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Glucose-dependent insulinotropic polypeptide (GIP) activation of protein kinase B (PKB) and its contribution… Winter, Kyle D. 2004

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Glucose-dependent Insulinotropic Polypeptide (GIP) Activation of Protein Kinase B (PKB) and its Contribution to Pancreatic p-cell Survival B y K y l e D . W i n t e r B . S c . ( H o n s . ) , Q u e e n ' s U n i v e r s i t y 2 0 0 2 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T F O R T H E D E G R E E O F M A S T E R O F S C I E N C E i n T H E F A C U L T Y O F G R A D U A T E S T U D I E S ( D e p a r t m e n t o f P h y s i o l o g y ) W e a c c e p t th i s t hes i s as c o n f o r m i n g to the r e q u i r e d s t a n d a r d T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A J u l y 2 0 0 4 © K y l e D . W i n t e r , 2 0 0 4 Library Authorization In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. KirlE toviT6R i s/o7/2001 Name of Author (please print) Date (dd/mm/yyyy) Title of Thesis: f^ larjttg. tJLyyJflir ft^mAA. (6T-f\ yU^Ai*. ^Q-fnAc:. K w l ft revets) Degree: j ^ s W c £ S r ^ a , H.Sr,. Y e a r : TSXft Department of jPr\iAS^ o\?c The University of British Vancouver, BC Canada Columbia^ ABSTRACT In addition to their well-documented insulinotropic actions, the incretin hormones Glucose dependent insulinotropic polypeptide (GIP) and Glucagon Like Peptide-1 (GLP-1), have recently been shown to exhibit non-insulinotropic effects on pancreatic (3-cell proliferation, survival and apoptosis. GIP regulates pancreatic (3-ceIl secretion by binding to its cognate Family B, G protein-coupled receptor and elevating intracellular cAMP and Ca 2 +. The hypothesis for the studies described in this Thesis was that GIP can induce pancreatic (3-cell survival through activation of Protein Kinase B (PKB). Using a (3-ceIl line, INS-1, and in human islets, it was possible to correlate GIP receptor activation with PKB phosphorylation through lipid signaling (arachidonic acid production). Through molecular biological and pharmacological approaches, GIP was shown to activate PKB. It was also shown that GIP can activate known downstream targets of PKB, namely GSK3a/|3, FKHR and BAD. These events were demonstrated to be functionally relevant for (3-cell survival. Studies using the INS-1 cells also displayed the ability of GIP to improve cell survival within a glucolipotoxic environment. GIP treatment reduced the activity of the pro-apoptotic enzyme caspase-3 in both INS-1 cells and human islets. This reduction was shown to be dependent on the PI3K pathway. Further studies verified the protective effect of active PKB for INS-1 cells. Using an animal model of obesity-related diabetes (fa/fa Zucker rat) and a combination of techniques (oral glucose tolerance testing, caspase-3 activity assay, immunoblotting) it was also shown that long-term DP IV (P32/98) treatment can improve the survival of islets and improve glucose tolerance. These findings display a novel role for GIP in regulating (3-cell survival. ii T A B L E OF C O N T E N T S A B S T R A C T ii T A B L E O F C O N T E N T S i i i LIST OF F IGURES vi A B B R E V I A T I O N S vii A C K N O W L E D G E M E N T S ix P U B L I C A T I O N S x C H A P T E R 1: I N T R O D U C T I O N 1 1.1 T H E I N C R E T I N C O N C E P T 1 1.1.1 GIP Discovery 1 1.1.2 GIP Distribution and Secretion 2 1.1.3 Biological Actions of GIP 4 1.1.4 GLP-1 Discovery 7 1.1.5 GLP-1 Secretion and Biological Actions 7 1.1.6 GIP and GLP-1 Receptors and Signal Transduction 9 1.1.7 Regulation of (3-cell Growth and Survival 13 1.1.8 Pathophysiology of the Incretins 15 1.2 D1PEPT1DYL P E P T I D A S E IV 17 1.2.1 Structure, Distribution and Function 17 1.2.2 DP IV regulation of Incretins 18 1.2.3 DP IV Inhibition in Type II Diabetes 18 1.3 P R O T E I N K I N A S E B , 19 1.3.1 Introduction 19 1.3.2 Phosphorylation and Regulation of P K B 20 1.3.3 P K B substrates and functions 21 1.4 THESIS I N V E S T I G A T I O N . . . 