all tubes. After another 24 hour incubation (day 3), antibody bound and free radioactive insulin were separated by centrifugation with dextran-coated charcoal (232) and plasma insulin concentrations were quantified. Intraperitoneal Glucose Tolerance Test. To measure glucose tolerance without the influence of the enteroinsular axis, Intraperitoneal Glucose Tolerance Tests (EPGTTs) were performed. The protocol is similar to that mentioned for OGTTs, except that D-glucose was administered by intraperitoneal injection (1.0ml syringe, 23 gauge needle; 40% glucose; injection volume according to body mass). Peptide Bioassay. Bioassays were performed according to the same protocol mentioned for OGTTs, except with the following modification: animals were given an oral glucose bolus (lg/kg) with a concurrent subcutaneous peptide (8 or 10 nmol/kg) or 0.9% saline (as a control) injection at t=0 (0.5ml insulin syringe; lOpM peptide solution, injection volume according to body mass). 2.9 Real-time Reverse Transcription-Polymerase Chain Reaction (Real-time RT-PCR) Real-time RT-PCR was employed to measure GEPR and GLUT2 mRNA expression in vivo. Total R N A was isolated from rat islets using Trizol® according to the protocol supplied by Invitrogen. Briefly, Trizol reagent (1 ml/100 islets) was added to isolated islets and total R N A was quantified using the fluorescent Ribogreen reagent (Molecular Probes, Eugene, OR, USA). Following R N A isolation and quantification, 1 ug of R N A was subjected to one-step real-time (or quantitative) RT-PCR. Both cDNA synthesis and PCR were performed in a single tube using gene specific primers, total RNA, and all components necessary for RT-PCR. The GEP receptor RT-PCR reaction mix consisted of 2X Thermoscript™ reaction mix, 200nM rat GEPR 5' forward primer (5 '-CCG CGC TTT T C G T C A TCC - 3'), 200nM GEPR 3' reverse primer (5 '-CCA C C A A A T GGC TTT G A C TT-3'), 200nM GEPR probe colabeled with the fluorescent dyes F A M and T A M R A (5'-CCC A G C A C T G C G TGT TCT CGT A C A G G - 3 ' ) , 40 U/ul RNaseOUT, 5 m M MgCl2, Thermoscript™ Plus/Platinum® Taq enzyme mix, and RNase-free H 2 0 . The G L U T 2 RT-PCR reaction mix consisted of 2X Thermoscript™ reaction mix, 200nM rat GLUT2 5' forward primer (5'-GGG T C A T C A G A G A C T GTG T G A G G - 3'), 200nM GLUT2 3' reverse primer (5'-GGG A G C A C C T G G TTC CCT T-3'), 200nM GLUT2 probe colabeled with the fluorescent dyes TET and T A M R A (5'-T G A GCT GCC T A A A A T C C A G G A A C A G A C CA-3') , 40 U/ul RNaseOUT, 5 m M MgCh, Thermoscript™ Plus/Platinum® Taq enzyme mix, and RNase-free H 2 0 . RT-PCR was carried out in duplicate in the PE Applied Biosystems 7700 sequence detection system. The cycling program included a cDNA synthesis step at 50°C for 30 minutes, and 45 two-step PCR cycles, which included a denaturation step at 94°C for 15 seconds followed by an annealing/extension step at 60°C for 1 minute. Fluorescence was measured real-time and was used to calculate a cycle threshold (Ct), the point at which the reaction is in the exponential phase and is detectable by the hardware. A GIPR mRNA and a GLUT2 cDNA standard curve was used to quantify initial R N A copy number. Rather than use G A P D H as an internal control, we determined that it was more accurate to normalize values to total R N A using the Ribogreen reagent. 2.10 Pancreas Perfusion ZDF rats were anaesthetized (sodium pentobarbital 65 mg/kg) and pancreata were isolated as previously described (20). Briefly, the pancreas was isolated, all minor vessels ligated, and the abdominal aorta was perfused at a rate of 4 ml/min with a perfusate that consisted of a modified Krebs-Ringer bicarbonate buffer, 3% dextran, 0.2% B S A gassed with 5% CO2 balance O2 to achieve pH 7.4. Following a 10 minute equilibration period, the perfusion continued with 4.4 m M glucose for 4 minutes followed by 8.8 m M glucose for the remainder of the experiment. GIP (20pmol/l) was introduced into the perfusion system from 20-40 minutes. Samples were collected at 1 minute intervals from the portal vein and then stored at -20°C until analysis. 2.11 Pancreatic Insulin Content Determination Rats were anaesthetized (sodium pentobarbital 65 mg/kg) and the pancreas was excised, blotted dry, weighed, and then homogenized in 5 ml of ice cold 2N acetic acid. Homogenates were filtered through a 1 mm nylon mesh and then 1 ml aliquots were boiled in a 100°C water bath (10 minutes) and centrifuged (15,000 rpm, 4°C; 10 minutes). Supernatants were then collected and stored at -20C until analysis. Samples were normalized for protein concentration (BCA) and insulin concentration was determined using an in house RIA (STZ rat; 1:1,000 final dilution). 2.12 Data Analysis Data are expressed as mean ± standard error of the mean (S.E.M.), with the sample size indicated in the figure legends. For animal studies, an n-1 is one animal, and a minimum of 3-6 animals were used in each group. For in vitro experiments, a minimum of three independent experiments were performed. Student's unpaired t-test or one-way analysis of variance (one-way A N O V A ) were performed where appropriate, followed by the post-hoc tests, Tukey test, and Dunnet's t-test. Statistical significance was determined using Prism data analysis software (GraphPad, San Diego, CA); P<0.05 was considered significant. Area under the curve was calculated using the algorithm provided in the Prism software package. Chapter 3 - Results 3.1 - [DAla2]GIP Treatment in Type 1 Diabetes The incretins play important physiological roles in the maintenance of blood glucose homeostasis and incretin analogs are currently being tested for the treatment of type 2 diabetes. In addition to their insulinotropic actions, both GIP and GLP-1 have been shown to stimulate P-cell proliferation and neogenesis, and inhibit apoptosis. It was shown previously that the GIP analog, [DAla 2]GIP, exhibits increased biological activity in vivo compared to native GIP, owing to a more prolonged half-life a s a result of its resistance to DPIV degradation (140). The aim of the current study was to determine whether treatment with the GIP analog, [DAla 2]GIP, prior to and during the time of diabetes induction would decrease apoptosis and thereby protect against P-cell destruction and subsequent diabetes. 3.1.1 - Bioactivity of [DAla2]GIP and GIP(l-42) Comparative assays were performed to analyze the bioactivity of [DAla ]GIP and GIP(l-42) in vitro and in vivo. To compare the effectiveness of the peptides in vitro, cAMP production studies were performed in a P-cell line (PTC-3 cells). Biological activity of GIP(l-42) and [DAla 2]GIP were not significantly different (Fig. 1). [DAla 2]GIP showed equivalent cAMP stimulating potency as GIP(l-42) on pTC-3 cells (EC50 values, OmM glucose: [DAla 2]GIP: 13.34 ± 0.95 nmol/1, GIP(l-42): 16.55 ± 1.25 nmol/1; E C 5 0 values, l l m M glucose: [DAla 2]GIP: 13.65 ± 0.88 nmol/1, GIP(l-42): 16.78 ± 1.7 nmol/1; Fig. 1). To compare the effectiveness of GEP( 1-42) and [DAla2]GEP in vivo, a bioassay was performed on conscious unrestrained male Wistar rats (-300 g). The animals were fasted overnight and then an oral glucose tolerance test (OGTT) was performed with concurrent intraperitoneal saline or peptide (8 nmol/kg) injection. Blood glucose levels were measured at indicated times in samples obtained from the tail vein. Consistent with previous findings (140), [DAla2]GEP demonstrated markedly enhanced biological activity in vivo relative to GEP(l-42), which can be attributed to its DPIV resistance. Intraperitoneal injection of 8nmol/kg [DAla2]GEP resulted in a more pronounced reduction in the glycemic profile than native GEP during the oral glucose tolerance test (10 min OGTT blood glucose value: [DAla2]GEP: 6.6 ± 0.2 mmol/1, GEP(l-42): 8.1 ± 0.3 mmol/1, saline control: 8.6 ± 0.6 mmol/1; Fig. 2). Figure 2 (inset) shows that the integrated glucose profiles (over 30 minutes) in response to administration of [DAla2]GEP are significantly reduced compared to those in response to GEP(l-42) or saline administration. A .1 1 2 5 ' o •o 100-o CL Q. B 75-< o 50-re re E T f f • GIP(1-42) a [DAIa2]GIP -12 -11 -10 -9 -8 -7 Log [Peptide] -6 o 125-1 -a 100H o Q. Q_ 7 5 H < o 50-re I 25H re ^ 0 • GIP(1-42) a [DAIa2]GIP -13 -12 -11 -10 -9 -8 Log [Peptide] -7 -6 Figure 1: Bioactivity of [DAla2]GIP and GIP(l-42) in vitro. A: cAMP production in PTC-3 cells by GIP(1-42) (squares) and [DAla2]GIP (triangles) in OmM glucose conditions. E C 5 0 values: GIP(l-42), 16.55 ± 1.25 nmol/1; [DAla2]GIP, 13.34 ± 0.95 nmol/1. Each data point represents the mean ± S.E.M. of three independent experiments. B: cAMP production in PTC-3 cells by GIP(l-42) (squares) and [DAla2]GIP (triangles) in l lmM glucose conditions. E C 5 0 values: GIP(l-42), 16.78 ± 1.7 nmol/1; [DAla2]GIP, 13.65 ± 0.88 nmol/1. Each data point represents the mean ± S.E.M. of 3 independent experiments. 41 i 1 1 I I I I 0 10 20 30 40 50 60 70 Time (min) Figure 2: B ioassay o f [ DAla2]GIP and G IP( 1 -42) i n c onscious W istar r ats. I mmediately following o ral glucose administration (lg/kg), [DAla2]GIP (8nmol/kg), GIP(l-42) (8nmol/kg), or 0.9% saline (control) were administered via intraperitoneal injection at time 0. Whole blood glycemia was measured from tail vein samples in [DAla2]GIP-treated (upward triangles), GIP(l-42)-treated (downward triangles), and control (squares) animals. Each data point represents the mean ± S.E.M. of 4-8 animals. The inset shows the integrated plasma glucose responses for the OGTT. Statistical significance: * = PO.05, **= PO.01 and *** = PO.001 for the [DAla2]GIP-treated group versus saline control; # = PO.05 for the GIP(l-42)-treated group versus saline control and A = P<0.05 for the [DAla2]GIP-treated group versus the GIP(l-42)-treated group. 3.1.2 - The Multiple Low-Dose Streptozotocin-Induced Diabetic Rat The multiple low-dose streptozotocin (MLD-STZ) model is considered to mimic many of the characteristics of type 1 diabetes in human patients. P-cell apoptosis is responsible for the development of diabetes in this model, and we therefore chose it to investigate the therapeutic potential of [DAla ]GEP in type 1 diabetes. In order to ensure a stable diabetic phenotype was achieved, a dose-response investigation of the development of MLD-STZ-induced diabetes was performed. Male Wistar rats (325-375g) were administered 5 sub-diabetogenic doses of streptozotocin (20mg/kg, 25mg/kg, or 30mg/kg) via intraperitoneal injection and then monitored thereafter for diabetes induction (Fig. 3). The animals administered 30mg/kg of STZ began to show signs of diabetic complications (ie: weight loss) and therefore, were sacrificed early to avoid undue suffering. A minimum effective dose of 25mg/kg STZ was established based on glucose tolerance and fed morning blood glucose values and was used for all subsequent experiments. A • 20mg/kg STZ • 25mg/kg STZ • 30mg/kg STZ 10 20 Time (days) B 25-i 20-5-% 15-O 10* S o o m • 20mg/kg STZ • 25mg/kg STZ • 30mg/kg STZ 25 50 75 100 Time (min) 125 —i 150 Figure 3: Responses of Wistar rats to multiple low-doses of STZ in vivo. Multiple (5) sub-diabetogenic doses of STZ were administered to animals on day 0: 20mg/kg (squares), 25mg/kg (upward triangles), and 30mg/kg (downward triangles). Morning fed blood glucose levels were measured (A) and an oral glucose tolerance test (lg/kg; OGTT) performed on day 5 (B). Each data point represents the average value of 2 animals. To determine i f treatment with native GIP (GIP(l-42)) or the GIP analog, [DAla 2]GIP, could protect against streptozotocin (STZ)-induced p-cell death, Wistar rats were pretreated twice daily with either peptide (8nmol/kg) for 1 week prior to and 4 weeks after administration of multiple low-doses of STZ (MLD-STZ; 25mg/kg). Sham animals were administered lOmM citrate buffer (pH 4.0) alone to serve as controls for STZ administration. Fed morning blood glucose values and oral glucose tolerance were measured to monitor the progression of the disease state in the control animals and to detect any improvements in the treated groups. Surprisingly, treatment with [DAla 2] GIP potentiated STZ-induced diabetes. [DAla2]GIP-treated animals displayed increased fed b lood glucose r elative toeontrols; morning blood glucose levels for the three week time period post STZ exposure averaged 25.4 ± 1.8 mmol/1 in [DAla2]GIP-treated and 19.8 ± 0.8 mmol/1 in control animals (Fig. 4). In addition, [DAla2]GIP-treated animals displayed marked exacerbation of blood glucose and insulin responses during oral glucose challenges at days 5 and 28 (Day 5: peak OGTT blood glucose values 20.7 ± 0.8 vs 13.4 ± 1.3 mmol/1, respectively; Fig. 5; insulin values 0.69 ± 0.1 vs 1.68 ± 0.2 ng/ml, respectively; Fig. 5) (Day 28: peak OGTT blood glucose values 23.7 ± 0.9 vs 14.7 ±1 .5 mmol/1, respectively; Fig. 6; peak insulin values 0.39 ± 0.1 vs 0.76 ± 0.04 ng/ml, respectively; Fig. 6). For comparative purpose, animals were also treated with GIP(l-42) to determine whether native GIP protected against MLD-STZ-induced diabetes. In contrast to the above findings, GIP(l-42)-treated animals showed significantly improved fed blood glucose levels relative to controls, which progressively improved over the course of treatment (Day 25: 6.9 ± 0.8 vs 22.5 ± 2.3 mmol/1, respectively; Fig. 4). Although, the GIP(l-42)-treated animals did not display significantly improved glucose tolerance on day 5 relative to control animals (30min OGTT blood glucose values 12.7 ± 0.4 vs 12.9 ± 0.2 mmol/1, respectively; Fig. 5), glucose and insulin responses during the oral glucose challenge significantly improved by day 28 (60min OGTT blood glucose values 8.8 ± 0.8 vs 13.9 ± 1.7 mmol/1, respectively; Fig. 6; peak OGTT insulin values 2.1 ± 0.5 vs 0.76 ± 0.04 ng/ml, respectively; Fig. 6). These results indicated that pre-treatment of animals prior to STZ treatment did not act in a protective manner and in the case of the longer acting analog, actually exacerbated the response. However, with GIP(l-42) there was an improvement in fed glucose levels that could be due to subsequent islet regeneration. Figure 4: Effect of treatment with [DAla2]GIP or GIP(l-42) on morning fed blood glucose levels in the multiple low-dose STZ Wistar rat model. Wistar rats were exposed to multiple low doses of STZ (25mg/kg) and treated either with [DAla2]GIP (8nmol/kg), GIP(l-42) (8nmol/kg), or 0.9% saline (controls) for 1 week prior to the administration of STZ (days 0-4), and thereafter until the animals were sacrificed. Fed morning blood glucose values measured daily in STZ controls (squares), [DAla2]GIP-treated STZ-induced diabetic animals (upward triangles), and GIP(l-42)-treated STZ-induced diabetic animals (downward triangles). Each data point represents the mean ± S.E.M. of 3-4 animals. *Statistical significance (PO.05) for the [DAla2]GIP-treated group versus STZ control, "statistical significance (PO.05) for the GIP(l-42)-treated group versus STZ control. 0 25 50 75 100 125 150 Time (min) Figure 5: Effect of treatment with [DAla2]GIP or GIP(l-42) on glucose and insulin responses in OGTTs performed on day 5 in the multiple low-dose STZ Wistar rat model. A lg/kg OGTT was performed on Wistar rats exposed either to multiple low-doses of STZ (25mg/kg) or citrate buffer alone (sham) and treated either with GIP(l-42) (8nmol/kg), the GIP analog [DAla2]GIP (8nmol/kg), or 0.9% saline (controls). R ats were treated twice daily for 1 week prior to the administration of STZ (days 0-4) and thereafter until the animals were sacrificed. Blood glucose (A) and plasma insulin levels (B) were measured during an OGTT in sham (diamonds), STZ controls (squares), GIP(l-42)-treated STZ-induced diabetic animals (upward triangle) and GIP analog [DAla2]GIP-treated STZ-induced diabetic animals (downward triangle). Each data point represents the mean ± S.E.M. of 3-4 animals. *Statistical significance (P<0.05) and **statistical significance (PO.01) for the [DAla2]GIP-treated group versus STZ control; "statistical significance (PO.05 for the GIP(l-42)-treated group versus STZ control. B Time (min) Figure 6: Effect of treatment with [DAla2]GIP or GIP(l-42) on glucose and insulin responses in OGTTs performed on day 28 in the multiple low-dose STZ Wistar rat model. A lg/kg OGTT was performed on Wistar rats exposed to multiple low-doses of STZ (25mg/kg) and treated either with GIP(l-42) (8nmol/kg), the GIP analog [DAla2]GIP (8nmol/kg), or 0.9% saline (controls). Rats were treated twice daily for 1 week prior to the administration of STZ (days 0-4) and thereafter until day 28, at which time the OGTT was performed. B lood g lucose (A) a nd p lasma i nsulin 1 evels (B) w ere measured d uring a n O GTT i n s ham (diamonds), STZ controls (squares), GIP(l-42)-treated STZ-induced diabetic animals (upward triangle) and GIP analog [DAla2]GIP-treated STZ-induced diabetic animals (downward triangle). Each data point represents the mean ± S.E.M. of 3-4 animals. *Statistical significance (PO.05) and **statistical significance (PO.01) for the [DAla2]GIP-treated group versus STZ control; Statistical significance (PO.05 for the GIP( 1 -42)-treated group versus STZ control. 