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A characterization of glucose-induced downregulation of the glucose dependent insulinotropic polypeptide… Olver, Amy Virginia 2006

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A CHARACTERIZATION OF GLUCOSE-INDUCED DOWNREGULATION OF THE GLUCOSE DEPENDENT INSULINOTROPIC POLYPEPTIDE RECEPTOR (GIPR) by A M Y VIRGINIA OLVER BSc, The University of Guelph, 2004 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF - MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Cellular and Physiological Sciences) THE UNIVERSITY OF BRITISH COLUMBIA December 2006 © Amy Virginia Olver, 2006 ABSTRACT The primary role of the incretin hormone, glucose-dependent insulinotropic polypeptide (GIP) is to potentiate the secretion of insulin in a glucose-dependent manner. In humans and rat models with type 2 diabetes, however, this action is impaired as a result of attenuated GIP receptor (GIPR) expression in the pancreatic P-cell. It has been previously reported that glucose strongly downregulates GIPR mRNA expression in rat insulinoma FNS-1 (832/13) cells and rat islets in a concentration-dependent manner. Since chronic hyperglycemia was proposed to be a primary cause for GIP resistance one objective of my studies was to attempt reversal of this process by pharmacologically lowering blood glucose. However, treatment studies on the effects of the dipeptidyl peptidase (DPIV) inhibitor P32/98 and a DPIV-resistant analogue D-Ala2-GIP on VDF and ZDF rats respectively, showed only moderate improvements in glucose homeostasis, and therefore were not appropriate models for determining the potential reversal effects of lowering blood glucose on islet GIPR expression. The mechanism for the glucose-induced GIPR downregulation is not clear; however it has been previously reported that peroxisome proliferated activated receptor a (PPARa), an important transcription factor involved in fatty acid metabolism, plays an important role. The second objective of this study therefore, was to investigate the effects of a PPARa overexpression system on GIPR expression in INS-1 (832/13) cells. Unexpectedly PPARa did not significantly upregulate our gene of interest; however these studies and others suggest that stimulation of PPARa alone is not sufficient for regulation, and thus also requires an increase in availability of PPARa's co-activator retinoid X receptor (RXR). The third objective of this thesis was to examine the regulation of GIPR in adipose tissue and to determine Ill whether GIPR is differentially expressed in fat and pancreatic islet. Glucose concentration-dependent studies on INS-1 (832/13) P-cell lines and 3T3L1-adipocytes as well as an in vivo characterization study of lean (Fa/?) versus fatty (fa/fa) VDF rat models, collectively demonstrated a tissue-specific pattern of GIPR expression. In all, the findings of this thesis therefore prompt new questions and provide a basis for future experimentation. IV T A B L E OF C O N T E N T S A B S T R A C T . . i i T A B L E OF C O N T E N T S iv LIST O F T A B L E S : v i i i L IST O F F I G U R E S .. . . . ix LIST O F A B B R E V I A T I O N S x i A C K N O W L E D G E M E N T S .xiv C H A P T E R 1.0: INTRODUCTION 1 1.1 E N T E R O I N S U L A R A X I S A N D T H E I N C R E T I N C O N C E P T 1 1.2 G L U C O S E D E P E N D E N T I N S U L I N O T R O P I C P O L Y P E P T I D E (GIP) 2 1.2.1. Discovery of GIP 2 1.2.2. GIP Structure and Secretion 3 1.3 T H E GIP R E C E P T O R (GIPR) 6 1.3.1. GIPR Discovery and Structure... 6 1.3.2. Regulation of GIPR Expression 7 1.3.3. GIPR Signaling 8 1.4. B I O L O G I C A L A C T I O N S OF GIP. 9 1.4.1 Gastric A c i d Secretion 9 1.4.2. Adipose Tissue and Fat Metabolism 10 1.4.3. GIP's Role in Islet and p-cell Regulation 12 1.4.4. Other Biological Effects 14 1.5 G L U C A G O N L I K E PEPTIDE-1 (GLP-1) A N D R E C E P T O R 15 1.5.1 Discovery and Characteristics of the GLP-1 and the GLP-1 Receptor 15 1.5.2 Biological Actions of GLP-1 17 1.6 M E T A B O L I S M O F T H E I N C R E T I N S . . 19 1.6.1. Function of Dipeptidyl peptidase IV (DPIV) 19 1.6.2. Therapeutic Approaches to Improve GIP Action 20 1.7 P A T H O P H Y S I O L O G Y OF GIP F U N C T I O N 21 1.7.1 Character izat ion o f G I P Func t ion in Patho log ica l M o d e l s 21 1.7.2. Poss ib le Explanat ions for the Reduced Responsiveness to GIP .23 1.7.3. Glucose-dependent Downregu la t ion o f G I P R Express ion 24 1.8 T H E R E G U L A T O R Y R O L E O F N U T R I E N T S O N G E N E E X P R E S S I O N 25 1.8.1 G lucose 25 1.8.2. L i p i d s 26 1.9 P E R O X I S O M E P R O L I F E R A T O R A C T I V A T E D R E C E P T O R S (??ARs)............... 27 1.9.1. P P A R F a m i l y Structure and Genera l Func t ion 27 1.9.2. P P A R Tissue D is t r ibut ion 28 1.9.3. Regu la t ion o f P P A R a 29 1.9.4. P P A R a Funct ion and Pathophys io logy . 29 1.10 T H E S I S I N V E S T I G A T I O N 31 CHAPTER 2.0: METHODOLOGY ...33 2.1 C H E M I C A L S 33 2.2 C E L L C U L T U R E 33 2.3 C E L L T R E A T M E N T 34 2.4 T R A N S F O R M A T I O N A N D T R A N S F E C T I O N . . . . . '....36 2.5 A N I M A L S 37 2.6 P R O T O C O L F O R D A I L Y M O N I T O R I N G A N D D R U G A D M I N I S T R A T I O N T O T H E V A N C O U V E R D I A B E T I C F A T T Y ( V D F ) R A T : A S T U D Y W I T H T H E D I P E P T I D Y L P E P T I D A S E I N H I B I T O R P32/98 38 2.7 P R O T O C O L F O R A D M I N I S T R A T I O N O F G IP , D-Ala 2 -GIP A N A L O G U E , O R V E H I C L E T O Z U C K E R D I A B E T I C F A T T Y ( Z D F ) R A T S 38 2.8 O R A L G L U C O S E T O L E R A N C E T E S T S ( O G T T ) A N D R A D I O I M M U N O A S S A Y S (R IA ) 39 2.9 Q U A N T I F I C A T I O N O F P P A R a A N D G I P R P R O T E I N E X P R E S S I O N VI OF B Y WESTERN BLOT A N A L Y S I S 39 2.10 TISSUE E X T R A C T I O N 41 2.10.1 Islet Isolation .41 2.10.2 Adipose Tissue Isolation 43 2.11 RNA ISOLATION.. . 43 2.12 REVERSE TRANSCRIPTION A N D R E A L - T I M E P O L Y M E R A S E CHAIN REACTION : 44 2.13 D A T A A N A L Y S I S ; 46 CHAPTER 3.0 THE EFFECT ON GIPR EXPRESSION OF LOWERING BLOOD GLUCOSE IN HYPERGLYCEMIC RATS 47 3.1 PROJECT RATIONALE : 47 3.2 RESULTS 48 3.2.1 The Effect of 12-Week DPIV Inhibitor P32/98 Treatment in V D F (/a/fa) Rats 48 3.2.2 The Effect of Continuous Administration of GIP or D-Ala 2-GIP Analogue on Development of Insulin resistance and P-cell Responsiveness in ZDF Rats 53 3.3 DISCUSSION.. . . . . . . . 56 CHAPTER 4.0 THE EFFECTS OF PPARa OVEREXPRESSION IN THE INS-1 (832/13) P-CELL LINE 61 4.1 PROJECT R A T I O N A L E . . 61 4.2 RESULTS. . . . '. 61 5.2.1 The Effects of PPARa Overexpression in the INS-1 (832/13) P-CellLine .61 4.3 DISCUSSION 65 Vll CHAPTER 5.0 CHARACTERIZATION OF GIPR mRNA EXPRESSION IN ADIPOSE TISSUE AND 3T3-L1 ADIPOCYTES 69 5.1 PROJECT RATION A L E . . 69 5.2 RESULTS .' 69 5.2.1 Characterization of Epididymal Fat Pad GIPR Expression in Fatty (fa/fa) versus Lean (Fa/?) V D F Rats 69 5.2.2 Glucose Concentration-Response Study on GIPR mRNA Expression in.3T3-L1 Adipocytes —72 5.2.3 3T3-L1 Adipocyte Responses to Clofibrate Treatment Under High and Low Glucose Conditions .73 5.3 DISCUSSION !. 74 CONCLUSIONS A N D FUTURE DIRECTIONS.. 77 REFERENCES 79 viii LIST OF T A B L E S Table 1: Real-time PCR primer and probe sequences .45 ix LIST OF FIGURES Figure 1: Food Intake, water consumption, and body weight of V D F (fa/fa) rats in a 12-week P32/98 treatment study 49 Figure 2: Morning blood glucose levels of V D F (fa/fa) rats in a 12-week P32/98 treatment study 50 Figure 3: Blood glucose concentrations in oral glucose tolerance tests on V D F (fa/fa) rats in a 12-week P32/98 treatment study 51 Figure 4: Plasma insulin concentrations during oral glucose tolerance tests on VDF (fa/fa) rats in a 12-week P32/98 treatment study 52 Figure 5: GIPR mRNA levels in extracts from islets and peri-renal fat pads of VDF (fa/fa) rats in a 12-week P32/98 treatment study 53 Figure 6: Body weight of 5 week old ZDF rats during a 4-week continuous infusion of either GIP, D-Ala 2-GIP analogue or saline control 54 Figure 7: Morning plasma glucose levels of 5 week old ZDF rats during a 4-week continuous infusion of either GIP, D-Ala 2-GIP analogue or saline control 54 Figure 8: Insulin tolerance tests were performed on 5 week old male ZDF rats altera 4-week continuous infusion of either GIP, D-Ala 2-GIP analogue or saline control 55 Figure 9: GIPR mRNA expression in epididymal fat pads of 5 week old ZDF rats after a 4-week continuous infusion of either GIP, D-Ala 2 - GIP analogue, or saline control 56 Figure 10: A Western blot of PPARa protein expression in INS-1 (832/13) cell extracts with or without transient transfection of human C M V - P P A R a 62 Figure 11: The effect of human PPARa overexpression and elevated glucose on PPARaand GlFR mRNA expression in INS-1 (832/13) cells '." .63 Figure 12: The effect of human PPARa overexpression and elevated glucose on UCP-2 mRNA levels in INS-1 (832/13) cells 64 Figure 13: Blood glucose concentrations during oral glucose tolerance tests on VDF (fa/fa) and lean (Fa/?) rats; .70 X Figure 14: GIPR mRNA levels in isolated islets of V D F (fa/fa) versus lean (Fa/?) V D F rats.. 71 Figure 15: GIPR mRNA levels in epididymal fat pads of V D F (fa/fa) versus lean (Fa/?) rats '. 71 Figure 16: The effect of glucose on GIPR mRNA levels in INS-1 (832/13) cells and differentiated 3T3-L1 adipocytes 72 Figure 17: GIPR mRNA expression in 3T3-L1 adipocytes after 24 hour treatment of 1 m M clofibrate 73 xi LIST OF ABBREVIATIONS 1-Oct octamer-1 A A arachidonic acid A C C acetyl CoA carboxylase A O X acyl-CoA oxidase aP adipocyte fatty acid binding protein ADP adenosine diphosphate A N O V A analysis of variance ATP adenosine triphosphate B C A bicinchoninic acid BSA bovine serum albumin cAMP cyclic adenosine monophosphate C C K cholecystokinin cDNA complementary deoxyribonucleic acid C/EBP C C A A T Box/Enhancer-binding protein ChIP chromatin immunoprecipitation CHO Chinese Hamster Ovary C M V cytomegalovirus CPT-1 carnitine palmitoyl transferase-1 CRE-BP1 cAMP response element-binding protein Ct cycle threshold C-terminal carboxy-terminal Cys Cysteine D M E M Delbucco's modified eagle media DMSO dimethylsulfoxide D N A deoxyribonucleic acid dNTP deoxyribonucleotide triphosphates DPIV dipeptidyl peptidase 4 E C L enhanced chemi-luminescence EDTA ethylenediaminetetraacetic acid EGTA ethylene glycol tetra acetic acid E M S A electromobility shift assay EPAC Rapl guanine nuclear exchange factor E R K extracellular regulated protein kinase F A M 1-Dimethoxytrityloxy-3 - [0-(N-carboxy-(di-O-pivaloyl-fluorescein)-3-aminopropyl)]-propyl-2-0-succinoyl-long chain alkylamino FAO fatty acid oxidase FAS fatty acid synthase FAT/CD36 fatty acid translocase FATP fatty acid transport protein FBS fetal bovine serum FFA free fatty acid GIP gastric inhibitory polypeptide/glucose-dependent insulinotropic polypeptide GIPR GIP receptor Gin glutamine Glu glutamate Gly glycine GLP glucagon-like peptide GPCR G-protein coupled receptor GRE glucose response element GSIS glucose-stimulated insulin secretion H E K human endothelial kidney HIT-T15 hamster insulinoma I B M X 3-isobutyl-1-methylxanthine IP3 inositol trisphosphate IR immunoreactive IV intravenous K A T P inwardly rectifying potassium channel (Kir) 6.2 Kb kilobase Kd dissociation constant kDa kilodaltons L B Luria-Bertani L P L lipoprotein lipase Lys lysine M A P mitogen activate protein mRNA messenger ribonucleic acid NH3-terminal amino-terminal . OGTT oral glucose tolerance test Opti-MEM optimum modified eagle media P A L palmitate PBS phosphate buffered saline PC prohormone convertase PCR polymerase chain reaction PDX-1 pancreas duodenum homeobox-1 PEPCK phophoenolpyruvate carboxy-kinase PI3K phosphtidylinositol 3-kinase PK pyruvate kinase P K A protein kinase A PKC protein kinase C P L A 2 phospholipase A 2 PLC phospholipase C PMSF phenylmethylsulfonylfluoride PPAR peroxisome proliferator activated receptor PPRE peroxisome proliferator response element Pro proline PUFA polyunsaturated fatty acid RGS regulators of G-protein signaling RIA radioimmunoassay RIM Rab3 interacting molecule RIN38 rat insulinoma cell line RIPA radioimmunoprecipitation buffer R N A ribonucleic acid rPCR real-time RT-PCR RT reverse transcription RT-PCR reverse transcription polymerase chain reaction R X R retinoid X receptor SDS-PAGE sodium dodecylsulphate-polyacrylamide gel electrophoresis S E M standard error of mean SRE sterol regulatory element SREBP/ADD1 sterol regulatory element protein/adipocyte determination differentiation-dependent factor 1 T A M R A 1 -Dimethoxytrityloxy-3 - [0-(N-carboxy-(Tetramethyl-rhodamine)-3 -aminopropyl)]-propyl-2-0-succinoyl-long chain alkylamino Taq T. aquaticus D N A polymerase TBST tris-buffered saline with 0.5% Tween 20 TIF 2 transcriptional intermediary factor 2 UCP-2 uncoupling protein 2 U N G • uracil N-glycosylase USF/MLTF upstream stimulatory factor/major late transcription factor UTR untranslated region V D C C voltage-dependent calcium channels VDF Vancouver diabetic fatty VIP vasoactive intestinal polypeptide WT wild-type ZDF Zucker diabetic fatty xiv A C K N O W L E D G E M E N T S Firstly, I would like give a very appreciative thanks to Chris Mcintosh for his patience, expertise and kindness over the past two years. I would also like to thank my previous and present lab colleagues John Han, Maddy Speck, Rick, and Scott Widenmeier for their help and suggestions throughout the duration of my project. Thanks to Su Jin Kim for her intelligence and time spent helping me overcome those all too common roadblocks that make success in research so celebratory. To Cuilan Nian my friend and teacher who made each day in the lab all that more interesting, Shia Shia. To our 4 t h year student Eiman Zargaran creator of our now abducted mascot PP, who frequently put up with my music choices and brought good conversation to the bench. Thanks to all the graduate students in the physiology department like Rhonda Wideman, who made me feel at home and among friends when I didn't know anybody. I should also include my good Portugese/Scottish/Canadian friend Bruno De Fonseca from the Proud lab who was always there with a smile and ready for a good chat. To my fellow hikers, perhaps we will have to have a reunion at the top of the Lions, but maybe no time soon ©. To the "Johnson's Angels" or more appropriately my closest friends here in Vancouver; Jen Beith who was brave enough to join me on the wild side snowboarding down black diamonds mostly on our behinds in the dark and surfin' the waves in Tofino. To Emilyn Alejandro karoke extraordinaire whose kindness, BBQ's on the roof, and frequent leg slap/feet stomp responses to her own jokes will be missed. To my good friend Kristin Jeffrey who always knew how to provoke thought and challenge me to vocalize my opinions and ideas. Thanks to my uncle Chris Crowley,and his family who allowed me to escape the city by opening their home to me on Bowen Island. Of course thanks to my family back home, my grandma Loretta Crowley, my mom Pauline Crowley, dad Ivan Olver, and siblings Denise, Nick, and Ben Olver who not only supported my move out to the wild West but patiently listened to all my exiting adventures throughout. A special thanks of course goes out to Trevor Topping my best friend and confidante for all his love, support and share of enthusiasm for exploration. Lastly, I'd like to dedicate this work to my late grandfather Francis Crowley who has forever given me a love and appreciation for the simpler things in life. 1 CHAPTER 1: INTRODUCTION 1.1 ENTEROINSULAR AXIS A N D THE INCRETIN CONCEPT Bayliss and Starling (1903) pioneered the field of endocrinology by discovering that an intestinal extract was capable of stimulating pancreatic juices; they called the active substance secretin. Shortly after, Moore and colleagues (1906) proposed that this intestinal extract was not only capable of stimulating internal pancreatic secretion but also influenced blood sugar levels. It was not until 1921, after Banting and Best discovered insulin (Banting & Best, 1922), that Zunz and Labarre were able to prove this by showing that intravenous infusion of a crude secretin preparation into dogs caused a reduction in serum glucose levels (Zunz & Labarre, 1926). To account for this effect, Labarre and colleagues suggested that the gut extract was composed of two parts: one for stimulating the exocrine pancreas and the other for stimulating an internal secretion from the endocrine pancreas. The term incretin was coined for the latter (Labarre & Still, 1930). The actual physiological effects of this substance were poorly understood until the development of the radioimmunoassay (RIA) in 1960. The RIA enabled investigators to measure blood insulin levels and it was then that they discovered that glucose given orally caused a much higher increase in serum insulin than an equal dose administered intravenously (Elrick et al, 1964; Mclntyre et al, 1964). It was therefore concluded that there must be some form of communication between the gastrointestinal system and the endocrine pancreas that modulates the secretion of insulin. This endocrine connection between the gut and the pancreatic islets was termed the enterosinsular axis (Unger & Eisentraut, 1969). Several years later, Creutzfeldt extended the definition of the enteroinsular axis to also encompass both neural and substrate communication. Substrates 2 in particular were capable of stimulating the release of incretins, defined as hormones that: 1) are secreted from the gut in response to nutrients and 2) enhance insulin secretion only under elevated blood glucose conditions (Creutzfeldt 1979; Creutzfeldt & Ebert 1985). 