23 C H A P T E R 2: M E T H O D O L O G Y 24 2.1 R E A G E N T S 24 2.2 P L A S M I D D N A C O N S T R U C T S 24 2.3 C E L L C U L T U R E A N D T R A N S F E C T I O N 25 2.4 C H A R A C T E R I Z A T I O N O F S I G N A L - T R A N S D U C T I O N P A T H W A Y S B Y W E S T E R N B L O T A N A L Y S I S 25 in 2 .5 A R A C H I D O N I C A C I D R E L E A S E 2 6 2 . 6 H U M A N I S L E T I S O L A T I O N A N D C U L T U R E 2 7 2 .7 D E T E R M I N A T I O N O F C E L L S U R V I V A L 2 8 2 .8 C E L L D E A T H A S S A Y 2 8 2 . 9 A P O P T O S I S A S S A Y 2 8 2 . 1 0 C A S P A S E - 3 A S S A Y 2 9 2.11 S T U D I E S O N 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 Z U C K E R R A T ( V D F ) 3 0 2 . 1 2 A N I M A L S 31 2 . 1 3 P R E P A R A T I O N O F V D F P L A S M A 3 2 2 . 1 4 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 3 2 2 . 1 5 P R O T O C O L F O R M O N T H L Y A S S E S S M E N T O F G L U C O S E T O L E R A N C E 3 2 2 . 1 6 I S O L A T I O N A N D C U L T U R E O F I S O L A T E D R A T I S L E T S 3 3 2 . 1 7 W E S T E R N I M M U N O B L O T T A N A L Y S I S 3 3 2 . 1 8 D A T A A N A L Y S I S 3 4 CHAPTER 3: REGULATION OF PKB SIGNALING BY GIP 35 3.1 P R O J E C T R A T I O N A L E 3 5 3 .2 R E S U L T S 3 5 3.2.1 G I P s t i m u l a t e s p h o s p h o r y l a t i o n o f P K B a n d the d o w n - s t r e a m targets B a d ( S e r 136) , G S K 3 cx/|3 ( S e r 2 1 / 9 ) a n d F H R K / A F X ( S e r 2 5 6 / 1 9 6 ) i n (3 ( I N S - 1 ) c e l l s . 3 5 3 . 2 . 2 G I P - m e d i a t e d a c t i v a t i o n o f P K B i n v o l v e s G - p r o t e i n |3y s u b u n i t a c t i v a t i o n o f P L A 2 a n d A r a c h i d o n i c A c i d P r o d u c t i o n 3 8 3 .2 .3 G I P - m e d i a t e d a c t i v a t i o n o f P K B i n v o l v e s a c t i v a t i o n o f P I 3 K i n a s e a n d is C a 2 + - d e p e n d e n t 41 3 .2 .4 G I P - i n d u c e d p h o s p h o r y l a t i o n o f G S K 3 a / p \ B A D a n d F H R K i s P O K i n a s e - d e p e n d e n t i n I N S - 1 c e l l s 4 4 3.3 D I S C U S S I O N 4 6 i v C H A P T E R 4: I N C R E T I N M E D I A T E D P R O T E C T I O N O F 0 - C E L L S A G A I N S T C E L L D E A T H : GIP P R O T E C T I O N A G A I N S T G L U C O L I P O T O X I C I T Y - I N D U C E D C E L L D E A T H IN VITRO AND T H E E F F E C T O F O R A L L Y ADMINISTERED P32/98 ON p - C E L L S U R V I V A L IN V D F Z U C K E R RATS...52 4.1 P R O J E C T R A T I O N A L E 52 4.2 R E S U L T S 52 4.2.1 GIP and GLP-1 reverse glucolipotoxicity in INS-1 cells 52 4.2.2 GIP and GLP-1 reduce |3 (INS-1) cell glucolipotoxicity 53 4.2.3 GIP and GLP-1 reduce glucolipotoxicity in human islets 55 4.2.4 GIP and GLP-1 promote cell survival via P K A and PI3K 56 4.2.5 Effects of P32/98 treatment on oral glucose tolerance 59 4.2.6 D P IV inhibitor treatment of V D F Zucker rats affects pro-survival protein expression 62 4.2.7 D P IV inhibitor treatment of V D F Zucker rats improves (3 cell survival 63 4.3 D I S C U S S I O N 66 C H A P T E R 5: CONCLUSIONS 70 R E F E R E N C E S 72 v L I S T O F F I G U R E S Fig. 1. G I P stimulates P K B phosphorylation in a time (A) and (B) concentration dependent manner in INS-1 (832/13) cells. 36 Fig. 2. G I P stimulates phosphorylation of G S K 3 a / p \ B A D and F K H R . 37 Fig. 3. Forskolin, c A M P (A) and arachidonic acid (B) regulate P K B in INS-1 (832/13) cells. 39 Fig. 4. The effect of GIP on arachidonic acid release from INS-1 cells ( A , B , Q or transfected INS-1 cells (D). 40 Fig. 5. G I P mediated P K B phosphorylation is G[3y dependent in INS-1 cells. 41 Fig. 6. G I P regulation of P K B is P K A , P K C , M A P K and Tyrosine Kinase independent and PBKinase , P L A , and C a 2 + dependent in INS-1 cells. 43 Fig. 7. G I P activation of G S K 3 a / [ i is P K B dependent. 