3.1.3 - Peptide Dose-Response Investigation in the MLD-STZ Diabetic Rat It was considered possible that homologous desensitization of the GEP receptor, resulting from the high concentrations of long-acting peptide administered, may have contributed to the paradoxical responses in [DAla 2] GEP-treated animals. Agonist-induced homologous desensitization of receptors is a well-documented phenomenon (54,183,234,235). In fact, elevated concentrations of GLP-1 and GEP have been shown to inhibit cAMP generation in pancreatic P-cell lines (53,230,236) and insulin release from the perfused pancreas (127). We postulated that desensitization could result in potentiation of the diabetogenic action of STZ in the M L D model by inducing a state of unresponsiveness to both endogenous and exogenous GEP. The effect of lower doses of peptide on the acute development of diabetes in the MLD-STZ model was therefore tested. Prior to administration of multiple low doses of STZ (25mg/kg), Wistar rats were pretreated twice daily for 1 week with GEP(l-42) in doses designed to produce either supraphysiological or physiological circulating concentrations of peptide (2nmol/kg or 500pmol/kg, respectively). Considering the long-acting nature of the analog, and thus higher biological activity in vivo compared to GEP(l-42), lower concentrations of [DAla 2] GEP (lnmol/kg or 125pmol/kg, respectively) were chosen for the dose response study. Fed morning blood glucose values and oral glucose tolerance were measured to monitor potential protection against MLD-STZ-induced diabetes. The fed blood glucose values over the course of treatment did not differ significantly between the GIP(l-42)-treated and control animals (GEP(l-42)-treated fed blood glucose values on day 8: 500nmol/kg GIP(l-42): 25.4 ± 1.9 mmol/1, 2nmol/kg GEP(l-42): 26.1 ± 1.0 mmol/1; Control fed blood glucose values on day 8: 22.5 ± 3.7 mmol/1; Fig. 7). In addition, the glucose excursions during the OGTT did not significantly differ between the two groups of animals (GEP(l-42)-treated 30min OGTT blood glucose values: 500nmol/kg GEP(l-42): 19.1 ± 2.2 mmol/1, 2nmol/kg GIP(l-42): 18.4 ± 1.7 mmol/1; Control 30min OGTT blood glucose values: 16.4 ± 1.4 mmol/1; Fig. 7). These data suggest that desensitization does not appear to be the mechanism behind the lack of protective effects of native GEP against STZ action on the p-cell. The fed blood glucose values over the course of treatment also did not differ significantly between the [DAla2]GEP-treated and control animals ([DAla2]GEP-treated fed blood glucose values on day 8: 125pmol/kg [DAla2]GEP: 26.4 + 1.8 mmol/1, lnmol/kg [DAla2]GEP: 19.7 ± 2.7 mmol/1; Control fed blood glucose values on day 8: 22.5 ± 3.7 mmol/1; Fig. 8). In addition, glucose tolerance did not significantly differ between the two groups of animals ([DAla2]GEP-treated 30min OGTT blood glucose values: 125pmol/kg [DAla2]GEP: 17.1 ± 1.8 mmol/1, lnmol/kg [DAla2]GEP: 14.0 ± 1.7 mmol/1; Control 30min OGTT blood glucose values: 16.4 ± 1.4 mmol/1; Fig. 8). These data demonstrate that, in contrast to pharmacological doses of the peptide (Fig. 5,6), lower doses of [DAla 2] GIP did not result in exacerbation of diabetes (Fig. 8). Therefore, the much higher doses of [DAla2]GEP used for the experiments described in Section 3.1.2 could have resulted in desensitization to the positive effects of both exogenous and endogenous peptide. However, it is also possible that its paradoxical effect is due to other effects associated with the N-terminal modification. B 25-1 STZ Controls STZ + 500nmol/kg GIP(1-42) STZ + 2nmol/kg GIP(1-42) Time (min) Figure 7: Responses of Wistar rats exposed to multiple low doses of STZ to different doses of GIP(l-42). Wistar rats were exposed to multiple low doses of STZ (25mg/kg) and treated twice daily with either GIP(l-42) or 0.9% saline (controls). Rats were treated for 1 week prior to the administration of STZ (days 0-4) and thereafter until the animals were sacrificed (day 8). Fed morning blood glucose values (A) were measured daily and blood glucose responses during an lg/kg OGTT (B) were measured on day 5 in STZ controls (squares), 500nmol/kg GIP(l-42)-treated STZ-induced diabetic animals (upward triangles), and 2nmol/kg GIP(l-42)-treated STZ-induced diabetic animals (downward triangles). Each data point represents the mean + S.E.M. of 4 animals. *Statistical significance (PO.05) for the 500nmol/kg GIP(1-42)-treated group versus STZ control. STZ Controls STZ+ 125pmol/kg [DAIa2]GIP STZ + 1 nmol/kg [DAIa2]GIP oH 1 1 1 1 1 0 2 4 6 8 10 Time (days post 1st STZ injection) Figure 8: Responses of Wistar rats exposed to multiple low doses of STZ to different doses of [DAla2]GIP. Wistar rats were exposed to multiple low doses of STZ (25mg/kg) and treated twice daily with either the GIP analog [DAla2]GIP or 0.9% saline (controls). Rats were treated for 1 week prior to the administration of STZ (days 0-4) and thereafter until the animals were sacrificed (day 8). Fed morning blood glucose values (A) were measured daily and blood glucose responses during an lg/kg OGTT (B) were measured on day 5 in STZ controls (squares), 125pmol/kg GIP analog [DAla2]GIP-treated STZ-induced diabetic animals (diamonds), and lnmol/kg GIP analog [DAla2]GIP-treated STZ-induced diabetic animals (circles). Each data point represents the mean ± S.E.M. of 4 animals. 3.1.4 -The Effects of Concomitant GIP Stimulation of the P-cell on STZ Action To investigate whether direct effects of [DAla 2]GIP on the P-cell during STZ action, as opposed to effects resulting from pretreatment with [DAla ]GIP, may have contributed to exacerbation of the diabetic condition, the single high-dose streptozotocin (IP)-induced d iabetic rat w as e mployed. A d ose-response i nvestigation o f s ingle h igh dose-induced diabetes was initially performed in order to determine the optimal dose of STZ necessary to establish a moderate diabetic phenotype. Male Wistar rats (325-375g) were administered a single dose of streptozotocin (50mg/kg, 60mg/kg, or 75mg/kg) via intraperitoneal injection and then monitored thereafter for diabetes induction. A minimum effective dose of 75mg/kg STZ was established based on glucose tolerance and fed morning blood glucose values (Fig. 9) and was used for all subsequent experiments. 25-| 20-o g 15-u O 10-T3 O o 5-m 50mg/kg STZ 60mg/kg STZ 75mg/kg STZ 0.0 i 2.5 5.0 7.5 —I 10.0 Time (days) B o w o u (3 T3 O o CD •50mg/kg STZ •60mg/kg STZ •75mg/kg STZ Time (min) Figure 9: Responses of Wistar rats to treatment with a single high dose of STZ. Morning fed blood glucose levels (A) and an OGTT (B) performed on Wistar rats exposed to a single high dose of STZ on day 0. Morning blood glucose values were measured daily and blood glucose was measured during a lg/kg OGTT on day 5 in animals administered a single high dose of 50mg/kg STZ (squares), 60mg/kg STZ (upward triangles), and 75mg/kg STZ (downward triangles). Each data point represents the mean ± S.E.M. of 5 animals. Wistar rats (325-375g) were concomitantly treated with a single dose of STZ (75mg/kg) and either GIP(l-42) (lOnmol/kg) or [DAla 2]GIP (8nmol/kg), and peptide treatment continued twice daily thereafter until the animals were sacrificed. Fed morning blood glucose values and oral glucose tolerance were measured to document any diabetes-exacerbating effects in the treated groups. [DAla 2] GIP and GIP(l-42) action did not appear to interfere directly with STZ metabolism and intracellular action in the p-cell. [DAla2]GIP-treated and GIP(l-42)-treated animals displayed similar fed blood glucose relative to M L D - S T Z controls (Day 4: GIP(l-42) treatment: 14.5 + 2.3 mmol/1 vs 14.5 ± 1.9 mmol/1, respectively; Fig. 10; [DAla 2]GIP treatment: 17.8 ± 2.5 mmol/1 vs 21.8 ± 3.4, respectively; Fig. 11), and equivalent blood glucose responses during an oral glucose challenge (peak OGTT blood glucose values: GIP(l-42) treatment: 10.1 ± 0.7 vs 10.0 ± 0.7 mmol/1, respectively; Fig 10; [DAla 2]GIP treatment: 14.8 ± 1.6 vs 14.3 ± 1.7 mmol/1, repectively; Fig. 11). The glycemic profiles during the OGTTs between the STZ controls and either the GIP(l-42)-treated or [DAla2]GIP-treated STZ animals were almost identical. Neither treatment with [DAla ]GIP nor GIP(l-42) therefore had a marked effect on responses to STZ treatment, suggesting that neither peptide has any direct effect on either STZ metabolism or action in the p-cell. R ather, these findings lend credence to the possibility that pretreatment with the GIP analog is indirectly exacerbating diabetes by evoking effects in the p-cell that synergize with STZ and thereby, facilitate greater p-cell death. 20-1 E, 15-a> in o u 3 o T3 O O CO 10-STZ Controls STZ + GIP( 1-42) 1 2 3 4 T ime (days pos t STZ admin i s t ra t i on ) B • STZ Controls STZ + GIP(1-42) 50 75 100 Time (min) Figure 10: Effect of treatment with GIP(l-42) on fed blood glucose levels and glucose responses in OGTTs performed in Wistar rats exposed to a single high dose of STZ. Rats were administered an intraperitoneal injection of STZ (75mg/kg; day 0) and concommitantly treated with either a single injection of GIP(l-42) (8nmol/kg) or 0.9% saline (controls). Rats were continuously treated twice daily thereafter until sacrificed. Fed morning blood glucose values (A) and blood glucose responses during a lg/kg OGTT (B) were measured in GIP(l-42)-treated animals (triangles) and controls (squares). Each data point represents the mean ± S.E.M. of 10 animals. B STZ Controls STZ+ [DAIa2]GIP CQ 0 | | | l l l —| 0 25 50 75 100 125 150 Time (min) Figure 11: Effect of treatment with [DAla2]GIP on fed blood glucose levels and glucose responses in OGTTs performed in Wistar rats exposed to a single high dose of STZ. Rats were administered an intraperitoneal injection of STZ (75mg/kg; day 0) and concommitantly treated with either a single injection of [DAla2]GIP (8nmol/kg) or 0.9% saline (controls). Rats were continuously treated twice daily thereafter until sacrificed. Fed morning blood glucose values (A) and blood glucose responses during a lg/kg OGTT (B) were measured in DAla2]GIP-treated animals (triangles) and controls (squares). Each data point represents the mean ± S.E.M. of 10 animals. 3.1.5 - Regulation of Islet GLUT2 Expression by [DAla2]GIP In Vivo One possible explanation for the potentiated response to streptozotocin following pretreatment with [DAla 2]GIP in the MLD-STZ-induced diabetic rat model, is [DAla2]GEP-induced upregulation of GLUT2 glucose transporter levels. As mentioned in the Introduction (Section 1.2.5), a model has been proposed, based on several lines of evidence (205-214), that differential sensitivity to STZ is correlated with the level of P-cell GLUT2 expression. Accordingly, it was of interest to determine the effect of [DAla2]GEP on p-cell GLUT2 expression in vivo, in an attempt to explain the paradoxical findings. Islet GLUT2 mRNA and protein expression were determined in Wistar rats (325-375g) treated with [DAla2]GEP for 1 week. Following treatment, islets were isolated and real-time RT-PCR and Western blot analysis were carried out to measure mRNA and protein expression, respectively. As predicted, there was a significant increase in GLUT2 mRNA expression in [DAla 2]GIP -treated islets (336.1 ± 48.1%; Fig. 12) relative to controls, which appears to be linked to an increase in functional expression. Mean GLUT2 protein levels in [DAla2]GEP-treated islets were increased (131.7 + 22.8%; Fig. 13) relative to controls; however, the increase did not reach statistical significance. Figure 12: Effect of [DAla2]GIP treatment on islet GLUT2 mRNA expression in vivo. Male Wistar Rats (275-325g) were treated with the GIP analog [DAla2]GIP (8nmol/kg) or 0.9% saline (controls) for 1 week prior to the isolation of islets. Total pancreatic islet RNA was isolated from the islets and 1 ug RNA was subjected to one-step real-time RT-PCR. GIP receptor mRNA was e xpressed a s % of control values of GLUT2 mRNA. Data are expressed as percentage of controls and represent means ± S.E.M. of 4 animals. **Statistical significance (P S 0.01) for [DAla2]GIP-treated versus control animals. CI T l C2 T2 C3 T3 C4 T4 Figure 13: Effect of [DAla2]GIP treatment on islet GLUT2 protein expression. Male Wistar Rats (275-325g) were treated with the GIP analog [DAla2]GIP (8nmol/kg; Treated, Tl-4) or 0.9% saline (Controls, CI-4) for 1 week prior to the isolation of islets. Total cellular protein was isolated from the islets and ten microgram protein samples were separated by SDS-PAGE and membranes blotted with antibodies against GLUT2. Although data do not reach significance, [DAla2]GIP appears to upregulate GLUT2 protein levels in vivo. Blots were normalized with p-tubulin to compensate for loading differences. Data are expressed as percentage of control islet protein expression and represent mean ± S.E.M. of 4 animals. 3.1.6 - The Effects of [DAla2]GIP on Susceptibility to STZ-induced P-Cell Death In view of the obvious limitations in the multiple-low dose STZ model of type 1 diabetes for studying the effect of [DAla ]GIP, the single high-dose streptozotocin (IV)-induced diabetic rat was chosen as a well-characterized model of diabetes. A characteristic triphasic response in blood glucose levels occurs in response to an intravenous injection of STZ within the first 24 hours (217), and thus, documentation of changes in glucose homeostasis would easily be detectable. Therefore, we chose to use this model to investigate the ability of [DAla ]GJP to increase susceptibility to STZ-induced p -cell d eath i n v ivo, r ather t han t he i ntraperitoneal m odel o f s ingle h igh d ose STZ-induced diabetes. In order to establish a dose-response curve for STZ-induced diabetes in which to document the potentiation of diabetes with [DAla2]GEP treatment, three d oses o f S TZ w ere c hosen: 3 0 m g/kg (nondiabetic d ose), 4 5 m g/kg (moderately diabetic dose), and 60 mg/kg (severely diabetic dose). We hypothesized that treatment with [DAla 2] GEP would left-shift the STZ dose-response curve to induce diabetes in the nondiabetic animals and potentiate diabetes in the moderately and severely diabetic animals. Fed blood glucose values, glucose tolerance, insulin secretion, and pancreatic insulin content were the metabolic parameters measured to test this hypothesis. Surprisingly, [DAla2]GEP treatment protected against STZ-induced diabetes in the animals administered the lower doses of STZ (30mg/kg and 45mg/kg STZ). [DAla2]GEP-treated animals administered 45mg/kg STZ (TV) displayed significantly improved fed blood glucose levels relative to controls one day following STZ administration, and they remained significantly lower thereafter (45mg/kg STZ, Day 1: 9.2 ± 0.6 vs 18.6 ± 2.8 mmol/1, respectively; Fig. 15). [DAla ]GEP-treated animals administered 30mg/kg STZ showed a trend for improved fed blood glucose values over time, although values did not reach significance (30mg/kg STZ, Day 3: 7.4 ± 0.7 vs 9.4 ± 0.8 mmol/1, respectively; Fig. 14). In addition, [DAla2]GIP-treated animals administered the lower doses of STZ (30mg/kg and 45mg/kg STZ) displayed significantly improved blood glucose responses during the oral glucose challenge (30mg/kg STZ, 20 min OGTT blood glucose values: 11.9 ± 0.7 vs 14.0 ± 0:3 mmol/1, respectively, Fig.14; 45mg/kg STZ, 60 min OGTT blood glucose values: 13.0 ± 0.4 vs 16.8 ± 1.3 mmol/1, respectively; 120 min OGTT blood glucose values: 7.8 ± 0.9 vs 12.3 ± 1.4 mmol/1, respectively, Fig.15). Consistent with improved glucose tolerance, the [DAla2]GIP-treated animals administered 45mg/kg STZ showed increased insulin secretory responses during the OGTT (10 min OGGT insulin value 1.7 ± 0.6 vs 1.3 ± 0.1 ng/ml, respectively; Fig 16) and whole pancreas insulin content (0.136 ± 0.03 vs 0.074 ± 0.02 ng/mg protein, respectively; Fig. 16), values of which should reach statistical significance with increased sample size. Figure 14: Effect of treatment with [DAla2]GIP on fed blood glucose levels and glucose responses in OGTTs performed in Wistar rats exposed to a single dose (30mg/kg) of STZ. Rats were administered an intraveous injection of STZ (30mg/kg; day 0) and treated with the GIP analog [DAla2]GIP (8nmol/kg) or 0.9% saline (controls) by intraperitoneal injection twice daily (0800 and 1700) for 1 week prior to the administration of STZ and thereafter until the animals were sacrificed. Fed morning blood glucose values (A) and blood glucose responses during a lg/kg OGTT (B) were measured in [DAla2]GIP-treated animals (triangles) and controls (squares). Each data point represents the mean ± S.E.M. of 3 animals. Controls (45mg/kg STZ) Treated (45mg/kg STZ) Time (days) Controls (45mg/kg STZ) Treated (45mg/kg STZ) Time (min) Figure 15: Effect of treatment with [DAla2]GIP on fed blood glucose levels and glucose responses in OGTTs performed in Wistar rats exposed to a single dose (45mg/kg) of STZ. Rats were administered an intraveous injection of STZ (45mg/kg; day 0) and treated with the GIP analog [DAla2]GIP (8nmol/kg) or 0.9% saline (controls) by intraperitoneal injection twice daily (0800 and 1700) for 1 week prior to the administration of STZ and thereafter until the animals were sacrificed. Fed morning blood glucose values (A) and blood glucose responses during a lg/kg OGTT (B) were measured in [DAla2]GIP-treated animals (triangles) and controls (squares). Each data point represents the mean + S.E.M. of 3 animals. Controls (45mg/kg STZ) Treated (45mg/kg STZ) Figure 16: Effect of treatment with [DAla2]GIP on insulin responses in OGTTs and total pancreatic insulin content in Wistar rats exposed to a single dose (45mg/kg) of STZ. Rats were administered an intraveous injection of STZ (45mg/kg; day 0) and treated with the GIP analog [DAla2]GIP (8nmol/kg) or 0.9% saline (controls) by intraperitoneal injection twice daily (0800 and 1700) for 1 week prior to the administration of STZ and thereafter until the animals were sacrificed. Plasma insulin levels were measured during a lg/kg OGTT on day 3 (A) and pancreatic insulin content determinations were made for pancreata excised under anaesthesia on day 4 (B) in GIP analog [DAla2]GIP-treated STZ-induced diabetic animals (triangles) and STZ controls (squares). Each data point represents the mean ± S.E.M. of 3 animals. In contrast, in the animals administered the highest dose of STZ (60mg/kg STZ), treatment with [DAla 2]GIP slightly potentiated STZ-induced diabetes. [DAla 2]GIP-treated animals displayed increased fed and fasting blood glucose values relative to controls (60mg/kg STZ, Day 3 fed blood glucose values: 30.1 ± 1.2 vs 25.9 ± 0.5 mmol/1; Day 4 fasting blood glucose values: 22.4 ± 2.2 vs 12.9 ± 2.9 mmol/1; Fig. 17). In addition, [DAla2]GIP-treated animals displayed marked exacerbation of blood glucose responses during the oral glucose challenge (60mg/kg STZ, 10 min OGTT blood glucose values: 26.5 ± 2.5 vs 17.6 ±1 .1 mmol/1, respectively, Fig. 17). Consistent with induction of severe diabetes upon administration of the highest dose of STZ (60mg/kg STZ), the insulin secretory response during the OGTT was largely ablated in both the control and [DAla ]GIP-treated animals administered 60mg/kg STZ (peak OGTT insulin values: 0.617 ± 0.07 vs 0.543 ±0.126 ng/ml; Fig 18). In addition, whole pancreas insulin content was severely depleted and values did not differ between the treated and control groups (0.044 ± 0.004 vs 0.046 ±0.014 ng/mg protein, respectively; Fig. 18). Figure 17: Effect of treatment with [DAla2]GIP on fed blood glucose levels and glucose responses in' OGTTs performed in Wistar rats exposed to a single dose (60mg/kg) of STZ. Rats were administered an intraveous injection of STZ (60mg/kg; day 0) and treated with the GIP analog [DAla2]GIP (8nmol/kg) or 0.9% saline (controls) by intraperitoneal injection twice daily (0800 and 1700) for 1 week prior to the administration of STZ and thereafter until the animals were sacrificed. Fed morning blood glucose values (A) and blood glucose responses during a lg/kg OGTT (B) were measured in [DAla2]GIP-treated animals (triangles) and controls (squares). Each data point represents the mean ± S.E.M. of 3 animals. *Statistical significance (PO.05) for the [DAla2]GIP-treated group versus STZ control. 1.0(h • STZ Control •STZ+[DAIa2]GIP 25 50 75 Time (min) B 0.07-c "5 0.06-(A C o tein 0.05-eati o 0.04-eati Q. k. o 0.03-Pan E Pan O) 0.02-0.01-•*-> o \- 0.00-T Figure 18: Effect of treatment with [DAla2]GIP on insulin responses in OGTTs and total pancreatic insulin content in Wistar rats exposed to a single dose (60mg/kg) of STZ. Rats were administered an intraveous injection of STZ (60mg/kg; day 0) and treated with the GIP analog [DAla2]GIP (8nmol/kg) or 0.9% saline (controls) by intraperitoneal injection twice daily (0800 and 1700) for 1 week prior to the administration of STZ and thereafter until the animals were sacrificed. Plasma insulin levels were measured during an lg/kg OGTT on day 3 (A) and pancreatic insulin content determinations were made for pancreata excised under anaesthesia on day 4 (B) in GIP analog [DAla2]GIP-treated STZ-induced diabetic animals (triangles) and STZ controls (squares). Each data point represents the mean + S.E.M. of 3 animals. 