1.2 GLUCOSE DEPENDENT INSULINOTROPIC POLYPEPTIDE (GIP) 1.2.1. Discovery of GIP Brown and Pederson discovered that a crude preparation of porcine cholecystokinin (CCK) was more effective at inhibiting gastric acid secretion than more highly purified C C K , suggesting the presence of an additional bioactive factor in the crude preparation (Brown et al, 1969; 1970; Brown & Dryburgh, 1971). Shortly after, this factor was isolated and named for its actions as a gastric inhibitory polypeptide (GIP) (Pederson & Brown et al, 1972). Further investigation into this novel peptide led to the discovery of GIP's ability to potentiate the secretion of insulin (Dupre et al., 1973) in a dose-dependent manner (Pederson & Brown, 1976; Pederson et al., 1982). This effect was subsequently described in humans (Dupre et al, 1973; Elahi et al., 1979), dogs (Pederson et al., 1975) and rats (Ebert & Creutzfeldt, 1982; Pederson & Brown, 1976; 1978) as well as in isolated rat islets (Hinke et al, 2000a; Lynn et al, 2001) and several P-cell lines (Ehses et al, 2001; 2002; Kieffer et al, 1993; O'Harte et al, 1998). Perfusion of the rat pancreas demonstrated that the insulin-releasing action of GIP was only effective at glucose concentrations exceeding 5mM with maximal secretagogue activity occurring at approximately 16mM glucose. Additional studies defined a similar range of in vivo activity in rat (Tseng et al, 1996), as well as human (Dupre et al, 1973; 3 Elahi et al, 1979) and dog (Pederson et al, 1975). This glucose-dependence indicated that GIP fulfilled Creutzfeldt's criteria for an incretin hormone. Consequently, to reflect this newly discovered property of GIP, Pederson et al. (1976) gave gastric inhibitory polypeptide an alternate designation as glucose-dependent insulinotropic polypeptide (GIP). 1.2.2. GIP Structure and Secretion GIP is a 42 amino acid polypeptide belonging to the glucagon family of peptides that is highly conserved amongst humans with a 95% sequence identity to porcine and rat and 91%) homology to bovine and mouse GIP (Jornvall et al., 1981). The GIP gene spans lOkb and consists of 6 exons. Exon 1 encodes most of the 5' untranslated region (UTR), while exon 2 comprises the remainder of the 5'UTR and the secreted signal peptide. Exons 3, 4 and 5 encompass the hormone-coding region, and exon 6 encodes the 3' UTR (Inagaki et al., 1989). Several transcription factor binding sites have been identified on the GIP promoter including AP-1 and AP-2 elements in humans, which are target sites for transcription factors regulated by protein kinase A (PKA) and protein kinase C (PKC) (Inagaki et al, 1989). In the hamster insulinoma (HIT T15) cell line two binding sites, believed to be required for basal GIP promoter activity, were identified for the cAMP response element-binding protein 1 (CRE-BP1). The GIP promoter nucleotide -158 in particular appears to be particularly important for this cAMP inducibility (Someya et al, 1993). Further mutational analysis in the neuroendocrine cell line STC-1 also identified potential transcription factor binding sites for GATA-4 and ISL-1 that were later confirmed by electrophoretic mobility shift analysis (EMSA) to be involved in cell-specific expression of the GIP gene (Jepeal el al, 2003). In intact GIP producing cells 4 and the STC-1 cell line, the important pancreatic development gene PDX-1, was also found to play a critical role in GIP expression, after E M S A and CHiP analysis confirmed a Pdx- l /DNA interaction on the regulatory region of the GIP promoter (Jepeal et al, 2005). The biologically active GIP protein is the end product of endoproteolytic processing of its precursor preproGIP, and subsequently formed proGIP. Recent studies in neuroendocrine AT-20 cell lines and GH4 gut cell lines have shown that prohormone convertase PC 1/3 (the hormone responsible for the cleavage of intestinal proglucagon) is sufficient for cleavage of proGIP to GIP. The other prohormone convertase PC2 however was neither present in intestinal GIP-expressing cells, nor was required for proGIP cleavage (Ugleholdt et al, 2006). The primary site of GIP synthesis is the enteroendocrine K-cell localized within the intestinal epithelia of the duodenum and jejunum in humans as well as the ileum in rats and dogs (Buchan et al, 1982; 1978; Polak et al, 1973). However, GIP expression has also been detected in the stomach (Cheung et al, 2000; Yeung et al, 1999) and duct cells of the submandibular glands (Tseng et al, 1995), though the action of extra-intestinal GIP is not well understood. More recently, significant co-expression of proglucagon and proGIP genes have been found in single human duodenal cells (Theodorakis et al, 2006) suggesting enteroendocrine cells express both GIP and GLP-1. This observation supports previous work by Vilsboll and colleagues (2003) who reported that GIP and GLP-1 are released simultaneously and equally contribute to the insulinotropic effect. 5 In the enteroendocrine K-cells, secretion of GIP is stimulated by the intestinal absorption of nutrients such as glucose, amino acids and free fatty acids (Alam & Buchanan, 1993). Long chain, highly saturated fatty acids (Lardinois et al, 1988; Ross & Shaffer et al, 1981) and triglycerides (Brown & Otte et al, 1978; Pederson al. 1975) are the most potent stimulators of GIP release. Nutrient stimulation results in a 5-6 fold increase in human GIP plasma levels (Pederson et al, 1994) from basal circulating levels of 12-92 p M to postprandial levels of 35-235 p M (Alam & Buchanan, 1993). This stimulatory effect is limited to oral intake, as intravenous injection of glucose into the bloodstream does not result in an incretin response (Elahi et al, 1981; Sykes et al, 1980). The K-cells are capable of rapid, nutrient-responsive GIP secretion owing to their cellular polarity. It is thought that glucose is absorbed at the apical surface of the K-ce l l via a Na+-dependent transport mechanism (Sykes et al, 1980) and its presence is detected in part through the glucose sensor, glucokinase (Cheung et al, 2000). Secretory granules containing GIP are stored close to the basolateral surface of the K-ce l l , allowing for rapid granule exocytosis and release, of GIP into the bloodstream (Kieffer et al., 1994). The exact mechanism of K-ce l l secretion and the ion channels involved are still poorly understood. Many endocrine cell types contain ATP-dependent K + channels ( K + A T P ) which consist of 8 subunits of SUR and Kir subtype. However, in a mouse enteroendocrine derived GIP-producing STC-1 K-ce l l line, very low expression of both Kir and SUR subunits were detected, and these cells were found to be insensitive to K + A T P channel activators such as sulfonylureas and K + channel openers (Ramshur et al, 2002). Previous in vivo findings have also showed that sulfonylurea treatment had no effect on either GIP or GLP-1 secretion but yet still demonstrated enhanced insulin release (Groop et al, 1983; Groop et al, 1985; 1987; Fukase et al, 1995). Taken together, these results suggest that K-cell secretion may be independent of K + A T P , and thus further investigation is warranted to elucidate a mechanism of secretion (Ramshur et al, 2002). 1.3 THE GIP RECEPTOR (GIPR) 1.3.1. GIP Receptor Discovery and Structure Once GIP is released from the K-cells it exerts its actions via interaction with the GIP receptor (GIPR) in targeted tissues. The GIPR was initially identified via 1 2 5I-GIP binding studies in hamster IN 111 cells, human insulinomas and mouse P-TC3 cells that had a K d for GIP in the low nM range (Amiranoff et al, 1984,1985; Kieffer et al, 1993; Maletti et al, 1983; 1987). Using the ultraviolet irradiation procedure the 1 2 5I-GIP binding studies characterized the receptor as a 64 kDa direct cross-linked GIP-protein complex. Further SDS-PAGE analysis of this complex, isolated and characterized the GIPR as a 59 kDa monomer with intrachain disulfide bridges. The GIPR was also found to be a glycoprotein after carbohydrate moieties such as N-acetylglucosamine, mannose, and possibly sialic acid were found present (Amiranoff et al, 1986). The GIPR was cloned from a cDNA library of the rat brain (Usdin et al 1993) and later from rat islets (Wheeler et al, 1995), a hamster insulinoma (HIT-T15) cDNA library (Yasuda et al, 1994) and human insulinoma cDNA (Volz et al, 1995).. In humans, the GIPR gene is comprised of 14 exons spanning 13.8kb; 12 of these encode the protein sequence while the 1st encodes the 5' UTR flanking promoter region region and the 14 th encodes the 3'UTR. In contrast, rats have 15 exons spanning 10.2kb; the additional 15 th exon encodes the 3' UTR while the 5'UTR is located on exon 1 and 7 the remainder encodes the protein (Boylan et al, 1999, Wolfe et al, 1999, Yamada et al, 1995). Despite this difference, the rat GIPR gene still contains ~ 81% homology to human (Yamada et al, 1995). The 466 amino acid GIPR belongs to the secretin/VIP family of receptors as a seven transmembrane G-protein coupled receptor (GPCR) (Gremlich et al, 1995; Usdin et al, 1993; Wheeler al, 1995). It contains a large extracellular amino-terminal region with consensus sequences for both glycosylation (necessary for proper expression) and the facilitation of GIP binding (Gelling et al, 1997), while the carboxy-terminal end of the receptor has been demonstrated to play a key role in the expression and orientation of the receptor (Wheeler et al, 1999). Moreover, the GIPR has phosphorylation sites present in the serine/threonine rich C-terminus and third cytoplasmic loop (Wheeler et al, 1999). Collectively, the link between extracellular stimulation and intracellular signal transduction is believed to be facilitated by the first transmembrane helix of the receptor (Gelling et al, 1997; Buggy et al, 1995; Couvineau et al, 1995; Gaudin et al, 1995; Jiippner et al, 1994; Van Eyll et al, 1996; Wilmen et al, 1997; Vilardaga et al, 1995). Like many GPCRs, the GIPR is also capable of undergoing reversible receptor desensitization (Fehmann et al, 1991; Hinke et al, 2000). 1.3.2. Regulation of GIPR Expression The GIPR contains a T A T A / C A A T - less promoter where the first 100 bp upstream of the 5' flanking region were found to be sufficient for transcription in a rat insulinoma cell line 2 (RIN38) (Boylan et al, 1999) and human tissues (Baldacchino et al, 2005). Serial deletion analysis indicated that the promoter region between -85 and -40 8 is required for maximal promoter activity and, consistent with other TATA-less promoters, this region also contains a high guanine cytosine (GC) content as well as multiple Spl binding sites (Boylan et al, 2006; Baldacchino et al, 2005). Spl is a member of a family of transcription factors that each contain three highly conserved zinc finger D N A binding domains that bind to GC-rich sequences to regulate gene expression (Cook et al, 1999). Transcription factors Spl and Sp3 have been shown to bind to the Spl motifs of the GIPR promoter in RIN38 cells (Boylan et al, 2006) and human tissues (Baldacchino et al, 2005) and in turn lead to an increase in GIPR transcriptional activity (Boylan et al, 2006). Other binding motifs that have been identified on the GIPR promoter are octamer-1 binding factor (OCT-1) (Verrijzer et al, 1992; Boylan et al, 1999) and a cAMP response element (Meyer et al, 1993). The GIPR is expressed in a variety of tissues including stomach, intestine, adipose tissue, adrenal cortex, heart, lung, endothelium, telencephalon, diencephalon, brain stem, cerebellum, pituitary, and the pancreas (Usdin et al, 1993; Yasuda et al., 1994; Zhong et al, 2000). It is not known however whether GIPR gene regulation differs across tissues, since most studies have been done in islets, or islet cell lines. This broad expression of the GIPR in various tissues suggests that it is likely a protein involved in the regulation of many functions, yet to be elucidated. 1.3.3. GIPR Signaling The binding of a hormone to a GPCR induces a conformational change in the receptor and subsequent activation of a coupled specific heterotrimeric G-protein. Once G-proteins are activated, they often dissociate into their various subunits and stimulate 9 enzymes for second messenger activation or act on proteins and ion channels directly (Hedin et al, 1983). The binding of GIP to the GIPR stimulates a conformational change in the third intracellular loop causing a dissociation of the coupled heterotrimeric G-protein into two components. Gsa is one component that is coupled to adenylate cyclase, the enzyme responsible for initiating the c A M P / P K A signaling pathway (Lu et al, 1993; Miura et al, 1997). The other two subunits of the heterotrimeric G-protein, G P and y, act as a dimer in PTC-3 cells, to liberate arachidonic acid from the lipid membrane via activation of phospholipase A2 (PLA2) (Ehses et al, 2001). GIP has also been shown to activate various other pathways including the Raf-Mekl/2-extracellular signal-regulated kinase (ERK)l/2 and the phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (PKB) pathways. Further discussion of the implications of GIP signal-transduction in the P-cell will be discussed in Section 1.4.3. Since the GIPR is a member of a large receptor family the possible cross-reactivity of other members was examined (Mcintosh et al, 1996). Although, both the GLP-1R agonist exendin (9-39) and the GLP-1R antagonist exendin-4 (1-39) were 19 S capable of displacing I labeled GIP from the GIPR, this only occurred at micromolar concentrations. Other hormones, including glucagon, GLP-1, GLP-2, VIP, and secretin had no displacement effect (Wheeler et al, 1995). 1.4. BIOLOGICAL ACTIONS OF GIP 1.4.1 Gastric Acid Secretion GIP's inhibition of acid secretion in the extrinsically denervated stomach led to its initial discovery (Pederson & Brown, 1972), but this action was later found to be minimal in innervated preparations (Andersen et al.,, 1978; Arnold et al, 1978a; El Munshid et al, 10 1980; Maxwell et al, 1980; Soon-Shiong et al, 1979; Yamagishi & Debas, 1980). To account for this, Mcintosh et al (1981) proposed that GIP was acting indirectly through another substance, and that GIP may stimulate secretion of somatostatin from gastric D-cells. Indeed, Mcintosh and colleagues (1981) were able to demonstrate that GIP stimulated somatostatin release from the D-cells of both the gastric corpus and antrum, suggesting an indirect mode of action through both direct inhibitory effects of somatostatin on the parietal cell and inhibition of gastrin release. Both the inhibitory action of GIP on acid secretion by the denervated stomach (Soon-Shiong et al, 1984) and the stimulatory effect of GIP on somatostatin secretion (Mcintosh et al, 1981) were abolished upon the addition of a cholinergic agonist; thus potentially accounting for the lack of effect of GIP in the innervated stomach. 1.4.2. Adipose Tissue and Fat Metabolism In adipose tissue, GIP has been shown to exhibit both lipogenic and lipolytic actions. One such anabolic action of GIP is to induce an increase in 2-deoxy-glucose uptake in isolated rat adipocytes (Hauner et al, 1988). The anabolic actions of GIP in fat are said to be insulinomimetic as they appear to enhance insulin activity. In 3T3-L1 adipocytes (Eckel et al, 1979) and explants of rat epididymal adipose tissues (Knapper et al, 1995), a significantly greater lipoprotein lipase activity was seen after co-incubation of GIP and insulin than either hormone alone. Similarly co-incubation of rat epididymal fat pads with GIP and insulin also enhanced insulin effects on fatty acid incorporation into adipose tissue. The importance of the anabolic actions of GIP were further exhibited in GIPR"7" mice fed a high fat diet, since they demonstrated decreased diet-induced obesity and, when leptin-deficient mice were crossed with the GIPR knockout line 11 (ob/ob, GIPR"7"), they developed less obesity than the regular ob/ob mouse (Miyawaki et al, 2002). Collectively, these studies suggest that GIP is an important regulator of fat deposition and possibly a target for anti-obesity therapy. In contrast to GIP's lipogenic actions, GIP has also been demonstrated to exhibit lipolytic effects (Hauner et al, 1988). In 3T3-L1 adipocytes, GIP treatment induced a lipolytic release of glycerol via activation of cAMP production. In the presence of insulin however, these actions were antagonized in a wortmannin-sensitive manner (Mcintosh et al, 1999). Moreover, perifusion of isolated rat adipocytes with medium containing GIP demonstrated increased glycerol release, supporting a lipolytic role (Getty-Kaushik et al, 2006). It has been proposed that these lipolytic actions elevate the level of free fatty acids sufficiently to optimize insulin secretion from the pancreas by priming the cell (Mcintosh et al, 1999). This is further suggested in the GIPR"7" mice, which depict a delay in first phase insulin secretion (Miyawaki et al, 1999). Although levels of free fatty acids were not measured in the GIPR"7" mice it has been reported that fat is used as a preferential substrate in these animals (Miyawaki et al, 2002), thus a lack of free fatty acids may attribute to impairment in first phase insulin secretion. Taken together, one possible explanation for both the lipogenic and lipolytic roles of GIP in adipose tissue is the notion of segregated pools of cAMP within fat cells. These pools could either be coupled or uncoupled to certain pathways that could determine the metabolic outcome (Honeyman et al, 1979; Schimmel et al, 1984). This is not well studied however, and thus much still remains to be discovered about GIP's role in fat metabolism. 