45 Fig. 8. Proposed coupling of the GIP receptor to P K B activation. 47 Fig. 9. The effect of glucolipotoxicity (A) coupled with varying G I P (B) or GLP-1 (C) concentrations on the survival of (3—INS-1 cells. 53 Fig. 10. G I P and GLP-1 reduce glucolipotoxicity in INS - I (832/13) cells. 54 Fig. I I . G I P and GLP-1 reduce glucolipotoxicity in isolated human islets. 56 Fig. 12. G I P and GLP-1 reduce glucolipotoxicity in INS-1 (832/13) cells. 57 Fig. 13. The P I 3 K / P K B module is involved in the GIP-mediated reduction in glucolipotoxici ty-induced caspase-3 activity in INS - I (832/13) cell. 59 Fig. 14. Oral glucose tolerance tests ( O G T T ) administered to both D P IV inhibitor treated (n=4, filled triangle) and vehicle-treated control (n=3, filled square) V D F rats after four ( A , B ) , eight (C,D) and twelve (E,F) weeks of treatment. 61 Fig. 15. Protein expression is enhanced in V D F rats (n=3) treated for twelve weeks with the D P IV inhibitor P32/98 (P) relative to vehicle-treated controls (C). 63 Fig. 16. Caspase-3 activity (A) and P A R P cleavage (B) were reduced in V D F rats (n=3) treated for twelve weeks with the D P IV inhibitor P32/98 (P) relative to controls (C). 64 Fig. 17. G I P and GLP-1 reduce V D F plasma-induced glucolipotoxicity. 65 VI A B B R E V I A T I O N S A A Arachidonic Ac id A C T H Adrenocorticotropic hormone A M C 7-amino-4-methyIcoumarin A N O V A Analysis of variance [3ARKct (3-adrenergic receptor kinase C-terminus B S A Bovine serum albumin p n Intracellular C a2 + C a M K Calmodulin-dependent kinase c A M P Cycl ic adenosine monophosphate c A M P - G E F c A M P guanine nucleotide exchange factor C C K Cholecystokinin Cdk Cyclin-dependent kinase C H O Chinese hamster ovary C I C R Calcium induced calcium release C K Casein kinase C R E c A M P response element C R E B C R E binding protein D A G Diacylglycerol D M E M Dulbecco's modified eagle media D P IV Dipeptidylpeptidase IV E G F Epidermal growth factor E R K 1 / 2 Extracellular regulated kinase-1/2 F F A Free fatty acid F K H R Forkhead transcription factor G F P Green flurescent protein G H Growth hormone GIP Glucose-dependent insulinotropic polypeptide G I P R ' GIP receptor knockout G LP-1/2 Glucagon-like peptide-1/2 G L T X Glucolipotoxicity G P C R G-protein coupled receptor G S K 3 Glycogen synthase kinase-3 H E L S S Haloenolactone suicide substrate H P A Hypothalamus-pituitary-adrenal IGF-1 Insulin-like growth factor-1 I K K Inhibitor N F - K B kinase IP 3 Inositol-1,4,5 triphosphate IPLA 2(3 ATP-stimulatable, Ca2 +-independent cytosolic P L A 2 isoform (3 J N K Jun N-terminal kinase K ATP A T P sensitive K + channel M A P K Mitogen-activated protein kinase M K K / M e k M A P K kinase N A A D P Nicotinic acid adenine dinucleotide phosphate N S C C Non-specific cation channel vii P A C A P Pituitary adenylate cyclase activating protein PC 1/3 Proconvertase-1/3 P D G F Platelet-derived growth factor PDK-1 3-Phosphoinositide-dependent kinase P K X - 1 Pancreatic duodenal homeobox transcription factor P13K Phosphatidylinositol 3-kinase P K A Protein kinase A P K B Protein kinase B P K C Protein Kinase C P L A 2 Phospholipase A 2 P L C Phospholipase C P T E N Phosphatidylinositol 3'-phosphate and protein phosphatase RIA Radioimmunoassay RIP Rat insulin promoter R p - c A M P Adenosine 3' ,5'-cyclic phosphorothioate-RP R T K Receptor tyrosine kinase Src Oncogene Src S T Z Streptozotocin V I P Vasoactive intestinal polypeptide vii i ACKNOWLEDGEMENTS On this journey, like any journey, there were hoops to jump through and hills to climb, and I could not have done all of it without a great deal of support. I would like to begin by thanking both Chris and Ray for their assistance in and outside the laboratory. I have probably learned as much about character as I have concerning specific laboratory science, for which 1 am very proud. Both of you teach in separate yet effective manners, forcing me to push