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The enteroinsular axis in glucose dependent insulinotropic polypeptide receptor knockout (GIPR-/-) miee Pamir, Nathalie 2002

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T H E E N T E R O I N S U L A R AXIS IN G L U C O S E D E P E N D E N T INSULINOTROPIC POLYPEPTIDE RECEPTOR K N O C K O U T (GIPR-/-) M I C E . by Nathalie Pamir B.Sc , The University of Istanbul, Turkey, 1997 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES Department of Physiology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A May 2002 © Nathalie Pamir, 2002 v In p resen t ing this thesis in partial fulfi lment of the r equ i remen t s for an a d v a n c e d deg ree at the Univers i ty of British C o l u m b i a , I agree that the Library shall make it freely available for reference and s tudy. I further agree that p e r m i s s i o n for ex tens ive c o p y i n g of this thesis for scholar ly pu rposes may be granted by the head of m y depa r tmen t o r by his o r her representat ives . It is u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n of this thesis for financial gain shall no t be a l l o w e d w i t h o u t my wr i t t en p e r m i s s i o n . T h e Univers i ty of British^ C o l u m b i a V a n c o u v e r , C a n a d a D e p a r t m e n t D E - 6 (2/88) Abstract The incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), are gut hormones that act via the enteroinsular axis to potentiate insulin secretion from the pancreas in a glucose-dependent manner. Both GLP-1 and GIP receptor knockout mice (GLP-1 R-/- and GIPR-/- respectively) have been generated to investigate the physiological importance of this axis. Studies in this thesis were carried out on GIP receptor knockout mice (GIPR-/-). Although reduced GIP action is a component of type 2 diabetes, GIP receptor-deficient mice exhibit only moderately impaired glucose tolerance. Thus, the present thesis was directed at investigating possible compensatory mechanisms that take place within the enteroinsular axis in the absence of GIP action. Fasting and 20 t h minute OGTT serum GIP levels as well as duodenojejunal GIP content were altered in GIPR-/- mice. Total serum GLP-1 levels and serum DPIV activity in GIPR knockout mice were not significantly different from those in control animals, either before or during a glucose tolerance test. However, insulin responses to GLP-1 in pancreas perfusions and static islet incubations were significantly greater in GIPR -/- than in +/+ mice (P<0.05), and GLP-1 induced c A M P production was also elevated in the pancreatic islets of the knockout animals (P<0.05). Additionally, pancreatic insulin content and insulin gene expression were reduced in GIPR-/- mice compared to wild type (+/+) mice (P< 0.05). There was, however, no discernible difference in GLP-1 receptor mRNA levels. Immunohistochemistry studies revealed a normal distribution and localization of endocrine cells within the pancreatic islets of GIPR-/- mice. Surprisingly, these studies showed an increase in islet area, when ii compared to total pancreatic area, in the -/- mice (P<0.05) with less intense staining for insulin. In conclusion, the GIPR-/- mouse exhibits increased islet size and (3 cell sensitivity to GLP-1 despite a decrease in pancreatic insulin protein content and gene expression. These findings suggest a critical role for GIP in normal islet and (3 cell function and development. iii TABLE OF CONTENTS A B S T R A C T i i T A B L E O F C O N T E N T S iv L I S T O F F I G U R E S v i A B R E V I A T I O N S v i i A C K N O W L E D G M E N T v i i i I N T R O D U C T I O N '. 1 THE ENTEROINSULAR AXIS 1 Glucose Dependent Insulinotropic Polypeptide (GIP) 5 Glucagon-Like peptide-1 (GLP-I) 14 DIPEPTIDYL PEPTIDASE IV (DPIV) 25 INCRETINS AND TYPE 2 DIABETES MELLITUS 26 GLP-1 receptor knockout mice 28 GIP receptor knockout mice 30 O B J E C T I V E S A N D H Y P O T H E S I S O F T H I S T H E S I S 31 T H E E N T E R O I N S U L A R A X I S I N T H E G I P R - / - M I C E 33 BACKGROUND 33 METHODS 34 Preparation of Genomic DNA from mouse tissue 34 Southern Blotting and Hybridization of Genomic DNA 35 Labeling of DNA by Random Oligonucleotide-Primed synthesis 36 Oral Glucose Tolerance Test 37 Measurement of Fasting and 20lh minute OGTT Blood Glucose, Plasma Insulin, GIP, and GLP-1 37 Measurement of Immunoreactive GIP 38 DPIV Activity Assay 38 Measurement of Duodenojejunal GIP and GLP-1 content 39 In vitro Mouse Pancreas Perfusions 40 Isolation and Culture of Pancreatic Islets 40 Static Stimulation of Pancreatic Islets for Insulin 41 Static Stimulation of Pancreatic Islets for cAMP 42 Pancreatic Insulin Measurements 42 Isolation and Measurement of Islet Insulin and GLP-1 Receptor mRNA by Reverse Transcriptase, Real-Time Polymerase Chain Reaction (PCR) 43 lmmunohistochemistry 44 Data Analysis 45 RESULTS 46 Oral Glucose Tolerance Test 47 Plasma Insulin, GIP, GLP-1 and DPIV levels for fasting and 20"' minute OGTT 48 Duodeno-jejunal GLP-1 and GIP content 52 Insulin Responses from pancreas perfusions of GIP, Glucose, and Arginine 54 Insulin Responses to GLP-1 57 Intracellular cAMP Production 60 Pancreatic insulin content and islet insulin and GLP-1 mRNA content 62 lmmunohistochemistry :-. 64 D I S C U S S I O N 70 iv F U T U R E D I R E C T I O N S 81 GLUCAGON AND SOMATOSTATIN SECRETION AND BIOSYNTHESIS 81 T H E ENTEROINSULAR AXIS O F G I P R +/- MICE 81 ISLET (3 CELL SIGNALING 82 DUODENAL AND INTESTINAL IMMUNOHISTOCHEMISTRY 82 S U M M A R Y 83 R E F E R E N C E S 84 v LIST OF FIGURES Figure 1- The enteroinsular axis 4 Figure 2- Homology for GIP amino acid sequence between species 10 Figure 3- Postranslational processing of proglucagon in pancreas, intestine, and brain 16 Figure 4- Passive transfer by denaturing agarose blotting for southern blots 36 Figure 5- Southern blot with Eco RI digested genomic D N A 46 Figure 6- Oral Glucose Tolerance Test 48 Figure 7- Fasting and 20 th minute OGTT blood glucose and plasma insulin levels 49 Figure 8- Fasting and 20 th minute OGTT plasma GLP-1, DPIV and GIP levels 51 Figure 9- Duodeno-jejunal GIP and GLP-1 content 53 Figure 10- Insulin response to pancreas perfusions with different secretogogues 55 Figure 11 - Insulin response from perfused pancreas to GLP-1 58 Figure 12- Static stimulation of WT and GIPR-/- mice islets 59 Figure 13- cAMP production in the islets of WT and GIPR-/- mice 61 Figure 14- Pancreatic insulin content and islet insulin gene expression 63 Figure 15- GLP-1 receptor gene expression in WT and GIPR-/- mice 64 Figure 16- Quantitative expression of islet area over total pancreatic area 66 Figure 17- Insulin immunostained pancreatic islets 67 Figure 18- Glucagon immunostained pancreatic islets 68 Figure 19- Somatostatin immunostained pancreatic islets 69 vi Abreviations OGTT IVGTT GIP GLP-1 GIPR-/-GLP-1R-/-WT RIA DPIV RT A U C C C K P K A PKC DPIV C C K - P Z PGK V D F Oral Glucose Tolerance Test Intravenous Glucose Tolerance Test Glucose-dependent Insulinotropic Polypeptide/Gastric Inhibitory Polypeptide. (In this manuscript GIP 1 . 4 2 will be referred as GIP) Glucagon-Like Peptide-1 (In this manuscript GLP-1 7 . 3 6 will be referred as GLP-1) Glucose-dependent Insulinotropic Polypeptide Receptor knockout Glucagon-Like Peptide-1 Receptor knockout Wild Type, GIPR+/+ Radioimmunoassay Dipeptidyl Peptidase IV Room Temperature Area Under the Curve Cholesystokinin Protein kinase A Protein kinase C Dipeptidyl Peptidase IV Cholesystokinin-pancreozymin Phosphoglycerate kinase Vancouver diabetic fatty zucker rat v i i Acknowledgment I'm thankful to Dr. Pederson for both taking me as a graduate student and for being a wonderful supervisor. A l l the techniques he taught to me were very valuable and extremely important in the completion of this thesis. Besides professional and academic supervision he also gave me all the support and help I needed as aforeign student far from home. From my first day in the lab Francis Lynn had the patience and the kindness to teach me the basics of a lab life and new techniques. He has made remarkable contributions to the development of my research skills, to my English and to my new life in Canada. His friendship is one of the most important outcomes of my graduate studies. My lab colleagues, Jan Ehses, Cuilan Nian, Simon Hinke and Andrew Pospisilik are important members of my new Canadian family. They always made me feel at home, and I feel fortunate to have worked with them. Chris Mcintosh is the most positive man I have met. He always encouraged me to pursue my studies and reassured me that results would come. I'm looking forward to working with him as a PhD. Student. My friend, my love and my husband Ahmet Gurcan, he is the main reason for me to be able to write these pages. I'm very grateful that I have a lifetime to thank him for his belief in me. viii INTRODUCTION The Enteroinsular Axis In the beginning of the twentieth century, Bayliss and Starling (1902) reported the possible existence of a substance released from the gut after ingestion of nutrients that modulates carbohydrate disposal by generating endocrine pancreatic responses. Subsequently, in 1906 Moore et al. postulated that the "internal secretion of the pancreas might be stimulated by a chemical excitant produced by the duodenal membrane". Their attempts to treat diabetic patients with hog duodenal mucous membrane acid extracts was however unsuccessful. Two decades later this approach was pursued by Zunz and La Barre (1929), who rendered dogs hypoglycemic by i.v. injections of secretin-free intestinal extracts. In 1932, La Barre proposed that the intestinal extract contained an active substance called 'incretin' that might enhance the endocrine secretion of the pancreas by its humoral activity. Loew et al. (1939, 1940a, 1940b) after trying elaborately different methods failed to lower blood glucose levels with intestinal mucosal extracts from dogs or hogs. For the following 20 years until the development of the radioimmunoassay technique for insulin, research on possible intestinal blood glucose lowering factors stagnated. In 1960, Yalow and Berson enabled the measurement of the circulating levels of insulin by the development of an insulin radioimmunoassay (RIA). RIA studies from two different goups, Mclntyre et al. (1964) and Elrick et al. (1964) demonstrated in 1 humans that a glucose load that acts directly on the intestine (oral glucose or intrajejunal administration) is a more potent stimulator of insulin than glucose given intravenously. It was concluded in another study conducted on healthy subjects and patients with end to side portcaval shunts, that after intrajejunal glucose infusion, an increased insulin response and glucose clearance was due to the incretin originating from the intestinal mucosa, and not from the liver (Mclntyre et al., 1965). The contribution of incretins to insulin secretion was quantified by Perley and Kipnis (1967) who found that intestinal stimulation accounts for more than 50 percent of the insulin secretion following a meal in diabetic and non-diabetic subjects. In 1969, Unger and Eisentraut proposed the term enteroinsular axis to describe the hormonal connection between pancreatic islet cells and the gastrointestinal tract. Ten years later Creutzfeldt (1979) enlarged the definition by including the neural and substrate influences along with the hormonal connection (Figurel). Two new terms, incretin and enteroinsular axis, ushered in a new age of research in the understanding of glucose metabolism. Two criteria were defined by Creutzfeldt to identify a substance as an incretin in the enteroinsular axis 1. The substance must be released in response to nutrients, particularly carbohydrates present in the lumen of the gut 2. When administered at physiological concentrations the insulinotropic action must be glucose concentration dependent According to these criteria, an incretin is a gut hormone released after a meal that has insulinotropic effects on the pancreas in the presence of elevated blood glucose. Thus, a true incretin cannot by itself stimulate insulin secretion and therefore cause 2 hypoglycemia. Several hormones were not accepted as incretins since they did not meet the requirements (Creutzfeldt, 1979, 1992). Today two hormones of the enteroinsular axis are considered as incretins: glucose-dependent insulinotropic polypeptide/gastric inhibitory polypeptide (GIP ,.4 2), and truncated forms of glucagon-like peptide-1 (GLP-1 7 . 3 6 and GLP-1 7.3 7) (Fehmann et al, 1995; Pederson, 1994). The work presented in this thesis concerns only the hormonal aspects of the enteroinsular axis, thus the incretins GIP and GLP-1. Therefore, the introduction will focus on the endocrine part of the enteroinsular axis. 3 Figure 1- The enteroinsular axis. The hormonal, neural and substrate directed communication of the gut with the endocrine cells of the pancreas. Reproduced from Creutzfeldt, 1979. 