3.1.7 - Inhibition of STZ-induced P-Cell Death In Vitro In an attempt to unravel the underlying mechanisms involved in the in vivo responses to [DAla ]GIP in the STZ (TV; 45 mg/kg)-induced diabetic rat, an examination was made of the ability of [DAla 2] GEP and GEP(l-42) to inhibit apoptosis in INS-1(832/13) cells exposed to STZ. It has previously been shown that GEP(l-42) exerts a concentration-dependent reversal of STZ-induced cell death in ENS-1(832/13) cells via an anti-apoptotic mechanism of action (237). Therefore, to examine the ability of [DAla ]GEP to inhibit STZ-induced P-cell death, cells were pretreated with either peptide (2nM) for 10 minutes prior to a 30-minute exposure to STZ (2mM) and a 24 hour recovery period, after which time caspase 3 activity was measured. [DAla2]GEP and GEP(l-42) were both able to protect completely against the proapoptotic (caspase-3 activating) effects of STZ. STZ-induced apoptosis under control conditions was associated with 3.3-fold increases in caspase 3 activity, whereas following [DAla ]GEP and GEP(l-42) treatment, changes in caspase 3 activity were only 1.6-fold (Fig. 19). •o o z. <D ° -^ i 5 -£ c 3 E » o , 1 — CO Om ° c E -a 1« 5 Basal Buffer [DAIa2]GIP GIP(1-42) STZ Figure 19: [DAla2]GIP and GIP(l-42) stimulation of INS-1(832/13) cells protects against STZ-induced apoptotic cell death. Cells were serum starved 24 hours before and during the experiment, and lOOnM [DAla2]GIP or GIP(l-42) were added 10 min prior to exposure to STZ (2mM) to assess affects on caspase-3 activity. Caspase-3 activity was quantified using the substrate, Z-DEVD-AMC, over 30 min, and corrected using for total protein concentration using the BCA protein assay. Data are expressed as mean ± S.E.M. and experiments are representative of n=4, in which * and # represent PO.05 vs. respective controls. 3.2 - GIP Receptor Expression in Type 2 Diabetes The decreased incretin effect observed in type 2 diabetes has been attributed to a loss of the GEP component of the enteroinsular axis since GLP-1 maintains insulinotropic activity in these individuals; however, the mechanism responsible for the diminished response to GEP has not been fully elucidated. Our laboratory has shown that GEP receptor (GEPR) mRNA levels are down-regulated in the pancreatic P-cell of an animal model of type 2 diabetes and that this is correlated with a decreased ability of the islets in these animals to respond to physiological concentrations of GEP (195). It was also shown that elevated glucose levels are able to significantly reduce GEPR expression significantly in vivo and in vitro (197). An aim of the current study was to examine whether normalizing hyperglycemia in vivo would reverse the down-regulation of GEP receptor expression in type 2 diabetes. The elucidation of the factors controlling the down-regulation of GEPR expression in type 2 diabetes may substantially increase our knowledge of the pathogenesis of this disease and serve to improve the efficacy of future treatment regimens. 3.2.1 - Rat Pancreatic Islet GIP Receptor Expression in Zucker Rats To determine the effects of different degrees of elevated blood glucose levels on GEPR mRNA expression levels in vivo, real-time RT-PCR was performed on islets isolated from Zucker lean, Zucker obese, and Zucker diabetic fatty (ZDF) male rats. Fed blood glucose and glucose tolerance were measured to document the degree of hyperglycemia in the different Zucker strains. Consistent with previous findings, obese Zucker rats displayed glucose intolerance and ZDF rats displayed severe diabetes at 12 weeks of age, as demonstrated in the oral glucose tolerance test (20 min OGTT values: lean Zucker: 8.18 ± 0.25 mmol/1, obese Zucker: 11.58 ± 0.56 mmol/1, and ZDF: 18.30 + 0.76 m mol/1; F ig. 20). I n a ddition, Z DF r ats w ere c hronically h yperglycemic, Z ucker obese rats exhibited normoglycemia and mild postprandial hyperglycemia, and Zucker lean rats were normoglycemic (Fed morning blood glucose levels: ZDF: 28.1 ± 0.4 mmol/1, Zucker obese: 5.7 ± 0.3 mmol/1, Zucker lean: 5.5 ± 0.2 mmol/1); therefore, these rat strains are good models to document the effects of different levels of blood glucose on GEPR expression. Zucker Lean Zucker Obese Zucker Diabetic Fatty (ZDF) <H 1 1 1 1 1 1 0 25 50 75 100 125 150 Time (min) Figure 20: Responses to oral glucose tolerance tests performed on Zucker lean, Zucker obese, and Zucker Diabetic Fatty (ZDF) rats at 12 weeks of age. Whole blood glycemia was measured during a lg/kg OGTT at indicated times from the tail vein samples in lean Zucker (squares), obese Zucker (upward triangles), and ZDF (downward triangles) rats. *Statistical significance (PO.05), **statistical significance (PO.01), and """•statistical significance (PO.001) for the ZDF versus lean Zucker rats; #statistical significance (PO.05) and ##statistical significance (PO.01) for the obese versus lean Zucker rats. Each data point represents the mean ± S.E.M. of 4 animals. GEPR mRNA levels were almost completely ablated in the ZDF rats (94.3 ± 3.8 %) and reduced by 48.8 ± 22.8 % in the Zucker obese rats (relative to expression levels in Zucker lean rats (Fig. 21)). These data corroborate previous findings by Lynn et al demonstrating a significant down-regulation of GEPR mRNA expression levels in the Vancouver Diabetic Fatty (VDF) rat. Interestingly, the degree of down-regulation in islet GEPR mRNA expression was positively correlated with the severity of hyperglycemia in the animals, suggesting that elevated glucose levels is a major factor leading to reduced GEPR mRNA expression. Figure 21: GIP receptor mRNA levels in the islets of Zucker lean, Zucker obese, and Zucker Diabetic Fatty (ZDF) rats. After islets were isolated, RNA was extracted from the islets, and 1 ug pancreatic islet RNA was subjected to one-step real-time RT-PCR. GIP receptor mRNA was expressed as percentage of Zucker lean islet GIPR mRNA. Data are expressed as means ± S.E.M. of 3-4 animals. *Statistical significance (P £ 0.05) for the ZDF versus Zucker lean rats. 3.2.2 - Glucose Regulation of GIP Receptor mRNA Expression in Vivo To determine whether down-regulation of the GIPR is secondary to chronically elevated blood glucose levels, the effects of lowering blood glucose levels on GIPR mRNA expression in vivo were examined in phlorizin-treated and control ZDF rats (14-16 weeks of age). Fed blood glucose values were measured to monitor the effects of phlorizin treatment on glucose normalization. As shown in Figure 22, phlorizin treatment effectively lowered blood glucose levels; morning blood glucose levels over the course of the experiment averaged 28.1 + 0.4 mmol/1 in control ZDF, 10.4 + 0.5 mmol/1 in phlorizin-treated ZDF, and 5.5 ± 0.2 mmol/1 in control Zucker lean rats (Fig. 22). The gain in body weights of ZDF rats remained unchanged with phlorizin treatment (control ZDF: 2.2 ± 0.7g/day, phlorizin-treated ZDF: 1.8 ± 0.7g/day, consistent with previous findings (201). Following two weeks of treatment with phlorizin, pancreatic islet R N A was subjected to real-time RT-PCR to determine whether islet GIPR mRNA expression changed upon normalization of blood glucose levels in the ZDF rats. Phlorizin-treatment reversed the down-regulation of GIPR mRNA expression in ZDF rats (Fig. 23) and restored GIPR expression to levels found in lean control animals (ZDF controls: 16.9 + 5.0 % of lean controls; ZDF phlorizin-treated: 83.0 ± 17.9 % of lean controls; Fig 23). These data suggest that chronic hyperglycemia negatively regulates GIPR mRNA expression in the ZDF rat. It is of note that there exists a small difference in the measured GIPR mRNA expression levels in the ZDF rats between the two independent studies: the current study and those described in Section 3.2.1 (5.7 ± 3.8% vs 16.9 ± 5.0 % of lean controls). This discrepancy could be the result of multiple factors, such as variability between animals, difference in age (~2 weeks), stress during animal handling, or effects of vehicle delivery twice daily (60% propylene glycol). ZDF Controls ZDF Phlorizin-Treated Lean Controls Time (days) Figure 22: Fed morning blood glucose levels in lean control, Zucker Diabetic Fatty (ZDF) control, and Zucker Diabetic Fatty (ZDF) phlorizin-treated rats. Fed morning blood glucose values were measured three times weekly in lean control animals (downward triangles), ZDF control animals administered 60% propylene glycol (squares) and ZDF phlorizin-treated animals administered 0.4g/kg phlorizin in 60% propylene glycol (upward triangles) by intraperitoneal injection twice daily (0830 and 2030) for two weeks. Each data point represents the mean ± S.E.M. of 4-5 animals. Figure 23: GIP receptor mRNA levels in the islets of lean control, Zucker Diabetic Fatty (ZDF) control, and Zucker Diabetic Fatty (ZDF) phlorizin-treated rats. GIP receptor mRNA was measured in lean control, ZDF control animals administered 60% propylene glycol (squares), and ZDF phlorizin-treated animals administered 0.4g/kg phlorizin in 60% propylene glycol (upward triangles) by intraperitoneal injection twice daily (0830 and 2030) for two weeks. After islets were isolated, RNA was extracted from the islets, and 1 ug pancreatic islet RNA was subjected to one-step real-time RT-PCR. Phlorizin-treatment reversed the down-regulation ofGIPRmRNA expression in ZDF rats. GIPRmRNA expression is expressed as percentage of lean control islet GIPR mRNA Data represent means ± S.E.M. of 3-5 animals. *Statistical significance (P £ 0.05) for the ZDF control versus lean control rats and "statistical significance (P 5 0.05) for the ZDF phlorizin-treated versus ZDF control rats. 3.2.3 - GIP Responsiveness in Phlorizin-Treated ZDF Rats In order to determine whether restoration of GIPR mRNA expression was correlated with restoration of biological responses to GIP, the glucose lowering potency and insulinotropic activity of GIP in phlorizin-treated ZDF rats and their respective controls were examined. An IP glucose tolerance test with concomitant administration of GIP (intraperitoneal injection; 8nmol/ml; injection volume according to body mass) was performed following 11 days of treatment to assay the glucose lowering potency of GIP. Phlorizin-treated ZDF rats demonstrated significantly improved glycemic control in response to GIP, during the intraperitoneal glucose challenge, relative to ZDF control rats (peak IPGTT blood glucose values: ZDF control: 28.9± 1.2 mmol/1, phlorizin-treated ZDF: 18.5 ±1 .4 mmol/1; Fig. 24). The change in blood glucose levels from basal during the IPGTT revealed that blood glucose clearance in response to GIP(l-42) was more rapid in the phlorizin treated ZDF rats (7.1 ±1 .0 mmol/1) compared to ZDF controls (12.4 ±1.2 mmol/1), reaching significance at 30 min (Figure 24 (inset)). As well, the integrated glucose responses in the phlorizin-treated ZDF rats were significantly reduced in the phlorizin-treated ZDF rats (AUC: 1372.8 ± 63.6 mmol/1 x 120min) compared to the ZDF controls (AUC: 2911 ± 215.3 mmol/1 x 120min) (PO.001). These results therefore suggest that GIP responsiveness is restored in the phlorizin-treated ZDF rats. However, in the absence of a saline control for comparative purposes, it is not possible to dissect the improved glucose tolerance in phlorizin-treated ZDF rats resulting from restoration of GIP's biological action from the improvement resulting from normalization of blood glucose levels (168-172). Nonetheless, a more rapid glucose clearance in the treated animals compared to the controls (Fig. 24) supports a role for improved responsiveness to GEP in the improved glycemic control. To determine whether GEP's biological activity was restored at the level of the pancreas, the insulinotropic activity of GEP was measured in the perfused pancreas. Although blunted, GEP evoked a characteristic biphasic insulin response in the phlorizin-treated ZDF rats. As demonstrated in Figure 25, 20 pmol/1 GEP(l-42) in the presence of 8.8mM glucose evoked a 2.6 fold increase in early insulin secretion from the phlorizin-treated perfused ZDF pancreas (Insulin Secretion at 22min: 2.4 ± 0.4 ng/ml; Fig. 25) relative to the ZDF control perfused pancreas (Insulin Secretion at 22 min: 0.9 ± 0.2 ng/ml, respectively; Fig. 25). Lnsulin secretion from the phlorizin-treated ZDF perfused pancreas remained elevated for the 20 minute GEP(l-42) infusion and insulin concentrations fell to those seen in the ZDF control perfused pancreas upon removal of the peptide (Phlorizin-treated ZDF perfused pancreas insulin secretory response: 23min: 1.5 ± 0.8 ng/ml, 40 min: 1.4 ± 0.2 ng/ml, 50min: 0.7 ± 0.1 ng/ml; Control ZDF pancreas: 23min: 0.9 ± 0.2 ng/ml, 40min: 0.7 ± 0.1 ng/ml, 50min: 0.6 ± 0.1 ng/ml; Fig. 25). The area under the curve during the peptide infusion is significantly larger for the phlorizin-treated ZDF rats (1.9 ± 0.3 fold; Fig 25 (inset)) relative to controls. These data suggest that GEP responsiveness at the level of the P-cell is restored upon blood glucose normalization in the ZDF rat. I 1 1 1 1 1 1 0 25 50 75 100 125 150 Time (min) Figure 2 4: G IP responsiveness b ioassays p erformed o n ZDF c ontrol a nd ZDF phlorizin-treated rats. A lg/kg IPGTT was performed with concurrent administration of GIP(l-42) (8 nmol/kg) via intraperitoneal injection at time 0 was performed and whole blood glucose levels were measured at indicated times from the tail vein samples in ZDF control animals administered 60% propylene glycol (squares) and ZDF phlorizin-treated animals administered 0.4g/kg phlorizin in 60% propylene glycol (upward triangles) by intraperitoneal injection twice daily (0830 and 2030) for 11 days. The inset shows the change in blood glucose levels from basal in response to GIP(l-42) during the IPGTT. Each data point represents the mean ± S.E.M. of 4-5 animals. *Statistical significance (PO.05), **statistical significance (PO.01), and ***statistical significance (PO.001) for the ZDF controls versus ZDF phlorizin-treated rats. Figure 25: Insulin secretion from perfused pancreata of control and phlorizin-treated ZDF rats. Pancreata from ZDF control animals administered 60% propylene glycol (squares) and ZDF phlorizin-treated animals administered 0.4g/kg phlorizin in 60% propylene glycol (upward triangles) by intraperitoneal injection twice daily (0830 and 2030) for two weeks were subjected to a low-to-high (4.4 - 8.8 mM) glucose perfusion and a 20 minute infusion with GIP(l-42) (20pmol/l) in the presence of 8.8 mM glucose. Pancreata were perfused at 4ml/min with Krebs buffer. After a 10 minute equilibration period, pancreata were perfused with 4.4 mM glucose for 4 minutes, and then 8.8mM glucose for the remainder of the experiment; GIP(l-42) was introduced into the system at 20 minutes via a side arm infusion pump. Samples were collected every minute and assayed for insulin by radioimmuoassay. The inset shows the integrated plasma glucose responses for the pancreas perfusions during the 20 minute peptide infusion (20-40min), values are expressed relative to 8.8mM glucose. Each data point represents the mean ± S.E.M. of 5-8 animals. *Statistical significance (P<0.05) for the ZDF controls versus ZDF phlorizin-treated rats. Chapter 4 - Discussion [DAla2] GIP Therapy in Type 1 Diabetes The therapeutic potential of the hormone, glucose-dependent insulinotropic polypeptide (GIP), for type 1 diabetes has largely been unstudied. Recently it has been shown that GEP stimulates P-cell growth, differentiation, and survival (58,82,83), and inhibits apoptosis (59,83), providing impetus for potential application of this peptide in type 1 diabetes. Since stable glycemic control is difficult to manage with insulin, a therapy that induces insulin biosynthesis and islet neogenesis is appealing as a treatment alternative to insulin or in conjunction with insulin. Pospisilik et al showed the potential for dipeptidyl peptidase EV (DPFV) inhibitors as a therapeutic strategy in type 1 diabetes through the demonstration of enhanced glucose homeostasis, p-cell protection, and possibly, islet neogenesis in the STZ-induced diabetic rat (237). Taken together, these findings provided the rationale for an investigation into the effects of [DAla ]GEP treatment on an animal model of type 1 diabetes, the multiple low-dose (MLD-STZ)-induced diabetic rat. The ability of [DAla 2] GEP to reduce or reverse the p -cell loss of experimental diabetes would have obvious therapeutic implications. Suprisingly, instead of protecting against diabetes induction, treatment with [DAla ]GEP in the multiple low-dose STZ-induced diabetic rat actually led to more severe diabetes. Increased fed blood glucose levels over the course of the experiment (Fig. 4) and worsening of glucose tolerance immediately following and 4 weeks post STZ administration (Fig. 6) indicate deterioration of metabolic function (likely insulin-deficiency) in the [DAla2]GEP-treated animals relative to the MLD-STZ controls. Comparative studies of the biological activities of [DAla2]GEP and GEP(l-42) both in vitro (Fig. 1) and in vivo (Fig. 2) established that both peptides exhibited the expected actions on cyclic A M P production and insulin secretion, indicating that the paradoxical observations in the MLD-STZ rat were specifically due to [DAla 2] GEP treatment. In contrast to the effects of the [DAla 2] analog, treatment with GIP(l-42) significantly improved the diabetic phenotype after 4 weeks of treatment relative to MLD-STZ controls (Fig. 4,6). Although not specifically studied, it is possible to speculate on the underlying basis for the improved diabetic phenotype by extrapolation from the results of Pospisilik et al (237). Treatment with the DPEV inhibitor P32/98 was proposed to partially preserve P-cell number and islet insulin content in STZ-treated animals mainly through enhancement of biologically active incretin concentrations (237). It was further speculated that the increased levels of biologically active incretins may have also increased islet neogenesis. In the current MLD-STZ study, fed blood glucose values (Fig. 4) and glucose and insulin responses during the OGTT showed marked improvements 4 weeks post-STZ administration (Fig. 6) in the GEP(l-42) treated animals, relative to those measured immediately following STZ administration (Fig. 5), also indicating islet regenerative rather than protective effects against STZ action. Such an action would be compatible with studies showing that GLP-1 has the ability to stimulate P-cell neogenesis, P-cell replication, and islet size in animal models of diabetes (111,112) and provide support for the proposed utilization of GEP(l-42) as a treatment modality in type 1 diabetes. In view of the marked difference in responses to [DAla 2]GIP and GIP(l-42) in the MLD-STZ diabetic rat, it was felt to be important to develop an understanding of potential underlying mechanisms, and several avenues were therefore explored. Initially, receptor desensitization was proposed to be a potential factor implicated in the diabetic effects of [DAla 2]GIP. Rapid and reversible homologous desensitization in response to supraphysiological concentrations of GLP-1 and GIP has been demonstrated for their respective receptors (234). As such, i f it could be demonstrated that lower concentrations of [DAla 2]GIP showed decreased exacerbation o f MLD-STZ diabetes (Fig. 8), it would suggest that pharmacological concentrations of [DAla 2]GIP may have caused receptor desensitization and thus, insensitivity to both endogenous and exogenous GIP action at the pancreatic p-cell. However, the fact that M L D - S T Z rats administered pharmacological concentrations of GIP(l-42) did not demonstrate exacerbation of diabetes (Fig. 4,5) suggests that other factors associated with the N -terminal modification may account for the diabetic effects of treatment with [DAla ]GIP. In fact, resistance of the receptor to rapid desensitization is a favourable characteristic i f GIP analogs are to be used as potential therapies for diabetes. Further studies will need to be pursued to clarify the potential role for desensitization in the [DAla ]GIP-treated MLD-STZ rats. Inbred strains of mice show different degrees of susceptibility to the diabetic effects of STZ injected as a single high dose or as multiple sub-diabetogenic doses, indicating the importance of genetic factors in the control of STZ activity (238). Rossini et al showed that strain differences in the degree of hyperglycemia elicited when mice are administered multiple low doses of STZ does not correlate with the degree of insulitis triggered (238). Additionally, Cardinal et al demonstrated that strain differences in STZ sensitivity appear to be due to intracellular events within the p-cell, as STZ metabolite accumulation was associated with STZ sensitivity (239). Thus, there appear to be unidentified factors affecting susceptibility to the direct cytotoxic effects of STZ. Accordingly, we sought to determine whether GIP receptor signaling or [DAla ]GIP-mediated up-regulation of p-cell gene expression was stimulating a factor(s) responsible for increasing susceptibility to MLD-STZ-induced diabetes. Whereas pretreatment with [DAla ]GIP potentiated subsequent MLD-STZ-induced diabetes, concomitant administration of [DAla 2]GIP (Fig. 11) or GIP(l-42) (Fig. 10) with a single high dose of STZ had no effect on the induction of diabetes relative to their respective controls. These findings were interpreted as suggesting that GIP receptor signaling does not interfere with direct STZ action and/or metabolism at the p-cell per se, but that [DAla ]GIP indirectly exacerbates diabetes by evoking effects on p-cell gene expression that synergize with STZ and thereby, facilitate greater P-cell death. Specifically, it was proposed that [DAla 2]GIP treatment in the MLD-STZ diabetic rat exacerbates STZ-induced P-cell death via up-regulation of GLUT2 transporters in the p-cell. This proposal was based on several lines of evidence indicating that differential sensitivity to STZ was correlated with the level of GLUT2 expression and empirical evidence suggesting that GLUT2 is a key target structure for STZ-induced p-cell toxicity (206,240-242). Although there is no direct evidence demonstrating that STZ is taken up by the P-cell through the glucose transporter GLUT2, studies using GLUT2 knockout mice demonstrated that STZ toxicity toward p -cells d epends on t he e xpression o f G LUT2 (243) a nd i nsulinoma (RTN) c ell lines engineered for overexpression of GLUT2 demonstrated increased susceptibility to STZ-induced destruction compared to P-cell lines not expressing this transporter (244). This idea is further supported by the fact that insulin-treated rats, which show decreased expression of P-cell GLUT2, were substantially more resistant to STZ (245) and human islets, which are much more resistant to STZ than rodent islets, express lower levels of GLUT2 (246). Consistent with this model, [DAla2]GEP increased GLUT2 expression in vivo (Fig. 12,13). These findings suggest that up-regulation of GLUT2 expression may increase the capacity for transport of STZ into the P-cell and thus, may facilitate greater P-cell death in [DAla2]GIP-treated animals. The patterns of GLUT2 mRNA and protein expression in vivo differed in that [DAla2]GEP-stimulated changes in islet GLUT2 mRNA expression were 2.6-fold greater than [DAla2]GIP-stimulated changes in GLUT2 protein content. A similar observation has been reported for glucose-induced GLUT2 expression in Wistar rat islets (247). Several mechanisms could account for the differential response of mRNA and protein induction, including rates of mRNA and protein turnover. In fact, accumulation of large amounts of protein beyond a certain physiological threshold may result in regulated degradation, thereby preventing protein crowding. Accordingly, GLUT2 protein expression may be regulated in such a manner as to prevent over expression of this protein beyond a physiological threshold. It is also possible that there are differences in GLUT2 protein distribution. However, in the current study, total cellular protein was measured, as it has b een previously reported that GLUT2 surface expression in P-cells is via the constitutive pathway and 95% of GLUT2 is expressed on the plasma membrane (248). The current finding, of increased GIP-induced GLUT2 expression, contrasts with a previous report in the literature demonstrating that P-cell GLUT2 mRNA (75) in RTN 1046-38 cells was unaffected by GIP treatment. The reason for the complete lack of response for GIP on GLUT2 expression in the study of Wang et al (75) may lie in the cell line used. Wang et al used the clonal rat insulinoma P-cell line RTN 1046-38 (passages 10-20), which may exhibit cell-specific effects in response to GIP that differ compared to the cell line used in this report, INS-1(832/13) cells (passages 50-65). Importantly, the RTN 1046-38 cell line is essentially glucose-unresponsive (75), therefore effects of GIP on gene expression may be very different from the glucose-responsive INS-1(832/13) cell line. Regardless, consistent with the proposed model that susceptibility to STZ is positively correlated with the level of GLUT2 expression, the current findings suggest an underlying mechanism for the [DAla ]GIP-mediated exacerbation of MLD-STZ-induced diabetes. Additionally, GIP may play a role in the up-regulation of p-cell GLUT2 transport activity. Cheeseman et al showed that vascular infusion of GIP produced a significant increase in the Vmax for intestinal basolateral membrane glucose transport (249) and that this up-regulation was correlated with increased glucose flux by the tissue (250). Therefore, in vivo [DAla 2]GIP may increase P-cell GLUT2 transport activity and thus the Vmax for STZ transport across the P-cell. Further experimentation is necessary to answer this important question. Finding an increased expression of islet GLUT2 in response to [DAla ]GIP treatment and exacerbation of MLD-STZ-induced diabetes in the same animals, although provocative, does not prove a cause and effect relationship. That said, [DAla ]GIP treatment surprisingly decreased STZ susceptibility in the single high-dose STZ-induced diabetic rat. [DAla2]GEP treatment in the single high-dose STZ-induced diabetic rat significantly protected against the metabolic deterioration mediated by both the lowest (30mg/kg; Fig. 14) and moderate (45mg/kg; Fig. 15) doses of STZ, as indicated by improved fed blood glucose values and glucose tolerance. In addition, improvements in both insulin responses during the OGTT and pancreatic insulin content (Fig. 16) in animals administered 45mg/kg STZ suggests protection against p-cell destruction. Reversal of STZ-induced stimulation of caspase-3 activity in INS-1(832/13) cells with [DAla 2] GEP (Fig. 19) supports an anti-apoptotic mechanism of action. Consistent with the present study, STZ-induced P-cell apoptosis has also been reported in vivo in the MLD-STZ model (148,117) and in vitro in cultured insulinoma cell lines (83,237,251,252). These protective effects of [DAla 2] GEP in STZ-induced diabetes indicate the diabetic effects of this peptide may be specific to the MLD-STZ model. It has been previously shown that a gradual decrement of both GLUT2 protein and mRNA expression occurred in the islets of MLD-STZ-treated mice, and that the extent of reduction increased with the number of STZ injections administered and increased over time, after the last (fifth) STZ injection (240,242). Consistent with these findings, [DAla2]GEP-stimulated up-regulation of GLUT2 expression may have restored the decreased GLUT2 protein levels in the islets of MLD-STZ-treated rats, thereby enhancing capacity for STZ transport into the p-cell with each subsequent injection, and thus facilitating greater STZ-induced p-cell death. Although this effect of [DAla2]GEP treatment is detrimental in MLD-STZ-induced diabetes, there is data to support the notion t hat p harmacological m eans t o s timulate e xpression o f t he G LUT2 g ene w ould prove to be beneficial for the treatment of type 1 diabetes. During the prediabetic period of type 1 diabetes, there is an impaired glucose-induced insulin secretion in rodent models of type 1 diabetes (253-256), suggesting that the glucose-sensing apparatus in p-cells may be selectively impaired during diabetes development. Studies have shown that during diabetes in the B B rat and the NOD mouse, there is selective loss of GLUT2 from the p-cell membrane preceding clinical onset (240,257). It is conceivable that compared to the MLD-STZ diabetes model, NOD mouse, and B B rat, the impaired response to a glucose load during the clinically silent prediabetic period in patients with type 1 diabetes (258) m ay r esult from 1 oss or dysfunction o f molecules involved i n glucose transport. Consequently, these data support the potential utilization of [DAla2]GLP as a treatment option in type 1 diabetes. On the other hand, the protective effects of [DAla 2] GIP in STZ-induced diabetes also indicate that other factors, in addition to up-regulation of GLUT2 expression, may be involved in the increased susceptibility to MLD-STZ diabetes in [DAla2]GEP-treated rats. Several pieces of evidence in the literature are also inconsistent with increased GLUT2 being the sole factor: firstly, strains of mice with different susceptibility to STZ did not show differences in 3-O-methylglucose transport or immunoreactive GLUT2 protein levels (239); secondly, human fetal islets grafted into nude mice were not destroyed by injections of STZ, in spite of adequate uptake of the drug by human tissue (259); and thirdly, although the presence of GLUT2 may account for part of the specific vulnerability of the B-cell, GLUT2 is also present in the liver and kidney, tissues that are relatively resistant to STZ damage. In fact, the much greater sensitivity of the B-cell to STZ is probably due to its very low level of antioxidant enzyme expression and activity (260,261), which leaves it unable to inactivate reactive oxygen species (ROS) generated by STZ (208). Thus, alternative mechanisms probably also contribute to the diabetic effects of [DAla 2]GIP in the MLD-STZ diabetic rat. Interestingly, [DAla 2] GIP treatment slightly worsened the diabetic phenotype elicited by the highest single dose (60mg/kg; Fig 17,18) of STZ. The type of cell death triggered by the different doses of STZ may underlie these paradoxical findings. Cell death may occur by either of two mechanisms: necrosis caused by ischemic, chemical, physical, or thermal cell injury; and apoptosis, a programmed cell death, which is required for the normal maintenance of development and homeostasis in a cell system, an organ, or a whole individual (262). Lennon et al demonstrated that cell death induced by a variety of agents may take the form of either apoptosis or necrosis and the intensity of the injury may determine which pathway is triggered (263). Apoptotic cell death was found to occur at low levels of these agents, while at higher levels necrosis was the death mechanism triggered (263). Hence, cells that are merely injured by agents, and not killed directly, have the capacity to activate programmed cell death, whereas cells receiving greater injuries do not, and undergo uncontrolled, irreversible cell death. In support of this concept, it has been reported that high concentrations of hydrogen peroxide induced necrotic cell death while low concentrations induced apoptosis, indicating that the degree of cytotoxicity triggered by hydrogen peroxide determines which pathway of cell death is triggered (264). In addition, Saini et al demonstrated that STZ induces apoptosis at low doses (15mM) and necrosis at high doses (30mM) in the pancreatic Pcell line, INS-1 (252). Hence, an argument can be raised that [DAla 2]GIP protects against apoptotic cell death, elicited by the low and moderate doses of STZ, and potentiates necrotic cell death, elicited by the high dose of STZ. In an attempt to support the idea, an investigation into the effects of [DAla 2]GIP treatment in the presence of low (15mM) and high (30mM) doses of STZ was undertaken in vitro, as used by Saini et al (252). However, the P-cell line used, ENS-1(832/13), was too sensitive to the toxicity of STZ at these concentrations, and all the cells died. Nonetheless, the present report has shown evidence indirectly •y supporting an anti-apoptotic mechanism for the protective effects of [DAla ]GEP in the STZ-induced diabetic rat. Moreover, the lack of protection against, and slight worsening of diabetes induced by the highest dose of STZ suggests a detrimental role for [DAla 2] GEP in necrotic cell death. From the current results it is clear that further studies are warranted, both to understand the different responses obtained with [DAla 2] GEP and GEP(l-42) in different STZ models and to develop a better understanding of the mechanisms underlying the beneficial effects of GIP in type 1 diabetes. Firstly, it is unclear as to whether the •y diabetogenic effect of the long-acting analog, [DAla ]GEP, was due to its time of action or to the specific substitution of a D-alanine at the two position. A comparison of the diabetogenic effects i n t he M LD-STZ m odel a mong a s eries o f a nalogs with d ifferent substitutions at the 2-position in GEP and susceptibilities to DPIV degradation should provide important information regarding importance of in vivo half life. Similarly, by testing the effects of a series of analogs with different substitutions at the 2-position but similar DPFV-resistance it should be possible to establish the importance of the specific amino acid substitution to the diabetogenic effect. As well, a study examining GLUT2 protein expression over the course of STZ administration in the MLD-STZ diabetic rat will provide important information regarding the ability of [DAla ]GIP treatment to restore islet GLUT2 protein levels, and thus facilitate STZ transport into and destruction of P-cells. Furthermore, identification of the differences in cellular responses to the two peptides resulting in the differential susceptibility to STZ is more complicated, but gene and protein array studies on islets isolated from [DAla 2]GIP and GEP(l-42) animals could provide significant information on specific pathways that differ. To develop a better understanding of the mechanisms underlying the positive effects of long-term treatment with GIP(l-42) on P-cell regeneration in the STZ-induced diabetic rat, islet neogenesis, P-cell replication, and apoptosis should be examined by immunohistochemistry. It is important to note that treatment should commence immediately following the last STZ administration, in order to exclude any potentiation of M L D STZ action from masking the full potential of GIP's P-cell regenerative effects. Additionally, the p-cell protective effects of [DAla 2]GIP in the single high dose STZ (i.v.) diabetic rat should be examined by immunohistochemistry and a T U N E L assay to establish in vivo the role of anti-apoptotic effects. In addition, a 48 hour infusion study of GEP(l-42) post STZ administration would provide information on the efficacy of GIP treatment in the prevention of STZ-induced p-cell destruction. Similar in vivo studies on the effect of [DAla 2] GIP and GIP(l-42) in the MLD-STZ diabetic mouse and other models of type 1 diabetes, such as the B B rat or NOD mouse could also provide important information regarding specificity of the observed effects and anti-diabetic potential of GIP treatment in this disease. A number of uncertainties remain regarding the mode of action of STZ and modulation of its action, including the conclusive demonstration of STZ transport into the P-cell via GLUT2 and the potential stimulation of STZ transport into the P-cell by [DAla 2]GIP and GIP(l-42), coupled with studies on the up-regulation of GLUT2 protein expression and/or GLUT2 transport activity. In conclusion, the findings in these studies exemplify the importance of the pleiotropic effects of GIP receptor signaling and shed light on the possibility of utilizing GIP therapy in type 1 diabetes. The GIP analog, [DAla 2]GIP, was demonstrated to exhibit both paradoxical diabetic and anti-diabetic effects in two animal models of experimentally-induced diabetes, the multiple low-dose streptozotocin diabetic rat and the single high dose streptozotocin diabetic rat, respectively. While synergistic action with direct cytotoxic effects of STZ was discounted as a potential mechanism, increased P-cell GLUT2 expression was implicated as one of the underlying mechanisms in [DAla2]GIP-mediated exacerbation of MLD-STZ-induced diabetes. Anti-apoptotic effects of [DAla 2]GIP offered a basis for the protective effects of this peptide against STZ-induced P-cell destruction. Importantly, treatment with the native hormone, GIP(1-42), significantly improved the diabetic phenotype after 4 weeks of treatment relative to MLD-STZ controls, supporting a potential role for GIP therapy in type 1 diabetes. Further experimentation is warranted to reconcile the paradox involving diabetic and anti-diabetic effects of the [DAla 2] analog in type 1 diabetes, and to illuminate the beneficial effects of GIP in the treatment of this disease. GIP Receptor mRNA Expression in Type 2 Diabetes The development of P-cell dysfunction is fundamental to the pathogenesis of type 2 diabetes. One of the p-cell defects linked to type 2 diabetes is a reduced incretin effect, specifically involving GIP-stimulated insulin secretion. Chronic hyperglycemia is believed to be a major factor contributing to the tissue dysfunction, also known as the glucose toxicity hypothesis. Consistent with this hypothesis, one of the focuses of studies described in this Thesis was to investigate whether the loss of GIP responsiveness at the level of the p-cell in rats is an acquired defect secondary to the chronic hyperglycemia in type 2 diabetes. Therefore, we addressed this question indirectly by investigating whether this defect could be reversed with normalization of blood glucose levels in the ZDF rat. To corroborate Lynn et al's data that GIP receptor (GIPR) mRNA expression is down-regulated by glucose in a concentration dependent manner in INS-1(832/13) cells (197), we have shown that GIPR mRNA expression was down-regulated in proportion to the degree of hyperglycemia present in vivo. ZDF rats, which are severely diabetic (Fig. 20), displayed a 94% reduction in GIPR mRNA expression (Fig. 21), and Zucker obese rats, which exhibit a less severe glucose intolerance (Fig. 20), displayed a 48% reduction in GIPR mRNA expression relative to lean controls (Fig. 21). Lynn et al previously observed a 75% decrease in V D F rats (195). This difference in the degree of GIPR down-regulation may be attributed to the severity of diabetes exhibited in the different substrains of the Zucker rat; the ZDF rats displayed higher peak blood glucose values by approximately 5mM during an IPGTT and higher fed blood glucose values by approximately 8mM, compared to the V D F rats (data not shown). These data lend support to the idea that graded levels of hyperglycemia lead to progressive losses of GIPR expression. The causal role of