myself above and beyond even my own expectations. Between debating potential experiments, discussing intriguing results or arguing over which beer one prefers, I have really appreciated care and thought involved. In the laboratory, I have benefited from your balance of freedom and guidance. I could not have gotten the ball rolling, let alone the seen the day this thesis was completed without Jan and Pops. They have been a lasting influence on my work as a scientist and as a person. They showed me the ropes for both experimental as well as social procedure at U B C . Even their departure to bigger and better things has not halted our communication and their tutor. I must thank both my family and friends for supporting my decision to enter the world of graduate school. They have been there for me at eveYy step, helping me attain my goals while keeping my ego in check. A large thank you goes to the Mcintosh lab support staff I have relied upon for intellectual dialogue, their technical abilities and for my sanity. I would like to recognize Cuilan, Maddy, Su-Jin and Shalea for all their assistance over the past two years. I could not have done any of this without your work. M y final acknowledgement goes to all of the graduate students in the department. It is comforting to know that there are always people who understand your particular struggles and triumphs. Good luck and best wishes to all of you! i PUBLICATIONS Winter, K . D. , J. A . Ehses, S. J. K i m , G . Eeson, R. A . Pederson and C. H . S. Mcintosh (2004). Glucose-dependent Insulinotropic Polypeptide (GIP) Activates P K B via a PBKinase and P L A 2 dependent mechanism in (3 (INS-1) cells. J. Biol. Chem. (Paper in Press). Winter, K. D., J. A . Ehses, S. J. K i m , S. Piteau, G . Warnock, R. A . Pederson and C. H . S Mcintosh (2004). Glucose dependent Insulinotropic Polypeptide (GIP) and long term DP IV inhibitor P32/98 treatment prevents glucolipotoxic mediated pancreatic |3 cell death. Diabetologia (Paper in Press). x C H A P T E R 1: I N T R O D U C T I O N 1.1 T H E I N C R E T I N C O N C E P T 1.1.1 GIP Discovery During the late 1870s Claude Bernard showed that although large doses of glucose administered to dogs intravenously (i.v.) resulted in glucosuria, similar doses of oral glucose did not (Bernard 1877). Based on this evidence and the discovery of secretin in 1902, Moore, Edie, and Abram suggested in 1906 that the duodenum might supply a "chemical excitant for the internal secretion of the pancreas" (Moore et al. 1906). Twenty years later La Barre and colleagues demonstrated that i.v. injection of a crude secretin preparation produced hypoglycemia in dogs via stimulation of the endocrine pancreas (Zunz and La Barre 1929; La Barre and Still 1930). The term "incretin", was introduced to explain the fact that two active components could be identified in secretin preparations: an incretin that stimulates the internal secretion of the pancreas, and an excretin, that stimulates the exocrine pancreas (La Barre and Still 1930). However, the relationship between the pancreas and the intestinal origin of incretins went largely unstudied until the development of the insulin radioimmunassay (RIA) allowed the demonstration that a much greater insulin response to oral glucose was observed when compared to i.v.glucose responses (Elrick et al. 1964; Mclntyre et al. 1964). Based on the difference between the data points, Perley and Kipnis estimated that around 50% of the insulin secreted after oral glucose is due to gastrointestinal elements (Perley and Kipnis 1967). Unger and Eisentraut'(1969) coined the term "Enteroinsular Axis" (Unger and Eisentraut, 1969) to describe the communication between the islets of Langerhan and the small intestine. This includes nutrient, neuronal, and hormonal gut signals that act on the islets and stimulate the secretion of insulin ((3-cells), glucagon (cx-cells), somatostatin (5-cells) and pancreatic