4 Glucose Dependent Insulinotropic Polypeptide (GIP) GIP was first isolated in 1970 by Brown and Pederson as an 'enterogastrone'. An enterogastrone is an endocrine substance secreted in response to fat and its digestive products present in the intestinal lumen with a primary function of inhibiting gastric acid secretion (Kosaka and Lim, 1930). GIP was isolated from impure preparations of cholesystokinin -pancreozymin that were tested for their ability to stimulate gastric acid secretion in vagally denervated canine Heidenhain pouch bioassays (Brown et al., 1970, Brown and Pederson, 1970). Interestingly, the more impure preparation was found to be more effective in inhibiting gastric acid secretion. It was concluded that the C C K - P Z preparation contained an active substance that inhibits gastric acid secretion and it was given the name Gastric Inhibitory Polypeptide, GIP (Brown et al. 1970). Studies demonstrating that impure preparations of C C K exhibited insulinotropic activity were first provided by Dupre and Beck (1966). Subsequently, it was shown that the insulinotropic action of these preparations could be removed by increasing the purity. This finding was very similar to what had been observed by Brown et al. for the gastric inhibitory action of the CCK-PZ extracts (Rabinovitch and Dupre, 1972). Shortly after, in 1973, the insulinotropic action of GIP was reported by Dupre et al. They infused purified preparations of porcine GIP in humans in the presence of glucose and observed that a greater amount of insulin was released when compared to glucose infusion alone. The same study also showed that GIP had no insulinotropic action during the euglycemic state suggesting that the insulin stimulatory action was glucose dependent. Insulin release was observed in the presence of at least 5.5 mM glucose in perfused rat pancreas (Pederson, 5 and Brown, 1976; Jia et al., 1995). The glucose- dependent insulinotropic action of GIP was further verified in humans by glucose clamp studies (Elahi et al, 1979). The glucose-dependency of GIP action provides an important protection against hypoglycemia that might occur due to inappropriate insulin release in response to a low carbohydrate, high fat meal. This led Brown and Pederson (1976) to the alternate designation of GIP as glucose dependent insulinotropic polypeptide. G I P Secretion Polak et al. (1973) showed using a conventional antiserum that immunoreactive GIP was localized in the duodenum and jejenum in man and dog. Later, immunoreactive GIP cells were also located in the upper small intestine of humans, dogs, pigs, rats, and ruminants (Bufa et al., 1975; Buchan et al., 1982). Tseng et al. (1993) detected by Northern blot analysis an 800-nucleotide GIP transcript with a dense distribution through out the duodenum and jejenum and with strong attenuation in the ileum. It has been shown by Polak et al. (1982) in man and dog that GIP is primarly present in the cells of the midzone of the duodenal villi with a lesser intensity in the jejenum. Buchan et al. (1978) identified the cell of origin for GIP as the K cell of the intestinal mucosa, defined by the typical appearance of the secretory granules indicative of an endocrine cell. The development of an RIA for GIP was a turning point in the investigation of the physiology of GIP (Kuzio et al., 1974). The use of different antibodies resulted in widely varied fasting and postpandrial plasma GIP measurements. Kuzio et al. (1974) reported fasting GIP levels in healthy subjects of 237±14 pg/ml rising to 1200 pg/ml after a mixed meal. Although other researchers have reported different values ranging from 60-460 6 pg/ml for fasting and from 170-1470 pg/ml after a meal, the percentage increase relative to basal values was comparable in each case (Kuzio et al, 1974; Morgan et al., 1978; Jorde et al, 1983; Cataland et al, 191'4; Sarson et al, 1980; Burhol et al, 1980). Despite the variability of reports, it has been shown that GIP levels rise 5 to 6 fold within an hour of ingestion of a mixed meal and the levels stay elevated for the next 2-3 hours. To determine the effects of carbohydrate ingestion on GIP secretion Cataland et al (1974) administered lg/kg glucose orally to healthy subjects and measured plasma GIP levels. The plasma glucose and insulin profile was accompanied by a similar plasma GIP profile. Shortly after, Pederson et al. (1975) in dogs and Falko et al. (1980) in humans reported a dose dependent relationship between glucose ingestion and plasma GIP levels. Studies with i.v. injection of glucose resulted in no changes in circulating GIP levels, therefore it was concluded that the luminal presence of carbohydrates is necessary to stimulate GIP secretion (Pederson et al, 1975; Cataland et al, 1974; Andersen et al, 1978). Furthermore, small intestinal perfusions with glucose, sucrose, and galactose resulted in GIP secretion, whereas, fructose, mannose, and lactose perfusions had no effect (Sykes et al, 1980). Evidence was reported by Kieffer et al (1994, 1995) in studies on canine endocrine cells and on a tumor derived cell line that glucose acts directly at the level of K cells to stimulate GIP release. The intracellular mechanisms of this stimulation are not clear as yet, however it has been demonstrated that sodium-dependent transport of the carbohydrates is necessary, and when glucose transport is inhibited so is glucose stimulated GIP secretion (Hopfer, 1987) Intraluminal triglycerides have been also shown to stimulate GIP release in man, dogs, and rats (Cleator, 1975; Krarup et al, 1985; Pederson, 1975). The delays caused by 7 lipids on the absorption and on gastric emptying results in a different GIP secretory profile than with carbohydrates. Hence, fat ingestion results in delayed but more potent and prolonged secretion of GIP (Falko et al, 1975; Brown and Otte, 1978). It has been shown that only long chain fatty acid were able to stimulate GIP secretion whereas, medium and short chain fatty acids have little effect (O'Dorisio et al., 1976; Ross and Shaffer, 1981). Although the first aproaches in the early 1970s by Brown et ai, and Cleator and Gourlay failed to detect the stimulation of GIP secretion by proteins, later on, it was shown that, in addition to glucose and fat, the intraluminal administration of protein also stimulates GIP secretion (Brown, 1974; Cleator and Gourlay, 1975). Thomas et al. (1976, 1978) observed GIP secretion when they perfused the duodenum with an amino acid mixture containing arginine, histidine, isoleucine, lysine, and threonine. The rise in serum GIP levels occurred within 5 min of administration of the perfusate and reached peak values within 30 min. In contrast perfusate containing methionine, phenylalanine, tryptophan, and valine, a mixture known to stimulate C C K release, failed to stimulate GIP secretion (Thomas et al., 1978). Structure of GIP The complete amino acid sequence of porcine G I P M 2 was first described by Brown and Dryburgh (1971). Later, it was shown that this initial sequencing did not correspond exactly to the structure of GIP due to some impurities in the extract. The sequence was corrected by Jornwall et al. in 1981. The human GIP cDNA was sequenced by Takeda et al. (1987) and the complete GIP peptide sequence was reported by Moody 8 et al. (1984) from human intestinal tissue. Shortly after, the rat GIP gene was also isolated and sequenced (Inagaki et al, 1989). The mouse GIP sequence was reported recently by Schieldrop et al. in 1996. Comparison of GIP sequences among species revealed more than 90% conservation at the amino acid level. The human sequence differs by only two amino acids from the porcine and rat sequences and by three amino acids from mouse and bovine sequences (Figure2). It is also important to note that size differences exist in the GIP gene among species, the human GIP gene spans about 10 kbp wheras rat GIP gene is only 8.2 kbp (Inagaki et al., 1989; Higashimoto and Liddle, 1993). It has been also shown that these size differences are mainly due to the arrangements and variations in the lengths of the introns (Higashimoto et al., 1992; Inagaki et al., 1989). The understanding of the structure of the preproGIP gene and the computer analysis of the amino acid sequence of GIP have shown that structural homology exists between GIP and other members of the secretin family including secretin, glucagon and glucagon-like peptides (GLP-1 and GLP-2). (Bell, 1986; Campbell and Scanes, 1992; Tseng etal, 1993). 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Human Tyr A l a G l u G l y . Thr P h e He S e r A s p Tyr S e r lie A l a Met Porcine Rat Mouse 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Human A s p Lys lie His G i n G i n A s p P h e V a l A s n Trp L e u L e u A l a Porcine A r g Rat A r g Mouse A r g 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Human G i n L y s G l y Lys Lys A s n A s p Trp L y s His A s n lie Thr G i n Porcine S e r Rat L e u Mouse A r g S e r Figure 2- Homology for GIP amino acid sequence between species. This compara i son is based on human sequence , only amino ac ids that differ from the human sequence are s h o w n . S e q u e n c e s are from M o o d y et al. (1984), Jornval l et al. (1981) Higashimoto etal. (1992), and Schie ldrop et al. (1996) for human, porcine, rat and m o u s e respectively. The GIP Receptor The GIP receptor is a member of the secretin-VIP family of G protein-coupled receptors with seven transmembrane domains, an intracellular carboxy (C) terminus and an extracellular amino (N) terminus that is involved in ligand binding (Usdin et al, 1993; Gelling et al, 1997; Ulrich et al, 1998; Fehmann et al, 1995). In 1993 Usdin et al analyzed a partial cDNA from rat cerebral cortex and a full length cDNA from a rat tumor cell line (RINm5F) encoding a putative seven transmembrane G-coupled receptor 10 of 455 amino acids and demonstrated a 44% homology with the glucagon receptor and 40% homology with the GLP-1 receptor. The GIP receptor was first shown to be present in hamster insulinoma cells, and hamster |3 cell line and later in human insulinomas and (3-TC3 cell lines (Maletti et al, 1984; Amiranoff et al, 1984, 1985). These studies suggested that GIP acts directly on the (3 cell of the pancreas to stimulate insulin release. Multiple studies have shown that stimulation of the GIP receptor results in activation of the adenylyl cyclase- c A M P pathway resulting in elevated intracellular c A M P levels in islet tumor cell lines and pancreatic islets leading to insulin secretion (Amiranoff et al, 1984; Siegel and Creutzfeldt, 1985; Wheeler et al, 1995). In addition, it has been also shown by several groups that GIP binding to its receptor increases intracellular Ca 2 + levels in HIT-T 15, RINm5F, and COS cell lines (Wheeler et al, 1995; Wahl et al, 1992; Usdin et al, 1993; Lu et al, 1993 a). It is believed that this increase is partly due to the increased Ca 2 + uptake and or direct stimulation of Ca 2 + channels. Although there is no evidence of activation of PI3 kinase directly by GIP, Kubota et al. (1997) recently reported that GIP activates mitogen activated protein kinase via a wortmannin (a phosphatidylinositol 3-kinase inhibitor)-sensitive pathway. Ehses et al. (2001) suggested a new signalling pathway for GIP in the (3-TC3 cell line in which GIP stimulates phospholipase A2 and arachidonic acid production leading to the secretion of insulin via a Ca 2 + independent pathway. Studies with Northern blot analysis, RT-PCR, and in situ hybridization have reported a ubiquitous distribution of GIP receptor mRNA in rat tissues, specifically in pancreas, stomach, intestine, adipose tissue, adrenal cortex, heart, lung and endothelium 11 of major vessels (Usdin et al, 1993; Yasuda et al., 1994). Interestingly, although GIP receptor mRNA transcripts are present in several regions of the brain, multiple studies were unable to detect GIP mRNA or GIP itself in the brain (Tseng et al, 1993; Higashimoto et al, 1992; Buchan et al, 1982). GIP Actions on the pancreas The actions of GIP on pancreatic |3 cells has been discussed previously. Additionally, GIP also has effects on other endocrine cells of the pancreas. GIP has been reported to stimulate glucagon release from islet a cells (Pederson and Brown, 1978). This effect was observable only when glucose levels were below 5.5 mM in perfused rat pancreas. Studies in humans by Elahi et al. (1979) have shown that although GIP was able to stimulate glucagon release in the presence of higher glucose concentrations, glucose-inhibited glucagon release was not restored by GIP. However, GIP was able to stimulate glucagon release in perifused mouse islets (Opara and Go, 1991). Verchere (1991) reported that stimulation of isolated rat islets with GIP following 2 days of incubation increases glucagon, insulin and somatostatin release. It seems that the action of GIP on a cells is variable between species. GIP has also been shown in the perfused rat pancreas to be a weak stimulator of somatostatin release from islet 8 cells (Schmid et al., 1990). No conclusion has been reached on whether this stimulation is physiologically significant or not. 12 Extrapancreatic actions of GIP As discussed earlier, GIP receptors are widely distributed in the body. Although, the physiological importance is yet unclear this suggests that GIP might have other roles in addition to enterogastrone and incretin actions. Recently, attention has focused on the actions of GIP on adipose tissue. The actions of GIP on lipid metabolism have been reviewed by Morgan et al. (1996) and Wolfe et al. (1999). Wasada et al. (1981) have shown in dogs that GIP increases the clearance of injected chylomicron triglycerides. Similar results have been reported from porcine GIP infusion into rats receiving intraduodenal lipid (Ebert et al., 1991). However, in humans exogenous porcine GIP and endogenous GIP released after a mixed meal had no effect on the clearance rate of soy oil given by i.v. injection (Jorde and Burhol, 1984). Furthermore, treatment of rat adipocytes with GIP improved insulin stimulated glucose uptake and increased insulin receptor affinity (Starich et al., 1985; Hauner et al., 1988). It has been also shown that GIP increases lipoprotein lipase activity in a concentration dependent manner in cultured 3T3-L l cell line (Eckel et al., 1981). Using the same cell line, Mcintosh et al. (1999) suggested that GIP stimulates lipolysis and this could be inhibited by insulin via a wortmannin-sensitive pathway. Taken together these results strongly implicate GIP in the regulation of fat metabolism. Although there is no evidence of GIP receptor mRNA in the liver, GIP has been reported to inhibit glucagon stimulated hepatic glucose production in human and rat liver (Hartmann et al., 1986; Elahi et al., 1986). The mechanism of this action is still unclear but it may be an indirect effect or signaling via glucagon receptors (Wolfe et al., 1999). 13 Glucagon-Like peptide-1 (GLP-1) V Following the discovery of GIP, it was observed that, despite the removal of GIP action by immunoneutralization, the incretin effect was still conserved (Ebert et al, 1983). The possibility of a second incretin was resolved in the early 1980's with the development of recombinant D N A technology that enabled the isolation of cDNA encoding the preproglucagon from the pancreata of the anglerfish (Lund et al., 1983). The strong resemblance between the glucagon related peptide section of preproglucagon and GIP led this group to conclude that glucagon related peptide might be the second incretin in the enteroinsular axis (Lund et al., 1982). It was concluded that the anglerfish glucagon related peptide-1 is a homolog of mammalian GLP-1. It has been shown by Bell et al. (1983) in hamsters that GLP s are encoded by the glucagon gene and are a product of post translational processing of proglucagon (160 aminoacid length peptide; PG1-160). Upon analysis of the glucagon gene in different species, two carboxy-terminal glucagon like sequences were identified: GLP-1 and GLP-2 (Bell et al, 1983; Lopez et al., 1983; Heinrich etal, 1984). GLP-1 is widely distributed throughout the body, the majority being located in L cells of the small and large intestine, pancreatic a cells, and in the brain (hypothalamus, thalamus, and septal regions)(Mojsov et al, 1990; Orskov et al, 1992, 1994; Shimizu et al, 1987; Jin et al, 1988). The L cells are mainly localized in the distal jejenum and ileum with an increasing abundance in the colon and rectum (Eissele et al, 1992). Despite this wide distribution, the majority of circulating GLP-1 is expressed from the proglucagon gene in the a cells and in the L cells by a tissue-specific process (Figure3) (reviewed by Kieffer and Habener, 1999, Fehmann et al, 1995; Orskov, 1992). 