12 1.4.3. GIP's Role in Islet and P-cell Regulation A primary function of GIP in the P-cell is its ability to potentiate glucose-stimulated insulin secretion (GSIS). GSIS involves transport of glucose into the P-cell and metabolism resulting in an increase in the ATP/ADP ratio. Once the ratio is elevated it induces closure of K + A T P channels leading to an overall reduction in K + efflux and depolarization of the cell membrane. Depolarization subsequently induces the opening of voltage gated Ca 2 + channels in the plasma membrane and an increase cytosolic Ca 2 + levels to stimulate insulin exocytosis. GIP potentiates response to glucose by increasing intracellular cAMP and subsequent activation of P K A . The activated kinase is then believed to work in partnership with the elevated ATP/ADP ratio to close the K + A T P channel (Light et al, 2002). Insulin exocytosis may also be regulated by other GIP induced pathways. In PTC-3 cells, exogenous arachidonic acid has been shown to stimulate insulin secretion in a glucose dependent manner, as well as increasing cytosolic C a 2 + in pancreatic islets (Metz et al, 1988). Later studies also reported that arachidonic acid was coupled to insulin secretion (Turk et al, 1993; Simonsson et al, 2000) and was believed to be activated through an ATP-sensitive Ca independent PLA2 (Ramanadham et al, 1994; Simonsson et al, 2000). Interestingly, GIP has been shown to liberate arachidonic acid from the lipid bilayer in PTC-3 cells via P L A 2 activation, thus it is likely that this is an alternative pathway for GIP induced insulin secretion (Ehses et al, 2001). GIP has also been found to activate phospholipase C (PLC), an enzyme that increases the production of second messenger IP3 and subsequently opens the IP3 receptor of the endoplasmic reticulum (ER) for calcium release (Lu et al, 1993; Yasuda et al, 1994). This pathway however 13 has not been observed in normal P-cells and therefore warrants further investigation. There is also evidence for a Ca2+-independent mechanism of insulin secretion in which cAMP, directly activates an EPAC that forms a cAMP-GEFII-Rim 2 complex and stimulates insulin exocytosis (Kasima et al, 2001). More prolonged effects of GIP on the insulin secretory process have been reported. This includes increased P-cell gene expression of the glucose regulating proteins hexokinase and GLUT-1 (Wang et al, 1996), as well as insulin mRNA (Lu et al, 1993a; Fehmann & Goke, 1995) and protein expression (Wang et al, 1996) levels. The effects on insulin are diminished in the GIPR_ /" mouse (Pamir et al, 2003). Recent studies by Kim et al. (2005) have demonstrated that GIP, through a c A M P / P K A dependent pathway downregulates cell surface expression of Kvl .4 , a subfamily member of Kv channels important for repolarization of the cell membrane. It is thought that by delaying the repolarization of the cell, the voltage-dependent Ca channels will remain open longer for C a 2 + influx, thus prolonging the potential for insulin release. GIP has also been reported to affect other K + channels such as the ATP-dependent channel Kir6.2, via P K A phosphorylation and subsequent opening probability of the channel. The physiological relevance of this event remains unclear (Beguin et al, 1999). While the major role of GIP is thought to be potentiation of GSIS, mounting evidence suggests that GIP also has important protective effects under pro-apoptotic and gluco-lipotoxic conditions in the P-cell. K im and colleagues (2005) demonstrated in INS-1(832/13) cells and C57/BL6 and Bax"7" mouse islets, that under gluco-lipotoxic conditions, GIP induced a decrease in the pro-apoptotic factor bax via nuclear exclusion of Foxol. The protective effect of GIP was also seen after a 2 week continuous infusion 14 of GIP into V D F rats. Histochemical and TUNEL staining illustrated that the islets from GIP treated rats had decreased bax protein, increased pro-survival protein bcl-2, and an overall reduction in apoptotic cells (Kim et al, 2005). GIP has also been shown to enhance P-cell proliferation and survival through c A M P / P K A and the mitogen-activated protein kinase (MAP) kinase ERK1/2 pathways in INS-1 (832/13) cells (Ehses et al, 2002; Trumper et al, 2001; Kim et al, 2005). GIP also demonstrates actions on other cell types within the endocrine pancreas. It was shown to stimulate the release of glucagon from isolated, cultured human and rat islets (Fujimoto et al, 1978; Verchere, 1991), although only at glucose concentrations below 5.5mM (Elahi et al, 1979; Pederson & Brown, 1978). This low glucose requirement appears to be species specific however, since GIP-induced glucagon secretion can also occur at high glucose in mice (Opara & Go, 1991). Lastly, GIP is also capable of weakly stimulating somatostatin secretion from the pancreatic 8-cells (Schmid etal, 1990; Verchere, 1991). 1.4.4. Other Biological Effects In addition to GIP's insulinotropic and pro-survival effects on the pancreatic islet GIP has also been found to have roles in other tissues. Studies on the GIPR 7" mouse have recently shown a decline in bone formation parameters with a subsequent increase in bone-degrading osteoclasts (Tsukiyama et al, 2006). Postprandially, GIPR"7" mice also demonstrated elevated plasma C a 2 + levels, an indicator of increased bone resorption. GIP 2+ therefore is believed to be an important link between bone deposition and dietary Ca (Bollag et al, 2001; 2000; Tsukiyama et al, 2006). 15 There is also evidence for involvement of GIP in food-dependent Cushing's Syndrome, a disease characterized by hypersecretion of Cortisol. Normally Cortisol release is regulated by the levels of adrenocorticotropin hormone (ACTH) released from the hypothalamus. In food-dependent Cushing's syndrome, however, it is thought that irregular expression of GIPR in the adrenal cortex causes a chronic postprandial increase in cAMP leading to an inappropriate release of Cortisol and further tumorogenesis (Crough et al, 2000; Lacroix et al, 1992; Chabre et al, 1998; Swords et al, 2005). The extensive expression of GIPR in other tissues suggests that there may be other as-yet undiscovered roles; however, the function of GIPR signaling in these areas is still relatively unknown. 1.5 G L U C A G O N LIKE PEPTIDE-1 (GLP-1) A N D THE GLP-1 RECEPTOR 1.5.1 Discovery and Characteristics of GLP-1 and the GLP-1 Receptor Early studies found that in vitro or in vivo immunoneutralization of GIP only partially reduced the insulin-releasing effects of GIP or endogenous incretins (Ebert et al, 1982; Lauritsen et al, 1981). This prompted studies to investigate the, possible existence of a second incretin. In the early 1980's Lund and Habener identified a GIP-like peptide encoded within the preproglucagon gene of the pancreata of anglerfish (Lund et al, 1982). Preproglucagon mRNAs were also cloned from gut tissue of anglerfish (Lund et al, 1981), humans (Novak et al, 1987) and rats (Drucker et al, 1989) further supporting the possibility of an incretin hormone. The proglucagon gene was later found to be expressed in pancreatic a-cells, various regions of the brain and L-cells of the ileum and ascending colon (reviewed in 16 Kieffer & Habener, 1999) and encodes multiple peptides including glucagon, GLP-1 and GLP-2. Proglucagon undergoes post-translational processing by prohormone convertases (PC), a class of enzymes that are expressed in tissue-specific fashion and cleave specific basic amino acid residues. In the a-cells, PC2 is present and responsible for the liberation of glucagon, while PC 1/3 present in the intestinal L-cell cleaves the precursor to produce GLP-1 and GLP-2, oxyntomodulin and glicentin (Kieffer & Habener, 1999). Before prohormone convertases were discovered however researchers proposed that the bioactive peptide could be cleaved from the prohormone during post-translational processing; however the exact cleavage sites were only hypothesized. The end product of post-translational processing was expected to be cleaved at a Lysine-Arginine bond yielding a peptide of 37 or 36 amino acids. As a result 1-37 and 1-36 GLP-1 peptide isoforms were the first to be synthesized and tested, but rendered only a weak stimulus to insulin secretion (Ghiglione et al., 1984). It was later discovered in 1986 that the biologically active GLP-1 was truncated in the N-terminus by posttranslational processing in the intestinal L-cells (Drucker et al., 1986; Mojsov et al., 1986). As a result GLP-1 (7-36) and (7-37) isoforms were found to be the source of glucose-dependent insulinotropic action in the isolated perfused pancreas of rats (Mojsov et al., 1986), pigs (Hoist et al., 1987) and in humans (Kreymann et al., 1987) and thus were classified as incretins. L-cells are primarily located in the ileum, an area distal of most nutrient absorption. Stimulated K-cells in rat and dog are therefore believed to play a role in L-cell activation in a feed-forward mechanism (Damholt et al., 1999; Elliot et ai, 1993; Hermann-Rinke et al., 1995; Roberge & Brubaker et al., 1993); a similar pathway however has not been identified in humans. Moreover, as eluded to earlier, there is also 17 evidence for co-expression of GLP-1 and GIP in duodenal cells that may also contribute to GLP-1 release. . GLP-1 functions and signals in a similar manner to GIP by activating a seven transmembrane G-coupled GLP-1 receptor (GLP-1 R) and stimulating adenylate cyclase/ P K A pathway, phospholipase C/PKC (Drucker et al, 1987; Thorens et al, 1992; Wheeler et al, 1993) and increasing cytosolic free C a 2 + (Holz et al.,1995; Lu et al, 1993). GLP-1 has also been shown to enhance P-cell proliferation through intracellular pathways such as Akt, and transactivation of the epidermal growth factor (EGF) via src kinase (Brubaker etal, 2004). Since the cloning of the GLP-1 R from a rat pancreatic islet cDNA library (Thorens et al, 1992), the receptor has been found in brain, heart, intestine, hypothalamus, intestine, stomach and within the a, P, and 8-cells of the islets of Langerhans (Wei, et al, 1995; Wheeler et al, 1993; Dunphy et al, 1998; Campos et al, 1994; Bullock et al, 1996). Interestingly, no sequence corresponding to the cloned rat GLP-1 R has been found in the liver or skeletal muscle, despite reports of GLP-1 induced effects in these tissues (Wheeler et al, 1993; Bullock et al, 1996). 1.5.2 Biological Actions of GLP-1 In the P-cell, GLP-1 potentiates insulin secretion, in a glucose-dependent manner, similar to GIP (MacDonald et al, 2002; Light, et al, 2002). Like the GIPR"7" mouse, the GLP-1 R";" mouse exhibits only a modest reduction in incretin effect, likely due to compensatory increases in GIP (Scrocchi et al, 1996). Unlike the GIPR"'" mouse however, the GLP-1 R"7" mouse exhibits abnormal fasting glucose levels suggesting an important role for GLP-1 in maintaining glucose homeostasis in the fasted state (Scrocchi 18 et al, 1996). In other pancreatic endocrine cells, GLP-1 has also been shown to stimulate somatostation secretion from 5-cells but inhibit glucagon secretion from a-cells (Komatsu et al, 1989; D'Alessio et al, 1989). Like GIP, GLP-1 also demonstrates pro-survival characteristics. Continuous GLP-1 treatment of apoptosis-induced INS-1 cells (Buteau et al, 1999) and partial pancreatectomized rat models of type 2 diabetes (Xu et al, 1999) showed marked improvements in P-cell proliferation and neogenesis. In terms of the extra-pancreatic tissues, GLP-1 has a variety of functions including inhibition of gastric emptying and gastrin secretion (Wettergren et al, 1993). In metabolic tissues such as the liver, skeletal muscle and adipose tissue GLP-1 exerts insulinomimetic effects such as glycogenesis and lipogenesis for an overall anabolic effect (Kieffer & Habener, 1999). The mechanism by which GLP-1 elicits these actions is unknown, and will be difficult to elucidate considering no GLP-1 receptor has yet been identified in liver and skeletal muscle. There has been some speculation on the existence of an alternatively spliced receptor or an alternative GLP-1 receptor of a different gene locus (Kieffer & Habener, 1999). It is also possible that GLP-1 may be inducing anabolic actions on these metabolic tissues indirectly through either hormones such as insulin or through metabolic controlling regions of the brain. In the mouse brain, Turton and colleagues (1996) demonstrated that infusion of GLP-1 into the third intracerebral ventricle, acted as an anorexigenic agent by reducing food intake. Similarly systemic administration of GLP-1 in healthy or diabetic rats (Larsen et al, 2001; Rodriquez de Fonseca et al, 2000) as well as in both healthy humans (Gutzwiller et al, 1999) and patients with type 2 diabetes (Gutzwiller et al, 1999) resulted in an increase in satiety and a marked decrease in food intake. The new 19 therapeutic GLP-1 analogue Byetta has also demonstrated similar reductions in food intake in human patients (Combettes et al, 2006). The exact mechanism of GLP-1 action is unclear; however high levels of GLP-1 R have been found in the arcuate and paraventricular nucleus of the hypothalamus (Shughrue et al, 1996). Studies measuring hypothalamic activity using a manganese-enhanced magnetic resonance imager reported a reduction in signaling at the paraventricular nucleus and an increase in the ventromedial hypothalamic nucleus, upon intraperitoneal injection of GLP-1 into C57/BL mice (Chaudhri et al, 2006). Furthermore, ablation of the vagal-brainstem-hypothalamic pathway was reported to diminish GLP-1 's anorexigenic effects (Abbott et al, 2005). Taken together, this would suggest that GLP-1 exerts its satiety effects through a hypothalamic pathway 1.6 M E T A B O L I S M OF THE INCRETINS 1.6.1 Function of Dipeptidylase IV (DPIV) The biological half-life of GIP, measured using radioimmunoassay employing C-terminally directed antibodies, was originally determined to be approximately 20 minutes (Brown et al, 1975; Elahi et al, 1979). It was later discovered that circulating GIP was composed of both a biologically active form, G I P 1 - 4 2 , and a truncated inactive form, GIP3. 42. Taking this into account exogenously administered GIP has been reported to have a half life of ~2 min in rodents (Kieffer et al, 1995), ~7 minutes in normal humans and ~5 minutes in type 2 diabetes patients (Deacon et al, 2000). Similarly infused GLP-1 also has a rapid rate of degradation with a half-life of ~2 minutes in rodents (Kieffer et al, 1995), healthy humans and people with type 2 diabetes (Deacon et al, 1995). This rapid degradation of GIP and GLP-1 is due to a protease, dipeptidyl peptidase IV (DPIV), that 20 is found both in a membrane-bound form as well as circulating in the bloodstream. DPIV has a high specificity for peptides containing an N-terminal proline, alanine or serine residue in the penultimate position (Hinke et al, 2000&; Pospisilik et al, 2001; Yaron & Naider, 1993). DPIV is expressed ubiquitously; however highest concentrations are seen in the brush border areas of the kidney, endothelium, blood, and intestinal epithelia (Yaron & Naider, 1993) locations in which it can readily cleave GIP and GLP-1 and potently diminish the incretin effect (Mentlein et al, 1993; Kieffer et al, 1995; Pauly et al, 1996; Deacon et al, 2000; Hansen et al, 1999; Pederson et al, 1996). 1.6.2. Therapeutic Approaches to Improve GIP Action Studies on DPIV inhibitors, such as P32/98, MK-0431 and vildagliptin have provided evidence and support for their use as a viable treatment option for type 2 diabetes (Pauly et al, 1999; Pederson et al, 1998; Pospisilik et al, 2002; 2001; Ahren et al, 2002; Sudre et al, 2002; Reimer et al, 2002; Mcintosh et al, 2006). Their administration decreases incretin hormone degradation and prolongs their effects on insulin secretion. Indeed, DPIV inhibitor treatment of Vancouver diabetic fatty (VDF) rats have shown overall improvement in glucose tolerance (Pederson et al, 19986) and significant decreases in hyperglycemic episodes (Pospisilik et al, 2002). The DPIV inhibitor Sitagliptin (Januvia) was recently approved by the American food and drug administration for clinical use, after it demonstrated improved overall P-cell function in both fasting and postprandial states (Raz et al, 2006).. Another potential therapeutic approach that would enhance incretin action is to engineer a synthetic DPIV-resistant GIP incretin analogue. D-Ala 2-GIP is one such peptide that was created by performing a single amino acid substitution for L-alanine 21 (Hinke et al, 20006). This small alteration prevents DPIV cleavage, and thus prolongs its half-life in the circulation, while still maintaining native function. Perfusion studies on normal and obese rats with diabetes showed that administration of this compound improved overall glucose tolerance more effectively than GIP itself (Hinke et al, 2002). Numerous other DPIV-resistant GIP analogues have also been developed and tested including N-AcGIP, and related palmitate derivatives, GIP(Lys(37)PAL and N-AcGIP (Lys(37)PAL. Daily injection of these compounds for a 14-day treatment regime, in obese diabetic (ob/ob) mice, demonstrated significantly decreased non-fasting plasma glucose levels, enhanced GSIS, and overall improvement in insulin sensitivity (Irwin et al, 2005). There are also numerous GLP-1 analogues being developed at the present time, including the most well known exendin-4 and synthetic version of it, Byetta (Exenatide). Exendin-4 alone has been shown to significantly reduce streptomycin induced p-cell apoptosis in mice and maintain overall glycemic control in these animals (Li et al, 2003). Byetta a compound presently released on the market has also exhibited efficacy in glycemic control, by augmenting insulin secretion, reducing glucose production, and lowering fasted and postprandial blood glucose (Combettes et al, 2006). 