polypeptide (PP cells). Creutzfeldt further re-defined the term incretin to include the hormonal component of the enteroinsular axis that 1) must be released by nutrients, particularly carbohydrates, and 2) must stimulate insulin secretion 1 in the presence of elevated glucose levels at physiological concentrations (Creutzfeldt 1979; Creutzfeldt and Ebert 1985). Although GIP qualifies as an incretin today, it was originally isolated from impure preparations of cholecystokinin-pancreozymin as a potent inhibitor of gastric acid and pepsin secretion ( C C K - P Z ) by Brown, Pederson and colleagues (Brown et al. 1969, 1970; Brown and Dryburgh 1971). The name gastric inhibitory polypeptide or " G I P " being introduced to describe its enterogastrone effects. GIP was only considered to have glucose-regulating effects once similar preparations of C C K were demonstrated to have insulinotropic activity (Rabinovitch and Dupre 1972; Dupre et al. 1973). Dupre and colleagues subsequently showed that intravenous infusions of GIP with glucose resulted in significantly increased insulin secretion when compared to glucose infusions alone (Dupre et al. 1973). In addition, this glucose-dependence was observed with in vivo studies in dogs (Pederson et al. 1975) and humans (Elahi et al. 1979) and in the perfused rat pancreas (Pederson and Brown 1976). Inappropriate stimulation of insulin secretion by GIP was therefore avoided by the requirement of a glucose threshold. To reflect the novel physiological function of GIP, the new name 'glucose-dependent insulinotropic polypeptide' was coined (Brown and Pederson 1976). These days, GIP and glucagon-like peptide-1 (GLP-1) are recognized to be the two main incretins that form the hormonal portion of the enteroinsular axis. Gastrin and secretin have been discredited as incretins because gastrin is not secreted in response to oral glucose and secretin does not promote insulin secretion at physiological concentrations. Additionally, since C C K fails to have significant insulinotropic effects in humans, it is also no longer considered an incretin. Today it is believed that GIP and GLP-1 are responsible for 50-70% of the postprandially secreted insulin (Fehmann et al. 1995). 1.1.2 GIP Distribution and Secretion GIP has been reported to be only present in K-cells of the proximal intestine in humans (Polak et al. 1973; Buchan et al. 1978), whereas in the rat and dog expression extends through to the ileum (Buchan et al. 1982). Immunoreactive GIP and GIP m R N A , 2 detected by in situ hybridization, have also been localized to the ductal cells of the submandibular salivary gland in rats. (Tseng et al. 1993, 1995). The role of GIP in the salivary gland and support for this finding in humans has not been established. Most recently, R T - P C R has been used to demonstrate the presence of GIP m R N A in human and mouse stomach extracts and distinct gastric GIP cells were identified by in situ hybridization and immunohistochemical analysis (Yeung et al. 1999). Of note in that study, a human GIP promoter driven transgene also resulted in reporter expression in the mouse pancreas. Although not all studies agree, GIP has been shown to be secreted in response to oral glucose (Cataland et al. 1974; Pederson et al. 1975), fat (Falko et al. 1975), protein (Wolfe et al. 2000) and amino acids (Thomas et al. 1978). Circulating concentrations of GIP have been reported to increase from fasting levels of 12-92 p M to 35-235 p M during a mixed meal (5-6 fold on average) depending on the antiserum used for measurement (Alam and Buchanan 1993). The discrepancy in ranges was attributed to low specificity of antisera raised against porcine GIP used in human or rat experiments (Alam and Buchanan 1993). In accordance with its role as an incretin, oral glucose increases IR-GIP secretion in humans (Cataland et al. 1974), dogs (Pederson et al. 1975), and rats (Pederson et al. 1982) whereas i.v. glucose