hyperglycemia in loss of GIPR expression in the ZDF rat is strongly suggested by restoration after 2 weeks treatment with phlorizin, a drug that normalizes blood glucose without increasing plasma insulin or changing plasma free fatty acid or triglyceride levels. Phlorizin treatment effectively lowered blood glucose in the ZDF rat over the course of the experiment to levels comparable to lean controls (Fig. 22). Because previous studies have shown that phlorizin selectively lowers glucose without affecting plasma triglyceride and free fatty acid levels (170,201), we omitted analysis of these parameters from the present experiments. After two weeks of treatment, islet GIPR mRNA expression levels in the ZDF rat were restored to 83% of lean control values (Fig. 23), suggesting that GIPR down-regulation is secondary to chronic hyperglycemia. The present study is consistent with reports in another model of hyperglycemia, the 90% pancreatectomized rat, which have shown that gluocotoxicity is reversible and that restoration of glucose control partially improves P-cell function. Weir and colleagues demonstrated that hyperglycemia causes dedifferentiation of the P-cell in the 90% pancreatectomized rat, and that the observed changes in gene expression, including decreased expression of genes that optimize P-cell function and increased expression of normally suppressed genes, are reversible with normalization of blood glucose levels by phlorizin (168,169). Furthermore, their results show that graded levels of chronic hyperglycemia lead to progressive loss of p-cell differentiation, lending further support to our findings. For instance, the mRNA expression of several genes important for glucose-stimulated insulin secretion (GLUT2, glucokinase, insulin, ion channels) and genes involved in transcription of insulin and various metabolic enzymes (PDX-1, Nkx6.1, Pax6, Beta2) were gradually decreased in the presence of increasing levels of hyperglycemia (169). Interestingly, GEPR mRNA expression appears to be more severely down-regulated by chronic hyperglycemia in the ZDF rat (94% of lean values) compared to the down-regulation of gene expression in the pancreatectomized rat (40-60% of sham values). It is important to note, however, that the hyperglycemia in the pancreatectomized rat was not as severe as that exhibited in the ZDF rat, with fed blood glucose levels reaching only 16mM compared to 28mM glucose in the latter model. Therefore, it appears that the greater the severity of hyperglycemia, the greater the down-regulation in gene expression. Restoration of GEPR expression was correlated with restoration of pancreatic GEP responsiveness. Insulin secretion in response to 20pM GEP in the presence of 8.8mM glucose was significantly improved in the perfused pancreas of the phlorizin-treated ZDF rats (Fig. 25), and is reflected as a 1.9-fold increase in the area under the curve of the insulin secretion from the treated animals' pancreata. These findings indicate that GEP insensitivity in the pancreatic islet of ZDF rats can be reversed with normalization of blood glucose levels. Although GEPR protein levels were not measured in the present study, Lynn e t a 1 w ere able t o d emonstrate t hat d own-regulation i n G EPR m R N A w as correlated with decreased protein levels in islets (195) and in clonal p-cells that induction of GEPR mRNA expression is directly linked to similar increases in cell surface expression using radioligand saturation binding curves (197). Thus, we suspect that phlorizin-mediated changes in mRNA expression are likely to be associated with similar changes in functional expression of the GEPR. In support of our study, Pospisilik et al demonstrated amelioration of GIP insensitivity in V D F pancreata after 12 weeks of treatment with the DPIV inhibitor, P32/98 (265). Taken together, i f a similar down-regulation of receptor expression occurs in humans, these findings offer hope that the GIP resistant state can be reversed in type 2 diabetic patients. In fact, treatment with sulphonylureas was reported to improve P-cell responsiveness to GIP in type 2 diabetic patients (139). Furthermore, this improved GIP responsiveness was correlated with improved glucose homeostasis, supporting the idea that this P-cell defect may be reversed with tight metabolic control in patients with type 2 diabetes. Not only is the GIP resistance implicated in type 2 diabetes, but the incretin effect of GIP is also lost in diabetic patients with phenotypes and etiologies distinct from obese type 2 diabetics (266), in type 1 diabetics (267,268), in the perfused STZ-induced diabetic r at p ancreas (269), and i n i solated d iabetic B B r at i slets (270). T he f act t hat abnormal enteroinsular signaling is also present in different forms of diabetes, each with an islet defect distinct from typical type 2 diabetes, lends support to our hypothesis that chronic hyperglycemia m ay b e the c ommon m etabolic abnormality responsible for the defective response to GIP. In fact, any maneuver that lowers glucose levels in diabetes appears t o be a ssociated w ith a n i mproved i ncretin e ffect. K osaka et a 1 d emonstrated improved insulin secretion in response to meals or oral glucose after hyperglycemia was reduced by diet, sulfonylurea treatment, or insulin administration in patients with overt maturity-onset diabetes (271), providing strong support that hyperglycemia plays a role in the reduced incretin effect in type 2 diabetes. Based on the preliminary nature of the data, no conclusions can be made regarding the molecular basis by which normalization of glucose leads to restoration of GIPR mRNA expression. However, Lynn et al recently demonstrated a novel pathway for stimulation of GIPR expression at normal glucose levels through fat-stimulated P P A R a activation (197). Interestingly, P P A R a is unable to reverse the GIPR down-regulation associated with hyperglycemia (197). The findings that high glucose levels lead to down-regulation of PPARa in isolated islets and INS(832/13) cells (200) and that P P A R a is down-regulated in pancreatectomized (168) and ZDF rats (199) provided a link between the above observations. Therefore, in the presence of hyperglycemia, decreased levels of P P A R a expression may lead to down-regulation of GIPR expression. Consistent with this hypothesis, the reduction in P P A R a expression is reversible upon glucose normalization (168), which may explain the observed increase in GIPR expression and the restoration of GIP responsiveness in the phlorizin-treated ZDF rat islets. However, whether GIPR expression is directly or indirectly stimulated by P P A R a has yet to be elucidated. The physiological significance of PPARa-regulated GIPR expression is ambiguous at the present time. P P A R a only stimulates GIPR expression at low glucose levels, which at first seems paradoxical because GIP only stimulates insulin release at higher glucose levels. However, GIPR signaling stimulates cAMP production (54) and activation of M A P kinase (56) and PLA2 (55) at OmM glucose, suggesting multifaceted action of GIP at the P-cell. Recently, GIP has been shown to stimulate fatty acid oxidation within the p-cell (272), and thus it has been proposed that GIP may act to enhance ATP levels in the p-cell to potentiate glucose-stimulated insulin secretion. However, this hypothesis should be verified with experimentation to clarify GIP's role in P-cell fat metabolism. A question that remains is whether reversal of GIPR down-regulation can slow or prevent the progression of type 2 diabetes. Type 2 diabetes is characterized by progressive increases in fasting and postprandial plasma glucose concentrations, which typically develop over a period of several years (273). It is widely accepted that impairments in both insulin secretion and insulin action play a critical role in the pathogenesis of diabetes (273). Impaired glucose tolerance (IGT) represents an intermediate stage in the progression from normal glucose tolerance (NGT) to type 2 diabetes (274). Certain individuals, with a genetic predisposition to type 2 diabetes, are insulin resistant and have a relatively low insulin secretion but maintain NGT and never progress to develop diabetes. These individuals are able to maintain NGT in the presence of the development of insulin resistance by compensating with increased insulin secretion (274,275). However, in addition to a primary defect in insulin secretion, progression from NGT to IGT and then to diabetes is associated with a further impairment in insulin secretion and only a modest decline in the existing insulin resistance, such that insulin secretion is inappropriate for the degree of insulin action. The loss of early insulin secretion leads to the development of postprandial hyperglycemia and IGT (274). The mechanism of glucotoxicity then acts as a self-perpetuating factor and contributes to the worsening of insulin secretion. Eventually, the defect in insulin secretion reaches a critical threshold and clinical hyperglycemia and diabetes develops (274). These findings demonstrate the critical role of impairments in early insulin secretion in the pathogenesis of type 2 diabetes and that strategies aimed at enhancing early insulin secretion may be effective approaches to stemming the disease. The slow course of the progression to diabetes suggests that interventions can be instituted at an early stage to either slow or prevent t he d evelopment o f t he d isease. T he fact t hat G IP i s i mportant i n first p hase insulin secretion (233) and GIP insensitivity at the pancreatic P-cell is secondary to the elevated glucose levels in type 2 diabetes suggests that down-regulation of the GEPR may be implicated in the progression of this disease. As such, pharmacological interventions aimed at preventing the down-regulation of the GIPR may be an effective approach to enhance insulin secretion and thus, slow or prevent the development of clinical diabetes. Studies aimed at answering whether GEPR dysfunction is implicated in the pathogenesis of diabetes are certainly warranted. GIP therapy has the potential to play a significant role as a treatment option for type 2 diabetes. Present treatment regimens for hyperglycemia in type 2 diabetes include several different classes of oral antihyperglycemic agents: thiazolidinediones, sulphonylureas, biguanides, and oc-glucosidase inhibitors. Each of these classes is effective in lowering blood glucose concentrations and they act by different mechanisms: thiazolidinediones are insulin sensitizers, sulphonylureas are insulin secretogogues, biguanides decrease hepatic glucose output, and oc-glucosidase inhibitors delay carbohydrate absorption (276). Although tight glycemic control reduces the risk of microvascular complications in type 2 diabetics, most patients do not maintain glycosylated hemoglobin (<7%), even when treated intensively with sulphonylureas, biguanides, or insulin monotherapy (276). The difficulty in normalizing blood glucose values arises in part because of a progressive decline in p-cell function (276). As a result, combination therapy with two or more agents with different, complementary mechanisms of action has arisen as an option to achieve glycemic goals. Unfortunately, none of these treatment options act directly on the P-cell to combat the progressive decline in pancreatic function seen in type 2 diabetes. However, the recent advances made on the utility of DPIV inhibitors and GLP-1, not only in the treatment of diabetes in animal models (277-281) but also in humans (282-285), offers promise to circumvent this problem and improve P-cell function over time. In addition, in light of the effects of GEP in the regulation of p-cell function and our finding that GEP responsiveness at the P-cell can be restored upon glucose normalization in type 2 diabetes, the use of DPEV-resistant GEP analogs alone or in combination with traditional oral therapy may b e an effective treatment strategy for type 2 diabetes. In fact, recent studies on the additive effects of combination therapy with metformin and GLP-1 (286) or metformin and DPIV inhibitors (287) on glycemic control suggests that combination therapy of GEP and DPEV inhibitors is a good candidate for type 2 diabetes. Based on the observations made in this report on the reversibility of GEP dysfunction at the pancreatic p-cell in type 2 diabetes, it remains to be seen whether DPIV-resistant GEP analogs alone or in combination therapy can advance to clinically useful anti-diabetic therapeutics. Demonstration of phlorizin-induced prevention of, or recovery from a GEP insensitive state is novel and of great importance to the field of diabetes research. Future studies to further characterize the metabolic control of GEPR down-regulation in diabetes are certainly justified. Firstly, in order to determine whether GEPR down-regulation is a characteristic p-cell defect in all diabetic states, r egardless of phenotype and etiology, future studies should be performed in the STZ-induced diabetic rat and type 1 diabetic animal models, such as the B B rat and the NOD mouse. The observation of GEPR down-regulation in these animal models would corroborate our hypothesis that GEPR expression is negatively regulated by chronic hyperglycemia and is secondary to the metabolic milieu in diabetes. Secondly, a longitudinal study on the development of GIPR down-regulation in the ZDF rat should be performed to monitor the progressive down-regulation of the GIPR in relation to the development of IGT and diabetes. Such information will prove informative for preventative therapeutic interventions. Thirdly, to determine i f GIPR down-regulation is implicated in the pathogenesis of diabetes, prevention of GIPR down-regulation should be examined in the ZDF rat. In order to avoid global changes in p -cell function induced by treatments that normalize blood glucose, gene therapy should be instituted to express the GIPR gene in the p-cells of ZDF rats. The GIPR gene should be expressed after birth using a virus vector and the metabolic deterioration of the animals monitored longitudinally to investigate whether prevention of GIPR down-regulation slows or prevents diabetes. Fourthly, the mechanisms by which P P A R a up-regulates and glucose down-regulates expression of the GIPR gene should be thoroughly investigated to develop an understanding of the molecular basis for GIPR expression and thus, indicate potential therapeutic targets for diabetes treatment. Roduit et al demonstrated that down-regulation of P P A R a by high glucose leads to a down-regulation of genes normally controlled by this nuclear transcription factor (200), and thus, it would be of interest to determine i f ZDF rat islet P P A R a mRNA levels are up-regulated in a parallel fashion with GIPR mRNA levels in response to glucose normalization. Additionally, it is important to determine w hether t he a ction o f P P A R a o n GIPR e xpression is a d irect e ffect o r i f i t occurs via activation.of another or in conjunction with other transcription factors on the GIPR gene promoter. Finally, the long-term effects of DPFV Inhibitor therapy on the reversal of GIPR down-regulation should be examined in the V D F rat. As previously mentioned, Pospisilik et al demonstrated restoration of pancreatic GIP sensitivity in the V D F rat pancreas after 12 weeks of treatment with the DPIV Inhibitor P32/98 (265), and thus, an investigation is currently being undertaken to determine whether amelioration of a GIP insensitive state is linked to restoration of GIPR expression. In addition, GIP therapy would be particularly useful after restoration of GIP function at the level of the p-cell, and c ould i mprove i nsulin s ecretion a nd glucose t olerance i n a n additive m anner w ith traditional oral therapy to combat the diabetic state. Accordingly, a long-term study of combination therapy with a DPIV-resistant GIP analog and an anti-hyperglycemic agent in ZDF rats is warranted. In conclusion, GIPR mRNA expression was measured in ZDF, Zucker obese, and Zucker 1 ean r ats. It w as f ound t hat g raded h yperglycemia 1 eads top regressive d own-regulation of the GIPR. Accordingly, it was hypothesized that normalization of glucose levels would lead to reversal of the down-regulation of GIPR mRNA expression in the ZDF rat islet. Treatment for two weeks with phlorizin, a drug that normalizes blood glucose without increasing plasma insulin or changing plasma free fatty acid or triglyceride levels, led to restoration of GIPR expression, suggesting that GIPR down-regulation is secondary to the chronically elevated blood glucose levels of type 2 diabetes. Pancreas perfusions performed in phlorizin-treated ZDF rats demonstrated improved GIP sensitivity at the pancreatic islet. Taken together, these findings suggest that GIPR down-regulation in type 2 diabetes is secondary to chronic hyperglycemia and that tight glycemic control leads to restoration of GIPR expression and subsequently, to improved GIP sensitivity at the pancreatic islet. Demonstration of recovery of biological activity of GIP is of fundamental importance in type 2 diabetes and indicates great potential for the use of GIP analogs in the treatment of this disease. References 1. Bayliss W M , Starling E H (1902). Mechanism of pancreatic secretion. J Physiol (Lond) 28:235-334. 2. Moore B, Edie ES, Abram JH (1906). On the treatment of diabetes mellitus by acid extract of duodenal mucous membrane. Biochem J 1:28-38. 3. Zunz E, La Barre J. (1929). Contributions a l'etude des variations physiologiques de la secretion interne du pancreas: relations entre les secretions externe et interne du pancreas. Arch Int Physiol Biochim 31:20-44. 4. La Barre J, Still E U . (1930) Studies on the physiology of secretin. Am J Physiol 91:649-653. 5. Loew ER, Gray JS, Ivy A C . (1940). Is a duodenal hormone involved in carbohydrate metabolism? Am J Physiol 129:659-663. 6. Mclntyre N , Holsworth DC, Turner DS. (1964). New interpretation of oral glucose tolerance. Lancet 2:20-21. 7. Elrick H , Stimmler L, Hlad CJ, Arai Y . (1964). Plasma insulin responses to oral and intravenous glucose administration. J Clin Endocrinol Metab 24:1076-1082. 8. Perley MJ , Kipnis D M . (1967). Plasma insulin responses to oral and intravenous glucose. Studies in normal and diabetic subjects. J Clin Invest 46:1954-1962. 9. Creutzfeldt W. (1979). The incretin concept today. Diabetologia 16:75-85. 10. Unger R H , Eisentraut A M . (1969). Entero-insular axis. Arch Intern Med 123:261-266. 11. Kosaka T, Lim RKS. (1930). Demonstration of the humoral agent in fat inhibition of gastric acid secretion. Proc Soc Exp Biol Med 27: 890-891. 12. Brown JC, Pederson R A . (1970a). A multiparameter study on the action of preparations containing cholecystokinin-pancreozymin. Scand J Gastroenterol 5: 537-541. 13. Brown JC, Mutt V , Pederson R A . (1970b). Further purification of a polypeptide demonstrating enterogastrone activity. J Physiol (Lond) 209: 57-64. 14. Brown JC, Dryburgh JR. (1971). A gastric inhibitory polypeptide. II. The complete amino acid sequence. Can J Biochem 49:867-872. 15. Dupre J, Beck JC. (1966). Stimulation of release of insulin by an extract of intestinal mucosa. Diabetes 15:555-559. 16. Rabinovitch A , Dupre J. (1972). Insulinotropic and glucagonotropic activities of crude preparation of cholecystokinin-pancreozymin. Clin Res 20:945. 