1.7 PATHOPHYSIOLOGY OF GIP FUNCTION 1.7.1 Characterization of GIP Function in Pathological Models The incretin effect in a healthy individual accounts for at least 50% of overall insulin release associated with a meal (Unger & Eisentraut, 1969). In people with type 2 diabetes however, a remarkable reduction in the incretin response is seen (Nauck et al, 22 1986). To elucidiate the basis for this loss of function, plasma GLP-1 and GIP levels were measured after a mixed meal in a healthy individuals, and patients with impaired glucose tolerance, or diabetes. Collectively, compared to the healthy individuals, people with diabetes and to a lesser degree those with impaired glucose tolerance, demonstrated a minimal decrease in GIP secretion while a significant reduction in GLP-1 secretion (Vilsboll et al, 2001; Toft-Nielsen et al, 2001). This impairment in GLP-1 release however only accounts for partial loss of incretin effect seen in the diabetic phenotype, as pancreatic responsiveness to the incretin hormones is also severely reduced (Lynn et al, 2001; Elahi et al, 1994; Krarup et al, 1987; Nauck et al, 1993a). An investigation was performed to compare the insulinotropic actions of exogenously administered GIP and GLP-1 in healthy people and patients with diabetes under hyperglycemic clamp conditions. The study found that individuals with diabetes were significantly less responsive to GIP than controls (Nauck et al, 1993), while GLP-1 function appeared to remain intact in all subjects (Nauck et al., 1993; Elahi et al, 1994; Kieffer & Habener, 1999; Vahl & D'Alessio, 2003; Perry & Greig, 2003; Hoist et al, 2002)). Pancreatic perfusion studies in the Zucker diabetic rat, and its substrain the Vancouver diabetic fatty rat, have shown similar results, with an ablated pancreatic response to exogenous GIP. and a sustained response to GLP-1 (Lynn et al, 2001). Collectively, the attenuated incretin effect exhibited in people with diabetes is attributed to both incretin hormones, with an impairment in GLP-1 secretion and a decline in responsiveness to GIP. To account for the reduction in GIP effect, circulating GIP levels were examined, and found to differ among different studies (Vahl & D'Alessio, 2003; Perry & Greig, 2003; Hoist, 2002; Jones et al, 1989a; 1989b; Fukase et al, 1993; Ahren et al, 1997; Vaag et al, 23 1996). Overall most studies claim that insufficient or excess levels of circulating GIP are not a limiting factor in people with type 2 diabetes. 1.7.2. Possible Explanations for the Reduced Responsiveness to GIP Currently there is no available information on the underlying basis for GIP resistance in human P-cells, and this has driven research to investigate possible causes. One possible explanation is a reduction in hormone-receptor interaction, either caused by a genetic defect, such as a point mutation in the binding site, or a structure-related problem that reduces the binding affinity of the receptor for the ligand. Point mutations in critical regions of the receptor such as the amino-terminus could alter the affinity of the ligand-receptor interaction. Using genomic D N A samples from subjects with or without diabetes two missense mutations were identified, Glyl98Cys in exon 7 and Glu354Gln in exon 12. CHO cells expressing the former mutation demonstrated half the maximal cAMP response after GIP stimulation, while the exon 12 mutation showed no change. Allelic frequency of these two mutations however, did not correlate between control and subjects with diabetes (Kubota et al, 1996). Similarly another group by mutational analysis compared the allelic frequency of the two amino acid substitutions A207 V and E354Q found in control subjects and patients with type 2 diabetes; however no significant correlation was found. Furthermore CHO. cells expressing these mutations showed no difference in GIP-induced cAMP production (Almind et al, 1998). Overall, no studies to date have been able to conclusively link type 2 diabetes with any GIPR mutations. The reduction. in GIP responsiveness therefore could also be explained by defective signal transduction. One study sought to examine the integrity of GIP's major 24 pathway, c A M P / P K A in lean (Fa/?) versus fatty (fa/fa) V D F rat islets. Treatment of islets with forskolin, a potent cAMP activator showed no difference in cAMP responses between lean and fatty islets, while GIP. treatment revealed a significantly attenuated cAMP response in obese rat islets. This would suggest that the defect in GIP signaling cascade is likely upstream of cAMP activation (Lynn et al, 2001). It is noteworthy to mention that impaired GIP actions may not be exclusively associated with the P K A / c A M P pathway as there is also evidence for GIP signaling through the GPy-PLA2 pathway (Ehses et al, 2001). Lynn and colleagues sought to investigate whether the decline in GIPR function of VDF and ZDF rodents was attributed to a downregulation in GIPR expression. Real-time quantitative Polymerase Chain Reaction (RT-PCR) analysis confirmed these suspicions by demonstrating a significant downregulation in GIPR mRNA levels in ZDF rat islets (Piteau, 2004) and GIPR mRNA and protein levels in obese V D F rat islets compared to lean controls (Lynn et al, 2001). To this point however the mechanism of attenuation is unclear. Elevated levels of circulating GIP may be one possible explanation for GIPR attenuation. However, elevated circulating levels of GIP have not been consistently shown in V D F rats or people with type 2 diabetes (Creutzfeldt et al, 1979; 1985; 1992; Pederson et al, 1994), and thus hyperGIPemia is unlikely to be the primary cause for GIPR downregulation. 1.7.3. Glucose-dependent Downregulation of GIPR Expression It had been previously reported that several G-protein coupled receptors such as the GLP-1 receptor need appropriate glycosylation to be efficiently expressed at the cell surface (Couvineau et al, 1996). Lynn and colleagues therefore examined the expression 25 of several forms of GIPR with mutated glycosylation sites in transfected H E K cells. Competitive GIP binding studies demonstrated a diminished cell surface expression of the GIPR in the mutants compared to control; however receptor affinity did not greatly differ. Measurements of GIP-stimulated cAMP production demonstrated significantly greater responses in cells transfected with the wildtype receptor than the mutant forms. It was concluded that abnormal glycosylation of the receptor (a possible product of hyperglycemia) could lead to diminished cell surface expression (Lynn, 2003) Hyperglycemia is a prominent characteristic of the diabetic phenotype, as such, there is mounting evidence to suggest that chronically elevated levels of glucose are toxic to the P-cell. Lynn et al. (2003) therefore, sought to examine whether glucose influenced the regulation of GIPR mRNA expression. In the INS-1(832/13) (3-cell line, glucose was found to significantly downregulate GIPR mRNA expression in a time and dose-dependent manner (Lynn et al, 2003). This dose-dependent correlation was also demonstrated in a hyperglycemic clamp study of Wistar lean rats that showed a similar glucose-induced suppression of GIPR expression in the islet (Lynn et al, 2003). Collectively these studies suggest that glucose plays a significant role in the regulation of GIPR expression and warrants further investigation. 1.8 THE R E G U L A T O R Y ROLE OF NUTRIENTS ON GENE EXPRESSION 1.8.1 Glucose Nutrients, including glucose, have been demonstrated to play important roles in the regulation of expression of specific genes. Elevated glucose stimulates the gene transcription of a number of enzymes involved in controlling and storing an. abundance of nutrients, while lower concentrations of glucose appear to stimulate the transcription of 26 genes involved in protection and adaptation against starvation and diminished energy stores (Foufelle et al., 1998). Examples of glucose-induced gene expression of metabolic enzymes include fatty acid synthase (FAS) in adipose tissue and pyruvate kinase (PK) in the liver and P-cell (Towle et al., 1995; Vaulont & Kahn, 1994). The exact mechanism by which glucose regulates gene transcription is not well understood. It is known, however, that phosphorylation of glucose is required for its regulatory actions, whether it be to glucose-6-phosphate (Foufelle et al., 1998) or xylulose-5-phosphate, an intermediate in non-oxidative glucose pathways (Doiron et al., 1996). Two glucose response elements (GRE) have been identified on the PK (Thompson & Towle, 1991) and SI4 (Shih & Towle, 1992) gene promoters both of which have a canonical sequence that is two sequences of C A N N T G separated by 5 nucleotides. The exact mechanism by which phosphorylated glucose metabolites up-regulate gene transcription however is still unclear. In a limited number of cases, exposure to high glucose conditions or glucose metabolites has . been shown to suppress the transcription of genes such as phosphoenolpyruvate carboxykinase (PEPCK) in hepatocytes (Cournarie et al., 1999) and the transcription factor peroxisome proliferator activated receptor a (PPARa) in INS-1(832/13) P-cells (Roduit et al., 2000). The mechanisms involved are again unclear. 1.8.2. Lipids Similar to the effects of glucose, elevated free fatty acids also tend to decrease gene transcription. Specifically, polyunsaturated fatty acids (PUFAs) have been reported to decrease FAS, acetyl CoA carboxylase ( A C Q , liver PK and apolipoprotein A (apo-A) in hepatocytes (Duplus et al., 2000). Free fatty acids also upregulate important metabolic 27 factors including adipocyte lipid binding protein (aP), PEPCK (Antras-Ferry et al, 1994; 1995; Amri et al, 1991) and PPARa mRNA expression in rat islets (Zhou et al, 1998). 1.9 PEROXISOME PROLIFERATOR A C T I V A T E D RECEPTORS (PPARs) 1.9.1. PPAR Family Structure and General Function Peroxisome proliferator activated receptors (PPARs) were first identified in hepatocytes in 1990 as a result of studies on carcinogenic and hypolipidaemic peroxisome proliferators (Issemann & Green, 1990). PPARs were later cloned from hamster (Aperlo et al, 1995), human (Sher et al, 1993) and xenopus (Dreyer et al, 1992) tissues. In the latter, three isoforms of PPAR were identified: a, y, and p75 (Dreyer et al, 1992). These isoforms were identified as members of the large nuclear receptor family and they have been characterized as ligand-activated transcription factors that act as fatty acid sensors to convert nutrient signals to changes in gene expression. PPARs consist of multiple domains: the A /B domain, that is responsible for ligand-independent activation, a two finger D N A binding region, a D domain that acts as a protein hinge and E and F domains that are responsible for ligand binding (Beamer et al, 1997; Gearing et al, 1994; Krey et al, 1993; Zhu et al, 1995). The ligand-independent A /B domain contains a transcriptional activating function (AF-1) within, which are phosphorylation sites for signaling kinases such as insulin activated ERK-M A P K (Diradourian et al, 2005). The natural ligands for the ligand-dependent E/F domains of PPARs have not been identified with certainty. However, PUFAs and insulin sensitizing thiazolidinediones, have a higher affinity for PPARy and (3/8 while ligands for PPARa include saturated fatty acids, lipid lowering drug fibrates (Desvergne & Wahli, 1999), leukotriene B4 (Devchand et al, 1996) and oleoylethanolamide (Fu et al, 2003). 28 Once activated by a ligand, PPARs heterodimerize with a retinoid X receptor (RXR) at the ligand binding domain of the E/F domain before binding to a peroxisome proliferator response element (PPRE) on a gene promoter (Desvergene et al, 1999). The PPRE consists of a canonical repeat sequence of A G G T C A separated by one nucleotide (Tugwood et al, 1992). Different PPAR isoforms are believed to gain their specificity by acting on slightly different areas within the PPRE sequence (Bishop-Bailey et al, 2000). In vitro binding studies have also shown that PPAR -a, ~p/5 and -y/RXR heterodimers have either equal affinity for PPREs on genes such as acyl CoA oxidase (AOX) or preferential affinity for certain genes such as PPARy/RXR and the adipogenic aP2 promoter (Brun et al, 1996). How the different PPAR isoforms achieve preferential affinity for a promoter is not well understood; however overexpression of PPARS has been shown to compete with PPARa and the thyroid hormone receptor for R X R binding (Jow & Mukherjee, 1995). Thus cell type and local cellular environment may also play an important role in determining which specific PPAR pathways are initiated. 1.9.2. PPAR Tissue Distribution A l l three PPAR isoforms have distinct expression patterns. PPARp/5 is ubiquitously expressed in most tissues, while PPARy is found mostly in adipose tissue and parts of the large intestine. PPARa on the other hand is highly expressed in highly catabolic tissues such as liver, heart, kidney, muscle, and brown adipose tissue and is also the most predominant PPAR isoform in the p-cell, although PPARy is also present in lower amounts (Braissant et al, 1996; Ouali et al, 1998; Zhou et al, 1998). 29 1.9.3. Regulation of PPARa PPARa in particular is of interest because it is an important transcription factor responsible for the upregulation of enzymes involved in lipid oxidation and maintenance of lipid homeostasis in the (3-cell (Desvergne & Wahli, 1999). The regulation of PPARa gene expression, although not well understood, is increased by STAT 3, a downstream target of the leptin JAK/STAT pathway (Zhou et al, 1998) and, as mentioned earlier, the ability of glucose to attenuate PPARa expression suggests the presence of a glucose response element (Roduit et al, 2000). In addition to ligand activation by fatty acids, the regulation of PPARa function has also been shown to involve phosphorylation events (Shalev et al, 1996). Phosphorylation of PPARa by P K A at the D N A binding domains enhances its activity (Lazennec et al., 2000), while phosphorylation by E R K - M A P K or J N K - M A P K at the ligand independent transcription binding site reduces PPARa activity (Barger et al, 2000) and can also act as a signal for ubiquitin mediated degradation (Diradourian et al, 2005). Although phosphorylation is associated with enhancing stabilization and transcription, it is not known whether it also exerts any control on nuclear localization or trafficking signals. 1.9.4 PPARa Function and Pathophysiology PPARa has three major biological roles. The first is to improve fatty acid incorporation into the cell by increasing gene expression of proteins such as fatty acid translocase (FAT/CD36), fatty acid transport protein (FATP) and lipoprotein lipase (LPL) (Schoonjans et al, 1996; Motojima et al, 1998). Secondly, PPARa is responsible for initiating the peroxisomal P-oxidation pathway, via activation of A O X (Tugwood et al, 1992). Lastly, PPARa is also responsible for increasing mitochondrial P-oxidation 30 through the upregulation of catabolic mitochondrial proteins, carnitine palmitoyl transferase -1 (CPT-1) (Brandt et al, 1998) and uncoupling protein 2 (UCP-2) (Roduit et al, 2000). The evidence supports a previous theory of Prentki and colleagues whereby insufficient insulin secretion from the P-cell in type 2 diabetes is due to a condition of glucolipotoxicity involving glucose-induced abnormal lipid partitioning. In his studies, chronically elevated glucose was found to lead to an increase in malonyl CoA, an important molecule that regulates the flux of free fatty acid oxidation and esterification. Upregulation of malonyl Co A in turn, inhibits a limiting factor for P-oxidation in the P-cell, CPT-1 (McGarry et al, 1992; Prentki et al, 2002). In parallel, elevated glucose, as mentioned earlier, will also decrease PPARa levels causing a downregulation of additional factors necessary for P-oxidation. As a result, this imbalance of lipid metabolism will cause free fatty acids to undergo esterification and accumulate within the P-cell leading to apoptotic events, activation of reactive oxidative species (ROS) pathways, and further impair insulin secretion (Prentki et al, 2002). There is however an additional, or perhaps alternative role, for PPARa in the regulation of insulin secretion that is related to GIPR expression. In INS-1(832/13) cells, palmitate, a potent PPARa activator, and WY14643, a specific PPARa agonist have been shown to increase expression of both PPARa and GIPR mRNA under low (5.5mM) glucose conditions, with decreased expression of both under high (20mM) glucose conditions (Lynn et al, 2001; 2003). Similarly, INS-1(832/13) cells transfected with the ~2kb 5' rat GIPR promoter poly-linked to the luciferase vector showed a significant increase in GIPR promoter activity upon PPARa agonist activation (Lynn et al, 2003). Under low glucose 31 conditions, expression of a dominant negative form of PPARa, or administration of the PPARa antagonist MK-886, to INS-1(832/13) cells produced decreases in GIPR expression (Lynn et al., 2003). Taken together, these studies support the involvement of PPARa in the stimulation of GIPR expression and a novel pathway by which glucose indirectly reduces its effect (Lynn et al., 2003), thus contributing to the overall reduction in insulin release. This of course could be acting in parallel with the adverse effects of lipid that together play a role in the etiology of this multi-factorial disease. 