does not. Support for the idea that glucose directly acts on the K-cel l was first suggested by the demonstration of a requirement for sodium-dependent active transport of monosaccharides (Morgan et al. 1979). The mechanisms underlying such nutrient-induced secretion have been difficult to examine due to the relatively low frequency of K-cells in the duodenum, although studies on a sub-clone of an intestinal tumour (STC-1) cell line also supported direct glucose effects on the K-cell (Kieffer et al. 1995A). Recent evidence has begun to highlight similarities between K-cells and pancreatic [3-cells. Both cell types express glucokinase and inward rectifying K + A T P channel subunits (Cheung et al. 2000; Ramshur et al. 2002). However, studies on STC-1 cells expressing insulin under control of the GIP promoter suggested that insulin secretion (and presumably also GIP secretion) were independent of glycolysis and the K + A T P channel (Ramshur et al. 2002). 3 Several studies have suggested that the ingestion of fat in humans could be a more potent stimulant for GIP secretion that glucose (Brown et al. 1975; Morgan 1996). In fact, fat results in a more prolonged elevation of the incretin, although in the absence of elevated glucose, GIP is not insulinotropic (Cleator and Gourlay 1975; Pederson et al. 1975; Falko et al. 1975; Morgan 1996). Studies on the effectiveness of protein or partially digested proteins as stimulants of GIP secretion have yielded varying results. Thomas and colleagues reported that an amino acid mixture served as a potent stimulus for GIP and insulin secretion in humans (Thomas et al. 1978). Conversely, meals of cod or steak, that are high in protein, failed to stimulate GIP secretion (Cleator and Gourlay 1975; Sarsan et al. 1980). A recent study contended that peptone can stimulate GIP secretion in the rat (Wolfe et al. 2000). Neural components to incretin secretion have also been identified. Rats subjected to bilateral subdiaphragmatic vagotomy in conjunction with gut transection, where gut input neurons are severed, displays complete abolishment of fat induced GLP-1 release (Rocca and Brubaker 1999). However, it is not clear if this neural component is required for both GIP and GLP-1 secretion, or just one of the two incretins. Various studies have shown that parasympathetic and sympathetic nerves can inhibit, stimulate or produce no change on GIP secretion (Mcintosh 1991). 1.1.3 Biological Actions of GIP A s previously mentioned, the demonstration that exogenously administered GIP inhibits gastric acid secretion in the denervated dog stomach supported the classification of GIP as an entrogastrone (Brown et al. 1969, 1970; Pederson and Brown 1972). However, due to the findings that GIP was just a weak inhibitor of gastric acid secretion in an intact stomach in humans (Maxwell et al. 1980) and dog (Soon-Shiong et al. 1979) questions arose as to its physiological role. Mcintosh et al. (1981) demonstrated that GIP was able to stimulate somatostatin release in the perfused rat stomach, with vagal stimulation or acetylcholine inhibiting this release. This finding may explain the reduced capacity of GIP to act on the innervated stomach, and implies that paracrine effects of somatostatin may be important in mediating GIP 's actions. GIP m R N A is reportedly 4 expressed in the rat and human stomach (Yeung et al. 1999) further suggesting a role for GIP in regulating gastric physiology. In light of these data, the current view is that the enterogastrone effects of GIP may be species-specific and may act additively with other enterogastrones to facilitate inhibition of gastric acid physiologically. The status of GIP in the regulation of gastrointestinal motility is also uncertain. GIP infusions in the canine stomach were shown to inhibit both pentagastrin and acetylcholine stimulated motor activity (Pederson and Brown 1972; Brown et al. 1975), while gastric relaxation was