14 The intestinal processing of PG-1 160 leads to glicentin (PG1-69), oxyntomodulin (PG33-69), intervening peptide 2, GLP-1 (1-37), and GLP-2. (Figure3). It has been shown that GLP-1 undergoes further proteolytic cleavage between the sixth and seventh amino acid leading to the predominant end product of intestinal PG1 processing: GLP-1 (7-36) (Schmidt et al, 1985; Mojsov et al, 1986; Orskov et al, 1989). This truncated form has greater homology with GIP and enhanced insulinotropic activity (Schmidt, 1985; Hoist, 1987). It has been shown by several groups that GLP-1 (7-36) is the principal GLP-1 related peptide secreted after a meal from the gut and it corresponds to 80% of circulating GLP-1 levels in humans (Fehmann et al, 1995) . The pancreatic a cell processing of PG-1 leads to different post-translational end products with pancreatic glucagon (PG33-61) as the main end product (Figure3). Other products are secreted in equimolar amounts: glicentin related pancreatic polypeptide (PG1-30), intervening peptide-1 (PG64-69), and the major proglucagon fragment (PG72-158) corresponding to GLP-1 and GLP-2 (Figure3)(Orskov et al, 1994; Hoist et al, 1994; Mojsov etal, 1986). Proglucagon processing in the brain is similar to that in the intestine, leading to oxyntomodulin, glincentin, GLP-1 and GLP-2 (Figure3) (Larsen et al, 1997; Yoshimoto et al, 1989). The in situ hybridization studies revealed proglucagon mRNA in the cell bodies of paraventricular nucleus, medulla oblongata, dorsal and ventral part of medullary reticular nucleus. It is believed that the processing of the proglucagon occurs within these cells (rewieved by Kieffer and Habener, 1999). 15 P R O G L U C A G O N 1 30 33 61 64 69 72 78 108 111 123 126 158 NH GRP GLUCAGON IP-1 GLP-1 IP-2 GLP-2 P P A N C R E A S 30 33 61 64 69 GRP P GLUCAGON IP-1 MAJOR PROGLUCAGON FRAGMENT I 72 158 GLP-1 IP-2 GLP-2 I N T E S T I N E A N D B R A I N GLICENTIN 30 33 69 72 108 111 123 126 158 GRP P GLUCAGON IP-1 , GLP-1 IP-2 GLP-2 O X Y N T O M O D U L I N Figure 3- Postranslational processing of proglucagon in pancreas, intestine, and brain. Glucagon is the main product resulting from processing of proglucagon in a cells. In intestine and brain GLP-1 and GLP-2 are formed following similar enzymatic cleavages. More than 80% of the circulating GLP-1 levels in humans correspond to GLP-1 (7-37 amide) form produced mainly in the intestine. Adapted from Fehmann ef al. (1995) and Mojsov etal. (1986) GLP-1 Secretion As discussed previously, small quantities of GLP-1 are produced in a cells and cosecreted with glucagon, whereas the main portion of circulating GLP-1 originates in intestinal L cells. It has been shown that ingestion of nutrients stimulates intestinal GLP-1 secretion, although, the L cells of the distal intestine are not directly exposed to nutrients 16 (Elliott et al, 1993; Morgan et al, 1993; Orskov et al, 1994). As wil l be discussed below, this led to the suggestion that GLP-1 secretion is a combined result of nutrient, hormonal and neural stimulation. Circulating GLP-1 levels have been shown to rise within 15-30 min. and reach peak levels 90 min. after ingestion of a mixed meal in normal man (Orskov et al, 1994). As is the case with GIP, reported GLP-1 levels differ from study to study according to the antisera used, with reported peak levels of about 6 times the basal levels reaching up to 50 p M (Orskov et al, 1994). To investigate the mechanism of GLP-1 secretion, in vitro techniques such as development of intestinal cell lines, and tumor derived cell lines expressing the glucagon gene products (i.e. GLUTag, and STC-1) have been used (Brubaker et al, 1992; Drucker et al, 1992). In vitro studies demonstrated that P K A and P K C are important intracellular regulators of GLP-1 secretion. P K A has been shown to stimulate GLP-1 gene expression and secretion in intestinal cell cultures of canine, rat and mouse ileum, and in GLUTag tumor cell line (Damholt et al, 1998; Saifia et al, 1998; Drucker et al, 1994, Brubaker et al, 1998). P K C has also been shown to stimulate GLP-1 release in various experiments with intestinal cell cultures and GLUT-ag and STC-1 cell lines, but it was unable to stimulate peptide synthesis (Drucker et al, 1994; Brubaker et al, 1998; Abello et al, 1994). Additionally, increased intracellular Ca 2 + was shown to inhibit GLP-1 release suggesting a role for calcium in the basal regulation of GLP-1 secretion (Brubaker, 1988). Oral ingestion or intestinal injection of glucose stimulates GLP-1 release in humans, pigs, dogs, rats, and, mice (reviewed by Kieffer and Habener, 1999; Fehmann et al, 1995). It has been shown that intestinal administration of glucose must be 17 accompanied by sodium in order to stimulate GLP-1 release (Hermann et al., 1995; Sasaki et al., 1993). This implies that glucose absorption is necessary to stimulate GLP-1 secretion and that this occurs through sodium/glucose transporters on intestinal villi. This is consistent with the information that intestinal infusion of the non-transportable carbohydrates (e.g. fructose, and lactose) does not stimulate GLP-1 release (Kong et al, 1999; Horowitz et al., 1996). In contradiction, recently, Rayner et al. (2000) compared the effects of glucose and fructose on GIP and GLP-1 secretion in healthy humans. Intraduodenal infusions of glucose and fructose stimulated similar levels of GLP-1 secretion. Furthermore, a recent study by Damholt et al. using canine L cells has shown that GLP-1 secretion is not affected by different concentrations of glucose (Damholt et al., 1998b). This implies that factors other than direct stimulation of L cells by glucose are in place since carbohydrates are digested before reaching the distal intestine. Additionally, the rapid rise in GLP-1 levels, after an oral glucose load suggests that glucose alone cannot account for direct GLP-1 stimulation. In addition to glucose, the ingestion of tryglycerides or fatty acids stimulates GLP-1 secretion from the gut. Studies in humans, dogs and rats have shown that as for glucose ingestion, intraluminal administration of fats and or triglycerides stimulates GLP-1 release (Hermann et al., 1995; Elliott et al, 1993, Layer et al., 1995). Additionally, it has been shown that fatty acids are able to act directly on L cells (Rocca and Brubaker, 1995; Brubaker, 1991). Interestingly, despite the increasing abundance of L cells in the distal intestine, studies have shown that administration of fat to the duodenum or to the ileum does not modify the magnitude of GLP-1 secretion (Roberge and Brubaker, 1993; Brubaker, 1991). This observation led Roberge and Brubaker (1993) to suggest that there 18 is a proximal-distal loop regulating nutrient stimulated GLP-1 secretion from the L cells. Neural, and endocrine factors are thought to contribute to this loop. Hence, the rapid rise in circulating GLP-1 levels after administration of a mixed meal may be partly explained by the operation of the proximal-distal loop. In support of this, it has recently been shown that disruption of the subdiaphragmatic vagus nerve in rats abolishes fat induced GLP-1 release (Rocca and Brubaker, 1999). A wide range of studies in different species have reported that proteins when administered alone do not stimulate GLP-1 secretion (Hermann et al, 1995; Layer et al, 1995). However, recently, Cordier-Bussat et al. (1998) have shown that peptones are able to stimulate GLP-1 secretion from the perfused rat intestine, and murine STC-1 cells. The same group has also been able to show an increase in proglucagon gene expression in STC-1 and GLUT-ag cell lines. In addition to nutrients, various hormones have been reported to feedback on GLP-1 secretion. Both in vitro and in vivo insulin has been reported to inhibit GLP-1 secretion (Matsuyama et al., 1975; Brubaker and Vranic, 1987). Intestinal somatostatin, a general endocrine inhibitor, has been reported to inhibit GLP-1 secretion (Brubaker, 1991, Damholt et al., 1998b). Interestingly, recent studies with immunocytochemistry and intestinal cell cultures in dogs have shown that GIP stimulates GLP-1 secretion (Damholt et al., 1998a, 1999). These studies have suggested that glucose stimulates GIP secretion from the proximal intestine, which in turn acts on L cells to stimulate GLP-1 release: the concept of proximal-distal loop. However, the existence of a similar loop was not shown in humans. In addition Rocca and Brubaker have recently shown that disruption of several hepatic vagal branches abolishes GIP induced GLP-1 release and 19 stimulation of subdiaphragmatic vagus nerve also stimulates GLP-1 secretion (Rocca and Brubaker, 1999). Taken together, these data suggest GIP stimulated GLP-1 secretion. The pathway of this stimulation is yet still unclear. It might be a paracrine interaction between K and L cell or might involve vagus mediated signaling, (reviewed in Kieffer and Habener, 1999). The GLP-1 receptor GLP-1 receptors are present on tumor derived (3 and a cell lines, pancreatic islets, lung membranes, stomach, intestine, heart, kidney and brain (Shimizu et al., 1987; Richter et al, 1990; Wei and Mojsov, 1995; Bullock et al, 1996; Yamato et al, 1997; Gros et al, 1992). Despite existence of reports on the actions of GLP-1 on liver, adipose, and skeletal tissue, investigations involving RT-PCR and in situ hybridization have failed to find GLP-1 receptors on these tissues (Wei and Mojsov, 1995; Bullock et al, 1996). Intra-islet distribution of GLP-1 receptors has also been studied. Bullock et al. (1996) reported the colocalization of insulin and GLP-1 receptor mRNA. They were unable to determine whether the mRNA was present in the a cells or not. Recently, Heller et al. (1997b), using GLP-1 receptor and islet hormone specific antibodies on dispersed islet cells, reported that the GLP-1 receptor was present on 90% of insulin positive cells, 76% of somatostatin positive cells and 20% of glucagon positive cells. As has been previously discussed for the GIP receptor, the GLP-1 receptor is also a member of the G protein coupled family of receptors including glucagon, VIP, secretin, GIP, P A C A P , GHF, calcitonin and PTH (reviewed in Kieffer and Habener). The 463 amino acid receptor has eight hydrophobic segments with an extracellullar N terminus 20 responsible for ligand binding and an intracellular C terminus responsible for intracellular signaling (reviewed in Kieffer and Habener). Ligand-binding studies have shown that GLP-1 receptors have a specificity for GLP-1 in the nanomolar range whereas other peptides from the glucagon family do not bind to the receptor (Fehmann et al., 1994; Kieffer et al, 1996). Recently Moens et al. (1998) reported that glucagon binds to GLP-1 receptors on islet |3-cell cultures with very low affinity (millimolar range). As for all other members of VIP-secretin receptor family, the primary signaling pathway is believed to be via the adenylate cyclase-cAMP pathway. For the GLP-1 receptor this has been demonstrated in |3 cell lines, isolated (3 cells and isolated islets (Drucker et al, 1987; Ahren et al, 1996; Moens et al, 1996). The receptors present on the surface of the cells are coupled to stimulatory G proteins leading to the stimulation of adenylyl cyclase and a rise in intracellular c A M P levels. c A M P in turn stimulates the protein kinase A and c A M P dependent Ca 2 + pathways that both conribute to the rise of intracellular Ca 2 + leading to insulin secretion (reviewed in Kieffer and Habener, 1999 and in Gromada et al. 1998). Recently, it has been shown that activation of the c A M P / P K A pathway by GLP-1 improves the effectiveness of the K - A T P channel-independent action of glucose (Yajima et al, 1999). Studies have shown that activation of c A M P / P K A pathway in (3 cell results in increased intracellular Ca 2 + levels only in the presence of glucose (Yajima et al, 1999; Gromada et al, 1998). It has been proposed that GLP-1 contributes to (3 cell glucose responsiveness (glucose competence of the (3 cell) mainly by this process. 21 GLP-1 Actions on the pancreas The insulinotropic action of GLP-1 was first demonstrated in the isolated perfused pancreas of rat, pig and dog (Hoist et al, 1987; Mojsov et al, 1987; Kawai et al, 1989). Later, in humans it was shown that the presence of glucose and GLP-1 in the circulation stimulates insulin release (Kreymann etal., 1987). In vivo studies using the GLP-1 receptor antagonist exendin (9-39) have shown that GLP-1 accounts for an important portion of the incretin effect in rats, baboons and recently in man (reviewed in Kieffer and Habener, 1999). Several studies have shown that GLP-1 also exhibits glucose dependency, the threshold glucose dose varying between species and experiments (using different doses of GLP-1) between 2.8 and 5.0 mM (Fridolf and Ahren, 1991, Hargrove et al., 1995; Ahren et al., 1995) The glucose dependence of GLP-1 is an important measure to protect from the hypoglycemia caused by elevated circulating insulin levels. The interdependence between glucose and GLP-1 action implicates a synergism between glucose metabolism and activation of GLP-1 receptor signaling cascades leading to the glucose competence concept where glucose is required for GLP-1 action and GLP-1 is required to improve |3 cell responsiviness to glucose (Holz et al., 1993). GLP-1 has also been shown to stimulate proinsulin gene transcription and biosynthesis in islets and insulinoma cell line (Fehmann and Habener, 1992; Drucker et al., 1987). Recently, it has been also demonstrated that GLP-1 contributes to the proliferation and neogenesis of (3 cells in rodents by activation of cell proliferation and differentiation transcription factors such c-fos, c-jun, jun B (Xu et al., 1999; Edvell and Lindstrom, 1999; Susini etal, 1998). 