17. Dupre J, Ross SA, Watson D, Brown JC. (1973). Stimulation of insulin secretion by gastric inhibitory polypeptide in man. J Clin Endocrinol Metab 37:826-828. 18. Pederson R A , Schubert HE, Brown JC. (1975). Gastric inhibitory polypeptide. Its physiological release and insulinotropic action in the dog. Diabetes 24:1050-1056. 19. Elahi D, Anderson DK, Brown JC, Debas H T , Hershcopf RJ, Raizes GS, Tobin JD, Andres R. (1979). Pancreatic a- and 6-cell responses to GIP infusion in normal man. Am J Physiol 237: E185-E191. 20. Pederson RA, Brown JC. (1976). The insulinotropic actions of gastric inhibitory polypeptide in the perfused isolated rat pancreas. Endocrinology 99: 780-785. 21. Brown JC, Pederson RA. (1976). GI hormones and insulin secretion. In: James V HT (ed) 5 th International C ongress o n Endocrinology, v ol 2. Excerpta Medica, Hamburg, pp 568-570. 22. Jornvall H , Carlquist M , Kwauk S, Otte SC, Mcintosh C H , Brown JC, Mutt V . (1981). Amino acid sequence and heterogeneity of gastric inhibitory polypeptide (GEP). FEBSLetters 123: 205-210. 23. Higashimoto Y , Liddle RA. (1993). Isolation and characterization of the gene encoding rat glucose-dependent insulinotropic polypeptide. Biochem Biophys Res Commun 193: 182-190. 24. Inagaki N , Seino Y , Takeda J, Yano H , Yamada Y , Bell GI, Eddy R L , Fukushima Y , Bryers M G , Shows TB, Imura H . (1989). Gastric inhibitory polypeptide: structure and chromosomal localization of the human gene. Mol Endocrinol 3: 1014-1021. 25. Polak J M , Bloom SR, Kuzio M , Brown JC, Pearse A G E . (1973). Cellular localization of gastric inhibitory polypeptide in the duodenum and jejunum. Gut 15: 284-288. 26. Buchan A M , Polak JM, Capella C, Solcia E, Pearse A G . (1978). Electronimmunocytochemical evidence for the K cell localization of gastric inhibitory polypeptide (GIP) in man. Histochemistry 56: 37-44. 27. Tseng CC, Jarboe L A , Landau SB, Williams E K , Wolfe M M . (1993). Glucose-dependent insulinotropic peptide: structure of the precursor and tissue-specific expression in the rat. Proc Natl Acad Sci USA 90: 1992-1996. 28. Yeung C M , Wong CK, Chung SK, Chung SS, Chow B K . (1999). Glucose-dependent insulinotropic polypeptide gene expression in the stomach: revealed by a transgenic mouse study, in situ hybridization and immunohistochemical staining. Mol Cell Endocrinol 154: 161-170. 29. Pederson RA. (1994). GIP. In Gut Peptides. Ed. Walsh J & Dockray G, pp 217-259. Raven Press, New York. 30. Kuzio M , Dryburgh JR, Malloy K M , Brown JC. (1974). Radioimmunoassay for gastric inhibitory polypeptide. Gastroenterology 66: 357-364. 31. Morgan L M , M orris B A , M arks V . (1978). R adioimmunoassay o f gastric inhibitory polypeptide. Ann Clin Biochem 15: 172-177. 32. Jorde R , B urhol P G, S chulz T B. (1983). F asting and p ostprandial p lasma GEP values in man measured with seven different antisera. Regul Pept 7:87-94. 33. Amland PF, Jorde R, Revhaug A , Myhre ESP, Burhol PG, Giercksky K E . (1984). Fasting and postprandial GEP values in pigs, rats, dogs, and man measured with five different GEP antisera. Scand J Gastroenterol 19:1095-1098. 34. Kieffer TJ, Pederson RA, Buchan A M J . (1992). Glucose-dependent insulinotropic polypeptide release from cultured porcine gut endocrine cells. Can J Physiol Pharmacol 70:Axiii-Axiv. 35. Cataland S, Crockett SE, Brown JC, Mazzaferri EL . (1974). Gastric inhibitory polypeptide (GEP) stimulation by oral glucose in man. J Clin Endocrinol Metab 39: 223-228. 36. Anderson DK, Elahi D, Brown JC, Tobin JD, Andres R. (1978). Oral glucose augmentation of insulin secretion. Interactions of gastric inhibitory polypeptide with ambient glucose and insulin levels. J Clin Invest 62: 152-161. 37. Sykes S, Morgan L M , English J, Marks V . (1980). Evidence for preferential stimulation of gastric inhibitory polypeptide secretion in the rat by actively transported carbohydrates and their analogues. J Endocrinol 85: 201-207. 38. Creutzfeldt W, Ebert R. (1988). The incretin concept. Adv Metab Disord 11: 333-367. 39. Ross SA, Shaffer RH. (1981). The importance of triglyceride hydrolysis for the release of gastric inhibitory polypeptide. Gastroenterology 80: 108-111. 40. Thomas FB, Mazzaferri EL, Crockett SE, Mekhjian HS, Gruemer HD, Cataland S. (1976). Stimulation of secretion of gastric inhibitory polypeptide and insulin by intraduodenal amino acid perfusion. Gastroenterology 70: 523-527. 41. Thomas FB, Sinar D, Mazzaferri EL , Cataland S, Mekhjian HS, Caldwell JH, Fromkes JJ. (1978). Selective release of gastric inhibitory polypeptide by intraduodenal amino acid perfusion in man. Gastroenterology 74: 1261-1265. 42. Usdin TB, Mezey E, Button DC, Brownstein MJ , Bonner TI. (1993). Gastric inhibitory polypeptide receptor, a member of the secretin-vasoactive intestinal peptide receptor family, is widely distributed in peripheral organs and the brain. Endocrinology 133: 2861-2870. 43. Gremlich S, Porret A , Hani EH, Cherif D, Vionnet N , Froguel P, Thorens B (1995). Cloning, functional expression, and chromosomal localization of the human pancreatic islet glucose-dependent insulinotropic polypeptide receptor. Diabetes 44: 1202-1208. 44. Yamada Y , Hayami T, Nakamura K , Kaisaki PJ, Someya Y , Wang CZ, Seino S, Seino Y . (1995). Human gastric inhibitory polypeptide receptor: cloning of the gene (GIPR) and cDNA. Genomics 29: 773-776. 45. Yasuda K , Inagaki N , Yamada Y , Kubota A , Seino S, Seino Y . (1994). Hamster gastric inhibitory polypeptide receptor expressed in pancreatic islets and clonal insulin-secreting cells: its structure and functional properties. Biochem Biophys Res Commun 205: 1556-1562. 46. Wheeler M B , Gelling RW, Hinke SA, Tu B, Pederson R A , Lynn FC, Ehses JA, Mcintosh CHS. (1995). Functional expression of the rat pancreatic islet glucose-dependent insulinotropic polypeptide receptor: ligand binding and intracellular signaling properties. Endocrinology 136: 4629-4639. 47. Boylan M O , Jepeal LI, Wolfe M M . (1999). Structure of the rat glucose-dependent insulinotropic polypeptide receptor gene. Peptides 20: 219-228. 48. Moens K , Heimberg H , Flamez D, Huypens P, Quartier E, Ling Z, Pipeleers D, Gremlich S, Thorens B, Schuit F. (1996). Expression and functional activity of glucagon, glucagon-like peptide-1, and glucose-dependent insulinotropic polypeptide receptors in rat pancreatic islet cells. Diabetes 45: 257-61. 49. Ottlecz A , Samson W K , McCann S M . (1985). The effects of gastric inhibitory polypeptide (GIP) on the release of anterior pituitary hormones. Peptides 6: 115-119. 50. Lacroix A , Bolte E, Tremblay J, Dupre J, Poitras P, Fournier H , Garon J, Garrel D, Bayard F, Taillefer R, Flanagan RJ, Hamet P. (1992). Gastric inhibitory polypeptide-dependent Cortisol hypersecretion - a new cause of Cushing's syndrome. N Engl J Med 327: 974-980. 51. Kogire M , Inoue K, Sumi S, Doi R, Yun M , Kaji H , Tobe T. (1992). Effects of gastric inhibitory polypeptide and glucagon on portal venous and hepatic arterial flow in conscious dogs. Dig Dis Sci 37: 1666-1670. 52. Bollag RJ, Zhong Q, Phillips P, Min L, Zhong L , Cameron R, Mulloy A L , Rasmussen H , Qin F, Ding, K H , Isales C M . (2000). Osteoblast-derived cells express functional glucose-dependent insulinotropic peptide receptors Endocrinology 141: 1228-1235. 53. Drucker DJ, Philippe J, Mojsov S, Chick WL, Habener IF. (1987). Glucagon-like peptide-1 stimulates insulin gene expression and increases cyclic A M P levels in a rat islet cell line. Proc. Natl. Acad. Sci USA. 84: 3434-3438. 54. Hinke S A , P auly R P, E hses J A , K erridge P , D emuth H I, M clntosh C HS, Pederson RA. (2000). Role of glucose in chronic desensitization of isolated rat islets and mouse insulinoma (0TC-3) cells to glucose dependent insulinotropic polypeptide. J Endocrinol 165: 281-291. 55. Ehses JA, Lee SST, Pederson R A , Mcintosh CHS. (2001). A new pathway for glucose-dependent insulinotropic polypeptide (GIP) receptor signaling -Evidence for the involvement of phospholipase A 2 , in GIP-stimulated insulin secretion. J Biol. Chem. 276: 23667-23673. 56. Ehses JA, Pelech SL, Pederson RA, Mcintosh CHS. (2002). Glucose-dependent insulinotropic polypeptide activates the Raf-Mekl/2-ERKl/2 module via a cyclic AMP/cAMP-dependent protein kinase/Rapl-mediated pathway. J Biol Chem 277: 37088-37097. 57. Lu M , Wheeler M B , Leng X H , Boyd A E 3rd. (1993). The role of the free cytosolic calcium level in beta-cell signal transduction by gastric inhibitory polypeptide and glucagon-like peptide 1(7-37). Endocrinology 132: 94-100. 58. Triimper A, Triimper K , Trusheim H , Arnold R, Goke B, Horsh D. (2001). Glucose-dependent insulinotropic polypeptide is a growth factor for beta (INS-1) cells by pleiotropic signaling. Mol Endocrinol 15: 1559-70. 59. Triimper A , Triimper K , Horsch I. (2002). Mechanims of mitogenic and anti-apoptotic signaling by glucose-dependent insulinotropic polypeptide in beta (INS-1) cells. J Endocrinol 174: 233-246. 60. Pederson R A , Brown JC. (1972). Inhibition of histamine-, pentagastrin-, and insulin-stimulated canine gastric secretion by pure "gastric inhibitory polypeptide". Gastroenterology 62: 393-400. 61. Arnold R, Creutzfeldt W, Ebert R, Becker HD, Borger HW, Schafmayer A . (1978). Serum gastric inhibitory polypeptide (GIP) in duodenal ulcer disease: relationship to glucose tolerance, insulin and gastrin release. Scand J Gastroenterol 13: 41-47. 62. Maxwell V , Shulkes A , Brown JC, Solomon TE, Walsh JH, Grossman MI. 1980). Effects of gastric inhibitory polypeptide on pentagastrin-stimulated acid secretion in man. Dig Dis Sci 25: 113-116. 63. Mcintosh CHS, Pederson R, Mueller M , Brown J. (1981a). Autonomic nervous control of gastric somatostatin secretion from the perfused rat stomach. Life Sci 29: 1477-1483. 64. Soon-Shiong P, Debas HT, Brown JC. (1984) Bethanechol prevents inhibition of gastric acid secretion by gastric inhibitory polypeptide. Am J Physiol 247: G171-175. 65. Bloom SR, Mortimer C H , Thorner M O , Besser G M , Hall R, Gomez-Pan A , Roy V M , Russell RC, Coy D H , Kastin AJ , Schally A V . (1974). Inhibition of gastrin and gastric-acid secretion by growth-hormone release-inhibiting hormone. Lancet 2: 1106-1109. 66. Mcintosh CHS, Pederson R A , Koop H , Brown JC. (1981b). Gastric inhibitory polypeptide stimulated secretion of somatostatinlike immunoreactivity from the stomach: inhibition by acetylcholine or vagal stimulation. Can J Physiol Pharmacology 59: 468-472. 67. Rossowski W J, C heng B -L , Ji ang N - Y , C oy D H . (1998). E xamination o f somatostatin involvement in the inhibitory action of GIP, GLP-1, amylin, and adrenomedullin on gastric acid release using a new SRIF antagonist analogue. Brit J Pharmacol 125: 1081-1087. 68. Meier JJ, Goetze O, Anstipp J, Hagemann D, Hoist JJ, Schmidt WE, Gallwitz B , N auck M A . (2004). G astric i nhibitory p olypeptide d oes n ot i nhibit gastric emptying in humans. Am J Physiol Endocrinol Metab 286: E621-625. 69. Schauder P, Brown JC, Frerichs H , Creutzfeldt W. (1975). Gastric inhibitory polypeptide: effect on glucose-induced insulin release from isolated rat pancreatic islets in vitro. Diabetologia 11: 483-484. 70. Siegel EG, Creutzfeldt W. (1985). Stimulation of insulin release in isolated rat islets by GIP in physiological concentrations and its relation to islet cyclic A M P content. Diabetologia 28: 857-861. 71. Amiranoff B, Vauclin-Jacques N , Laburthe M . (1984). Functional GIP receptors in a hamster pancreatic beta-cell line I n l l l : specific binding and biological effects. Biochem Biophys Res Commun 123: 671-676. 72. Kieffer TJ, Verchere CB, Fell CD, Huang Z, Brown JC, Pederson R A (1993). Glucose-dependent insulinotropic p olypeptide stimulated insulin release from a tumor-derived p-cell line (PTC-3). Can J Physiol Pharmacol 71: 917-922. 73. Fehmann HC, Goke B. (1995). Characterization of GIP(l-30) and GIP(l-42) as stimulators of proinsulin gene transcription. Peptides 16:1149-1152. 74. Lu M , Wheeler M B , Leng X H , Boyd A E , 3 r d . (1993). Stimulation of insulin secretion and insulin gene expression by gastric inhibitory polypeptide. Trans Assoc Am Physicians 106: 42-53. 75. Wang Y , Montrose-Rafizadeh C, Adams L , Raygada M , Nadiv O, Egan JM. (1996). GIP regulates glucose transporters, hexokinases, and glucose-induced insulin secretion in RIN 1046-38 cells. Mol Cell Endocrinol 116: 81-87. 76. Ebert R, Creutzfeldt W. (1982). Influence of gastric inhibitory polypeptide antiserum on glucose-induced insulin secretion in rats. Endocrinology 111: 1601-1606. 77. Ebert R, Unger R H , Creutzfeldt W. (1983). Preservation of incretin activity after removal of gastric inhibitory polypeptide (GIP) from rat gut extracts by immunoadsorption. Diabetologia 24: 449-454. 78. Tseng CC, Zhang X - Y , Wolfe M M . (1999). Effect of GIP and GLP-1 antagonists on insulin release in the rat. Am J Physiol 276: 1049-1054. 79. Gault V A , Flatt PR, Harriott P, Green BD, O'Harte F P M . (2003). Effects of the novel (Pro3)GEP antagonist and exendin (9-39) amide on GEP and GLP-1 induced cyclic A M P generation, insulin secretion, and postprandial insulin release in obese diabetic (ob/ob) mice: evidence that GEP is the major physiological incretin. Diabetologia 46: 222-230. 80. Pederson R A , Satkunarajah M , Mcintosh CHS, Scrocchi L A , Flamez D, Schuit F, Drucker DJ, Wheeler M B . (1998). Enhanced glucose-depependent insulinotropic polypeptide secretion and insulinotropic action in glucagon-like peptide 1 receptor -/- mice. Diabetes 47: 1046-1052. 81. Miyawaki K , Yamada Y , Yano H , Niwa H , Ban N , Ehara Y , Kubota A , Fujimoto S, Kajikawa M , Kuroe A , Tsuda K , Hashimoto H , Yamashita T, Jomori T, Tashiro F, Miyazaki J, Seino Y . (1999). Glucose tolerance caused by a defect in the enteroinsular axis: a study in gastric inhibitory polypeptide receptor knockout mice. Proc Natl Acad Sci USA 96: 14843-14847. 82. Kubota A , Yamada Y , Yasuda K , Someya Y , Ehara Y , Kagimoto S, Watanabe R, Kuroe A , Ishida H , Seino Y . (1997). Gastric inhibitory polypeptide activates M A P kinase through the wortmannin-sensitive and -insensitive pathways. Biochem Biophys Res Commun. 235: 171-175. 83. Ehses J A , Casilla VR, Doty T, Pospisilik JA, Winter K D , Demuth H U , Pederson R A , Mcintosh CH. (2003). Glucose-dependent insulinotropic polypeptide promotes beta-(INS-l) cell survival via cyclic adenosine monophosphate-mediated caspase-3 inhibition and regulation of p38 mitogen-activated protein kinase. Endocrinology 144: 4433-4445. 84. Adrian TE, Bloom A , Andersson M , Jornvall H , Mutt V , Boman H G (1978). Pancreatic polypeptide, glucagon, and insulin secretion from the isolated perfused canine pancreas. Diabetologia 14: 413-417. 85. Pederson R A , Brown JC. (1978). Interaction of gastric inhibitory polypeptide, glucose, and arginine on glucagon secretion from the perfused rat pancreas. Endocrinology 103: 601-615. 86. Nauck M A , Heimesaat M M , Orskov C, Hoist JJ, Ebert R, Creutzfeldt W. (1993) . Preserved incretin activity of glucagon-like peptide 1 [7-36 amide] but not of synthetic human gastric inhibitory polypeptide in patients with type 2 diabetes mellitus. J Clin Invest 91: 301-307. 87. Meier JJ, Gallwitz B, Siepmann N , Hoist JJ, Deacon CF, Schmidt WE, Nauck M A . (2003). Gastric inhibitory polypeptide (GEP) dose-dependently stimulates glucagon secretion in healthy human subjects at euglycemia. Diabetologia 46: 798-801. 88. Raben A , Andersen HB, Christensen NJ, Madsen J, Hoist JJ, Astrup A . (1994) . Evidence for an abnormal postprandial response to a high-fat meal in women predisposed to obesity. Am. J. Physiol. Endocrinol. Metab. 267: E549-E559. 89. Wasada T, McCorkle K , Harris V , Kawai K , Howard B , Unger R H . (1981). Effect of gastric inhibitory polypeptide on plasma levels of chylomicron triglycerides in dogs. J Clin Invest 68: 11016-11017. 90. Ebert R, Nauck M , Creutzfeldt W. (1991). Effect of exogenous or endogenous gastric inhibitory polypeptide (GIP) on plasma triglyceride responses in rats. Horm Metab Res 23: 517-521. 91. Eckel R H , Fujimoto W Y , Brunzell JD. (1979). Gastric inhibitory polypeptide enhances lipoprotein lipase activity in cultured preadipocytes. Diabetes 28: 1141-1142. 92. Knapper JM, Puddicombe S M , Morgan L M , Fletcher J M , Marks V . (1995). Investigations into the actions of glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 (7-36)amide on lipoprotein lipase activity in explants of rat adipose tissue. JNutr 125: 183-188. 93. Oben J, Morgan L , Fletcher J, Marks V . (1991). Effect of the enter-pancreatic hormones, gastric inhibitory polypeptide and glucagon-like polypeptide-1(7-36) amide, on fatty acid synthesis in explants of rat adipose tissue. J Endocrinol 130: 267-272. 94. Beck B, Max JP. (1983). Gastric inhibitory polypeptide enhancement of the insulin effect on fatty acid incorporation into adipose tissue in the rat. Reg Peptides 7:3-8 95. Hauner H , Glatting G, Kaminska D, Pfeiffer EF. (1988). Effects of gastric inhibitory polypeptide on glucose and lipid metabolism of isolated rat adipocytes. Ann Nutr Metab 32: 282-288. 96. Starich GH, Bar RS, Mazzaferri EL. (1985). GIP increases insulin receptor affinity and cellular sensitivity in adipocytes. Am J Physiol 249: E603-E607. 97. Dupre J, Greenidge N , McDonald TJ, Ross SA, Rubinstein D. (1976). Inhibition of action of glucagon in adipocytes by gastric inhibitory polypeptide. Metabolism 25:1197-1199. 98. Miyawaki K , Yamada Y , Ban N , Ihara Y , Tsukiyama K , Zhou H , Fujimoto S, Oku A , Tsuda K , Toyokuni S, Hiai H , Mizunoya W, Fushiki T, Hoist JJ, Makino M , Tashita A , Kobara Y , Tsubamoto Y , Jinnouchi T, Jomori T, Seino Y . (2002). Inhibition of gastric inhibitory polypeptide signaling prevents obesity. Nature Med 8: 738-742. 99. Mcintosh C H , Bremsak I, Lynn FC, Gi l l R, Hinke SA, Gelling R, Man C, McKnight G, Jaspers S, Pederson RA. (1999). Glucose-dependent insulinotropic polypeptide stimulation of lipolysis in differentiated 3T3-L1 cells: wortmannin-sensitive inhibition by insulin. Endocrinology 140: 398-404. 100. Anderson D K , Sun YS, Brunicardi FC, Berlin S A , Lebovitz HE, Elahi D (1984). Regulation of hepatic glucose production by gastric inhibitory polypeptide (GIP), insulin (I), and glucagon (GLUC). DigDis Sci 29: A5 101. Elahi D, Meneilly GS, Minaker K L , Rowe L D , Anderson DK. (1986) Regulation of glucose production by gastric inhibitory polypeptide in man. Can J Physiol Pharmacol (Suppl) 65:18 102. Lauritsen K B , Moody AJ , Christensen K C , Lindkaer Jensen S. (1980). Gastric inhibitory polypeptide (GIP) and insulin release after small-bowel resection in man. Scand J Gastroenterol 15: 833-40. 103. Lund PK, Goodman RH, Dee PC, Habener JF. (1982). Pancreatic preproglucagon cDNA contains two glucagon-related coding sequences arranged in tandem. Proc Natl Acad Sci USA 79: 345-349. 104. Bell GI, Santerre RF, Mullenbach GT: Hamster preproglucagon contains the sequence of glucagon and two related peptides. Nature 302: 716-718, 1983. 105. Kieffer TJ, Habener IF. (1999). The Glucagon-like Peptides. Endocrine Reviews 20: 876-913. 106. Mojsov S, Weir GC, Habener J. (1987). Insulinotropin: glucagon-like peptide-1 (7-37) co-encoded in the glucagon gene is a potent stimulator of insulin release in the perfused rat pancreas. J Clin Invest 79: 616-619. 