1.10 THESIS INVESTIGATION GIP's primary role is potentiate the secretion of insulin in a glucose-dependent manner. In humans with type 2 diabetes however, there is partial or complete loss of GIP-stimulated insulin secretion. This (3-cell resistance to GIP is also seen in type 2 diabetic rat models and believed to be due to a decline in GIPR expression. While the mechanisms of downregulation are unclear, in vitro and in vivo studies have demonstrated that chronically elevated glucose levels results leads to a concentration dependent attenuation of GIPR expression. Therefore it is hypothesized that chronic hyperglycemia is the primary cause of GIPR down regulation in type 2 diabetes. To examine this, the following aims will be addressed: Aim 1: To investigate whether improved glucose tolerance is associated with an increased islet GIPR expression in vivo. Aim 2: To determine whether GIPR downregulation results from, an indirect glucose-induced reduction in PPARa expression. 32 Aim 3: To examine whether GIP resistance is also a characteristic of adipose tissue and compare the regulation of GIPR expression in adipose tissue and 3T3-L1 adipocytes with that of islets and INS-1(832/13) cells during elevated glucose conditions. 33 CHAPTER 2.0: METHODOLOGY 2.1 C H E M I C A L S Chemical reagents were obtained from Amersham Pharmacia Biotech (Mississauga, ON), B D H Inc. (Toronto, ON), Fisher Scientific International (Pittsburgh, PA, USA), Gibco Life Technologies Inc. (now Invitrogen Canada, Burlington, ON), Merck (Darmstadt, Germany), Perkin-Elmer/Mandel Scientific/NEN Life Scientific Co. (Guelph, ON), Sigma (Oakville, ON) or V W R Canlab (Mississauga, ON) and tissue culture supplies from BD Falcon (San Jose, CA, USA). The DPIV inhibitor, P32/98 (di-[2S,3S]-2-amino-3-methyl-pentanoic-l,3-thiazolidine fumarate), was provided by Probiodrug (Halle, Germany) and the GIP analogue, D-Ala 2 -GIPl-42, was synthesized as previously described (Demuth, et al, 1990). Other specific sources of reagents are indicated throughout the sections describing experimental methodology. 2.2 C E L L . C U L T U R E Two types of cell lines were used for experimentation. INS-1(832/13) cells, originated from rat P-cell insulinoma cells transfected with the human insulin gene (Asfari et al, 1992; Hohmeier et al, 2000) and were provided by Dr. C. B. Newgard (Duke University, USA). Mouse 3T3-L1 preadipocytes were from they American Type Culture Collection; (ATCC). INS-1(832/13) cells were cultured in 1 ImM glucose RPMI 1640 (Sigma) medium supplemented with 24mM sodium bicarbonate, 2mM glutamine, 0.050mM P-mercaptoethanol,T0mM HEPES, ImM sodium pyruvate, and 10% fetal bovine serum (FBS) (Sigma). Twenty-four hours prior to experimentation, cells were cultured (2xl0 6 34 cells/well) in 6-well plates in the complete RPMI supplemented medium described above. Passages 46-60 were used for experiments. 3T3-L1 cells were cultured in DMEM-high glucose (Gibco) and supplemented with 44mM sodium bicarbonate, and 10% FBS (Sigma). Cells were cultured in 6-well plates and allowed to expand to 100% confluence. Cells were then induced to differentiate into adipocytes by supplementing the medium with 6.0x1 O^mM dexamethasone, O.lmM 3-isobutyl-l-methylxanthine (IBMX) and 0.016mM insulin for 72 hours. Medium was then changed back to regular 3T3-L1 medium plus 10% FBS for seven days. Observation of a large lipid droplet phenotype was considered successful differentiation. Once INS-1 (832/13) or 3T3-L1 cells were ready for experimentation, fresh medium was added as above, but in the absence of serum, and supplemented with 0.1 % bovine serum albumin (BSA; Sigma). Experimental agents were applied as described in the Results section. 2.3 C E L L T R E A T M E N T The compound W Y 14643 (Calbiochem, La Jolla, CA) was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 200mM and added directly to the experimental media to a final concentration of O.lmM. Clofibrate (Sigma; liquid form) was added directly to the media at a final concentration of ImM. For the light-sensitive compound 9-cis-retinoic acid (Sigma), all preparations were performed under limited light conditions. Since this compound is not water soluble and precipitates in aqueous solution, 0.002g of 9-cis-retinoic acid was completely dissolved in DMSO to a concentration of 50mM and then dispensed into 0.03OmL aliquots. Using an Eppendorf 35 Vacufuge™ speed vacuum, DMSO was fully evaporated for 5 hours at 30°C resulting in a bright yellow pellet. A 0.3g/mL P-methyl-cyclodextrin (Sigma) water solution was then added to the pellets at a ratio of 0.001 g 9-cis-retinoic acid: 0.2g P-methyl-cyclodextrin (as recommended by manufacturer) and incubated for 2 hours in a 37°C shaking water bath. This step is required to introduce a vehicle for transfer of 9-cis-retinoic acid into aqueous solution. Although after two hours a large portion of the pellets still remained, the supernatant had changed from colourless to a transparent yellowish colour suggesting some dissolution had occurred. The P-methyl-cyclodextran step was not continued for longer because it was already 1.5 hours longer than recommended and therefore it was not certain whether increasing duration would improve the solubility further. Since the pellet was not completely dissolved, the final concentration could only be estimated. Therefore the concentrations quoted in the Results section are estimates based on the volume added to the RPMI 1640 serum-free 0.1 % BSA medium. No precipitation was seen upon adding supernatant to the media, indicating successful transfer of 9-cis-retinoic acid into media. The same concentration of p-methyl cyclodextrin was also added to cells not receiving 9-cis-retinoic acid to serve as controls. Unfortunately, after 24 hours, cells receiving retinoic acid had all died, while the control cells remained viable. This response was unexpected since a much lower concentration of 9-cis-retinoic acid than the ImM used in experiments in the literature. The reason for this is unclear, but for future experiments requiring activation of PPARa's co-activator retinoid X receptor (RXR), a more stable and soluble compound is needed. Unfortunately at this time such a compound is not commercially available. 36 2.4 TRANSFORMATION A N D TRANSFECTION Plasmid transformation of bacteria was performed by growing 25-1 OOng of human C M V - P P A R a (GenBank: NM_005036) TransExpression vector (Panomics, Fremont, CA, USA) into 0.050mL of thawed DH-5a competent bacterial cells (Invitrogen) and gently mixed. Cells were then incubated on ice for 20 minutes before being heat-shocked for 1 min at 37°C followed by another freeze period of 2 minutes. A l l cells were then cultured onto an L B plate containing 50ug/mL kanamycin and allowed to grow for 24 hours at 37°C. One colony was selected and allowed to grow at 37 °C in kanamycin (50ug/mL)-treated 3mL L B broth for 8 hours before further diluting it 500 times and growing overnight at 37°C. Plasmid D N A was then isolated using the standard Qiafilter™ plasmid midi prep kit and protocol (Qiagen). Transient transfection studies were performed on INS-1(832/13) cells. Cells were plated 24 hours prior to transfection on a 6-well plate, at a concentration of 1.8xl06 cells/well. Following the standard lipofect 2000™ (Invitrogen) protocol, 2.5ug/well of human PPARa plasmid were incubated in antibiotic free Opti-Mem (Invitrogen) medium at a ratio of 2.5ug: O.lmL for 15 minutes at room temperature. Lipofectamine at 0.004mL/ug of DNA was then combined with the same volume of Opti-MEM at room temperature first, before being combined with the DNA- medium mix for an additional 15 minutes at room temperature. Afterwards, RPMI complete medium was replaced with 0.8mL/well of Opti-MEM and 0.2mL of the plasmid/ lipofectamine/ medium mix were added to each well for 5 hours. Medium was then replaced by INS-1(832/13) complete medium for the next 48-60 hours. 37 2.5 A N I M A L S A l l aspects of the animal studies including certification of the experimenter were in compliance with the guidelines of the Canadian Council on Animal Care, and approved by the University of British Columbia Council on Animal Care. The two animal models used for experimentation, were the Zucker Diabetic Fatty (ZDF) Rat (Charles River) and a substrain, the Vancouver diabetic fatty (VDF) rat maintained at the University of British Columbia Physiology Division. These animals were established from a colony that spontaneously developed a mutation resulting in a Gln269Pro substitution in the extracellular domain of the leptin receptor. Animals homozygous for this recessive missense mutation (fa/fa) demonstrate a global non-functional leptin receptor in both signaling and binding (Yamashita et al., 1997). This reduction in signaling capacity results in a hyperphagic severe obese phenotype. These rat models also display evidence of hyperinsulinemia, hyperlipidemia, insulin resistance and P-cell hyperplasia. The pathology of the V D F rats are milder than the ZDF rats, demonstrating modestly higher levels of fasting glucose compared to their lean littermate controls, as well as impaired glucose tolerance (review Mcintosh and Pederson, 1999). Interestingly, in both the ZDF, and VDF rat models as well human patients the postprandial incretin effect is attenuated, at least partly due to reduced GIP responsiveness (Perley & Kipnis, 1967; Nauck et al, 1993; Elahi et al. 1994; Lynn et al. 2001). 38 2.6 PROTOCOL FOR D A I L Y MONITORING A N D D R U G ADMINISTRATION TO THE V A N C O U V E R DIABETIC F A T T Y (VDF) RAT: A STUDY WITH THE DIPEPTIDYL PEPTIDASE INHIBITOR P32/98 At 12-14 weeks of age ~ 600g homozygous recessive (fa/fa) V D F rats were randomly allocated to either control (n=4 female, n=2 male) or treatment (n=4 female, n=2 male) groups. The control group received 1% water/cellulose solution twice daily by way of oral gavage for 5 days per week over a twelve week period, while the treatment group received O.Olg/kg of the DPIV inhibitor P32/98 with an identical protocol. In addition, each weekday morning, food consumption and water intake were also recorded. Three times a week, body weight was recorded and morning blood glucose from tail clips was measured using a glucose SureStep analyzer (Lifescan Canada, Burnaby, BC). Animals were housed on a 12 hour light/dark cycle, and water and regular chow were available ad libitum. 2.7 PROTOCOL FOR ADMINISTRATION OF GIP, D-ALA 2 -GIP A N A L O G U E OR V E H I C L E TO Z U C K E R DIABETIC F A T T Y (ZDF) RATS To take advantage of an ongoing drug treatment study, epididymal fat pads from these animals were collected as a preliminary screen to see whether it is possible to detect GIPR mRNA in rat adipose tissue. Treatments for this experimental protocol therefore were performed by Violet Yuen and Su-Jin Kim. Saline, GIP or analogue was administered for 4 weeks at an infusion rate of (lOpmol/kg/min) to 5 week-old ZDF rats (n=6/group) via intraperitoneal Alzet osmotic mini-pumps (Durect Corporation, Cupertino, CA). Morning blood glucose was measured in blood from tail clips using a glucose SureStep analyzer (Lifescan Canada, Burnaby, BC) and body weight was monitored throughout. At the end of the infusion period an insulin tolerance test was 39 performed by intraperitoneal injection of 0.5U/g of Humulin R (lOOU/mL) to overnight fasted rats. Post-injection, blood was collected at 0, 5, 10, 15, 20 and 30 minutes and blood glucose and insulin were measured from these samples as described above. One day later, the animals were sacrificed, and epididymal fat pad tissue removed. 2.8 O R A L GLUCOSE T O L E R A N C E TESTS (OGTT) A N D R A D I O I M M U N O A S S A Y (RIA) One to three days prior to drug or peptide administration to the VDF and ZDF rats, as well as on weeks 6 and 12 of the P32/98 study and week 4 of ZDF treatment with D-Ala -GIP analogue, animals were subjected to an oral glucose tolerance test (OGTT). This test involved an oral gavage administration of lg/kg of 40% dextrose solution after 16 hours of fasting and blood glucose measurements made from the tail veins using a SureStep analyzer at 0, 15, 30, 60, 90, and 120 minutes after bolus receipt. Blood samples (~0.3mL) were also collected at similar time points from the tail vein using heparinzed Caraway/ Natelson collecting tubes (Fisher Scientific, Pittsburgh, PA, USA). Plasma was then separated from red blood cells by centrifugation at 10,000 x g for 20 minutes at 4"C, and plasma (supernatant) was removed and frozen immediately at -20°C. Plasma insulin levels were measured by radioimmunoassay (RIA) with guinea pig anti-rat insulin serum and precipitating goat anti-guinea pig IgG serum reagent (Linco Research, St.Charles, Missouri, USA). 2.9 QUANTIFICATION OF PPARa A N D GIPR PROTEIN EXPRESSION B Y WESTERN BLOT A N A L Y S I S : For quantification of PPARa protein, INS-1 (832/13) cells plated into 6-well plates were first washed with ice cold lx PBS buffer and replaced with ~0.060mL/well 40 lysis buffer containing 0.5% Triton x-100 (BDH), 60mM B-glycerophosphate, 20mM MOPS, ph 7.2, 5mM EDTA, 5mM EGTA, ImM N a 3 V 0 4 , 20mM NaF. Protease inhibitors 1% Trasylol® (Bayer Pharmaceuticals, Etobicoke, ON, Canada) and ImM phenyl methyl sulfonyl fluoride (PMSF) added to the buffer on the day of the experiment. Cells lysates were then subjected to two 20 second sonication intervals at 4°C and centrifuged for 20 minutes at 12,000 x g for in 4°C. The supernatant containing the protein was then removed and stored at -20°C. Protein quantification was done using a Bicinchoninic Acid (BCA) protein assay (Pierce, Rockford, IL, USA) at about 5-10x dilution. A 4x loading buffer containing 5% P-mercaptoethanol was then added to each the protein sample for a final lx concentration. Samples were then denatured at 95°C for 5-10 minutes and 20-30 ug of protein/well were separated on a 15% SDS/polyacrylamide gel. After electrophoresis, protein was transferred onto nitrocellulose membranes (BioRad, Mississauga, ON, Can.) followed by a one hour shaker incubation period in blocking buffer ( lxTBS and 5% non-fat milk) at room temperature. Membranes were then washed three times at 10 minute intervals using TBST ( lx TBS with 0.5%> Tween 20 (Sigma)) followed by incubation in a 50mL Falcon tube with the primary antibody at 1/1000 PPARa (h-98; Santa Cruz Biotechnology Inc., CA, USA) dilution in 7mL of blocking buffer. Membranes incubations were then fastened to a mixer model 346 (Fisher) for 2 hours at room temperature, after which membranes were washed 3 times at 10 minute intervals. Secondary antibody anti-rabbit (Cedarlane Laboratories Inc.) was then added to lOmL of blocking buffer to make a final concentration of 1/2000, which was again shaken for 1 hour at room temperature. After secondary incubation, 41 membranes then underwent 3 x 1 0 minute washes, and protein was' visualized using chemi-luminescence (ECL) (Amersham-Pharmacia). To verify equal loading, P-actin levels were examined by incubating the membrane for one hour at 1/2000 antibody dilution (Santa Cruz biotechnology Inc., CA) in lx TBST on a shaker at room temperature. Protein was visualized as before. To estimate molecular weight, a pre-stained protein molecular weight marker (Fermentas Canada Inc., Burlington , ON) was used. Numerous attempts were made to show reproducible GIPR protein blots; including the use of a more potent detergent containing RIPA buffer, stringent washing methods and various primary antibodies. The primary antibody that appeared to be most effective and specific was an N-terminus targeted GIPR (courtesy of Dr. T.J. Kieffer). Unfortunately, the results from these studies depicted several inconsistent non-specific bands that could not be explained. Further western blot studies on GIPR therefore are warranted, to optimize GIPR protein visualization and antibody specificity. 