22 GLP-1 also has effects on the other endocrine cells of the pancreas. There is evidence that GLP-1 inhibits glucagon secretion in vivo. Perfused pancreas studies in pigs, rats and dogs resulted in up to an 80% decrease in pancreatic glucagon output (Orskov et al, 1988; Kawai et al, 1989). Later, these reports were supported with similar findings in human islet studies (Fehmann et al., 1995b). The glucagonostatic action of GLP-1 is believed to contribute to its glucose lowering effects. As has been previously introduced, GLP-1 receptors are also present on the surface of 6 cells. Perfused pancreas and islet studies have reported that GLP-1 stimulates somatostatin secretion (Kawai et al., 1989; Schmid, 1990). Additionaly, this effect has been shown in human islet studies (Fehmann et al., 1995b). It is widely discussed whether the glucagonostatic effects of GLP-1 are mediated directly on the a cell or via the stimulation of islet somatostatin. In either case GLP-1 has a role in intra islet hormone regulation. Extrapancreatic actions In addition to its insulinotropic action, GLP-1 has well documented extrapancreatic actions. As discussed previously, tryglicerides and fats are potent stimulators of GLP-1 secretion. The presence of partially digested fat in the ileum contributes to the ileal brake by slowing gastric emptying and jejunal motility (Layer and Hoist, 1993; Lin et al., 1997; Layer et al, 1995). It has been shown in humans that GLP-1 infusion in an amount equal to postpandrial plasma GLP-1 concentrations, results in inhibition of gastric acid secretion and gastric emptying (Willms et al., 1996; Schirra et al., 1996; Nauck et al., 1997b). The mechanisms underlying these effects remain unclear 23 and confusing since possible mechanisms differ from species to species. In rats, there is evidence suggesting that the gastric action of GLP-1 is mediated through inhibition of gastrin secretion and stimulation of gastric somatostatin (Eissele et al, 1990; Jia, 1994). Whereas in humans and pigs the involvement of central nervous system seems more probable (reviewed in Kieffer and Habener, 1999; Wettergren et al, 1998). Interestingly, despite the absence of evidence showing the existence of GLP-1 receptors on liver, fat and muscle tissue, GLP-1 has been shown to have glycogenic and lipogenic actions in these tissues (reviewed in Kieffer and Habener, 1999). The reported effects of GLP-1 on hepatic glucose metabolism are controversial. GLP-1 has been reported to improve or to not change insulin-independent hepatic glucose absorption (Gutniak et al, 1992; D'Alessio et al, 1994). This information led researchers to focus on the possible insulin like effects of GLP-1. Initial reports on diabetic models suggested that GLP-1 was able to increase peripheral glucose uptake (D'Alessio et al, 1994, 1995). However, subsequent studies in healthy man were unable to reproduce the insulin-like effects of GLP-1 (Ryan et al, 1998; Toftnielsen et al, 1996). Since all these studies were carried out with healthy subjects, the effects of GLP-1 on peripheral glucose disposal in diabetic state remains unclear. In this perspective a new study from, Sandhu et al. (1999) reported that in depancreatized dogs, GLP-1 was able to potentiate insulin action during a hyperinsulinemic clamp. A new area of interest is GLP-1 action in the brain. Multiple studies have reported that the intracerebral ventricular administration of GLP-1 decreases the food intake (reviewed in Kieffer and Habener, 1999; Turton etal, 1996). The intraperitoneal administration of GLP-1 to test the same intent was however unsuccesful suggesting that 24 GLP-1 action on the brain might be effected by locally produced GLP-1 (Turton et al., 1996). Furthermore, subcutaneous infusions of GLP-1 in humans did result in the reduction of food intake suggesting that intestinal GLP-1 might be crossing the blood-brain barrier (Toft-Nielsen et al, 1999). It is also still unclear whether the reduction of food intake is a result of food aversion or a satiety effect. Dipeptidyl Peptidase IV (DPIV) Both incretins after their secretion are metabolized through several processes; renal clearance, hepatic clearance, and proteolytic processing in the circulation. A plasma enzyme, dipeptidyl peptidase IV (DPIV), has been demonstrated to be responsible for rendering the incretins biologically inactive by removing the N terminal dipeptide. Mentlein et al. (1993) reported that incubation of GIP or GLP-1 with either serum or purified DPIV results in production of the inactive forms of both hormones; GIP 3 . 4 2 and GLP-1 9 . 3 6 . Later it was shown by Kieffer et al. (1995) that this degradation is very rapid with more than 50% of the circulating intact peptide hydrolysed to the inactive form in less than 2 minutes. Degradation by DPIV in the circulation is believed to be the primary mechanism for the physiological inactivation of incretins. DPIV is a serine protease most abundantly present in intestinal enterocytes, proximal tubule of kidney and placental tissue (Yaron and Naider, 1993). It has been also found in liver, stomach, spleen, lung, bone, testes, gall bladder, large intestine, vascular endothelium and pancreatic islets (Vanhoof et al., 1992; Poulsen et al., 1993; Mentzel et al, 1996). 25 The inhibition of DPIV activity plays an important role in the diabetes research over the past decade. It has been shown that in pigs the inhibition of DPIV activity potentiates the insulin response to GLP-1 (Deacon et al, 1998). More recently, Deacon et al. (2001) have shown in pigs with the use of valine pyrolidide (DPIV inhibitor) that DPIV inhbition reduces the degradation and clearance of GIP and potentiates its insulinotropic and antihyperglycemic effects. Mice with genetic disruption of the DPIV gene exhibit increased levels of bioactive GIP and GLP-1 along with enhanced glucose clearance after an OGTT (Marguet et al., 2000). Furthermore the oral administration of a DPIV inhibitor isoleucine thiazolidide to Zucker fatty rats has been reported to improve glucose tolerance mainly by increasing the plasma half-life of both incretins GIP and GLP-1 (Pederson etal, 1998). Very recently, Pospisilik et al, (2002) reported imporoved glucose tolerance and insulin sensitivity in long term P 32/98 (DPIV inhibitor) treated V D F (Vancouver Diabetic Fatty) Zucker rats. Ahren et al. (2000) reported improved glucose tolerance and insulin secretion by inhbition of DPIV with valine-pyrolidide in mice. Incretins and type 2 Diabetes Mellitus The incretin effect after an oral glucose load may account for up to 50% of insulin secretion. Alterations in GIP and GLP-1 secretion and/or modifications in incretin action are important issues to address in the investigation of the pathophysiology of type 2 (non-insulin dependent) diabetes mellitus. Fasting GIP levels have been reported to be normal or elevated in type 2 diabetic patients when compared with healthy individuals (Ebert et al, 1976; Mazzaferri et al, 26 1985; Osei et al., 1986). Postpandrial GIP secretion has also been reported to be normal, increased or decreased in type 2 diabetes patients (Ahren et al., 1997; El-Salhy, 1998; Nauck, 1986). Nauck et al. (1986) reported that although GIP secretion after an OGTT was not different between type 2 diabetic and normal patients the insulinotropic action of GIP was abolished in diabetic patients. This suggests an impaired responsiveness of the |3 cell to GIP stimulation. Creutzfeldt et al. (1983) identified two distinctive hyper- and hypo-secreters of GIP among 141 indiviuduals with type 2 diabetes that both exhibit loss of incretin effect. It has been argued that the (3 cells in the hypersecretors group are overstimulated perhaps leading to the impairment of the GIP receptors on the cell surface. Recent studies have demonstrated that GIP receptors exhibit similar characteristics as other G protein coupled receptors in terms of ligand binding, desensitization and internalization (Gelling et al., 1997; Wheeler et al., 1999; Tseng and Zhang, 1998). Tseng et al. (1996) reported that preincubation of L cells with high concentrations of GIP decreased c A M P production in response to subsequent GIP stimulation. The same group reported that supraphysiological infusions of GIP in rats resulted in the ablation of the incretin effect of GIP. A recent study on isolated rat islets and an insulinoma cell line reported desensitization of the GIP receptor after high dose GIP preincubation (Ffinke et al, 2000) Collectively, these findings suggest that elevated serum GIP levels in diabetic patients might induce chronic desensitization of the receptor leading to the loss of incretin effect. Furthermore, recently, Lynn et al. (2001) reported that GIP action was abolished in V D F rats due to decreased GIP receptor gene expression in the islets. This suggests down regulation of the pancreatic GIP receptor may contribute to the pathology of type 2 diabetes. 27 Studies in type 2 diabetic patients have shown that, while GIP loses its insulinotropic action, GLP-1 preserves its incretin effect. It is still controversial whether GLP-1 levels are normal or elevated in type 2 diabetes. Therfore it is not possible to conclude a role for over or under production of GLP-1 in type 2 diabetes, (reviewed in Kieffer and Habener, 1999) Recent studies in diabetic rodents with exendin 4 (a GLP-1 agonist) have shown reduced blood glucose, increased insulin secretion and insulin mRNA, and pronounced weight loss, and reduced adipose tissue content (Greig et al, 1999; Staffers et al, 2000; Szayna et al, 2000). Intravenous, subcutaneous, and oral administration of GLP-1 has been shown to improve both fasting and postpandrial glycemia in type 2 diabetics (Gutniak et al, 1994, 1996, 1997; Nauck et al, 1997a; Rachman et al, 1996; Willms et al, 1998). These findings strengthen the potential role of GLP-1 as a therapeutic agent in type 2 diabetes. The glucose dependence of incretin action differentiates GLP-1 from sulfonylureas and makes this peptide a good candidate for the treatment of type 2 diabetes (rewieved in Kieffer and Habener, 1999 and Wolfe et al. 1999). GLP-1 receptor knockout mice In 1996, to investigate the physiological importance of GLP-1 Scrocchi et al. generated GLP-1 receptor knockout mice. The mouse GLP-1 receptor gene was isolated and homologous recombination in mouse embryonic stem cells was used to generate mice with null mutations in both GLP-1 receptor alleles (Scrocchi et al, 1996). The targeting vector was designed to inactivate the GLP-1 receptor gene by replacing exons encoding for intra-membrane and intra and extracellular domains of the receptor. Since 28 1996, numerous studies have been pursued by different groups with the GLP-1 R-/- mice to identify the physiological importance of the lack of GLP-1 action. GLP-1R-/- mice exhibit modest impaired glucose tolerance and diminished plasma insulin levels, after oral and intravenous administration of glucose (Scrocchi et al., 1996). Interestingly the heterozygous GLP-1 receptor +/- mice also exhibited an abnormal glycemic response and reduced circulating insulin levels in response to a glucose challenge. These facts strongly suggest that besides its effects as an incretin on the (3 cell, GLP-1 also exerts actions peripherally, possibly through regulation of glucose disposal. However, no change in feeding behaviour nor in body weight was detected between GLP-1R-/- and wild type mice for 24 weeks (Scrocchi et al., 1996). This study was supported by the findings of Scrocchi and Drucker (1998), who examined the effects of aging and high fat diet on body weight in GLP-1R-/- mice. They were unable to relate GLP-1 to obesity. The glucose competence concept was also investigated in GLP-1R-/- mice. Flamez et al. (1998) perifused GLP-1R-/- mouse islets to assess insulin secretion in response to different concentrations of glucose. No difference in terms of insulin secretion has been observed between wild type and GLP-1R -/- mice. Accordingly, islet and pancreatic insulin content were found to be very similar. Flamez et al. concluded that pancreatic (3 cells exhibit preserved glucose competence in mice with a null mutation in the GLP-1 receptor. Pederson et al. (1998), however, reported a 30% decrease in pancreatic insulin content with an equivalent decrease in pancreatic insulin gene expression in GLP-1 R-/- mice. 29 Pederson et al. (1998) also reported that the absence of GLP-1 action was compensated for in part by upregulation of the GIP-insulin axis. The enhanced insulin response to pancreas perfusion and islet perifusion with GIP in the GLP-1R-/- mice suggests that some compensations occurs within the |3 cell. Flamez et al. (1999) reported that disruption of GLP-1 receptor signaling leads to reduced basal but enhanced GIP stimulated c A M P production and to altered basal and GIP stimulated cytosolic Ca 2 + accumulation. Thus, it appears evident that in the absence of GLP-1 action, GIP compensates with adaptive changes mainly occurring within the (3 cell. Very recently, Ling et al. (2001) have reported that the distribution of (3 cells is shifted from large to small and medium-sized islets in the absence of GLP-1R signaling Furthermore, GLP-1R-/- islets exhibit abnormalities in cell topography, with two to threefold more centrally located a cells detected in GLP-1R-/- islets. These alterations in a- and (3 -cell location indicate that basal levels of GLP-1 signaling are important to maintain normal cellular organization of the endocrine pancreas (Ling et al., 2001). GIP receptor knockout mice In 1999, Miyawaki et al. generated GIP receptor knockout mice. The exons 4 and 5 of the GIP receptor gene that encode the N-terminal extracellular region were replaced by phophoglycerate kinase cassettes. The embryonic stem cells were transfected with the construct and transmitted to germ lines. GIPR-/- mice have been shown to have impaired glucose tolerance after an OGTT accompanied by decreased insulin levels at the 15th and 30 t h OGTT time point. The intravenous administration of glucose did not alter the glycemic profile in the GIPR-/-30 mice. A high fat diet for three weeks in GIPR-/- mice resulted in increased plasma glucose levels during an OGTT but suprisingly the insulin secretory profile did not change. Weight gain for the high fat diet conditions was not different in wild type versus knockout animals (Miyawaki et al, 1999). Objectives and Hypothesis of this thesis GIPR-/- mice represent an important animal model to investigate the physiological importance of GIP and its contribution to the enteroinsular axis. The studies reported in this thesis had two principal aims: first, assessment of general characteristics that contribute to the enteroinsular axis of GIPR-/- mouse and second, investigation of compensatory mechanisms that might have taken place in the continuous absence of functional GIP receptors. The first aim was to investigate the physiological characteristics that are critical for the enteroinsular axis. It is important to outline the normal and abnormal profiles that might be present in the absence of a GIP-insulin axis. Plasma glucose, insulin and incretin levels were assessed under fasting and postpandrial conditions. Finally, duodeno-jejunal GIP and GLP-1 content were also measured. It is the main hypothesis of this thesis that GIPR-/- mice exhibit compensatory changes in the enteroinsular axis. However, it was difficult to predict where these changes might occur. Therefore, the experimental design of the second aim was developed based on the preliminary findings of the general physiological characteristics. (3 cell responsiveness to different secretogogues (different doses of glucose, and arginine) was assessed. In vitro insulin and cAMP responses to GLP-1 were measured. Pancreatic 31 insulin protein content and gene expression were investigated. RT-PCR was used to determine insulin and GLP-1 gene expression in pancreatic islets. lmmunohistochemistry was used to assess any alterations in the islet structure of the GIPR-/- mouse pancreas. 