107. Kreymann B, Ghatei M A, W illiams G , B loom S R. (1987). G lucagon-like peptide-1 7-36: A physiological incretin in man. Lancet 2: 1300-1303. 108. Fehmann H-C, Habener J. (1992). Insulinotropic hormone glucagon-like peptide-1 (7-37) stimulation of proinsulin gene expression and proinsulin biosynthesis in insulinoma f3TC-l cells. Endocrinology 130: 159-166. 109. Holz GGT, Kuhtreiber W M , Habener IF. (1993). Pancreatic beta cells are rendered glucose-competent by the insulinotropic hormone glucagon-like peptide-1(7-37). Nature. 361: 362-365. 110. X u G, Stoffers DA, Habener JF, Bonner-Weir S. (1999). Exendin-4 stimulates b oth b eta c ell r eplication a nd n eogenesis, r esulting i n i ncreased b eta cell mass and improved glucose tolerance in diabetic rats. Diabetes 48:2270-2276. 111. Stoffers DA, Kieffer TJ, Hussain M A , Drucker DJ, Bonner-Weir S, Habener JF, Egan JM. (2000). Insulinotropic glucagon-like peptide-1 agonists stimulate expression of homeodomain protein IDX-1 and increase islet size in mouse pancreas. Diabetes 49: 741-748. 112. Tourrel C, Bailbe D, Meile M-J, Kergoat M , Portha B. (2001). Glucagon-like peptide-1 and exendin-4 stimulate beta cell neogenesis in streptozotocin-treated newborn rats resulting in persistently improved glucose homeostasis at adult age. Diabetes 50: 1562-1570. 113. Hui H , Wright C, Perfetti R. (2001). Glucagon-like peptide-1 induces differentiation o f i slet h omeobox-1 -positive p ancreatic d uctal c ells i nto insulin-secreting cells. Diabetes. 50: 785-796. 114. De Leon DD, Deng S, Madani R, Ahima RS, Drucker D. (2003). Role of endogenous glucagon-like peptide-1 in islet regeneration after partial pancreatectomy. Diabetes. 52: 365-371. 115. Buteau J, Roduit R, Susini S, Prentki M . (1999). Glucagon-like peptide-1 promotes D N A synthesis, activates phosphatidylinositol-3-kinase and increases transcription factor pancreatic and duodenal homeobox gene 1 (PDX-1) D N A binding activity in beta (INS-1) cells. Diabetologia. 42: 856-864. 116. Drucker DJ. (2003). Glucagon-like peptides: regulators of cell proliferation, differentiation, and apoptosis. Mol Endocrinol. 17: 161-171. 117. L i Y , Hansotia T, Yusta B , Ris F, Halban PA, Drucker DJ. (2003). Glucagon-like peptide-1 receptor signaling modulates beta cell apoptosis. J Biol Chem. 276: 471-478. 118. Farilla L, Hui H , Bertolotto C, Kang E, Bulotta A , Di Mario U , Perfetti R. (2002). Glucagon-like peptide-1 promotes islet cell growth and inhibits apoptosis in Zucker diabetic rats. Endocrinology 143: 4397-4408. 119. Matsuyama T, Komatsu R, Namba M , Watanabe N , Itoh H , Tarui S. (1988). Glucagon-like peptide-1 (7-36amide): a potent glucagonostatic and insulinotropic hormone. Diabetes Res. Clin. Pract. 5: 281-284. 120. Fehmann H-C, Goke R, Goke B. (1995). Cell and molecular biology of the incretin hormones glucagon-like peptide-1 and glucose-dependent insulin releasing polypeptide. Endocrine Reviews 16: 390-410. 121. Wettergen A B , Schjoldager PE, Mortensen J, Myhre J, Christiansen J, Hoist JJ. (1993). Truncated GLP-1 (proglucagon 87-107-amide) inhibits gastric and pancreatic functions in man. DigDis Sci 38: 665-673. 122. Willms BJ, Werner C, Orskov C, Hoist JJ, Creutzfeldt W, Nauck M A . (1996). Gastric emptying, glucose-responses, and insulin secretion after a liquid test meal: effects of exogenous glucagon-like peptide-1 (GLP-l)-(7-36) amide in type 2 (noninsulin-dependent diabetic patients. J Clin Endocrinol Metab 81: 327-332. 123. Mentlein R. (1999). Dipeptidyl Peptidase IV (CD26) - role in the inactivation of regulatory peptides. Reg Peptides. 85: 9-24. 124. Mentlein R, Gallwitz B, Schmidt WE. (1993). Dipeptidyl peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon-like peptide-1(7-36) amide, peptide histidine methionine and is responsible for their degradation in human serum. Eur J Biochem 214: 829-835. 125. Kieffer TJ, Mcintosh C H , Pederson R A . (1995). Degradation of glucose-dependent insulinotropic polypeptide and truncated glucagon-like peptide 1 in vitro and in vivo by dipeptidyl peptidase IV. Endocrinology. 136: 3585-3596. 126. Deacon CF, Nauck M A , Meier J, Hucking K , Hoist JJ. (2000). Degradation of endogenous and exogenous gastric inhibitory polypeptide in healthy and in type 2 diabetic subjects as revealed using a new assay for the intact peptide. J Clin Endocrinol Metab. 85: 3575-3581. 127. Suzuki S, Kawaki K , Ohashi S, Mukai H , Yamashita K . (1989). Comparison of the effects of various C-terminal and N-terminal fragment peptides of glucagon-like peptide-1 on insulin and glucagon release from the isolated perfused pancreas. Endocrinology 125: 3109-3114. 128. Gault V A , Parker JC, Harriott P, Flatt PR, O'Harte F P M . (2002). Evidence that the major degradation product of glucose-dependent insulinotropic polypeptide (GEP), GIP(3-42), is a GEP receptor antagonist in vivo. J Endocrinology 175: 525-533. 129. Kolligs F, Fehmann HC, Goke R, Goke B. (1995). Reduction of the incretin effect in rats by the glucagon-like peptide 1 receptor antagonist exendin (9-39) amide. Diabetes 44: 16-19. 130. Knudsen L B , Pridal L . (1996). Glucagon-like peptide-1 (9-36)amide is a major metabolite of glucagon-like peptide-1 (7-36)amide after in vivo administration to dogs, and it acts as an antagonist on the pancreatic receptor. Eur J Pharmacology 318: 429-435. 131. Schmid R, Schusdziarra V , Aulehner R, Weigert N , Classen M . (1990). Comparison of GLP-1 (7-36)amide and GEP on release of somatostatin-like immunoreactivity and insulin release from the isolated rat pancreas. Z Gastroenterology 28: 280-284. 132. Suzuki S, Kawai K , Ohashi S, Mukai H , Murayama Y , Yamashita K . (1990). Reduced insulinotropic effects of glucagon-like peptide-l(7-36)amide and gastric inhibitory polypeptide in isolated perfused diabetic rat pancreas. Diabetes 39: 1320-1325. 133. Jia X , Brown JC, Ma P, Pederson RA, Mcintosh CHS. (1995). The effects of glucose-dependent insulinotropic polypeptide receptor and glucagon-like peptide-1(7-3 6) on insulin secretion. Am J Physiol 268: E645-E651. 134. Shima K , Hirota M , Ohboshi C. (1998). Effect of glucagon-like peptide-1 on insulin secretion. Reg Peptides 22: 245-252. 135. Siegel EG, Schulze A , Schmidt WE, Creutzfeld WE. (1992). Comparison of the e ffect o f G IP a nd G LP-1 (7-3 6)amide o n i nsulin r elease from r at p ancreatic islets. Eur J Clin Investigation 22: 154-157. 136. Nauck M A , Bartels E, Orskov C, Ebert R, Creutzfeldt W. (1993). Additive insulinotropic effects of exogenous synthetic human gastric inhibitory polypeptide and glucagon-like peptide-1-(7-36) amide infused at near physiological insulinotropic hormone and glucose concentrations. J Clin Endocrinol Metab 76: 912-917. 137. Tseng CC, Kieffer TJ, Jarboe L A , Usdin TB, Wolfe M M . (1996). Postprandial stimulation of insulin release by glucose-dependent insulinotropic polypeptide (GIP). Effect of a specific glucose-dependent insulinotropic polypeptide receptor antagonist in the rat. J Clin Invest 98: 2440-2445. 138. Ebert R, Illmer K , Creutzfeldt W. (1979). Release of gastric inhibitory polypeptide (GIP) by intraduodenal acidification in rats and humans and abolishment of the incretin effect of acid by GIP-antiserum in rats. Gastroenterology 76: 515-523. 139. Meneilly GS, Bryer-Ash M , Elahi D. (1993). The effect of glyburide on beta-cell sensitivity to glucose-dependent insulinotropic polypeptide. Diabetes Care 16: 110-114. 140. Hinke SA, Gelling RW, Pederson RA, Manhart S, Nian C, Demuth H-U, Mcintosh CHS. (2002). Dipeptidyl peptidase IV-resistant [D-Ala2]glucose-dependent insulinotropic polypeptide (GIP) improves glucose tolerance in normal and obese diabetic rats. Diabetes 51: 652-661. 141. Gault V A , Flatt PR, O'Harte F P M . (2003). Glucose-dependent insulinotropic polypeptide analogues and their therapeutic potential for the treatment of obesity-diabetes. Biochem Biophys Res Commun 308: 207-213. 142. Nauck M A , Niedereichholz U , Ettler R, Hoist JJ, Orskov C, Ritzel R, Schmiegei W H . (1997). G lucagon-like p eptide 1 i nhibition o f g astric e mptying outweighs its insulinotropic effects in healthy humans. Am J Physiol 273: E981-988. 143. Tisch R, McDevitt H . (1996). Insulin-dependent diabetes mellitus. Cell 85: 291-297. 144. Mathias D, Vence L , Benoist C. (2001). Beta cell death during progression to diabetes. Nature 414: 792-798. 145. Soeldner JS, Tuttleman M , Srikanta S, Ganda OP, Eisenbarth GS. (1985). Insulin-dependent diabetes mellitus and autoimmunity: islet-cell autoantibodies, insulin autoantibodies, and beta-cell failure. N EnglJMed 313: 893-894. 146. Gorsuch A N , Lister J, Dean B M , Spencer K M , McNally J M , Bottazzo GF, Cudworth A G . (1981). Evidence for a long prediabetic period in type 1 (insulin-dependent) diabetes mellitus. Lancet 26: 1363-1365. 147. O'Brien B A , Harmon B V , Cameron DP, Allan DJ. (1997). Apoptosis is the mode of beta cell death responsible for the development of JDDM in the nonobese diabetic (NOD) mouse. Diabetes 46: 750-757. 148. O'Brien B A , Harmon B V , Cameron DP, Allan DJ. (1996). Beta cell apoptosis is responsible for the development of IDDM in the multiple low-dose streptozotocin model. J Pathol 178: 176-181. 149. Mensah-Brown EP, Grujicic SS, Maksimovic D, Jasima A , Shahin A , Lukic M L . (2002). Downregulation of apoptosis in the target tissue prevents low-dose streptozotocin-induced autoimmune diabetes. Mol Immunol 38: 941-946. 150. Padanilam BJ. (2003). Cell death induced by acute renal injury: a perspective on the contributions of apoptosis and necrosis. Am J Physiol Renal Physiol 284: F608-F627. 151. Trudeau JD, Dutz JP, Arany E, Hi l l DJ, Fieldus WE, Finegood DT. (2000). Perspectives in diabetes: neonatal beta-cell apoptosis a trigger for autoimmune diabetes? Diabetes 49:1-7. 152. Finegood DT, Scaglia L , Bonner-Weir S. (1995). Dynamics of beta-cell mass in the growing rat pancreas: estimation with a simple mathematical model. Diabetes 44: 249-256. 153. Uchimura E, Kodaira T, Kurosaka K , Yang D, Watanabe N , Kobayashi Y . (1997) . Interaction of phagocytes with apoptotic cells leads to production of pro-inflammatory cytokines. Biochem Biophys Res Commun 239: 799-803. 154. Fadok V A , Bratton DL, Konowal A , Freed PW, Westcott JY, Henson P M . (1998) . Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-B, PGE2, and PAF.JClin Invest 101: 890-898. 155. Utz PJ, Hottelet M , Schur PH, Anderson P. (1997). Proteins phosphorylated during stress-induced apoptosis are common targets for autoantibody production in patients with systemic lupus erythematosus. J Exp Med 185: 843-854. 156. McGarry D. (2002). Dysregulation of fatty acid metabolism in the etiology of type 2 diabetes. Banting Lecture 2001. Diabetes. 51:7-18. 157. Leahy JL, Bonner-Weir S, Weir GC. (1992). p-cell dysfunction induced by chronic hyperglycemia: current ideas on mechanism of impaired glucose-induced insulin secretion. Diabetes Care 15: 442-455. 158. KosakaK, Kuzuya T, Akanuma Y , Hagura R. (1980). Increase in insulin response after treatment of overt maturity-onset diabetes is independent of the mode of treatment. Diabetologia 18:23-28. 159. Weir GC, Leahy JL, Bonner-Weir S. (1986). Experimental reduction of p-cell mass: implications for the pathogenesis of type 2 diabetes. Diabetes Metab Rev 2:125-161. 160. Leahy JL, Cooper HE, Deal DA, Weir GC. (1986). Chronic hyperglycemia is associated with impaired glucose influence on insulin secretion: a study in normal rats using chronic in vivo glucose infusions. J Clin Invest 77: 908-915. 161. Leahy JL, Bonner-Weir S, Weir GC. (1988). Minimal chronic hyperglycemia is a critical determinant of impaired insulin secretion after an incomplete pancreatectomy. J Clin Invest 81: 1407-1414. 162. Sako Y , Grill V . (1990). A 48-hour lipid infusion in the rat time-dependently inhibits glucose-induced insulin secretion and beta cell oxidation through a process likely coupled to fatty acid oxidation. Endocrinology 127: 1580-1589. 163. Capito K , Hansen SE, Hedeskov CJ, Islin H , Thams P. (1992). Fat-induced changes in mouse pancreatic islet insulin secretion, insulin biosynthesis and glucose metabolism. Acta Diabetol 28: 193-198. 164. Zhou Y P , Grill V . (1994). Long-term exposure of rat pancreatic islets to fatty acids inhibits glucose-induced insulin secretion and biosynthesis through a glucose fatty acid cycle. J Clin Invest 93: 870-876. 165. Zhou Y P , Grill V E . (1995). Long-term exposure to fatty acids and ketones inhibits beta-cell functions in human pancreatic islets of Langerhans. J Clin Endocrinol Metab 80: 1584-1590. 166. Lee Y , Hirose H , Ohneda M , Johnson JH, McGarry D, Unger R H . (1994). Beta cell lipotoxicity in the pathogenesis of non-insulin-dependent diabetes mellitus of obese rats: Impairment in adipocyte-beta cell relationships. Proc Natl Acad Sci USA 91: 10878-10882. 167. McGarry JD, Dobbins RL. (1999). Fatty acids, lipotoxicity and insulin secretion. Diabetologia 42:128-138. 168. Laybutt DR, Sharma A , Sgroi DC, Gaudet J, Bonner-Weir S, Weir GC. (2002). Genetic regulation of metabolic pathways in beta-cells disrupted by hyperglycemia. J Biol Chem. Ill: 10912-10921. 169. Jonas JC, Sharma A , Hasenkamp W, Ilkova H , Patane G, Laybutt R, Bonner-Weir S, Weir GC. (1999). Chronic hyperglycemia triggers loss of pancreatic beta cell differentiation in an animal model of diabetes. J Biol Chem 274:14112-14121. 170. Rossetti L, Shulman GI, Zawalich W, DeFronzo R A . (1987). Effect of chronic hyperglycemia on in vivo insulin secretion in partially pancreatectomized rats. J Clin Invest. 80: 1037-1044. 171. Leahy JL, Cooper HE, Weir GC. (1987). Impaired insulin secretion associated with near normoglycemia. Study in normal rats with 96-h in vivo glucose infusions. Diabetes 36: 459-464. 172. Leahy JL, Weir GC. (1991). Beta-cell dysfunction in hyperglycaemic rat models: recovery of glucose-induced insulin secretion with lowering of the ambient glucose level. Diabetologia 34: 640-647. 173. Nauck M A , Homberger E, Siegel EG, Allen RC, Eaton RP, Ebert R, Creutzfeldt W. (1986). Incretin effects of increasing glucose loads in man calculated from venous insulin and C-peptide responses. J Clin Endocrinol Metab 63: 492-498. 174. Creutzfeldt W. (2001). The entero-insular axis in type 2 diabetes-incretins as therapeutic agents. Exp Clin Endocrinol Diabetes 109 Suppl 2: S288-S303. 175. Vahl T, D'Alessio D. (2003). Enteroinsular signaling: perspectives on the role of the gastrointestinal hormones glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide in normal and abnormal glucose metabolism. Curr Opin in Clin Nut and Metab Care 6: 461-468. 176. Hoist JJ, Gromada J, Nauck M A . (1997). The pathogenesis of NEDDM involves a defective expression of the GIPR. Diabetologia 40: 984-986. 177. Nauck M , Stockmann F, Ebert R, Creutzfeldt W. (1986). Reduced incretin effect in type 2 (noninsulin-dependent) diabetes. Diabetologia 29: 46-52. 178. Elahi D, McAloon-Dyke M , Fukagawa N K , Meneilly GS, Sclater A L , Minaker K L , Habener JF, Andersen DK. (1994). The insulinotropic actions of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (7-37) in normal and diabetic subjects. Reg Peptides 51: 63-74. 179. Jones IR, Owens DR, Luzio S, Hayes T M . (1989). Glucose dependent insulinotropic polypeptide (GIP) infused intravenously is insulinotropic in the fasting state in type 2 (non-insulin dependent) diabetes mellitus. Horm Metabol Res 21: 23-26. 180. Jones IR, Owens DR, Moody A J , Luzio SD, Morris T, Hayes T M . (1987). The effects of glucose-dependent insulinotropic polypeptide infused at physiological concentrations in normal subjects and type 2 (non-insulin-dependent) diabetic patients on glucose tolerance and B-cell secretion. Diabetologia 30: 707-712. 181. Kubota A , Yamada Y , Hayami T, Yasuda K , Someya Y , Ehara Y , Kagimoto S, Watanabe R, Taminato T, Tsuda K , Seino Y . (1996). Identification of two missense mutations in the GIP receptor gene: a functional study and association analysis with NIDDM: no evidence of association with Japanese NEDDM subjects. Diabetes 45: 1701-1705. 182. Almind K , Ambye L, Urhammer SA, Hansen T, Echwald S M , Hoist J J, Gromada J, Thorens B, Pederson O. (1998). Discovery of amino acid variants in the human glucose-dependent insulinotropic polypeptide (GEP) receptor: impact on the pancreatic beta cell responses and functional expression studies in Chinese hamster fibroblast cells. Diabetologia 41: 1194-1198. 183. Tseng CC, Boylan M O , Jarboe L A , Usdin TB, Wolfe M M . (1996). Chronic desensitization of the glucose-dependent insulinotropic polypeptide receptor in diabetic rats. Am J Physiol Endocrinol Metab 270: E661-666. 184. Vaag A A , Hoist JJ, Volund A , Beck-Nielsen H . (1996). Gut incretin hormones in identical twins disconcordant for non-insulin-dependent diabetes mellitus (NEDDM) - evidence for decreased glucagon-like peptide-1 secretion during oral glucose tolerance ingestion in NEDDM twins, Eur J Endocrinol 135: 435-442. 185. Jones ER, Owens DR, Luzio S, Williams S, Hayes T M . (1989). The glucose dependent insulinotropic polypeptide response to oral glucose and mixed meals is increased in patients with type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia 32: 668-677. 186. Fukase N , Igarahi M , Takahashi H, Manaka H , Yamatani K , Daimon M , Tominaga M , Sasaki H . (1993). Hypersecretion of truncated glucagon-like peptide-1 and gastric inhibitory polypeptide in obese patients, Diabet Med 10: 44-49. 187. Ahren B, Larsson H , Hoist JJ. (1997). Reduced gastric inhibitory polypeptide but normal glucagon-like peptide response to oral glucose in postmenopausal women with impaired glucose tolerance. Eur J Endocrinol 137: 127-131. 188. Groop PH. (1989). The influence of body weight, age and glucose tolerance on the relationship between GIP secretion and beta-cell function in man. Scand J Clin Invest 49: 367-379. 189. Service FJ, Rizza R A , Westland RE, Hall LD, Gerich JE, Go V L . (1984). Gastric inhibitory polypeptide in obesity and diabetes mellitus. J Clin Endocrinol Metab 58: 1133-1140. 190. Nyholm B, Walker M , Gravholt C H , Shearing PA, Sturis J, Alberti K G , Hoist JJ, Schmitz O. (1999). Twenty-four-hour insulin secretion rates, circulating concentrations of fuel substrates and gut incretin hormones in healthy offspring of Type II (non-insulin-dependent) diabetic parents: evidence of several aberrations. Diabetologia 42: 1314-1323. 191. Meier JJ, Nauck M A , Siepmann N , Greulich M , Hoist JJ, Deacon CF, Schmidt WE, Gallwitz B. (2003). Similar insulin secretory response to a gastric inhibitory polypeptide bolus injection at euglycemia in first-degree relatives of patients with type 2 diabetes and control subjects. Metabolism 52: 1579-1585. 192. Ward W K , Bolgiano DC, McKnight B, Halter JB, Porte D Jr. (1984). Diminished B cell secretory capacity in patients with noninsulin-dependent diabetes mellitus. J Clin Invest 74: 1318-1328. 193. Fritsche A , Stefan N , Hardt E, Haring H , Stumvoll M . (2000). Characterisation of beta-cell dysfunction of impaired glucose tolerance: evidence for impairment of incretin-induced insulin secretion. Diabetologia 43: 852-858. 194. Vilsboll T, Krarup T, Madsbad S, Hoist JJ. (2002). Defective amplification of t he 1 ate p hase i nsulin r esponse t o glucose b y G IP i n o bese T ype II diabetic patients. Diabetologia 45: 1111-1119. 