reported for the cat (Jansson et al. 1978). Therefore, although GLP-1 has been repeatedly shown to inhibit gastric emptying (Schirra et al. 1996; Willms et al. 1996; Nauck et al. 1997), more work is required to verify this role for GIP. It has also been suggested that GIP could regulate the secretion of the other main incretin, G L P - 1 . The proximal location of K-cells versus the distal distribution of the L -cells (GLP-1 secreting cell) in the small intestine, and the apparent discrepancy in the kinetics of release (GLP-1 is secreted before nutrients enter the distal gut), supports this hypothesis. GIP was observed to cause intestinal GLP-1 secretion in vivo in rats (Roberge and Brubaker 1993) and in vitro in the perfused ileum (Dumonulin et al. 1995; Hermann-Rinke et al. 1995). Similar in vitro studies have validated these findings, as GLP-1 release from rat intestinal cells (Brubaker 1991; Huang and Brubaker 1995; Saifia et al. 1998) and canine L-cells (Damholt et al. 1998) were stimulated by GIP. Such evidence gave birth to the notion of a proximal-distal loop whereby nutrients in the duodenum stimulate the release of GIP, which circulates to the distal L-cells and promotes GLP-1 secretion. Interestingly, this hypothesis has failed to hold true in human studies (Nauck et al. 1993a; Raufman 1996; Schirra et al. 1997). The neuropeptide G R P (gastrin-releasing peptide) has been proposed to be a component of this loop, whereby GIP stimulates afferent (vagal) nerves to regulate G L P - 1 secretion via intestinal G R P neurons independent from direct GIP effects on the L-cells (Roberge et al. 1996). Following the initial demonstration that GIP was insulinotropic in humans (Dupre et al., 1973) numerous studies have confirmed its insulinotropic activity in vivo in dogs (Pederson et al. 1975) and rodents (Lynn et al. 2001), and in vitro in the perfused pancreas (Pederson and Brown, 1976), isolated islets (Siegel and Creutzfeldt 1985; Shima et al. 1988) and (3-cell lines (Kieffer et al. 1993; L u et al. 1993A; Montrose-5 Rafizadeh et al. 1994). The insulinotropic effect elicited by GIP in humans was found to be absent under euglycemic conditions (Dupre et al. 1973) in agreement with a role, along with G L P - 1 , as a major incretin (Creutzfeldt 1979). In the perfused rat pancreas (Pederson and Brown 1976) a glucose threshold for GIP insulinotropic action of approximately 5.5 m M has been observed. In addition, studies on a GIP receptor knockout mouse (GIPR _ / ) (Miyawaki et al. 1999) have clearly provided support for an important insulinotropic role of GIP despite the compensatory actions of GLP-1 (Pamir et al. 2003). While the physiological role of GIP during fasting has received little attention, its role in stimulating postprandial insulin secretion is unquestioned. GIP has even been reported to account for up to 70 % of the postprandial insulin response (Nauck et al. 1986, Tseng etal . 1996). Not only does GIP possess the ability to stimulate (3-cell insulin secretion, GIP has also been demonstrated to regulate (3-cell gene expression. It is well documented that GIP influences insulin gene (Lu et al. 1993a; Fehmann and Gbke 1995) and protein expression (Wang et al. 1996). Studies on G I P R ' mice in our laboratory have confirmed these effects: despite compensatory GLP-1 actions, islet insulin m R N A and protein levels were both shown to be significantly decreased in GIPR null mice (Pamir et al. 2003). It was additionally shown in our laboratory that the rat insulin promotor 1 (RIP 1) is activated by GIP in INS-1 cells (Ehses et al. 2003). Further (3-cell genes affected by GIP include G L U T - 1 and hexokinase I (Wang et al. 1996). Enhancement of glucose stimulated insulin secretion by GIP has been suggested to involve several additional mechanisms, including increasing m R N A copies of insulin and crucial glycolytic enzymes. GIP also elicits effects on other islet cell types. GIP