32 The Enteroinsular axis in the GIPR-/- mice. Background GIPR-/- mice were generated by Miyawaki et al. (1999) at the University of Kyoto in Japan. This group has published the details of the generation of mutant animals and the description of the targeting construct. This protocol leads to a null mutation where mutant animals were identified by the length of their GIP receptor. As has been pointed out in the Introduction, the exons coding the N terminal extracellular region of the GIP receptor gene were replaced by a PGK neo-cassette. The transmission of the mutant allele was confirmed by D N A blotting analysis using a 3' probe set in the external region of the targeting construct. The heterozygote mice islets did not respond to GIP indicating that the mutated GIP receptor does not have a dominant -negative effect but has lost its function. Three heterozygote (GIPR+/-) pairs were sent to our laboratory at U B C from the University of Kyoto in March 2000. To genotype the litters derived from GIPR+/-intercross, the genomic D N A was probed and identified by Southern blot analysis. Eco RI digested genomic D N A using a 3' external probe confirmed that homologous recombination results in a 3kb length GIP receptor instead of a 7 kb wild type. In our laboratory the Southern blot protocol was adapted and optimized by Francis Lynn. The GIPR-/- mouse colony developed in Japan exhibited impaired glucose tolerance after an OGTT along with a normal glucose excursion after an IVGTT (Miyawaki et al, 1999). The first step was to assess the physiological characteristics that 33 are thought to be important in the regulation of glucose homeostasis. Various protocols have been used to investigate glucose tolerance, plasma circulating insulin and incretin levels during an OGTT, and intestinal GIP and GLP-1 content. The possibility of a compensatory mechanism similar to what has been shown in GLP-1R-/- mice was also investigated (Pederson et al. 1998). The experimental approach was to characterize the beta cell insulin reponse and cAMP production in response to different secretagogues by pancreas perfusion, and static islet stimulation. The islet insulin and GLP-1 receptor gene transcription as well as insulin protein content were also measured. Immunohistochemistry was also performed to assess the morphological status of the islets in GIPR-/- mice. METHODS Preparation of Genomic DNA from mouse tissue Four week old mice were rendered unconscious under C 0 2 . A tail segment (5-8 mm) was cut and the ears were pierced according to a numbering system. Tissues were digested overnight at 55 °C in 700 u.1 of tail buffer, 50 m M Tris pH=8, lOOmM ethylenediamine tetraacetic acid (EDTA), 0.5% SDS and in 35 ul of 10 mg/ml proteinase K (Gibco). The next day digestion was stopped with a 5 M NaCl wash at 12,000 g for 5 min. The supernatant was precipitated in isopropanol with 30 min centrifugation at 12,000 g. The pellet was washed with 70% ethanol and air dryed. For D N A quantification the pellet was resuspended in 10 mM TrisHCl at 4 °C overnight. The 34 concentration was determined with sample absorbance at A 2 6 0 and A 2 6 0 / A 2 8 0 ratios. The D N A used for Southern blot had a A 2 6 0 / A 2 8 0 ratio lower than 1.6. Southern Blotting and Hybridization of Genomic DNA Ten u.g of genomic D N A was restriction digested overnight at 37 °C with ECO RI (Gibco Life Technologies). The reaction was stopped with 3M NaOAc and the digests were ethanol (100%) precipitated on dry ice for 10 min and centrifuged for 20 min at -4 °C. After ethanol wash the pellet was resuspended in 20 u.1 Tris/EDTA buffer, 10 mM TrisCl, 1 mM E D T A pH=8. The fragments were separated overnight at 15 V in a 0.7% agarose gel with T A E buffer, 40 mM Tris-acetate, 1 mM E D T A , 40 mM glacial acetic acid. The next day the gel was depurinated in 0.25 M HC1 for 15 min and neutralized with the transfer buffer 0.4 M NaOH. An alkaline transfer with a of positively charged membrane, where the transferred D N A becomes covalently linked to the membrane was chosen. The gel was transferred to a positively charged nylon membrane (Roche Diagnostic GmbH, Germany) in a pyramid type structure (Figure 4) for two hours. The membrane was then washed with 2X sodium chloride/sodium citrate (SCC), and baked in an oven at 120 °C for 30 min to immobilize the D N A on the membrane. The D N A was then hybridized with the 3' probe to allow detection of the segment on the membrane. After 15 min of prehybridization at 42 °C, the membrane was hybridized overnight with labeled 3' directed probe at 60 °C in A P H , 5X SCC, 5X Denhardts, (for 100X, 10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (Pentax fraction V : Miles lab), H 20 to 500 ml), 1% SDS, 150fxg/ml herring sperm DNA. The next day the membrane was washed first in 2XSCC with 0.1%SDS than 35 with 0 . 2 X S C C with 0.1%SDS at 42 °C and at room temperature. The final radioactivity was kept under 0.05 mrad/hr and the membrane was exposed for three days at -70 °C with the use of Kodak Biomax film. Weight Transfer Buffer Bridge Filter Paper Support Figure 4- Passive transfer by denaturing agarose blotting for Southern blots. This low-tech method for agarose transfer is by a passive, alkal ine, downward elut ion. The pyramid like sett ing prevents the shortcircuit between the membrane and the layers, and al lows a safe and complete transfer Labeling of DNA by Random Oliqonucleotide-Primed synthesis Random oligonucleotide-primed synthesis is an alternative to nick translation for producing uniformly radioactive D N A of high specific activity (Feinberg and Vogelstein, 1983). To allow double strand labeling, 100 ng gel purified wi ld type genomic D N A 36 fragment was combined with 4 u,g random hexanucleotides (Life Technologies, Burlington, ON, Canada) followed by 10 min boiling and cooling. Subsequently the mixture was incubated at 37 °C for 2 hours with 0.5 M 3dNTP mix (minus dATP) same volume of 10X Klenow fragment buffer (Gibco), 50 u,Ci [ct-32P] dATP (Amersham Pharmacia), and 6 U D N A polymerase large fragment (Klenow). The probe was purified with QIAQUICK PCR Spin kit, following the instruction manual. Oral Glucose Tolerance Test Age matched 9-14 weeks old male WT and GIPR-/- mice were used for this experiment. Animals were fasted overnight (16-18 hours). The next morning the mice were weighed and allowed 20 minutes of acclimatization to the laboratory. Blood samples were collected from the tail veins. Blood glucose was measured individually for each mouse using a handheld glucose meter (Surestep®, Lifescan INC., Burnaby, B C , Canada). Baseline, fasting glucose was measured before the administration of glucose. Just before the administration by gavage of 1 g/kg glucose as a 10% solution, each animal was rendered unconscious under C 0 2 for a very short period of time (less than 1 minute) to facilitate the feeding. Blood glucose was measured at the 10, 20, 30, 45, 60, and 120 minute- time points. Measurement of Fasting and 20 t h minute OGTT Blood Glucose. Plasma Insulin. GIP. and GLP-1. This experiment was designed to investigate the concurrent levels of insulin and incretins that are important in the control of glucose homeostasis. The 20 t h minute was 37 chosen as the peak period of glucose and hormone release. The OGTT protocol described above was used to assess plasma GIP, GLP-1, and insulin levels. To obtain a sufficiently large blood sample, four groups of 5-6 animals each were used. Blood was pooled at t=0 and t=20 min from each group. Plasma was than separated by centrifugation at 12000 xg for 15 min at 4 °C and stored at -20°C for RIA. Insulin was measured with a rat sensitive Linco kit (Linco St Charles, M O , USA) requiring 50 ul of plasma. Total GLP-1 (GLP-1,. 3 6 and GLP-1 7.3 6) was measured with a carboxy terminal directed Linco kit requiring 150 ui of plasma. To indirectly assess plasma active GLP-1 levels, plasma DPIV activity was measured. Plasma GIP levels were also measured as described below Measurement of Immunoreactive GIP Samples were diluted in assay buffer, 5% charcoal extracted plasma (CEP), 2% Trasylol, 0.4 M P 0 4 buffer (pH=6.5). GIP standards were serially diluted from 7.8 pg to 2000 pg to constitute the standard curve. On day 1 samples were incubated with GIP antisera (1:30,000) RK343F (Linda Morgan, University of Surrey, Guilford, Surrey). On the second day, radiolabeled GIP (5000 cpm/ tube) was added to the incubation tubes. On day 3, to separate the antisera bound GIP was separated from the unbound with 25% Polyethylene Glycol 8000 leading to a total volume of 1 ml in the assay tube. Radioactivity was assessed with a gamma counter,(LKB, Wallac 1277 , Gammamaster) DPIV Activity Assay A colourimetric assay was used to assess mouse plasma DPIV activity. H-Gly-Pro-pNA (Sigma G-0153-100 mg), a substrate for DPIV, is hydrolyzed to Gly-Pro and 38 - p N A whose yellow appearance can be monitored by spectrophotometer. A final concentration of 0.40 mM of substrate was prepared in 2.4% HEPES, 1.9% KC1, at pH=7.6. The standart, porcine kidney DPIV, (Probiodrug) was used at a serial dilution starting at 80 mU/ml. The formation of yellow product was measured at 405 nm with a kinetic reading for 30 min using an SP8 100 uv spectrophotometer (Pye Unicam). The sample activity values were calculated using linear regression based on the standards. Measurement of Duodeno- je juna l GIP and GLP-1 con ten t This experiment was designed to determine if there were any alterations in intestinal incretin content in the absence of GIP signaling. Seven male age-matched 12-14 week mice were anesthesized with 15% sodium pentobarbital (65 mg/kg) administered intraperitoneally. Segments of intestine (10 cm) starting from the pyloric antrum were isolated. Each segment was cut open and flushed with ice cold PBS to remove any intestinal contents. The clean segments were blotted to remove any liquid and weighed. The segments were boiled for 10 min in 5 times of their volume. The tissue was than homogenized in a polytron homogenizer. The homogenate was centrifuged at 10 000 g for 30 min. The supernatant was collected and stored in 4 °C. The pellet was boiled in 1M acetic acid for 10 min. To allow the homogenate to cool and equilibrate it was kept at 4°C for an hour prior to 30 min centrifugation at 10,000g. The supernatant was combined with the first supernatant and stored at -20 °C until assayed for GIP and GLP-1 as described previously. To normalize the hormonal content, tissue total protein content was also measured with the use of a Bicinchoninic Acid (BCA) kit (Pierce, Rockford, IL, USA) according to the instructions. 39 In vitro Mouse Pancreas Per fus ions The mouse pancreas perfusion was performed with some modifications similar to rat pancreas perfusion as previously described (Pederson et al., 1982). Briefly, overnight fasted male mice were anethesized by intraperitonial injection of 80 mg/kg sodium pentobarbital (Somnotol, M T C Pharmaceuticals, Cambridge, ON, Canada) prior to surgery. The abdominal aorta and the portal vein were cannulated with PE: 50 tubing (Cole-Parmer, Chicago). The perfusate consisted of a Krebs-ringer bicarbonate buffer supplemented with 3% dextran and 0.2% bovine serum albumin (BSA, Fraction V , RIA grade, Sigma) gassed with 95% C 0 2 to achieve and maintain pH=7.4. The flow rate was maintained at 1 mL/min and the outflow was collected at 1 min intervals. GIP (the final perfusate concentration being InM) and GLP-1 (the final perfusate concentration being 1 nM) were delivered by side arm infusion in the presence of 16.6 mM glucose, whereas arginine was mixed directly into the perfusate (final concentration 10 mM) containing 8.8 mM glucose. Iso la t ion and Cu l tu re of Pancreat ic Is lets Age matched male mice were anesthetized with Somnotol. The pancreas was exposed by midline laparotomy. The distal end of the pancreobiliary duct was clamped at the entrance to the duodenum. The common bile duct was punctured under a dissecting microscope with a 30 l / 2 gauge needle as close to the liver as possible. The pancreas was then inflated with ice cold collagenase (type V, 2mg/ml, Sigma) in Hank's Balanced Salt Solution (HBSS, GIBCO/Life Technologies, Burlington, ON, Canada) supplemented 40 with 10 mM HEPES, 2 mM L-glutamine, and 0.2% BSA, was injected into the common bile duct in order to inflate the pancreas. The pancreas was then removed and digested with collagenase in a 37°C water bath for 10 min. The digest was than dispersed mechanically with a Pasteur pipette, washed three times with 1 min centrifugation (1200 g) at each wash, and filtered through a 1 mm nylon screen, and washed again. Islets were separated by centrifugation at 1800 rpm for 20 min in a discontinuous dextran gradient (1.10 g/ml, 1.085 g/ml, 1.075 g/ml, 1.045 g/ml, and EC-Benz alone) diluted in EC Benz composed of 15 mM KC1, 15 mM K H 2 P 0 4 , 40 mM K 2 H P 0 4 , 10 mM NaHC0 3 , 0.08% Benzamidine, and 0.2 M D- glucose. Using a pasteur pipette the islets were picked from gradient interfaces and washed two times with ice cold HBSS. Hand picked islets were cultured overnight in RPMI 1640 with 8.8 mM glucose, 10% fetal calf serum (Cansera, Rexdale Ontario, Canada), 5% vol/vol antibiotics (50 U/ml of each penicillin G and streptomycin), 0.07% human serum albumin, 0.0025% human apotransferrin, 25 p M sodium selenite and 10 uM ethanolamine. Static Stimulation of Pancreatic Islets for Insulin Approximately 100 islets were collected after overnight culture, washed twice with Krebs-Ringer buffer, and incubated for 45 minutes in 4.4 mmol glucose containing Krebs-Ringer, 118 mM NaCl, 4.7 mM KC1, 2.5 mM CaCl 2 2H 2 0, 1.2 mM K H 2 P 0 4 , 1.2 m M MgSO 4 .7H 2 0, 24.9 mM N a H C 0 3 supplemented with 