2.10 TISSUE EXTRACTION i) Islet Isolation Rat islets in the study described in section 4.2.1 (DPIV inhibitor study) were isolated as described by Van Der Vliet et al, 1988, whereas for the study described in section 6.2.1. (Fat versus Lean study) islets were isolated using a modified mouse isolation procedure (Lacy et al, 1967; Salvalaggio et al, 2002) in an attempt to develop a more efficient and less stressful technique for the islets. In both experiments animals were anaesthetized with an intraperitoneal injection of 0.065g/kg Somnitol® (MTC Pharmaceuticals, Cambridge, ON, Canada) before a midline incision was made in the 42 abdominal area. In the DPIV inhibitor study, the common bile duct was cannulated with heparinized polyethylene tubing (PE50, Becton-Dickinson Co, Sparks MD,USA) and the pancreas inflated by injecting lOmL of a collagenase (0.00032g/mL,Type XI; Sigma) in Hank's balanced salt solution supplemented with lOmM HEPES, 2mM glutamine, and 0.2% BSA (Life Technologies, Burlington, ON, Canada). The pancreas was then removed and diced with scissors before addition to a further collagenase solution and subjected to 20 minute (for lean) or 10 minutes (for fatty) digestion by vigorously shaking in a 37°C water bath. Islet samples were then centrifuged, and the supernatant removed. A second digestion was then performed in a similar fashion for another 7 minutes by adding additional 25mL of collagenase solution. Islets were then filtered through a 1 mm nylon screen and separated from exocrine tissue by centrifuging at 1,200 rpm, for 4°C through a dextran gradient. In the experiments in section 6.2.1 however, the pancreas was inflated by directly injecting lOOOU/mL collagenase solution into the bile duct via a 21 gauge needle. As before the collagenase was dissolved in Hank's balanced salt solution and supplemented as described previously. The pancreas was then removed and shaken in an additional 25mL of collagenase solution for 13 minutes (Fat and lean) at 37°C, followed by a second digestion in an additional 25mL collagenase for a further 10 minutes at 37°C. Islets then underwent three washes, each time being centrifuged at 1200 rpm for 5 minutes and resuspended in Hank's solution. After the final centrifugation, the islets were resuspended in 5mM glucose, 1-0% FBS, RPMI 1640 medium (Gibco) and separated by a 70 micron filter (BD Falcon). Finally, in both experiments islets were hand picked under a compound microscope (Olympus America Inc., Center Valley, PA USA). 43 ii) A d i p o s e T i s s u e I so la t i on To extract adipose tissue, animals were anaesthetized as described earlier, and the tips of their epididymal fat pad and peri-renal fat pads were removed and immediately flash frozen in liquid nitrogen and stored at -70°C. 2.11 RNA ISOLATION R N A isolation from islets, INS-1 (832/13) cells, and 3T3-L1 adipocytes was performed using the standard Trizol® (Invitrogen) protocol. One mL of Trizol®/ ~2xl0 6 cells or 200 islets was used, and R N A was resuspended in 0.035mL lxTE buffer (Molecular probes, Eugene, OR, USA). RNA.from adipose tissue was first cut into 4mm cubes approximately 0.07g in weight and placed, while still frozen, in a 5 OmL Falcon tube on ice containing twice the amount of lysis buffer (RNeasy Lipid Tissue Mini Kit (Qiagen)) suggested in the protocol provided. The sample was then homogenized on ice by performing 5 second pulses for a total of 40 seconds on ice using the Kinematica homogenizer. The remaining isolation steps were then completed as instructed from the RNeasy Lipid Tissue Mini kit protocol, adjusting the volumes according to the increase in lysis buffer. Quantification of R N A was performed using the fluorescent Ribogreen reagent kit (Molecular Probes,) and FL600 microplate fluorescence reader (Bio-Tek) at a sensitivity of 75. R N A Quality control was checked by measuring the A260/280 ratio. DNase I treatment of all islet and cell samples was not performed, due to earlier problems with low RNA yields. To compensate for D N A contamination and carryover, Uracil N-glycosylase (Invitrogen) was added to the final PCR mixture to degrade any uracil containing nucleic acids. Furthermore due to the specificity of the TaqMan® probe 44 it is less likely that inappropriate primer-dimer binding occurred. This is also supported by the consistency of the data across the current studies and the correlation of results from repeated experiments with earlier studies. Moreover, values for the housekeeping gene P-actin were stable across treatments (quantification standard error ~ < 0.01) and would therefore correct for any uneven starting templates. 2.12 REVERSE TRANSCRIPTION A N D R E A L - T I M E P O L Y M E R A S E CHAIN REACTION Following R N A isolation and quantification 0.5ug-2pg of R N A (depending on the initial concentration) were subjected to reverse transcription. Total R N A was transcribed in a total volume of 0.020mL comprised of 0.5mM deoxynucleotide triphosphates (dNTPs)(Invitrogen), O.OOlmL random primer (Invitrogen), 200U Superscript II RNAse H - Reverse Transcriptase (Invitrogen), 40U RNAse inhibitor (Invitrogen), ImM dithiothreitol, 50mM Tris-HCl, 75mM KC1, and 3mM MgCl2. The standard reverse transcription programming cycles were used as indicated in the Superscript II® protocol. One tenth of the resulting cDNA was then used in real-time PCR reactions to measure levels of the housekeeping gene P-actin and either GIPR, PPARa, or UCP-2. The total 0.020mL PCR reaction mix contained lx buffer (Invitrogen), lOmM MgCb, 0.2mM dNTP mix (Invitrogen), 0.25U Uracil N-glycosylase. (Invitrogen), and 2.5U Taq Polymerase (Invitrogen). Although recommended, the more costly hot start Taq Polymerase was not used for experiments because no notable difference was seen when comparing the two enzymes. This is partly because all PCR reaction mixtures were prepared on ice, with regular Taq polymerase as the final ingredient, and secondly, the TaqMan® system allows for high specificity. The primers were used at a final 45 concentration of 500nM and the probes co-labeled with the reporter/quencher fluorescent dyes F A M and T A M R A respectively were used at a final concentration of 200nM. A l l primers and probes were synthesized by Sigma Genosys, (Oakville, ON, Canada) and design sequences were extracted from previously published work shown below. Table 1: Real-time PCR primer and probe sequences. GIPR Forward primer 5' -CCG CGC TTT TCG TCA TCC-3 ' Reverse primer 5' - C C A C C A A A T GGC TTT G A C TT-3' Probe 5'- CCC A G C A C T G C G TGT TCT CGT A C A GG-3' PPARa Forward primer 5' -AGT TTT TGC G G A C T A C C A GTA CTT A G G - 3 ' Reverse primer 5' -GAC T G A G G A G G G GCT GGA A-3 ' Probe 5'- CTC TGT C A T C A C A G A C A C CCT CTC TCC AGC-3 ' UCP-2 Forward primer 5' -CCT G A A A G C C A A CCT C A T GAC-3 ' Reverse primer 5' - C A A T G A C G G T G G TGC A G A AG-3 ' Probe 5'-A C G A C C TCC CTT GCC A C T T C A A C T TCT G-3' PCR reactions were carried out in triplicate in the PE Applied Biosystems 7700 sequence detection system using ABI prism 96-well optical reaction plates. The reaction cycle program included 10 minute preincubation at 37"'C to allow the U N G to degrade any uracil containing nucleic acids followed by a 10 minute at 95°C to deactivate U N G . The remainder of the reaction was a 40 cycle repetition involving a denaturing step of 94°C for 15 seconds and a 1 minute annealing/extension step at 60°C. Fluorescence was measured during the latter stage and the resultant threshold value (Ct) at which point the reaction is in exponential phase was used to calculate relative changes in expression compared to control. A l l reactions followed a typical sigmoidal reaction profile. Specificity of PCR products were verified by separating on a 0.8% agarose gel, and visualized using Eagle Eye software (Stratagene, La Jolla, CA) and a gel documentation system (Bio-Rad Laboratories, Inc, Hercules, CA). 46 Results for all samples were normalized to the housekeeping gene P-actin which was purchased as a pre-developed TaqMan® Assay reagent (20 x Rat ACTB) from ABI (Foster City, CA, USA). Samples quantified for P-actin were first diluted 50x in water before addition to the PCR mixture to ensure accurate quantification range. For each experiment and gene, including P-actin a standard curve was generated, involving 1 Ox serial dilutions of the respective gene cDNAs, to calculate relative expression and normalize for variations in amplification efficiency. The cDNAs used for standard curves were previously amplified using primers described above in regular reverse transcription PCR and identical volumes. General PCR steps were followed using the standard TaqMan® (Invitrogen) protocol. 2.13 D A T A A N A L Y S I S Data are expressed as means ± S.E.M with the number of individual experiments or animals presented in the figure legend. A l l data were analyzed with the Graphpad PRISM 4 programme (Graphpad, San Diego, CA, USA) using the standard non-parametric t-test and one-way A N O V A Newman-Keuls Multiple Comparisons test. Statistical significance was set at 5%. 47 CHAPTER 3.0 THE EFFECT ON GIPR EXPRESSION OF LOWERING BLOOD GLUCOSE IN HYPERGLYCEMIC RATS 3.1 PROJECT RATIONALE It has been previously shown that V D F and ZDF rats, rodent models of type 2 diabetes, exhibited diminished GIPR expression compared to their lean littermates (Lynn et al, 2001). This decline in GIPR expression can be attributed, at least in part, to the chronic hyperglycemia. This conclusion was supported by studies showing that INS-1(832/13) cells or rat islets incubated in high glucose also exhibit reduced GIPR expression (Lynn et al, 2003). Since elevated blood glucose is associated with decreased levels of GIPR expression, the question arises as to whether this pattern could be reversed, with lowering of blood glucose restoring GIPR expression? To address this question, a preliminary study was performed using the blood glucose lowering agent phlorizin. Phlorizin specifically lowers blood glucose by suppressing glucose reabsorption and increasing glucose excretion in the kidney via inhibition of the Na + -glucose co-transport channels in the proximal tubule. Treating adult ZDF rats with phlorizin for a two-week period almost completely normalized the GIPR content (Piteau, 2004). Unfortunately however, while phlorizin is effective at lowering blood glucose it also requires invasive administration and has potential toxic effects (Yki-Jarvinen et al, 1992). For this reason, other agents were considered, including DPIV-resistant incretin analogues and DPIV inhibitors, since they can be administered orally long term (Mcintosh et al, 2006). In previous studies, rodents that received oral DPIV inhibitor treatment were shown to demonstrate improvements in insulin sensitivity and glucose homeostasis (Mentlein et al, 1993; Kieffer et al, 1995; Pauly et al, 1996; 1999; Pederson et al, 1998; Pospisilik et al, 2002; Ahren et al, 2002; Sudre et al, 2002). 48 Likewise, the DPIV resistant GIP analogue D-Ala 2-GIP, also showed therapeutic potential in its ability to improve overall blood glucose homeostasis (Hinke et al., 2002). Studies on the effect on GIPR expression levels of treatment of diabetic animals with GIP analogues or DPIV inhibitors had not been performed. The current studies were designed to investigate whether GIPR expression could be restored in diabetic Zucker rats of the VDF or ZDF strains by either DPIV inhibitor (P32/98) or D-Ala 2-GIP analogue mediated improvements in glucose tolerance. 3.2.1 RESULTS: THE EFFECT OF 12-WEEK DPIV INHIBITOR (P32/98) T R E A T M E N T IN V D F (fa/fa) RATS In a 12-week study, 12-14 week old V D F rats were subjected to twice daily oral administration of either water (n=6) or DPIV inhibitor P32/98 (n=6). Throughout the study, food intake (figure l A ) , body weight (figure IB), and water consumption (figure 1C) gradually increased with age, but none of these parameters differed between the two treatment groups. Likewise morning blood glucose measurements from these animals also did not differ between groups, and they fluctuated in a similar manner. Unfortunately, the morning blood glucose data for the first 23 days of the study is not available due to a miscommunication from a former collaborator. From the data presented, however, morning blood glucose levels rose from a concentration of 4.5mM to an average of 5.7mM in the first 50 days, and then began to decline to their lowest points of approximately 4mM on the 75 t h day (Figure 2). The observed fluctuations in glucose levels are likely due to changes in the environment, as well as adaptation to the handlers. Unfortunately, these low morning blood glucose levels indicated that the animals did not demonstrate the expected impaired glucose values in control groups. 49 Figure 1: Food Intake, water consumption and body weight of VDF (fa/fa) rats in a 12-week P32/98 treatment study. Male and female rats 12-14 weeks of age were allowed food and water ad libitum, and adrriinistered orally either O.Olg/kg of P32/98 (n=6) or an equivalent volume of water (n=6) twice daily for 12 weeks. Data represent mean ±_ S.E.M. 50 The mild glucose intolerant phenotype was also evident in the oral glucose tolerance tests at week 0 where both P32/98 treated and control groups showed similar oral glucose challenge profiles (figure 3A). Despite slightly elevated fasting blood glucose levels however, peak glucose concentrations rose rather moderately to approximately 12-13mM. This indicates that these animals had much milder impairments in glucose tolerance when compared to earlier studies (Pederson et al., 19986; Pospisilik et al., 2002; Winter, 2004). By week 6 there was a small decrease in the glucose levels in the treated compared to non-treated groups was seen, with a statistically significant difference (p<0.001) at the 15 minute time point (figure 3B). The final OGTT at week 12 indicated a significantly (p<0.05) lower concentration of blood glucose at 60 minutes, while none of the values at other time points differed (figure 3C). 2 o "D g 4 -m 3-o Figure 2: Morning blood glucose levels of V D F (fa/fa) rats in a 12-week P32/98 treatment study. Male and female rats 12-14 weeks of age were were allowed food and water ad libitum and administered orally either P32/98 (n=6) or water (n-6) twice daily for twelve weeks. Glucose was determined as described in Materials and Methods. Data represent mean + S.E.M. 51 Week 6 20! 0 25 x 50 75 100 125 150 Time (min) Figure 3: Blood glucose concentrations in oral glucose tolerance tests on V D F (fa/fa) rats in a 12-week P32/98 treatment study. Male and female rats 12-14 weeks of age received either P32/98 (n=6) or water (n=6) orally twice daily for twelve weeks. At weeks 0, 6, and 12, rats were fasted overnight for 16 hours and blood glucose was measured in tail vein samples, before and after lg/kg of 40% dextrose solution was administered by gavage. Subsequent blood glucose concentrations were measured for two hours. Data represent mean +_ S . E . M . where asterisks indicate statistical significance * p<0.05, **p<0.001 by one-way A N O V A Newman-Keuls Multiple Comparison test. 52 Given the modest differences between glucose profiles of control and P32/98-treated groups in all three OGTTs, major changes in the plasma insulin levels would not be expected (figure 3). During the OGTTs on week 6, P32/98 treated animals showed slightly higher insulin levels at 30 minutes than the non-treated (figure 4A), although this not reach significance due the high variability. Slightly lower mean insulin levels were also observed at 30 minutes in P32/98 treated rats at week 12 (figure 4B). Part of the variation maybe due to differences in gender, since both females and males were present in the study, plus the small group size. Week 6 0.0-4—•—i 1 1——i— i i 0 25 50 75 100 126 150 Week 12 T , m e <m,n> (B) 3.0-j _ 2.sJ Time (min) (. Figure 4: Plasma insulin concentrations during oral glucose tolerance tests on VDF (fa/fa) rats in a 12-week P32/98 treatment study. Male and female rats 12-14 weeks of age received either P32/98 (n=6) or water (n=6) orally twice daily for twelve weeks. At week 6 and 12, rats were fasted overnight for 16 hours and blood samples were collected from the tail vein before and after lg/kg of 40% dextrose solution was administered by gavage. Plasma insulin levels were measured by radioimmunoassay at the corresponding time points. Data represent mean ± S.E.M. where asterisks indicate statistical significance * p<0.05, **p<0.001. 53 The GIPR islet mRNA expression in these animals, shown in figure 5A, revealed no difference between treated and non-treated rats. This is not surprising, considering only a modest change in glucose homeostasis was found. However, the peri-renal fat pads from the P32/98 treated animals showed a mild reduction in mean GIPR mRNA levels (figure Figure 5: GIPR mRNA levels in extracts from islets (A) and peri-renal fat pads (B) of VDF (fa/fa) rats in a 12-week P32/98 treatment study. Male and female rats 12-14 weeks of age received either P32/98 (n=6) or water (n=6) orally twice daily for twelve weeks. RNA was isolated using the RNAeasy Lipid Tissue Kit and GIPR mRNA expression was quantified using real-time RT-PCR. GIPR levels were normalized to P-actin. Data represent ± S.E.M. asterisks indicate statistical significance *p<0.05 by one-way ANOVA Newman-Keuls Multiple Comparison test. 3.2.2 RESULTS: THE EFFECT OF CONTINUOUS ADMINISTRATION OF GIP OR D-ALA2-GJP ANALOGUE ON DEVELOPMENT OF INSULIN AND P-CELL RESISTANCE AND p-CELLL RESPONSIVENESS IN ZDF RATS Five week old ZDF rats were chronically administered either GIP, D-Ala2-GIP analogue or saline for a 4 week duration, via implanted Alzet osmotic mini-pumps. 