195. Lynn FC, Pamir N , Ng EHC, Mcintosh CHS, Kieffer TJ, Pederson R A . (2001). Defective glucose-dependent insulinotropic polypeptide receptor expression in diabetic fatty Zucker rats. Diabetes 50:1004-1011. 196. Vilsboll T, Knop FK, Krarup T, Johansen A , Madsbad S, Larsen S, Hansen T, Pederson O, Hoist JJ. (2003). The pathophysiology of diabetes involves a defective amplication of the late-phase insulin response to glucose by glucose-dependent insulinotropic polypeptide - regardless of etiology and phenotype. J Clin Endocrinol Metab 88: 4897-4903. 197. Lynn FC, Thompson SA, Pospisilik JA, Ehses JA, Hinke SA, Pamir N , Mcintosh C H , Pederson RA. (2003). A novel pathway for regulation of glucose dependent insulinotropic polypeptide (GIP) receptor expression in beta cells. FasebJll: 91-93. 198. Desvergne B, Wahli W. (1999). Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr Rev 20: 649-688. 199. Zhou YT, Shimabukuro M , Wang M Y , Lee Y , Higa M , Milburn JL, Newgard CB, Unger R H . (1998). Role of peroxisome proliferator-activated receptor alpha i n disease o f pancreatic beta cells. P roc Natl Acad Sci USA 95: 8898-8903. 200. Roduit R, Morin J, Masse F, Segall L, Roche E, Newgard C B , Assimacopoulos-Jeannet F, Prentki M . (2000). Glucose down-regulates the expression of the peroxisome proliferator-activated receptor-alpha gene in the pancreatic beta cell. J Biol Chem 275: 35799-35806. 201. Harmon JS, Gleason CE, Tanaka Y , Poitout V , Robertson RP. (2001). Antecedent hyperglycemia, not hyperlipidemia, is associated with increased islet triglyceride content and decreased insulin gene mRNA level in Zucker diabetic fatty rats. Diabetes 50: 2481 -2486. 202. Roche E, Farfari S, Witters L A , Assimacopoulos-Jeannet F, Themelin S, Brun T, Corkey B E , Saha A K , Prentki M . (1998). Long-term exposure of beta-INS c ells to high g lucose concentrations increases anaplerosis, lipogenesis, and lipogenic gene expression. Diabetes 47: 1086-1094. 203. Kolb H . (1987). Mouse Models of Insulin Dependent Diabetes: Low-Dose Streptozotocin-Induced Diabetes and Nonobese Diabetic (NOD) mice. Diabetes/Metab Rev 3: 751-778. 204. Eisner M , Guldbakke B, Tiedge M , Munday R, Lenzen S. (2000). Relative importance of transport and alkylation for pancreatic beta-cell toxicity of streptozotocin. Diabetologia 43: 1528-1533. 205. Rossini A A , Like A A , Dulin WE, Cahill GF. (1977). Pancreatic beta cell toxicity by streptozotocin anomers. Diabetes 26: 1120-1124. 206. Gai W, Schott-Ohly P, Schulte im Walde S, Gleichmann H . (2004). Differential target molecules for toxicity induced by streptozotocin and alloxan in pancreatic islets of mice in vitro. Exp Clin Endocrinol Diabetes 112: 29-37. 207. Bannister B . (1972). The synthesis and biological activities of some analogs of streptozotocin. J Antibiotics 25: 377-385. 208. Wilson GL, Leiter EH. (1990). Streptozotocin interactions with pancreatic P-cells and the induction of insulin-dependent diabetes. Curr Top Microbiol Immunol 156: 27-54. 209. Bedoya FJ, Solano F, Lucas M . (1996). N-monomethyl-arginine and nicotinamide prevent streptozotocin-induced double strand D N A break formation in pancreatic rat islets. Experientia 52: 344-347. 210. Szkudelski T. (2001). The mechanism of alloxan and streptozotocin action in the beta cells of the rat pancreas. Physiol Res 50: 536-546. 211. Robbins MJ , Sharp R A , Slonim A E , Burr EM. (1980). Protection against streptozotocin-induced diabetes by superoxide dismutase. Diabetologia 18: 55-58. 212. Takasu N , Komiya I, Asawa T, Nagasawa Y , Yamada T. (1991). Streptozotocin- and alloxan-induced H2O2 generation and D N A fragmentation in pancreatic islets: H2O2 as mediator for D N A fragmentation. Diabetes 40: 1141-1145. 213. Masiello P, Cubeddu TL, Frosina G, Bergamini E. (1985). Protective effect of 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase, against streptozotocin-induced diabetes. Diabetologia 28: 683-686. 214. Masutani M , Suzuki H , Kamada N , Watanabe M , Ueda O, Nozaki T, Jishage K , Watanabe T, Sugimoto T, Nakagama H , Ochiya T, Sugimura T. (1999). Poly(ADP-ribose) polymerase gene disruption conferred mice resistant to streptozotocin-induced diabetes. Proc Natl Acad Sci USA 96: 2301-2304. 215. Burkart V , Wang Z-Q, Radons J, Heller B, Herceg Z, Stingl L , Wagner EF, Kolb H . (1999). Mice lacking the poly(ADP-ribose) polymerase gene are resistant to pancreatic beta-cell destruction and diabetes development induced by streptozotocin. Nat Med 5: 314-319. 216. Junod A , Lambert A E , Stauffacher W, Renold A E . (1969). Diabetogenic action of streptozotocin: Relationship of dose to metabolic response. J Clin Invest 48: 2129-2139. 217. Bell R H Jr, Hye RJ. (1983). Animal Models of Diabetes Mellitus: Physiology and Pathology. J Surg Res 35: 433-460. 218. Like A A , Rossini A A . (1976). Streptozotocin-induced pancreatic insulitis: a new model of diabetes mellitus. Science 193: 415-417. 219. Stosic-Grujicic SD, Maksimovic DD, Stojkovic M B , Lukic M L . (2001). Pentoxifylline prevents autoimmune mediated inflammation in low dose streptozotocin induced diabetes. Dev Immunol 8: 213-221. 220. Lukic M L , Al-Sharif R, Mostarica M , Bahr G, Behbehani K . (1991). Immunological basis of the strain differences in susceptibility to low-dose streptozotocin-induced diabetes in rats. In Lymphatic Tissues and In Vivo Immune Responses, Imhof, et al., Eds. (New York: Marcel Dekker), pp 643-647. 221. Tschop M , Heiman M L . (2001). Rodent obesity models: an overview. Exp Clin Endocrinol Diabetes 109: 307-319. 222. Mcintosh CHS, Pederson RA. (1999). Noninsulin-dependent animal models of diabetes mellitus, in Experimental Models of Diabetes, McNeill JHCRC Press L L C , New York, 337-398. 223. Moe OW, Berry CA, Rector FC Jr. (2001). Renal Transport of glucose, amino acids, sodium chloride, and water. In The Kidney, 6 t h ed. Section 9: Elements of normal renal structure and function, Vo l 1. Brenner B M Ed. USA, WB Saunders Company, pp 375-415. 224. Lee WS, Kanai Y , Wells RG, Hediger M A . (1994). The high-affinity NaVglucose cotransporter: reevaluation of function and distribution of expression. J Biol Chem 269: 12032-12039. 225. You G, Lee WS, Barros EJ, Kanai Y , Huo TL, Khawaja S, Wells RG, Nigam SK, Hediger M A . (1995). Molecular characteristics of Na+-coupled glucose transporters in adult and embryonic rat kidney. J Biol Chem 270: 29365-29371. 226. Kong CT, Yet SF, Lever JE. (1993). Cloning and expression of a mammalian NaVamino acid cotransporter with sequence similarity to NaVglucose cotransporters. J Biol Chem 268: 1509-1512. 227. Kuhn-Wache K , Manhart S, Hoffmann T, Hinke SA, Gelling R, Pederson R A , Mcintosh CHS, Demuth H U . (2000). Analogs of glucose-dependent insulinotropic polypeptide with increased dipeptidyl peptidase IV resistance. Adv Exp Med Biol 477: 187-195. 228. Thorens B, Sarkar H K , Kaback HR, Lodish HF. (1988). Cloning and functional expression in bacteria of a novel glucose transporter present in liver, intestine, kidney, and beta-pancreatic islet cells. Cell 55: 281-290. 229. Efrat S, Linde S, Kofod H , Spector D, Delannoy M , Grant S, Hanahan D, Baekkeskov S. (1988). Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene. Proc Natl Acad Sci USA 85: 9037-9041. 230. Hinke SA. Modulation of insulinotropic hormone bioactivity with a focus on GIP. [Ph.D. Dissertation], University of British Columbia, Vancouver. 231. Van der Vliet JA, Meloche R M , Field M J , Chen DJ, Kaufman DB, Sutherland DE. (1988). Pancreatic islet isolation in rats with ductal collagenase distension, stationary digestion and dextran separation. Transplantation 45: 493-495. 232. Pederson R A , Buchan A M , Zahedi-Asl S, Chan CB, Brown JC. (1982). Effect of jejunoileal bypass in the rat on the enteroinsular axis. Reg Peptides 5:53-63. 233. Lewis JT, Dayanandan B, Habener JF, Kieffer TJ. (2000). Glucose-dependent i nsulinotropic p olypeptide c onfers e arly p hase i nsulin r elease to o ral glucose in rats: deomonstration by a receptor antagonist. Endocrinology 141: 3710-3716. 234. Fehmann HC, Habener JF. (1991). Homologous desensitization of the insulinotropic glucagon-like peptide-I (7-37) receptor on insulinoma (HIT-T15) cells. Endocrinology 128: 2880-2888. 235. Murphy GJ, Hruby VJ , Trivedi D, Wakelam MJO, Houslay M D . (1987). The rapid desensitization of glucagon-stimulated adenylate cyclase is a cyclic AMP-independent process that can be mimicked by hormones which stimulate inositol phospholipid metabolism. Biochem J243: 39-46. 236. Gefel D, Hendrick GK, Mojsov S, Habener JF, Weir GC. (1990). Glucagon-like peptide-1 analogs: effects on insulin secretion and adenosine 3'5'-monophosphate formation. Endocrinology 126: 2164-2168. 237. ' Pospisilik J A , Martin J, Doty T, Ehses JA, Pamir N , Lynn FC, Piteau S, Demuth H-U, Mcintosh CHS, Pederson RA. (2003). Dipeptidyl peptidase IV Inhibitor Treatment Stimulates beta-cell survival and islet neogenesis in streptozotocin-induced diabetic rats. Diabetes 52: 741-750. 238. Rossini A , Appel M , Williams R, Like A . (1977). Genetic influence of the streptozotocin-induced insulitis and hyperglycemia. Diabetes 26: 916-920. 239. Cardinal JW, Allan DJ, Cameron DP. (1998). Differential metabolite accumulation may be the cause of strain differences in sensitivity to streptozotocin-induced P-cell death in inbred mice. Endocrinology 139: 2885-2891. 240. Reddy S, Young M , Poole CA, Ross J M . (1998). Loss of glucose transporter-2 precedes insulin loss in the nonobese diabetic and the low-dose streptozotocin mouse models: a comparative immunohistochemical study by light and confocal microscopy. Gen & Comp Endocrinology 111:9-19. 241. Wang Z, Gleichmann H. (1995). Glucose transporter 2 expression: prevention of streptozotocin-induced reduction of beta-cells with 5-thio-D-glucose. Endocrinology & Diabetes 103: 83-87. 242. Wang Z, Gleichmann H . (1998). GLUT2 in pancreatic islets: crucial target molecule in diabetes induced with multiple low doses of streptozotocin in mice. Diabetes 47: 50-56. 243. Hosokawa M , Dolci W, Thorens B. (2001). Differential sensitivity of GLUT1- and GLUT2-expressing beta cells to streptozotocin. Biochem Biophys ResCommun 289: 1114-1117. 244. Schnedl WJ, Ferber S, Johnson JH, Newgard CB. (1994). STZ transport and cytotoxicity. Specific enhancement in GLUT2-expressing cells. Diabetes 43: 1326-1333. 245. Thulesen J, Orskoc C, Hoist J J, Poulsen SS. (1997). Short term insulin treatment prevents the diabetogenic action of streptozotocin in rats. Endocrinology 138: 62-68. 246. De Vos A , Heimberg H , Quartier E, Huypens P, Bouwens L, Pipeleers D, Schuit F. (1995). Human and rat beta cells differ in glucose transporter but not in glucokinase gene expression. J Clin Invest 96: 2489-2495. 247. Ferrer J, Alvarez JF, Casamitjana R, Vilardell E. (1993). Signals derived from glucose metabolism are required for g lucose regulation of pancreatic islet GLUT2 mRNA and protein. Diabetes 42: 1273-1280. 248. Thorens B, Gerard N , Deriaz N . (1993). GLUT2 surface expression and intracellular transport via the constitutive pathway in pancreatic beta cells and insulinoma: e vidence f or a b lock i n t rans-Golgi n etwork e xit b y b refeldin A . J Cell Biol 123: 1687-1694. 249. Cheeseman CI, Tsang R. (1996). The effect of GIP and glucagon-like peptides on intestinal basolateral membrane hexose transport. Am J Physiol 271: G477-482. 250. Cheeseman CI, O'Niell D. (1998). Basolateral D-glucose transport activity along the crypt-villus axis in rat jejunum and upregulation induced by gastric inhibitory peptide and glucagon-like peptide-2. Exp Physiol 83: 605-616. 251. Morgan N G , Cable HC, Newcombe NR, Williams GT. (1994). Treatment of cultured pancreatic P-cells with streptozotocin induces cell death by apoptosis. Biosci Rep 14: 243-250. 252. Saini K S , Thompson C, Winterford C M , Walker NI, Cameron DP. (1996). Strepotozotocin at low doses induces apoptosis and at high doses causes necrosis in a murine pancreatic P-cell line, INS-1. Biochem Mol Biol Int 39: 1229-1236. 253. Eisenbarth GS. (1986). Type 1 diabetes mellitus. A chronic autoimmune disease. New EnglJ Med 314: 1360-1368. 254. Srikanta S, Ganda OP, Gleason RE, Jackson R A , Soeldner JS, Eisenbarth GS. (1984). Pre-type 1 diabetes: linear loss of beta cell response to intravenous glucose. Diabetes 33: 717-720. 255. Tominaga M , Komiya I, Johnson JH, Inman L , Alam T, Moltz J, Crider B , Stefan Y , B aetens D , M cCorkle K , O rci L , U nger R H . (1986). Loss o f i nsulin response to glucose but not arginine during the development of autoimmune diabetes in B B / W W rats: relationship to islet volume and glucose transport rate. Proc Natl Acad Sci USA 83: 9749-9753. 256. Reddy S, Bibby NJ, Fisher SL, Elliot RB. (1986). Longitudinal study of first phase insulin release in the B B rat. Diabetologia 29: 802-807. 257. Orci L, Unger R H , Ravazzola M , Ogawa A , Komiya I, Baetens D, Lodish HF, Thorens B. (1990). Reduced beta-cell glucose transporter in new onset diabetic B B rats. J Clin Invest 86: 1615-1622. 258. Gorsuch A N , Lister J, Dean B M , Spencer K M , McNally J M , Bottazzo GF, Cudworth A G . (1981). Evidence for a long prediabetic period in type 1 (insulin-dependent) diabetes mellitus. Lancet 26: 1363-1365. 259. Tuch BE, Turtle JR, Simeonovic CJ. (1989). Streptozotocin is not toxic to the human fetal B cell. Diabetologia 32: 678-684. 260. Tiedge M , Lortz S, Drinkgern J, Lenzen S. (1997). Relation between antioxidant enzyme gene expression and antioxidative defense status of insulin-producing cells. Diabetes 46:1733-1742. 261. Lenzen S, Drinkgern J, Tiedge M . (1996). Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues. Free Radic Biol Med 20:463-466. 262. Mauricio D, Mandrup-Poulsen T. (1998). Apoptosis and the pathogenesis of EDDM: a question of life and death. Diabetes 47: 1537-1543. 263. Lennon SV, Martin SJ, Cotter TG. (1991). Dose-dependent induction of apoptosis in human tumor cell lines by widely diverging stimuli. Cell Prolific. 203-214. 264. Gardner A M , X u FH, Fady C, Jacoby FJ, Duffey DC, Tu Y , Lichtenstein A . (1997). Apoptotic vs. nonapoptotic cytotoxicity induced by hydrogen peroxide. Free Radic Biol Med 22: 73-83. 265. Pospisilik JA. Regulation of glucose homeostasis by dipeptidyl peptidase IV: studies on the counter-regulation and long-term inhibition as diabetes therapy; [Ph.D. Dissertation], University of British Columbia, Vancouver. 266. Vilsboll T, Knop FK, Krarup T, Johansen A , Madsbad S, Larsen S, Hansen T, Pedersen O, Hoist JJ. (2003). The pathophysiology of diabetes involves a defective amplification of the late-phase insulin response to glucose by glucose-dependent insulinotropic polypeptide-regardless of etiology and phenotype. J Clin Endocrinol Metab 88: 4897-4903. 267. Greenbaum CJ, Prigeon RL , D'Alessio D A . (2002). Impaired p-cell function, incretin effect, and glucagon suppression in patients with type 1 diabetes who have normal fasting glucose. Diabetes 51: 951-957. 268. Krarup T, SaurbreyN, Moody A J , Kuhl C, Madsbad S. (1987). Effect o f porcine gastric inhibitory polypeptide on p -cell function i n type 1 and type 11 diabetes mellitus. Metabolism 36: 677-682. 269. Suzuki S, Kawai K , Ohashi S, Mukai H , Murayama Y , Yamashita K . (1990). Reduced Insulinotropic effects of glucagonlike peptide l-(7-36)-amide and gastric inhibitory polypeptide in isolated perfused diabetic rat pancreas. Diabetes 39: 1320-1325. 270. Curtis SB, Buchan A M , Pederson RA, Brown JC. (1992). Insulin response of cultured islets from diabetic and nondiabetic B B rats. Metabolism 41: 1047-1052. 271. KosakaK, Kuzuya T, Akanuma Y , Hagura R. (1980). Increase in insulin response after treatment of overt maturity-onset diabetes is independent of the mode of treatment. Diabetologia 18: 23-28. 272. Lynn FC. Regulation of glucose-dependent insulinotropic polypeptide (GIP) receptor expression in the pancreatic P-cell. [Ph.D. Dissertation], University of British Columbia, Vancouver. 273. Leahy JL. (1990). Natural history of p-cell dysfunction in NJUDM. Diabetes Care 13: 992-1010. 274. Pratley RE, Weyer C. (2002). Progression from IGT to type 2 diabetes mellitus: the central role of impaired early insulin secretion. Curr Diabetes Rep 2: 242-248. 275. Lehtovirta M , K aprio J, F orsblom C , E riksson J, T uomilehto J, G roop L . (2000). Insulin sensitivity and insulin secretion in monozygotic and dizygotic twins. Diabetologia 43: 285-93. 276. Braunstein S. (2003). New developments in type 2 diabetes mellitus: combination therapy with a thiazolidinedione. Clin Ther 25: 1895-1917. 277. Pospisilik JA, Stafford SG, Demuth H U , Mcintosh CHS, Pederson R A . (2002). Long-term treatment with dipeptidyl peptidase IV inhibitor improves hepatic and peripheral insulin sensitivity in the V D F Zucker rat: a euglycemic-hyperinsulinemic clamp study. Diabetes 51: 2677-83. 278. Pospisilik JA, Stafford SG, Demuth H U , Brownsey R, Parkhouse W, Finegood D T , Mclntosh C H S , Pederson R A. (2002). Long-term treatment with the dipeptidyl peptidase IV inhibitor P32/98 causes sustained improvements in glucose tolerance, insulin sensitivity, hyperinsulinemia, and P-cell glucose responsiveness in V D F (fa/fa) Zucker rats. Diabetes 51: 943-950. 279. Pederson RA, White H A , Schlenzig D, Pauly RP, Mcintosh CHS, Demuth H U . (1998). Improved glucose tolerance in Zucker fatty rats by oral administration of the dipeptidyl peptidase IV inhibitor isoleucine thiazolidide. Diabetes 47: 1253-1258. 280. Reimer M K , Hoist JJ, Ahren B. (2002). Long-term inhibition of dipeptidyl peptidase IV improves glucose tolerance and preserves islet function in mice. Eur J Endocrinol 146: 717-727. 281. Sudre B, Broqua P, White RB, Ashworfh D, Evans D M , Haigh R, Junien JL, Aubert M L . (2002). Chronic inhibition of circulating dipeptidyl peptidase IV by FE 999011 delays the occurrence of diabetes in male Zucker diabetic fatty rats. Diabetes 51: 1461-1469. 282. Ahren B, Simonsson E, Larsson H , Landin-Olsson M , Torgeirsson H , Jansson PA, Sandqvist M , Bavenholm P, Efendic S, Eriksson JW, Dickinson S, Holmes D. (2002). Inhibition of dipeptidyl peptidase IV improves metabolic control over a 4-week study period in type 2 diabetes. Diabetes Care 25: 869-875. 283. Nauck M A , Kleine N , 0rskov C, Hoist JJ, Willms B , Creutzfeldt W. (1993). Normalization of fasting hyperglycemia by exogenous glucagon-like peptide 1 (7-36) in type 2 (non-insulin-dependent) diabetic patients. Diabetologia 36: 741-744. 284. Gutniak M , Orskov C, Hoist JJ, Ahren B , Efendic S. (1992). Antidiabetogenic effect of glucagons-like peptide-1 (7-36) amide in normal subjects and patients with diabetes mellitus. N Engl J Med 326: 1316-1322. 285. Gutniak M , Linde B, Hoist JJ, Efendic S. (1994). Subcutaneous injection of the incretin hormone glucagon-like peptide 1 abolishes postprandial glycemia in NIDDM. Diabetes Care 17 (Suppl 9): 1039-1044. 286. Zander M , Taskiran M , Toft-Nielsen M - B , Madsbad S, Hoist JJ. (2001). Additive glucose-lowering effects of glucagon-like peptide-1 and metformin in type 2 diabetes. Diabetes Care 24: 720-725. 287. Yasuda N , Inoue T, Nagakura T, Yamazaki K , Kira K , Saeki T, Tanaka I. (2004). M etformin causes reduction of food intake and body weight gain, and improvement of glucose intolerance in combination with dipeptidyl peptidase IV inhibitor in Zucker fa/fa rats. J Pharmacol Exp Ther Mar 23 [Epub ahead of print]. 

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