0.1% B S A . After a brief centrifugation the medium was replaced with Krebs-ringer containing either 16.6 mM glucose alone or 16 mM glucose plus 10 nM GIP or 10 nM GLP-1. After 45 minutes of stimulation, islets were lysed by boiling for 5 min in 1 M acetic acid, and insulin RIA 41 (Linco, St. Charles, M O , USA), was conducted on all the supernatants according to the instruction manual. Static Stimulation of Pancreatic Islets for cAMP Each experimental condition was tested at the minimum in duplicate for each animal. After overnight culture, 15-18 islets were selected, washed twice with 3 min centrifugation at 1,200 g with 0.5 mL of Krebs-ringer supplemented with 0.2% BSA. To allow the islets to equilibrate in the experimental media, they were incubated at 37°C for 30 min. Thereafter, islets were stimulated for 30 min at 37 °C with either 0.5 ml of 0.5 mM I B M X (3-isobutyl-l-methylxanthine), 10 u M forskolin, or I B M X with either 10 nM GIP, or GLP-1 in the presence of 16.7 mM glucose. After a 2 minute centrifugation the medium was replaced with 0.05N HC1, and islets were lysed by boiling for 3 min. Samples were subsequently dried overnight by vacuum centrifugation (Speed-Vac, Sorvall, Farmingdale, N Y , USA) and stored at -20°C until assayed for c A M P (Biomedical Technologies, Stoughton, M A , USA) following the instructions in the manual. Pancreatic Insulin Measurements Age matched male animals were rendered unconscious with C 0 2 and exsanguinated. Upon removal the pancreas was blotted dry and weighed. The tissue was homogenized with a polytron homogenizer in 2 M acetic acid and boiled for 5 min. Homogenates were centrifuged at 15,000 rpm for 15 minutes and the supernatant stored at -70 °C. Total protein levels were measured with a Bicinchoninic Acid (BCA) kit 42 (Pierce, Rockford, IL, USA), and insulin was measured with a kit (Linco, St. Charles, M O , USA). Values were normalized to total protein content. Isolation and Measurement of Islet Insulin and GLP-1 Receptor mRNA by Reverse Transcriptase. Real-Time Polymerase Chain Reaction (PCR) Mouse islet R N A was isolated using Trizol (GIBCO). Following R N A isolation, 50 ng of islet R N A was subjected to reverse-transcription (RT). Total R N A was reverse transcribed in a volume of 10 ml containing, 0.5 mM deoxynucleotide triphosphate, 15 pmol gene specific primer targeted at the carboxy termini of the mouse GLP-1 receptor (ACC A A C A G G G A G G A C CGG) or the mouse Ins II (GTA GTT CTC C A G CTG G T A G A G GG) 100 U Superscript II RNAse H" Reverse Transcriptase (GIBCO), 10 U RNAse inhibitor (RNA Guard; Amersham-Pharmacia), 1 mM dithiothreitol, 50 mM Tris-HC1, pH 8.3, 75 mM KC1 and 3 mM M g C l 2 . Following RT, 10 ng (2 mL) of mouse cDNA was used in the real-time PCR reaction to measure insulin and GLP-1 receptor expression. The PCR reaction mix consisted of lx TaqMan Buffer A (PE Applied Biosystems, Foster City, C A , USA), 10 mM M g C l 2 , 200 mM dATP, dCTP, dGTP and 400 mM dUTP, 200 nM mouse GLP-1 receptor 5' forward primer (5 ' -CAG GGC TTG A T G GTG GCT ATC-3') or 200 nM mouse Ins II 5' forward primer (5' TGG A G G CCC G G G AGC) , 200 nM mouse GLP-1 receptor 3' reverse primer (5'- CGC T C C C A G C A T TTC C G -3') or 200 nM mouse Ins II 3' reverse primer (5'- A T C T A C A A T G C C A C G CTT C T G C - 3'), and 100 nM GLP-1 receptor probe co-labelled with the fluorescent dyes VIC and T A M R A (5'- A C T GCT TTG T C A A C A A T G A G G TCC A G A T G G -3') or 100 nM Ins II probe co-labelled with fluorescent dyes TET and 43 T A M R A (5 ' -ACC TTC A G A CCT T G G C A C T G G A G G TG-3') , 0.01 U/ml AmpErase" uracil N-glycosylase (UNG, PE-Applied Biosystems), and 0.025 U/ml of AmpliTaq Gold" (PE Applied Biosystems). PCR reactions were carried out in triplicate in the PE Applied Biosystems 7700 sequence detection system. The reaction profile included a 10 minute preincubation at 50 °C to allow the U N G to degrade any uracil containing nucleic acids and a further 10 minute incubation at 94 °C to activate the AmpliTaq Gold. Following these preincubations, a two-step PCR protocol was carried out, which included a denaturation step at 94 °C for 15s followed by a 1 minute annealing/extension step at 60 °C. Fluorescence was measured during the annealing/extension steps over 40 cycles and used to calculate a cycle threshold (Ct), i.e. the point at which the reaction is in the exponential phase and is detectable by the hardware. A l l reactions followed the typical sigmoidal reaction profile, and Ct was used as a measure of amplicon abundance (Freeman and Varana, 1999). lmmunohistochemistry Age matched male mice were fasted overnight, anesthesized under C 0 2 before being exsanguinated. The pancreases from wild type and knockout mice (n = 5) were isolated and cut into head and tail segments. Each segment was fixed separately in Bouin's solution for 1 hour at room temperature and washed three times with 70% ethanol. The tissue was embedded in paraffin wax, and kept at -20 °C prior to cutting. Three consecutive 5 um sections were taken 300 um apart and mounted on glass slides. The study was carried out blind; thus, the slides were coded to prevent identification of +/+ and -/- tissues prior to quantification. The sections were dewaxed in xylene (2X5 44 min) and cleared in petroleum ether for 5 min. (Fluka). The sections were then rehydrated in phosphate-buffered saline PBS (80 mM Na 2 HP0 4 , 1.47mM K H 2 P 0 4 , 2.86mM KC1, 137 mM NaCl). To control for intra-immunostain variability, all the sections were incubated in the same batch of solutions and stained at the same time. The insulin cells were detected by overnight incubation with a polyclonal rabbit antibody (1:100, Santa Cruz). The next day bound antibodies were localized by incubating sections with a biotinylated goat anti-rabbit secondary antibody (1:300 , Jackson Labs, PI) for 90 min at room temperature. Sections were then washed to remove unbound antibody and incubated in the Avidin Biotin Peroxidase complex (ABC, Vector Labs, Ontario) at a dilution of 1:1000 in PBS supplemented with 5% horse serum. The peroxidase reaction was developed with 2% diaminobenzidine tetrahydrochloride in 0.05 M Tris pH 7.5 with 0.2% H 2 0 2 . After counterstaining with hematoxylin for 5 min the sections were dehydrated through graded alchohol and coverslips applied with Permount (Fisher Scientific). The sections were analyzed using the NIH image software (shareware) and data analyzed as islet area over total pancreatic area. Once the quantification had been completed the source of the sections (head or body, +/+ or -/-) was identified and significance assessed. Data Analysis. A l l the data are expressed as mean ± standard error of the mean. Unpaired Student's't' test and Mann-Whitney U test (exclusively for immunocytochemistry) were used to compare the control values to GIPR-/- mice values, where P<0.05 was considered 45 statistically significant. The data analysis and area under the curve calculations were carried out using graph analysis software (Graphpad, Prism, San Diego, C A , U S A ) . RESULTS The mice were classified as homozygote dominant (WT), heterozygote (GIPR+/), and homozygote recessive (GIPR-/-) according to the length of their GIP receptor D N A : 7 kb alone, 7 and 3kb combined and 3 kb alone respectively (Figure 5). After one year of breeding a colony was established with sufficient animal numbers to allow the experiments to start. -/- -/- -/- -/- +/- +/+ +/+ +/+ +/- +/- +/+ ^HP Figure 5- Southern blot with Eco RI digested genomic DNA. The sizes of 7 kb (Wild Type and 3 kb (GIPR-/-) fragments are indicated. +/+ refers to GIPR+/+ (WT), +/- to heterozygote GIPR+/- and -/- to GIPR-/- knockout mice. 46 Oral Glucose Tolerance Test Oral administration of 1 g/kg glucose resulted in very similar blood glucose profile in both GIPR-/- and WT mice (Figure 6). The fasting values were normal (3.8 mM) and not different in WT and GIPR-/- mice. Both groups reached peak values of blood glucose at 15 min. The blood glucose values were 17.9±0.6 mM for WT and 22.7±0.7 mM for GIPR-/- (P<0.05). Although, statistically significant the peak glucose values for knockout mice were only 12% greater than wild type. For the 30 th minute, blood glucose values were 14.3±1.0 mM for WT and 17.8±1.5 mM for the GIPR-/- mice (P<0.05). The two curves merged after 40 minutes and both groups returned to their basal levels after 1 hour. 47 Figure 6- Oral Glucose Tolerance Test. B lood g lucose w a s measu red in response to oral g lucose load (1g/kg) in W T ( O , n=7) and G I P R - / - ( • , n=11) mice . V a l u e s are m e a n ± S E M . * denotes P<0.05 Plasma Insulin. GIP. GLP-1 and DPIV levels for fasting and 20 t h minute OGTT Fasting blood glucose was not different between groups (Figure 7A). This is consistent with the full profile OGTT data (Figure 6). Following an oral glucose challenge, 20 t h minute blood glucose levels were 18% higher in GIPR-/- mice than in 48 wild type mice (P<0.05) (Figure 7A). Fasting insulin levels did not differ between groups (Figure 7B). However, 20 t h minute insulin levels were 45% lower in GIPR-/- mice than in wild type mice; values were 1.6±0.3 ng/ml for WT and 0.9±0.1 ng/ml for GIPR-/-(P<0.05) (Figure 7B). 20 -, E, . CD CO O 3 10 O T3 O O CO 3+/+ 1-1-Fasting 20th minute C c ro E co CL X • -/-Fasting 20th minute Figure 7- Fasting and 20 minute OGTT blood glucose and plasma insulin levels. A: Blood glucose levels in WT ( • , n=4) and in GIPR-/- ( • , n=4) mice. B: Plasma insulin levels in WT and GIPR-/- mice. Values are mean ± SEM. * denotes P<0.05 49 To further assess the hormonal components of the enteroinsular axis in GIPR -/-mice, fasting and 20 t h minute plasma GLP-1, DPIV, and GIP levels were investigated. It was hypothesized that DPIV would be downregulated to compensate for the lack of GIP action. This would lead to greater circulating active GLP-1. Neither total GLP-1 nor DPIV levels in GIPR-/- mice differed from those in wild type animals either for fasting or for the 20 t h minute OGTT time point (Figure 8A, 8B). However, fasting plasma GIP levels were elevated (17%) in GIPR-/- mice (Figure 8C). Values were 2217±50 pg/ml in GIPR-/- and 1862±99 pg/ml in WT mice (P<0.05). For the 20 t h minute, plasma GIP levels were lower by 18% in GIPR-/- when compared to WT mice (Figure 8C). Values were 3266±258 pg/ml for GIPR-/- and 3973±260 ng/ml for WT (P<0.05). 50 A •5. socH & + / + Fasting B l i < H 51 > CL Q M y Fasting CZW-C 5000-, 4000-3000-2000-1000-0-Fasting 20th minute Figure 8- Fasting and 20 minute OGTT plasma GLP-1, DPIV and GIP levels. A: Plasma total GLP-1 levels in WT (O, n=4) and GIPR-/- ( • , n=4) mice. B: Plasma DPIV activity in WT (O, n=4) and GIPR-/- ( • , n=4) mice. C: Plasma GIP levels in WT (O, n=4) and GIPR-/- ( • , n=4). Values are mean±SEM. * denotes P<0.05 51 Duodenojejunal GLP-1 and GIP content It has been shown in dogs and rats that GIP release from the upper intestine in response to luminal glucose in turn stimulates GLP-1 release (Pederson et al. 1975; Roberge and Brubaker, 1993; Brubaker, 1991). To assess any alterations in intestinal cell distribution that might result from the absence of GIP signaling, incretin content was measured in duodenum and jejunum. Hormone levels were expressed over tissue total protein. Duodeno-jejunal GLP-1 levels were 50% reduced in knockout mice. GLP-1 levels were 10.5±1.5 pg/ug protein for WT and 5.05±0.4 pg/u.g protein (P<0.05) (Figure 9A). Interestingly GIP levels were also slightly reduced in GIPR-/- mice. Values were 67.5±8.2 pg/jig protein for WT and 59.7±4.8 pg/u.g protein, less than a 10% reduction(P<0.05) (Figure 9B). 52 A 15-2 o a- 10-O) .3. Q. +/+ 75-c B 2 50-Q. u> 3 O) Ct 25 Q. O +/+ Figure 9- Duodeno-jejunal GIP and GLP-1 content. A: Duodeno je juna l GLP-1 content in W T ( • , n=7) and in G I P R - / - ( • , n=7) mice . Hormonal content is normal ized over t issue total protein content. B: Duodeno-jejunal G I P content in W T ( • , n=6) and in G I P R - / - ( • , n=7) mice . V a l u e s are m e a n ± S E M , * denotes P<0.05. 53 Insulin Responses from pancreas perfusions of GIP. Glucose, and Arginine To ensure that GIP receptors were non-functional and GIP was ineffective at stimulating insulin secretion from the pancreases of -/- animals, pancreas perfusions were carried out with GIP in the perfusate (Figure 10A). As expected, 1 nM GIP generated no insulin response from the perfused knockout pancreases whereas WT mice responded with a first phase insulin peak followed by a sustained second phase insulin secretion (Figure 10A). To determine if absence of GIP receptors influenced the |3 cell responsiveness to glucose, low (4.4 mM) and high (16.7 mM) glucose perfusions were carried out (Figure 10B). Glucose- stimulated insulin secretion in WT and GIPR-/- mice were comparable with an area under the curve of 29.1 ±6.1 ng/ml and 33.7±4.5 ng/ml respectively (Figure IOC ). The peak values however of the first phase insulin secretion was significantly greater in GIPR-/- than in WT mice (P<0.05). The integrated insulin response as well as the peak value to high dose arginine (10 mM) perfusion in the presence of 8.8 mM glucose were not different between groups (Figure 10D and 10E). In conclusion, no significantly different insulin response profile was observed in response to insulin secretagogues, glucose and arginine in GIPR-/- versus WT mice. 54 55 4.4mM 8.8mM glucose 10mM Arginine +/+' -/-Figure 10- Insulin response to pancreas perfusions with different secretogogues. A: Insulin response to pancreas perfusion with GIP. WT (O, n=4) and GIPR-/- ( • , n=4) B: Insulin response to pancreas perfusions with low (4.4 mM) and high (16.7 mM) glucose. WT ( O , n=4) and GIPR-/- ( • , n=5) C: Integrated insulin values for glucose alone pancreas perfusion. D: Insulin response to pancreas perfusion with 10 mM arginine in the presence of 8.8 mM glucose. WT (O, n=4) and GIPR-/- ( • , n=4) E: Integrated insulin response for arginine perfusion. Arrows indicate the switch between conditions. 56 Insulin Responses to GLP-1 Pancreas perfusions were performed to assess any changes in the islet insulin response to GLP-1.The data are presented as percentage of the mean basal (in response to 4.4 mM glucose) insulin secretion. GIPR -/- mice exhibited a greater first phase and second phase insulin release in response to InM GLP-1 perfusion (Figure 11A). The first phase peak values were, 8.4±2 for WT and 26.7±10.9 fold basal for GIPR-/- (P<0.05). Furthermore, as is shown by the integrated insulin response, total insulin secretion during 1 nM GLP-1 perfusion was 60% greater in GIPR -/- mice compared to wild-type mice (P<0.05) (Figure 11B). The mean basal insulin secretion was not different between groups, 0.40±0.09 ng/ml in WT versus 0.42±0.03 ng/ml in GIPR-/- (Figure 11C). 