54 During the study no significant difference in body weight (figure 6) or morning blood glucose levels (figure 7) were observed between the three treatment groups. 400i *T 300H D) "5 •D 200H o m 100-• Control GIP Analog 10 Time (Days) — i — 20 Figure 6: Body weight of 5 week old male ZDF rats during a 4 week continuous infusion of either GIP, D-Ala 2-GIP analogue or saline control (n=6/group) via Alzet osmotic mini-pump. Data represent + S.E.M. o E <D (fl O u 3 O co E (A ro Q. Day Figure 7: Morning plasma glucose levels of 5 week old male ZDF rats during a 4 week continuous infusion of either GIP, D-Ala 2-GIP analogue, or saline control (n=6/group) via Alzet osmotic mini-pump. Glucose was determined as described in methods. Data represent mean + S.E.M. 55 Insulin tolerance tests were performed at the end of the study to assess the insulin sensitivity of these animals. Plasma glucose levels showed no significant differences between groups with an ~7mM drop in glucose after 30 minutes (figure 8). One control animal exhibited a fasting blood glucose level of 16.5mM compared to the average 8.2mM controls, therefore due to the unusually severe diabetic state of this animal, it was excluded from the test. 0-H 1 1 1 0 10 20 30 Time (min) Figure 8: Insulin tolerance tests were performed on 5 week old male ZDF rats after a 4 week continuous infusion of either GIP, D-Ala 2-GIP analogue or saline control (n=6/group) via Alzet osmotic mini-pump. At time 0 animals received an interperitoneal injection of 0.5U/g Humulin R (lOOU/mL). Glucose was measured as described in the methods. Data represent mean +_S.E.M. Despite the lack of effect of peptide treatment on plasma glucose, GIPR mRNA levels in the epididymal fat pads of the D-Ala 2-GIP analogue group were significantly reduced relative to groups treated with either saline or regular GIP (figure 9). 56 0_ o Figure 9: GIPR mRNA expression in epididymal fat pads of 5 week old male ZDF rats after a 4 week continuous infusion of either GIP, D-Ala 2-GIP analogue, or saline control (n=6/group) via Alzet osmotic mini-pump. R N A was isolated using RNAeasy Lipid Tissue Kit and GIPR mRNA levels were quantified using real-time RT- PCR. GIPR levels were all normalized to p-actin. Data represent mean + S.E.M. where asterisks indicate statistical significance * p<0.05 by student unpaired t-test. 3.3 DISCUSSION GIP's primary role is to potentiate the secretion of insulin in a glucose-dependent manner. However in patients with type 2 diabetes as well as ZDF and V D F rats this function is impaired. The attenuated incretin effect in the V D F rat is believed to be at least partially a result of glucose-induced downregulation of islet GIPR mRNA and protein expression (Lynn et al, 2001; 2003). Since it was previously shown that GIPR expression can be recovered by treatment with the blood glucose lowering agent phlorizin (Piteau, 2004), a less aggressive method was investigated: treatment with the DPIV inhibitor P32/98. As shown in figure 3A, the initial OGTT identifies the group of animals as having only mildly impaired glucose tolerance. Unfortunately, this left little room for 57 improvement and as a result, the success of DPIV treatment was difficult to assess. Previous P32/98 studies from this laboratory have reported a more severe diabetic V D F phenotype and as a result much more profound treatment effects were seen (Pederson et al, 1998b; Pospsilik et al, 2002; Winter, 2004). The V D F substrain displays a phenotype with variable severity (unpublished observations) and unfortunately, this particular group of animals exhibited a phenotype towards the mild end of the spectrum. A great deal of variation was also seen in the OGTT insulin profiles of these animals that may partly be attributed to the presence of both genders in the study. Although it could not be avoided at the time, generally females are excluded from studies involving glucose homeostasis in Zucker rats because of sex-specific differences in insulin sensitivity and differential signaling in other tissues (Guerre-Millo et al, 1985; Miyazaki et al, 2002; Heyener et al, 2002). Not surprisingly, islet and adipose tissue showed no significant changes in GIPR expression, although the reduction in mean adipose tissue GIPR mRNA levels is in agreement with the DPIV inhibitor study. However it is difficult to draw conclusions from a diabetic phenotype that was relatively weak to begin with. The D-Ala -GIP analogue treatment study, used a more severe model of diabetes, the ZDF rat, to hopefully establish a more distinguishable difference in glucose homeostasis between treated and non-treated groups. D-Ala 2-GIP is a DPIV-resistant GIP analogue rendering it biologically active for a longer period of time. The potential advantage of this therapy over DPIV inhibitors is its specificity. Chronic infusion of the D-Ala -GIP analogue over a 4 week duration however did not significantly change body weight, morning blood glucose or insulin sensitivity of these animals. Interestingly 58 despite little difference in glucose homeostasis of these animals, a significant reduction in GIPR expression was seen in the epididymal fat pads. Up to this point very little research has examined the GIPR's role in adipose tissue and especially its mechanism of regulation. Since the rat fat and islet GIPR are almost identical in structure (Gill, 2000), this regulation by glucose was thought to be regulated by a similar manner. It was therefore anticipated that GIP or D-Ala 2-GIP analogue induced decline in blood glucose would translate to an upregulation in GIPR expression in fat; however on the contrary a downregulation was seen and blood glucose was unchanged (figure 9). Unfortunately, islet GIPR mRNA expression could not be quantified in this experiment a direct comparison cannot be used to determine whether GIPR expression is differentially expressed between fat and islets. Interestingly, with adipose tissue, chronic D-Ala 2-GIP analogue treatment led to a moderate decline in GIPR expression, while the shorter lived GIP did not. This was despite a lack of difference in glucose levels between the two groups. The D-Ala -GIP analogue induced attenuation of GIPR expression in fat therefore may result from the chronic stimulation. Homologous desensitization of insulin responses to GIP in P-cell tumor lines was first reported by Fehmann and Habener (1991). Similarly, Tseng and colleagues (1996) demonstrated a decline in insulinotropic activity of GIP in vivo, following chronic infusion of GIP in anaesthetized rats. GIP pretreatment of mouse insulinoma PTC-3 cell lines (Tseng et al, 1996; Hinke et al., 2000) or primary rat islets (Hinke et al, 2000) was also shown to cause glucose-independent reduction in subsequent GIP-stimulated cAMP production. This attenuation may involve members of the regulators of G protein signaling (RGS) family (Tseng et al, 1998) and/or activation 59 of a negative feedback loop from downstream protein kinases such as P K C or P K A (Hinke etal, 2000). If plasma membrane levels of GIPR protein in adipose tissue are reduced by prolonged GIP action, then sustained in vivo treatment could lead to downregulation of GIPR expression, with reduced anabolic actions of GIP. Such a response to reduced GIP signaling is supported by the observation that GIPR"7" mice, sustain a leaner, more insulin sensitive phenotype compared to their controls, even when fed a high fat diet (Miyawaki et al, 2002). Additional evidence can be drawn from studies where the GIPR was chemically ablated by a specific GIPR antagonist (Pro3) GIP in leptin deficient ob/ob mice, which showed marked improvements in insulin-sensitivity and glucose tolerance despite the obese diabetic phenotype (Gault et al, 2005). From a therapeutic standpoint, chronic GIP treatment could potentially be a double-edged sword. On one side attenuation of GIPR function may act as a novel anti-obesity treatment, preventing fat storage and reducing insulin resistance in fat tissue. On the other hand, diminished GIPR function in the islet may have adverse effects on glucose-stimulated insulin secretion (Lynn et al, 2001), (3-cell survival (Ehses et al, 2003; Trumper et al, 2002), plus adverse effects on other tissues such as bone metabolism (Bollag et al, 2001; 2000; Tsukiyama et al, 2006). Therefore it will be imperative in the future to examine GIP analogue effects on islet GIPR expression, since little is known about chronic in vivo effects of GIP on its receptor. This concept may also be important for understanding the progressive development of type 2 diabetes, as one school of thought believes that high levels of GIP secreted in response to over-eating and excessive high fat diets (common in obesity-60 linked diabetes) leads to hyperinsulinemia, insulin resistance, and eventually hyperglycemia (Yamada & Seino, 2004). Therefore both hyperglycemia and hyperGIPemia could contribute to the downregulation of GIPR expression and overall impairment in glucose-stimulated insulin secretion observed in type 2 diabetes. Although there is evidence in support of a role for glucose as a significant regulatory molecule (Lynn et al, 2001) the possibility that chronically high levels of GIP also contribute in humans is controversial, because of the variability in reported levels with assays that measure both biologically active and inactive peptide (Jones et al, 1989; Bloom, 1975; Alam et al, 1992; Crockett et al, 1976; May et al, 1978; Ebert & Creutzfeldt, 1980). Nevertheless, the implications of GIP-induced desensitization and downregulation of the GIPR should be considered when testing potentially useful therapeutic systems based on increasing GIP action. 61 CHAPTER 4.0 THE EFFECTS OF PPARa OVEREXPRESSION IN THE INS-1 (832/13) p-CELL LINE 4.1 PROJECT RATIONALE It has been previously reported that incubation of INS-1 (832/13) cells with long chain saturated fatty acids or PPARa activators, such as WY14643, increase GIPR mRNA expression and increase GIPR promoter activity (Lynn et al, 2003; Lynn, 2003). Furthermore Lalloyer and colleagues (2006) have recently reported that PPAR"7" mice on an ob/ob background show significant downregulation of GIPR expression in parallel with decreased islet area and diminished glucose-dependent insulin secretion. Moreover like the GIPR, PPARa expression in INS-1(832/13) cells is also downregulated in a dose-dependent manner by glucose (Roduit et al, 2000). Lynn and colleagues (2003) initially proposed that the glucose-induced downregulation of GIPR expression may at least in part be a consequence of PPARa suppression. Our aim therefore was to investigate the effect, and glucose-dependence, of PPARa overexpression in INS-1 (832/13) P-cells on GIPR expression. 4.2.1 RESULTS: THE EFFECTS OF PPARa OVEREXPRESSION IN THE INS-1 (832/13) p-CELL LINE Transient transfection of INS-1 (832/13) cells with a PPARa construct under the control of a human C M V promoter resulted in a transfection efficiency of approximately 30%, similar to that seen previously for other genes (Ehses et al, 2003). This was sufficient to greatiy increase PPARa expression (figure 11), transfected samples showing strong PPARa protein bands at the anticipated 52 kDa region (Roduit et al, 2000), when analyzed by Western blots. 62 P P A R a Transfection + - + P P A R a (52 k D a ) ^ P-tubulin-> Figure 10: A Western blot of PPARa protein expression in INS-1 (832/13) cell extracts with or without transient transfection of human PPARa (under the control of C M V promoter). Similar results were obtained with 2 further analyses. In agreement with Roduit and colleagues (2000), as shown in figure 11 A , real time RT-PCR analysis revealed that glucose treatment of INS-1 (832/13) cells resulted in a concentration-dependent ( l l m M p<0.05; 25mM pO.OOl) downregulation of PPARa mRNA expression in non-transfected cells as shown previously (Lynn et al., 2001). Unexpectedly, however, cells transfected with the PPARa plasmid also responded in a similar pattern. In fact, at all three glucose concentrations, PPARa expression was lower than in the non-transfected cells, with significant difference at l l m M (p<0.05). With PPARa overexpression, however, GIPR mRNA levels were significantly decreased at 1 ImM and 25mM glucose (figure 1 IB). Interestingly, although PPARa and GIPR mRNA levels followed a similar trend, GIPR expression in the non-transfected cells at 25mM glucose was reduced to approximately 70%, while PPARa showed a 40-50%) reduction, suggesting additional factors may be involved. 63 Figure 11: The effect of human PPARa overexpression and elevated glucose on PPARa and GIPR mRNA expression in INS-1(832/13) cells. Non-transfected INS-1 (832/13) cells or cells transfected with a human CMV-PPARa plasmid were cultured for 24 hours in serum-ftee medium containing 0.1% BSA and 5, 11 or 25 mM glucose. RNA was isolated using the standard Trizol® method and mRNA quantified using real-time RT-PCR. PPARa and GIP-R mRNA levels were normalized to p-actin. Data represent mean + S.E.M. asterisks indicate statistical significance * p<0.05, ** p<0.001 relative to 5mM control, while # indicates statistical significance p<0.05 relative to glucose-matched control, by one way ANOVA Newman-Keuls Multiple Comparison test. n=3 To exclude the possibility that the PPARa expression vector was encoding a non-functional product, mRNA levels of uncoupling protein 2 (UCP-2), a known downstream target of PPARa, was quantified. As shown in figure 12, at 5mM glucose, transfection of PPARa modestly increased UCP-2 expression, although to a lesser extent than the specific PPARa agonist WY14643 (p<0.001). This mild response was surprising considering that UCP-2 is a direct downstream target of PPARa; however this may explain why no increase in GIPR expression was observed in the overexpression system. When cells were transfected with human PPARa vector and treated with WY14643 however, these treatments acted synergistically to enhance UCP-2 expression 1.9-fold 64 and 1.5-fold, compared to control (pO.OOl) and WY14643 alone (p<0.05) respectively. At 25mM glucose, UCP-2 expression decreased -25% compared to the 5mM control. This was expected considering its activator, PPARa is attenuated under elevated glucose conditions. Transfection of PPARa or treatment with WY14643 alone did not alter UCP-2 expression; however as before, the two treatments combined induced an increase in UCP-2 expression, although not significant. Figure 12: The effect of human PPARa overexpression and elevated glucose on UCP-2 mRNA levels in INS-1 (832/13) cells. Non-transfected INS-1 (832/13) cells or cells transfected with a human C M V - P P A R a plasmid were cultured for 24 hours in serum-free medium containing 0.1 % BSA and either 5 or 25mM glucose. R N A was isolated using Trizol® and UCP-2 mRNA quantified using real-time RT-PCR. UCP-2 levels were normalized to P-actin. Data represent mean + S.E.M. where asterisks indicate statistical significance * p<0.05, ** pO.OOl relative to 5mM control, and # indicates statistical significance p<0.05 relative to WY14643 treatment control by one-way A N O V A Newman-Keuls Multiple Comparison test. n=3 65 4.3 DISCUSSION Previous studies on INS-1(832/13) cells (Lynn et al., 2003) and ??ARa''-ob/ob mice (Lalloyer et al, 2006) provided evidence supporting an important role for PPARa in the regulation of GIPR expression. To examine this potential role further, INS-d(832/13) cells were transfected with a vector expressing the human PPARa gene. In comparison to the non-transfected controls, Western blot analysis confirmed an abundant increase in PPARa protein expression. In contrast, however, real-time RT-PCR, demonstrated a downregulation of PPARa mRNA expression in the transfected cells. The primers used for real-time RT-PCR were specific for the rat gene and were not compatible with the human GIPR sequence; therefore it is likely that the suppression was indicative of negative feedback on endogenous rat PPARa gene expression by the overexpressed human PPARa protein or the product of a PPARa-regulated gene. This would also explain the effect of PPARa overexpression on GIPR expression. It is generally assumed that transcription factors of higher organisms, such as humans, are capable of interacting with, and regulating, genes of lower orders, such as rats. Ammerschlaeger and colleagues (2004) however have demonstrated in genes related to peroxisome proliferation (an event that is known not to occur in humans), that there are differences between the potency of rat and human PPARa, on their respective PPREs and within different cell types. In primary rat hepatocytes and in the rat liver cell line FAO, overexpression of rat PPARa increased the promoter activity of peroxisomal enzyme A O X by 20-fold and 4.2-fold respectively; however overexpressing human PPARa in these same cell types induced a much lower response of 3.4 and 1.4-fold respectively. Not only was a clear difference seen between the origins of PPARa but also between the different cell types of the same 66 species of origin (Ammerschlaeger et al, 2004). Moreover, the rat A O X promoter has been found to be more responsive to human PPARa in murine cells than in rat (Hasmall et al, 2000; Sher et al, 1993), suggesting that co-signaling factors may be present and necessary for specificity. Taken together the origin of PPARa, PPRE, and the cellular environment, all appear to influence PPARa species specificity and selectivity. In INS-1 (832/13) cells therefore, it is possible that human PPARa was either unable to interact, or reacted only weakly with the promoter of the rat GIPR, and instead, expression of the latter was decreased due to both a glucose-dependent decline in PPARa as well as through attenuated endogenous rat PPARa. If this is the case, expression of rat PPARa does not appear to be downregulated to the same magnitude as GIPR expression with 25mM glucose, perhaps because in addition to the effect of glucose on the PPARa gene, the GIPR gene may also be downregulated by an additional factor, such as glucose itself. To date, however no glucose response elements have been identified on the GIPR promoter, and this possibility therefore will have to be investigated further. Moreover, studies overexpressing the rat PPARa will have to be performed to determine whether species-specificity involved. To confirm functionality of the PPARa vector, the effect of overexpression on a prominent downstream target of PPARa, UCP-2, was measured. Under basal conditions relatively small effects were again observed, but robust responses were obtained after addition of the PPARa activator WY14643. Moreover, under elevated glucose conditions human PPARa was not able to override the attenuating effect of glucose on PPARa and subsequently UCP-2 expression. Cell-specificity may, again, have played a part in restricting rat UCP-2 responses to increasing human PPARa levels; however the 67 synergistic effect of WY14643 and PPARa vector on rat UCP-2 expression suggests an additional explanation. Ravnskjaer and colleagues (2005) showed that in INS-E (3-cells (another subclone of INS-1 cells) increasing transcription or activation of PPARa was not sufficient for increasing downstream targets but required both together as well as activation and increased expression of PPARa's co-activator the retinoid X receptor (RXR). Co-transduction of INS-E cells with adenoviruses encoding PPARa and R X R and addition of their respective activators, synergistically stimulated downstream targets (Ravnskjaer et al., 2005). According to these studies, R X R is likely acting as the limiting factor in PPARa overexpression systems, a suggestion that could be tested by increasing R X R availability in the INS-1(832/13) cell system studied here. Currently the only commercially available means of increasing R X R is through retinoic acid treatment; however the solubility properties of this compound presented technical problems that could not be resolved, thus it is still not confirmed whether R X R is a limiting factor. From a mechanistic standpoint, it is noteworthy that there is only one consensus PPRE half-site (TGACCA) at position 597-609 of the rat GIPR promoter, similar to that found in the PPARoc-regulated A O X gene, which may be capable of mediating transcriptional activation of the GIPR promoter by the selective PPARa activator WY14643 (Lynn, 2003). PPARa may also play a more indirect role in regulation of GIPR expression than originally anticipated. One-way in which PPARa has been reported to exert indirect effects on transcription of other genes is through inhibition of the transcriptional intermediary factor 2 (TIF 2) in primary hepatocytes and hepatoma HepG2 cells (Gervois et al, 2001). TIF 2 elicits transcriptional regulation through a basic leucine zipper transcription factor C C A A T Box/Enhancer-binding protein P (C/EBP). 68 C/EBPP is a protein expressed abundantly in liver and fat cells (Birkenmeier et ai, 1989) that has been reported to have stimulatory effects on inflammatory mediators such as fibrinogen, as well as stimulatory and repressor effects on enzymes and receptors involved in glucose and lipid metabolism (McKnight et al., 1989). So far C/EBP response elements have been reported in the promoter regions of the leptin receptor (Gervois et al., 2001), GLUT-2 in liver and the pancreatic P-cell (Kim et al., 1998) and insulin in the P-cell (Lu et al., 1997). It is believed that fibrates and other PPARa activators elicit their effects partially by interfering with the C/EPB pathway via attenuation df TIF 2 action (Gervois et al., 2001). There is a consensus sequence for C/EBPP binding at position 359-373 in the GIPR promoter. Moreover, it has been reported that in pancreatic P-cell lines HIT-T15 and INS-1 (Lu etal., 1997) and ZDF rat islets (Seufert et al., 1998) high glucose conditions substantially upregulate C/EBPp expression. Thus we hypothesize a model by which, under high glucose conditions, suppression of PPARa, would prevent the inhibition of TIF 2 thus allowing it to' co-activate with elevated C/EBPP to form a complex that together would suppress GIPR expression. This proposed model raises many important questions that will require further investigation. 69 CHAPTER 5.0 CHARACTERIZATION OF GIPR mRNA EXPRESSION IN ADIPOSE TISSUE AND 3T3-L1 ADIPOCYTES 5.1 PROJECT R A T I O N A L E Compared to the P-cell, GIPR expression in fat is relatively understudied. This is unfortunate however considering the anatomical distribution of adipose tissue, potentially rendering it a much less invasive tissue for extraction and monitoring of GIPR expression during therapeutic treatments in humans. It is not known, however, whether the GIPR is differentially regulated in the P-cell and adipose tissue. Since islet GIPR expression is suppressed in fatty compared to lean V D F rats, we sought to examine whether GIPR expression in the epididymal fat pads of lean and fatty V D F rats exhibited a similar profile. It is also well established that glucose dose-dependently decreases GIPR expression in the P-cell (Lynn et al, 2001). We therefore sought to investigate whether glucose has the same effect in fat by performing glucose-concentration response studies in 3T3-LI adipocytes. 5.2.1 RESULTS: CHARACTERIZATION OF EPIDIDYMAL FAT P A D GIPR EXPRESSION IN F A T T Y (fa/fa) VERSUS L E A N (Fa/?) V D F RATS An OGTT was performed at the beginning of the study to confirm a marked difference in glucose homeostasis between fatty and lean V D F rats. As predicted, the fatty rats had significantly (p<0.001) higher blood glucose concentrations at all time points after a bolus of glucose was administered (figure 13). This confirms that impaired glucose tolerance was present in the fatty animals versus lean, thus providing an appropriate model for comparing GIPR mRNA expression between normal rats and those with impaired glucose control. Due to problems with islet isolation all lean rat samples 70 had to be pooled, as well as 2 of the fatty samples for a total n of 6 (Figure 14). However, this limited data were consistent with previous results of Lynn et al. (2001), with the fatty rats demonstrating greatly reduced mean levels of GIPR expression compared to lean (figure 14). 0 I I I I I | | 0 25 50 75 100 125 150 Time (min) Figure 13: Blood glucose concentrations during oral glucose tolerance tests on V D F (fa/fa) (n=8) and lean (Fa/?) (n=8) rats. Approximately 6 month old male rats were fasted overnight for 16 hours, fasting blood glucose was measured in blood collected from the tail vein and lg/kg of 40% dextrose solution was administered by gavage. Subsequent blood glucose concentrations were measured for two hours. Asterisks indicate statistical significance p<0.001 by one-way A N O V A Newman-Keuls Multiple Comparison test. With samples from the epididymal fat pads, GIPR mRNA expression showed no downregulation, but rather a mild upregulation (figure 15). 71 Figure 14: GIPR mRNA levels in isolated islets of V D F (fa/fa) (n=6) versus lean (Fa/?) (n=l) V D F rats. Islets were isolated from male V D F rats at ~6 months of age, four days after an OGTT had been performed. R N A was isolated using standard Trizol® methods and GIPR mRNA levels were quantified using real-time RT- PCR. GIPR levels are all normalized to P-actin mRNA levels. Data represent mean + S.E.M. 0. O Figure 15: GIPR mRNA levels in epididymal fat pads of V D F (fa/fa) (n=8) versus lean (Fa/?) rats (n=8). Epididymal fat pads were removed from male V D F rats at ~6 months of age four days after an OGTT had been performed. RNA was isolated using the Rneasy Lipid Tissue protocol and GIPR mRNA levels were quantified using real-time RT-PCR. GIPR levels are all normalized to P-actin mRNA levels. Data represent mean + S.E.M. 72 5.2.2 RESULTS: GLUCOSE CONCENTRATION-RESPONSE STUDY ON GIPR mRNA EXPRESSION IN 3T3-L1 ADIPOCYTES In agreement with studies by Lynn et al. (2003), INS-1(832/13) cells, GIPR mRNA levels were significantly downregulated by high glucose (figure 16A) with an approximate ~ 70 % reduction at 25mM glucose when compared to 5mM (pO.OOl). To examine whether glucose has a similar effect in an adipocyte cell line, glucose concentration-response experiments were performed on difFerentiated 3T3-L1 adipocytes. Surprisingly, the opposite response was seen, with a substantial upregulation in GIPR mRNA levels with increasing glucose concentrations, that reached significance (p<0.05) at 25mM compared to 5mM (figure 16B). This suggests that GIPR expression may be regulated in a tissue-specific manner. Figure 16: The effect of glucose on GIPR mRNA levels in INS-1 (832/13) cells (A) and difFerentiated 3T3-L1 adipocytes (B). Cells were incubated for 24 hours in serum-free medium containing 0.1% BSA and either 5, 11 or 25 mM glucose. RNA was isolated using the standard Trizol® method and GIPR mRNA levels were quantified using real-time RT-PCR. GIPR levels were normalized to P-actin mRNA levels. Data represent mean+jS.E.M. where asterisks indicate statistical significance *p< 0.05, **p<0.001 relative to 5 mM by one-way ANOVA Newman-Keuls Multiple Comparison test. n=3 73 5.2.3 RESULTS: 3T3-L1 ADIPOCYTE RESPONSES TO CLOFIBRATE TREATMENT UNDER HIGH AND LOW GLUCOSE CONDITIONS To investigate possible mechanisms by which expression of the fat GIPR is regulated, 3T3-L1 adipocytes were treated with the PPARa activator clofibrate under high and low glucose concentrations. Mean GIPR mRNA levels in 3T3-L1 adipocytes increased by -2.2 fold when the glucose concentration was raised from 5mM to 25mM (figure 17), although the increase did not reach significance. Clofibrate treatment induced a significant increase of -2.6 fold in GIPR expression at 5mM (p<0.05), but this response was not evident with cells cultured in 25mM glucose. c Figure 17: GIPR mRNA expression in 3T3-L1 adipocytes after 24 hour treatment of 1 mM clofibrate. Both cell types were treated in serum-free 0.1% BSA medium at either 5 mM or 25 mM glucose. RNA was isolated using standard Trizol® method, and GIPR mRNA was quantified using real-time RT-PCR. GIPR levels are all normalized to p-actin. Data represent mean + S.E.M. where asterisks indicate statistical significance * p<0.05, **p<0.001 relative to 5 mM control by one-way ANOVA Multiple Comparisons test. n=3 74 5.3 DISCUSSION The regulation of GIPR expression remains poorly characterized; however it was previously shown in studies on INS-1(832/13) P-cells and islets isolated from VDF rats that glucose has a strong suppressive effect on expression (Lynn et al., 2003). However, it is not known whether glucose has the same effect in adipose tissue. GIP serves a different function in adipocytes, involving both lipolytic (Mcintosh et al, 1999; Getty-Kaushik et al, 2006) and lipogenic actions that include promotion of fatty acid synthesis (Eckel et al, 1979) and fat storage (Knapper et al, 1995). To begin to characterize potential differences in GIPR regulation between tissues, a comparison between lean (Fa/?) and fatty if a/fa) V D F male rats was performed. Glucose intolerance was confirmed in the V D F rats by OGTTs and these rats showed a reduction in mean islet GIPR mRNA levels compared to their lean littermate controls. Unfortunately, however, since samples from the lean animals were pooled, no statistical significance could be calculated, although the trend was similar to that published previously (Lynn et al, 2001). In contrast, although not reaching significance, mean adipose, tissue GIP-R levels were slightly increased, suggesting that GIPR expression in fat may be regulated differently from the islet GIPR. To pursue this further, studies were performed on the effect of 24 h exposure of 3T3-L1 adipocytes to elevated glucose, revealing a concentration-dependent upregulation of GIPR expression. The results confirmed that expression of the GIPR is not downregulated by elevated glucose in fat, thus suggesting that it is regulated in a tissue-specific manner. It is noteworthy to mention however that under normal culture conditions 3T3-L1 adipocytes grow in 25mM glucose compared to the l l m M preferred 75 by INS-1(832/13). It is possible therefore that 3T3-L1 cells are healthier in a high glucose medium or that the regulation of GIPR expression differs from the natural adipocyte. Various other studies however have employed incubation conditions for 3T3-L1 adipocytes including 5mM glucose and demonstrated good viability and function with comparable leptin production and release (Zhang et al, 2002), as well as active glucose uptake and G L U T 4 translocation (Janez'er al., 2000). Furthermore, preliminary data from an APO Percentage™ apoptosis bioassay, showed no difference in the number of apoptotic 3T3-L1 cells cultured at 5mM compared to 25mM (data not shown). If in fact the GIPR is regulated in a tissue-specific manner what are the potential mechanisms controlling its differential regulation? As alluded to earlier, evidence suggests that PPARa may play a role in GIPR expression in INS-1 (832/13) cells. Studies were therefore performed on the effect of a selective PPARa activator, clofibrate. As shown in figure 17, administration at a concentration of ImM induced an increase in GIPR expression in 3T3-L1 adipocytes. Since PPARa is not the predominant isoform in fat, it was somewhat surprising to observe" such a profound change in GIPR expression Further experiments are required to establish such a role, however. Although clofibrate has been used as one of the more selective P P A R a agonists, in some cell types it induces expression of both P P A R a and PPARy (Muzio et al, 2003). Alternatively, the clofibrate induced increase in GIPR expression could have been a compensatory response to an increase in PPARa induced fatty acid oxidation.and lipid metabolism (figure 17). Since PPARy is the predominant PPAR isoform in adipose tissue it could well play a role in fat GIPR expression. In support of this, it has been shown that, like the GIPR, PPARy in fat 76 is also increased under elevated glucose conditions. In this case, as a result of an increase in sterol regulator element binding protein-1 (SREBP-1) (Hasty et al, 2000). 77 CONCLUSIONS AND FUTURE DIRECTIONS In rodent models and in humans with type 2 diabetes, the glucose-dependent insulinotropic effects of GIP are impaired; The effect in rodents has been attributed to a downregulation in GIPR protein and mRNA expression in the pancreatic islet. A clear mechanism for this attenuation remains to be identified, however Lynn and colleagues (2003), as well as work described in this thesis, illustrate that chronically elevated glucose plays an integral role in GIPR suppression. It is not clear however, whether glucose directly and/or indirectly mediates these effects, necessitating further studies to elucidate a mechanism for this glucose-induced downregulation. To begin with, an in depth study of the potential role of partial sequences for glucose response elements and C/ERB response elements should be performed using luciferase constructs to measure changes in GIPR promoter activity under media conditions. Using deletion analysis, sequences that are necessary for glucose-induced GIPR suppression could be identified. Confirmation of function could be established using E M S A and ChiP assays. In adipose tissue, studies from this thesis indicate that the GIPR is regulated in a tissue-specific manner. This glucose dose-dependent increase in GIPR expression however cannot be validated until both apoptosis assays and viability studies are performed to confirm the health and function of the 3T3-L1 adipocytes under experimental conditions. Once viability is confirmed, similar studies as described earlier can be performed to isolate and identify the region(s) responsible for mediating glucose and clofibrate regulation. In addition, an effective, specific GIPR antibody will need to be developed to measure changes in protein expression to corroborate results of real-time RT-PCR and luciferase assays. 78 In conclusion, understanding the mechanism of GIPR regulation in both islet and fat is crucial for our understanding of how the etiology of diabetes occurs, and how modern drug therapies such as GIP analogues, DPIV inhibitors, and GIP antagonists will affect long term glucose homeostasis. Furthermore, it is my expectation that one day, our knowledge and understanding of the GIPR will extend to other tissues of the body, where we can further learn and develop a comprehensive understanding of GIP's role in physiology. 79 REFERENCES Abbott, C. R., M . Monteiro, C. J. Small, A . Sajedi, K. L. Smith, J. R. C. Parkinson, M . A . Ghatei and S. R. Bloom (2005). 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