57 A 40- 4.4 16.7 mM glucose 1nM GLP-1 Fractions 50 _ 750-ra in ra .• 1 500-c "5 in S 250-O < 0.5-c 5 0.4-in c ra I 0.3-c ra a s 0.1 0.2-+/+ 0.0-Figure 11- Insulin response from GLP-1 perfused WT and GIPR-/- mice pancreas. A : The perfusion profile for 50 minutes with 4.4 m M glucose , 16.7 m M glucose , and 1nM G L P - 1 in the presence of high g lucose . W T (O, n=6) and G I P R - / - ( • , n=6) Ar rows indicate the scwitch between concentrat ions. B : The integrated insulin response . C : M e a n basal insulin va lues for W T and G I P R - / - mice during the perfusions. 58 To determine if these results were due to change in islet physiology, isolated islets were stimulated with low (4.4 mM) and high (16.7 mM) glucose alone or in the presence of either 10 nM GIP or GLP-1 (Figure 12). Insulin secreted over 45 minutes was 40% greater in response to GLP-1 stimulation for -/- (0.25±0.05 ng/ml) vs. +/+ (0.182±0.02 ng/ml) islets (P<0.05). As expected, GIP did not stimulate insulin release from -/- islets whereas it elicited a response comparable to GLP-1 stimulation from +/+ islets (0.10±0.01 vs. 0.18+0.05 respectively). The insulin response to 16.7 mM glucose was comparable in both groups. CC o 2 c 2 3 Ui CO c 0.3-1 -Zr 0.2 A 0.1 A I ** IZZW-0.0-4.4 16.7 16.7 +GIP 16.7 +GLP-1 Figure 12- Insulin responses to different stimulation of islets of WT and GIPR-/- mice. Islets were stimulated with 4.4 mM, and 16.7 mM glucose alone, and with high glucose in the presence of either 10 nM GIP or GLP-1. WT ( • , n=6) and GIPR-/- ( • , n=6). * denotes P<0.05 for GLP-1 stimulated insulin release between WT and GIPR-/-. *** denotes P<0.05 for 16.7 mM glucose stimulated insulin release between WT and GIPR-/-. ** denotes P<0.05 for 16.7 mM glucose, GIP and GLP-1 stimulated insulin release for GIPR+/+ mice . 59 Intracellular cAMP Production Both GIP and GLP-1 receptors are coupled to c A M P dependent signaling pathways in the beta cell (see Introduction for references). To correlate GLP-1-stimulated insulin release with receptor activation, cAMP production was measured in response to GLP-1 in isolated islets (Figure 13). A high concentration of forskolin (adenylate cyclase stimulator) was used as a positive control to stimulate near maximal levels of c A M P production. The c A M P values obtained from GIP and GLP-1 stimulation were normalized to forskolin-stimulated c A M P to account for differences in islet size and number. Interestingly, control (16.7 mM) cAMP levels were slightly increased in GIPR-/-mice compared to wild type (P<0.05). GLP-1 stimulated c A M P production was 100% increased in knockout animals (0.16±0.01 fold forskolin/islet) versus wild type (0.08+0.006 fold forskolin/islet) mice (P<0.05). 60 2 o G) W • mm Q_ = < (0 O »-0.2n 0.1 H X X ** 1 +/+ ]-/-o.o-16.7 16.7 +GIP 16.7 + G L P - 1 Figure 13- cAMP production in the islets of WT and GIPR-/- mice in response to 16.7 m M g lucose a lone , 10 n M G I P with 16.7 m M glucose , and 10 n M G L P - 1 with 16.7 m M glucose . W T ( • , n=5) and G I P R - / - ( • , n=4) B a s a l levels were 0 . 0 5 1 ± 0 . 0 0 3 (n=8) in GIPR+/+ vs . 0 . 0 5 9 ± 0 . 0 0 2 (n=8) in G I P R - / - . (*P<0.05). * denotes P<0.05 for basa l va lues between W T and G I P R - / - mice . G I P and G L P - 1 st imulated c A M P production w a s significantly different from basa l va lues in GIPR+/+ mice (**P<0.05).*** denotes P<0.05 for G L P - 1 st imulated c A M P production in GIPR+/+ versus G I P R - / - mice . V a l u e s are m e a n + S E M . 61 Pancreatic insulin content and islet insulin and GLP-1 mRNA content Both GIP and GLP-1 stimulate insulin gene transcription and protein synthesis in the beta cell (Drucker et al., 1987; Fehmann et al., 1995a). Thus, we hypothesized that absence of GIP action may lead to alterations in insulin gene transcription and therefore in pancreatic insulin content. The total insulin content from fed mice pancreata was significantly lower (40%) in GIPR-/- mice than in +/+ mice (1.1 ±0.1 ng/u.g protein vs. 0.75±0.07 ng/ug protein) (P<0.05, Figure 14A). These data are supported by findings that insulin mRNA levels were significantly reduced (40%) in isolated islets of GIPR -/- mice (1.06±0.09 relative to WT) when compared to control (0.74±0.11 relative to WT) (Figure 14B, P<0.05). 62 A ^ 1.5-c "55 o 2; 10-.E 0.5-3 </) C 0.0-+/+ B Figure 14- Pancreatic insulin content and islet insulin gene expression in WT and GIPR-/- mice. A . Pancrea t ic insulin content is expressed as a percentage of pancreat ic total protein content. W T ( • , n=5) and G I P R - / - ( • , n=5). B Islet insulin gene express ion in W T ( • , n=10) and G I P R - / - ( • , n=9). * denotes P O . 0 5 63 Interestingly, assessment of GLP-1 receptor mRNA levels revealed that, despite an increase in GLP-1 sensitivity, no increase in GLP-1 receptor mRNA was detected in the islets of GIPR-/- mice (Figure 15 ). Figure 15- GLP-1 receptor gene expression in the islets of W T ( • , n=11) and G I P R - / - ( • , n=12)mice . lmmunohistochemistry Immunhistochemical studies were carried out to assess the effect of GIP receptor deficiency on islet and pancreas morphology. Islet area over the total pancreatic area was 64 significantly increased (45%) in knockout versus wild-type mice (P<0.05), (Figure 16A). The head and tail analysis have shown that this increase was mainly due to the alterations occurring in the head of the pancreas where the islet area over total pancreatic area was dramatically altered whereas no significant difference has been observed for the tail segment (Figure 16B). Additionally, under identical experimental conditions, the staining intensity for insulin was reduced for -/- islets (Figure 17). This finding suggests that the insulin content is reduced in GIPR-/- islets. Glucagon and somatostatin staining have shown that the distribution of these cells within the islets is consistent with the literature, non beta cells occupy the periphery of the islet. (Figure 18 and 19). 65 A 0.03-. ra ra .2 0.02-O L Z Z 1 - / -Head Tail Figure 16- Quantitative expression of islet area over total pancreatic area in WT ( • , n=5) and GIPR-/- mice ( • , n=5). A: Islet area in the total pancreas in WT and GIPR-/-mice. B: The distribution of the Islet area in the head and tail of the pancreas in WT and GIPR-/- mice. 66 Figure 17- Pancreatic islets immunostained for insulin (peroxidase) in WT (top) and GIPR-/-(bottom) mice . 67 Figure 18- Glucagon immunostaining of WT (top) and GIPR-/- (bottom) mice islets. 6 8 Figure 19- Somatostatin immunostaining in WT (top) and GIPR-/- (bottom) mice islets 69 DISCUSSION Type 2 diabetes is a polygenic disease characterized by elevated blood glucose levels due to insulin resistance and impaired insulin secretion. In human type 2 diabetes, the insulinotropic action of GIP, but not GLP-1, is reduced (Jones et al, 1989, Nauck et al., 1993). In addition, postprandial circulating GIP levels in type 2 patients have been reported as being elevated, normal or reduced (Mazzaferri et al., 1985; Service et al., 1984; Nauck et al., 1986). These measurements were all made with C-terminal directed antibodies and therefore do not reflect the circulating levels of intact peptide. Hence, it is difficult to assess the contribution of serum GIP levels to the etiology of the disease. No consensus has been reached regarding the possibility that GIP receptor desensitization may contribute to the etiology of type 2 diabetes. However, this possibility was recently addressed in a model of type 2 diabetes, the V D F rat, in which it was demonstrated that GIP receptor expression is reduced (Lynn et al, 2001). Mice with a targeted disruption of the GIPR gene (GIPR-/-) provide a useful model for studying the potential implications of a lack of GIP signaling on glucose homeostasis and the development of type 2 diabetes. As has been discussed in the introduction, GIPR-/- mice are mildly glucose intolerant with normal fasting glucose and insulin levels (Miyawaki et al. 1999). However the oral glucose-stimulated insulin response is reduced while the insulin response to an intravenous glucose tolerance test (IVGTT) is intact, demonstrating a defective enteroinsular axis. Despite reduced insulin levels, glucose levels remain close 70 to the normal range. The mild degree of glucose intolerance suggests that, as with GLP-1R-/- mice, these animals compensate for the absence of incretin action. A role for GIP in the maintainance of glucose homeostasis in the fasting state is under investigation. A recent study by Baggio et al. (2000) reported that chronic exposure of GLP-1 R-/- and +/+ mice to a GIP receptor antibody (GIPR Ab), showed no differences in fasting glucose levels between groups. A similar study with GIPR Ab was also conducted in rats, and Lewis et al. (2000) have reported that inhibition of GIP action had no effect on fasting plasma glucose levels (Lewis et al., 2000). Accordingly, the Japanese study and the current thesis (Figure 7) report normal fasting plasma glucose and insulin levels in GIPR-/- mice demonstrating that absence of functional GIP receptors has no effect on the fasting state. However, it is also possible that this might be a consequence of compensatory changes. One such change might be an increased sensitivity to GLP-1. GLP-1 has been shown to regulate fasting glycemia by Baggio et al. (2000). It is unclear why fasting GIP levels are increased in GIPR-/- mice (Figure 8C). This increase might be partly explained by the absence of somatostatin-related regulation of the K cells. Somatostatin secreting D cells are distributed throughout the small intestine (Larsson et al., 1979). There is evidence that GIP secretion is inhibited by somatostatin (paracrine interaction), which in turn is stimulated by GIP (Jia et al., 1994; Rossowski et al, 1998). OGTT plasma GIP levels at the 20 t h minute, were decreased in GIPR-/- mice. These results might be partly explained by the reduced duodenal GIP content observed in GIPR-/- animals (Figure 8C), where even a potent stimulus would result in relatively reduced peptide secretion. On the other hand, if the peripheral sensitivity to GLP-1 is 71 increased, delayed gastric emptying may blunt the peak values of plasma GIP. Hence, this observation might only reflect a right shift on the time axis. Therefore, it is very important to assess circulating GIP and GLP-1 levels during an OGTT with shorter time intervals for blood sampling. This would allow a more complete profile for both incretins. In this thesis the restriction on animal numbers precluded an experimental design in which sufficient numbers of animals could be sacrificed at each time point in order to collect enough blood for glucose and hormone measurements. Although our GIPR-/- mice colony was developed from heterozygote pairs from the original Japanese colony, the OGTT profiles for the two colonies vary in terms of peak values and curve trends. The two hour OGTT profiles for GIPR-/- and +/+ mice are remarkably similar in the current study however, Miyawaki et al. reported more dramatic differences in GIPR-/- versus +/+ mice than was observed in the Vancouver colony. In both studies blood glucose reached peak values for both groups of animals at the 15 minute time point. In our study the curves merged after 40 minutes whereas in the Japanese study this did not occur until the 90 minute time period. Additionally, they reported a more pronounced increase in plasma glucose level for the 15th and 30 th minutes of OGTT than in our study. Interestingly, despite the variations in glucose profiles, the insulin profiles were very similar in the two groups of animals. Alterations in diet and environment, along with genetic shift from inbreeding might result in different adaptations occurring in the development of these mice as compared to other colonies. Despite the abscence of GIP receptors, the modest increase (18%) in the 20 th minute OGTT blood glucose levels does not correlate with the more profound reduction (55%) in plasma insulin levels observed in the GIPR -/- animals (Figure 7). However, in 72 the current study, it was found that the sensitivity of the pancreas to GLP-1 is increased in GIPR-/- mice (Figures 11-13), although no changes in total GLP-1 levels or in DPIV activity were observed (Figure 8A and B). As previously discussed in the introduction, controversy exists as to whether GLP-1 is capable of exerting insulin-like effects on peripheral tissue in addition to its well-studied insulinotropic effects. Studies have shown that GLP-1 improves glucose disposal in type 2 diabetes, by enhancing insulin-stimulated glucose utilization (Sandhu et al., 1999) and these effects have been shown to be independent of the amount of insulin secreted (D'Alessio et al., 1995; Gutniak et al., 2001). Recently, Ahren et al. (1999) reported that in mice, the effects of GLP-1 on glucose homeostasis were mainly insulin mediated and the use of a GLP-1 receptor antagonist, exendin-3 (9-39), reversed these actions (Ahren and Pacini, 1999). Additionally, Baggio et al. (2000) demonstrated in mice that basal levels of circulating GLP-1 are essential for glycemic control in the fasting and postpandrial state (Baggio et al., 2000). It is still to be determined whether these actions of GLP-1 could be explained by mechanisms other than insulinotropic action such as improvement of glucose dependent glucose uptake. Therefore, it is possible, that the relatively small change in glucose disposal observed in GIPR-/- mice results from increased extrapancreatic sensitivity to GLP-1 in peripheral tissues, thereby augmenting glucose clearance despite reduced insulin secretion, or as a result of increased insulin sensitivity of extrapancreatic tissues. Similar to GIPR-/- mice, GLP-lR-/-mice exhibited modest glucose intolerance, with upregulation, in this case, of the GIP component of the enteroinsular axis (Pederson et al, 1998). In keeping with this finding, the glucose excursion after an I.V. glucose 73 challenge, bypassing the enteroinsular axis, was normal in GIPR-/- mice (Miyawaki et al., 1999) suggesting that the incretin effect is responsible for both reduced circulating insulin levels (absence of GIP action) along with only modest changes in plasma glucose levels (due to enhanced GLP-1 action). Pancreas perfusions (Figure 11), static islet stimulation (Figure 12), and c A M P production (Figure 13) data presented here support our original hypothesis that the GLP-1 component of the enteroinsular axis in GIPR-/-mice is upregulated. This increased sensitivity to GLP-1 is more pronounced if we take into consideration the decrease in islet insulin gene transcription and total pancreatic insulin content in GIPR-/- mice (Figure 14). The fact that in vitro insulin responses to high glucose and arginine are similar, as well as islet GLP-1 receptor mRNA levels in GIPR-/- and in GIPR+/+ mice suggests that compensation occurs distal to the GLP-1 receptor in the beta cell. Both incretins act through G-protein coupled receptors and signal via the adenylyl cyclase-cAMP system (Wheeler et al., 1995; Drucker et al., 1987; Moens et al, 1996). This being the case, it could be hypothesized that permanent absence of the GIP receptor leads to increased availability of the cAMP signaling system to GLP-1 receptors. Reduced insulin gene expression and protein content, might be interpreted as a reduction in either islet size or beta cell number in the islet. We have shown that decreased islet size is not a valid explanation since GIP receptor null mice displayed increased islet area when compared to total pancreatic area (Figure. 17). This raises the possibility that other adaptive changes within the beta cell of GIPR-/- mice have taken place, since no differences in insulin responses to high glucose and arginine pancreas perfusions were observed between the two groups. 74 Both GIP and GLP-1 stimulate insulin gene expression (Fehmann et al. 1995a; Drucker et al., 1987). It might be predicted that, an increase in beta cell GLP-1 sensitivity in the absence of GIP action could protect against a decrease in islet insulin mRNA and protein levels. However, this study shows a 40% decrease in both insulin mRNA expression and pancreatic protein content. This is comparable to the 35% decrease that was shown by Pederson et al. (1998) in GLP-1 R-/- mice. Hence, absence of either of the incretins results in abnormalities within the |3 cell, leading to impaired insulin content. The compensation by GLP-1 or by GIP at the beta cell level, in GIPR-/- and in GLP-1R-/- mice respectively, is insufficient to regulate downstream signaling from the receptor, and subsequent activation of the transcription factors necessary for insulin biosynthesis. It was proposed that GLP-1 could regulate (3 cell function by maintaining it in a glucose competent state (Holz et al., 1993). However studies with GLP-1 R-/- mice have reported that these mice exhibit preserved glucose competence with preserved insulin storage capacity and glucose dependent insulin secretion. It has been shown by Pederson et al. (1998) that GLP-1 R-/- mice exhibit compensatory changes in the form of enhanced GIP action. Since GIP and GLP-1 activate the same type G coupled receptors and its intracellular effectors, it is possible that in GLP-1R-/- mice the enhanced GIP action could contribute to the well-preserved glucose responsiveness of the |3 cell. In this regard, we have shown that (3 cell glucose responsiveness to different doses of glucose was intact in GIPR-/- mice. An enhanced GLP-1-pancreatic axis might be responsible for preserving the (3 cell in a glucose competent state despite decreased insulin gene transcription and biosynthesis. 75 Recently, GLP-1 has been shown to be involved in the morphological developement of the islets of Langherhans. In immunohistochemical studies, Ling et al. (2001) demonstrated alterations in beta cell distribution and in islet size in GLP-1R-/-mouse pancreata which may in part explain the reduced insulin gene expression. Our studies on GIPR-/- mice, with the same objective, also showed alterations in islet morphology mainly occurring in the head (ventral bud) of the pancreas (Figure 16). Reduced insulin gene expression and insulin content correlated with less intensely stained islets in GIPR-/- mice (Figure 17). The glucagon and somatostatin staining of pancreatic islets (Figure 18 and 19) showed that a and 5 cells were localized on the periphery of the islet in both GIPR-/- and wild type animals; only the core of the islet staining for insulin. This suggests that an increase in either a or 5 cells cannot account for larger islets with less insulin content in GIPR-/- mice. It would seem paradoxical to have increased islet size along with reduced insulin content. This suggests that these |3 cells have been depleted of insulin. The current concept of the (3 cell mass is that it is dynamic, increasing and decreasing to maintain glucose homeostasis within a narrow physiological range (reviewed in Bonner-Weir, 2000). Hyperplasia and hypertrophy of the |3 cell mass is part of the continued turnover taking place during the lifespan. The turnover is dependent on changes in replication rate, cell death rate and in rates of differentiation from precursor cells. Studies with mice with disrupted insulin receptor substrate-1 (IRS-1) showed that the increased insulin demand to overcome the insulin resistance was answered by up to a 40 fold increase in |3 cell mass (Bruning et al. 1997). The increased (3 cell area over total pancreatic area observed in GIPR-/- mice (Figure 16) might be part of the compensatory 76 changes that occur in these mice. However to discuss |3 cell mass in detail one must take into consideration pancreatic volume as well as weight and three-dimensional localization. Very recently it has been shown that GLP-1 has growth hormone-like effects on pancreatic islets (Holz and Leech, 2001). Enhanced GLP-1 action on the GIPR-/- mouse pancreas could thus also result in increased islet size. The immunohistochemistry studies presented in this thesis support this hypothesis. Islet size was determined by insulin immunostaining and was expressed as a percentage of the total pancreatic area, therefore in this study only the core of the islet accounts for islet size. The increase in islet area over total pancreatic area does not take into consideration changes in a and 5 cell mass. It is important to note that GLP-1 receptors are abundantly present on (3 cells and their activation results also in the activation of growth, proliferation, and differentiation transcription factors such as c-jun, c-fos and jun B (Xu et al., 1999; Edvell and Lindstrom, 1999; Susini et al., 1998). Therefore, an upregulation of GLP-1 receptor signaling should mainly affect |3 cells. Hence, it is possible to partly explain the increase in islets size by the increased sensitivity of the (3 cell to GLP-1. However it is also important to determine whether a and 5 cells are affected by the upregulation of GLP-1 action. The development of the pancreas begins with the dorsal (tail) and ventral (head) protrusion of a region of the primitive gut epithelium (Edlund, 2001). The dorsal and ventral bud development of the pancreas as well as cell differentiation and specification processes remain unclear. The initiation and the development of the ventral and dorsal pancreatic buds are believed to be different. In mice the first endocrine cells appear in the 77 dorsal bud before the differentiation of the cells in the ventral bud (Lewis, 1996). Additionally, adult mice have fewer a cells in the ventral bud. In the current study it was observed that in GIPR-/- mice, islet area was increased mainly in the head (ventral) part of the pancreas. The developmental biology of the pancreas might account for the islet size difference observed between head and tail pancreatic segment of the GIPR-/- mice. As has been discussed in the introduction, two recent studies by Damholt et al. (1998 and 1999) proposed a role for GIP in the direct secretion of GLP-1. K cells are more abundantly distributed throughout the duodenum with a decreasing density in jejenum and ileum (Polak et al., 1982). L cells are mainly localized in the ileum with decreasing concentration in duodenum and jejenum (Eissele et al., 1992; Damholt et al., 1999). It is proposed that GIP secreted after a meal stimulates GLP-1 secretion. Immunohistochemical evidence suggests that in the canine upper intestine, K cells and L cells are adjacent to each other indicating a paracrine interaction (Damholt et al. 1999). In the current study, the absence of functional GIP receptors resulted in a 40% decrease in the duodeno-jejunal GIP and GLP-1 content. This implies either a relocalization of K and L cells and/ or a decrease in GIP and GLP-1 biosynthesis and storage. The data presented in this thesis are preliminary and further investigation is required to elucidate the effects of the absence of functional GIP receptors on the regulation and intestinal distribution of K and L cells Which incretin makes a more important contribution to the enteroinsular axis? This question has been discussed for more than 15 years. Studies with GLP-1R-/- and GIPR-/- mice should shed light in this debate. In GIPR-/- and GLP-1R-/- mice GIP and GLP-1 compensate for each other by enhancing the (3 cell sensitivity partly by altering 78 the cAMP-adenylyl cyclase pathway (Figure 13, Pederson et al. 1998, Flamez et al. 1999). The physiological significance of this compensation is still unclear. The ability of one component of the enteroinsular axis to compensate for a missing partner requires further investigation. GLP-1R-/- mice exhibit plateau like peak glucose levels following an OGTT and the glucose levels do not return to normal for 90 minutes. The GIPR-/- mice exhibit a single peak at 15 minute and the levels return to normal within 40 minutes. GIPR-/- mice also exhibit normal fasting glucose and insulin levels, while the absence of GIP action is reflected in a 50% decrease in 30 minute OGTT plasma insulin levels. These profiles suggest that impairment of glucose tolerance is more pronounced in GLP-1R-/- mice. It is important to note that this statement does not take into consideration different backgrounds of these mice (CD1 vs C57BL/6) but only a comparison between control and test animals. Baggio et al. (2000) suggests a role for GLP-1 but not GIP in the regulation of fasting glycemia but other reports indicate that neither fasting glucose levels nor insulin levels are altered in either GIPR-/-, or GLP-1R-/- mice (Pederson et al. 1998, Scrocchi and Drucker, 1998). GLP-1R-/- mice compensate for the absence of GLP-1 action partly by upregulating the GIP-insulin axis. As demonstrated in this thesis, GIPR-/- mice compensate for the absence of GIP action by increased (3 cell sensitivity to GLP-1. GIPR-/- and GLP-1 R-/- mice exhibit impaired glucose tolerance with different degrees of severity. It is arguable that the absence of one of the incretins is sufficient to suppress the enteroinsular axis by reducing glucose stimulated insulin release up to 50% in GLP-1 R-/- and GIPR-/- mice. However it is interesting that in both animal models the 79 |3 cell responsiveness to high and low glucose is preserved (Figure 10 and Pederson et al. 1998). Insulin gene expression and biosynthesis were reduced in comparable amounts in both animal models (current thesis and Pederson et al. 1998) suggesting that both incretins have similar effects on the (3 cell function. Pederson et al. (1998) have reported that first phase of the glucagon stimulated insulin secretion is altered in GLP-1R-/- mice. However they reported no effects of GLP-1 on pancreatic glucagon content. These parameters should be compared in GIPR-/- mice. In summary, this thesis demonstrates that disruption of the GIP component of the enteroinsular axis in mice results in alterations in the beta cell that lead to decreased insulin gene transcription and protein biosynthesis, increased islet sensitivity to GLP-1, and changes in islet structure. It was demonstrated that compensation for the absence of a functional GIP receptor occurs in part by up-regulation of the GLP-1 component of the enteroinsular axis. The physiological changes that take place in both the GIPR-/- and GLP-1R-/- strains of knockout mice suggest that the incretins act in concert to maintain glucose homeostasis. The balance between GIP and GLP-1, where chronic disruption of one results in upregulation of the other one, appears to be part of the homeostatic system in the enteroinsular axis. 80 FUTURE DIRECTIONS The research presented in this thesis was aimed at determinining general characteristics of GIPR-/- mice in order to have a better understanding of GIP physiology and its contribution to the enteroinsular axis. The few reported studies on GIPR-/- mice dictate a wide range of important topic still to be investigated. Several research directions are outlined below, which are based on the work presented in this thesis. Glucagon and Somatostatin secretion and biosynthesis Although GIP receptors are present on a and 6 cells, the effects of GIP on glucagon secretion are ambiguous (see introduction). GIPR-/- mice present an important opportunity to investigate the role of GIP on glucagon and somatostatin secretion and biosynthesis. In vitro pancreas perfusion and static islet stimulations could bring more clarity on this subject. The enteroinsular axis of GIPR +/- mice Although Miyawaki et al. (1999) reported that truncated GIP receptor did not have dominant-negative effect it is clear that GIPR+/- animals would have less functional receptors. This coincides with the findings of reduced GIP receptor gene expression in the fatty Zucker rats (Lynn et al. 2001). The enteroinsular axis of the GIPR+/- could be assessed with similar studies presented in this thesis. 81 Islet ft cell signaling The study of intracellular signaling in the isolated (3 cells could provide more information about the mechanisms of the compensation by the enhancement of GLP-1 -insulin axis in these animals. Transcription factors and the mechanisms that led to the gigantic islets are also in interest since the role of GIP in cell growth and proliferation is under investigation. Duodenal and intestinal lmmunohistochemistry Data presented here suggest that the morphology of the intestine has been modified. Immunocytochemistry studies with GIP and GLP-1 staining could provide more information about the distribution and localization of K and L cells in GIPR-/-mice. 82 SUMMARY The studies presented in this thesis were targeted at developing a better understanding of GIP physiology and its contribution to enteroinsular axis. Using in vivo and in vitro techniques in GIPR-/- mice, it was demonstrated that the absence of GIP action affects glucose homeostasis by altering glucose disposal and insulin gene transcription and secretion without changing the (3 cell responsiveness to different secretagogues. It has been also reported that these effects were alleviated in part due to the compensation by the enhancement of the GLP-1-insulin axis in this animal model. 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