stimulates glucagon secretion under low (<5.5 mM) glucose concentrations in the perfused rat pancreas (Pederson and Brown, 1978), however this has not been observed in humans (Nauck et al. 1993A; Meier et al. 2001). A role for GIP in the regulation of somatostatin secretion (from 5-cells) has also been suggested (Schmidt et al. 1990). The studies described in the present Thesis focused on the novel role of GIP as a survival factor for (3-cells. Very recently, work from our laboratory, and others, implicated GIP in the regulation of cell growth (Trumper et al. 2001) and survival 6 (Triimper et al. 2002, Ehses et al. 2003) in a p-cell line (INS-1). The phenotype of G I P R ' mice also support this hypothesis, as these mice display dysregulation of islet size (Pamir et al. 2003). Furthermore, the use of DP IV inhibitors in streptozotocin-induced diabetic rats has implicated a role for GIP in the (3-cell protective effects conferred by D P IV inhibitor treatment (Pospisilik et al. 2003). Although definitive studies on the G I P R ' mouse (vs wild type) have not been completed, GIP appears to play a similar role to GLP-1 in its ability to regulate (3-cell mass (Buteau et al. 1999; X u et al. 1999; Stoffers et al. 2000). In contrast to G L P - 1 , there has been little interest in studying the survival effects of GIP due to the resistance to its effects observed in type 2 diabetes. 1.1.4 GLP-1 Discovery The discovery of G L P - 1 resulted from the cloning of c D N A s encoding preproglucagon from the pancreata and intestine of anglerfish (Lund et al. 1981, 1982). This led to recognition of a sequence that bore strong homology to GIP, and the proposal of a second incretin hormone encoded by the proglucagon gene (Lund et al. 1981). The three main sites of expression of the proglucagon gene include the a-cells of pancreatic islets, the L-cells of the distal ileum, colon, and rectum, and the nucleus tractus solitarius in the hindbrain (vagal nerve nucleus) (reviewed by Kieffer and Habener 1999). Today it is known that the major product of the proglucagon gene in the pancreas is glucagon, and in the intestine and brain post-translational processing results in the production of GLP-1 (7-37 and 7-36 amide) and G L P - 2 . The tissue-specific expression of these genes is accomplished via prohormone convertase (PC) mediated post-translational cleavage at specific basic amino acid residues. The enzymes responsible for the formation of G L P s in the intestine are PC 1/3, while PC2 liberates glucagon in the a-cell (reviewed in Kieffer and Habener 1999). 1.1.5 Secretion and Biological Actions of GLP-1 G L P - 1 , similar to GIP, is released following nutrient ingestion in response to carbohydrates, fats, and proteins. However, GLP-1 release seems to follow a biphasic 7 model, with early release mediated by hormonal and neural inputs (15-30 min) and nutrient input directly regulating later secretion (30-60 min) (Kieffer and Habener 1999). The concept of a proximal-distal loop controlling GLP-1 secretion was mentioned above. Due to the rapid response of GLP-1 secretion, the presence of nutrients in the proximal intestine is thought to evoke GLP-1 secretion from distal L-cells in an endocrine and neurally mediated pathway (Kieffer and Habener 1999). After its secretion, GLP-1 is rapidly cleaved at the amino terminus (histidine-alanine) by D P IV resulting in the accumulation of G L P - 1 9 V l N H 2 (reviewed by Kieffer and Habener 1999). There is considerable interest in the anti-diabetic actions of G L P - 1 , owing to its preserved insulinotropic action in type 2 diabetics (Nauck et al 1993B). Because of this, more effort has been directed at identifying physiological actions of G L P - 1 , compared to GIP. Despite parallel actions on the stomach and pancreatic (3-cells, there are a few divergences in the reported biological actions of the two polypeptides. These differences could be genuine or from a lack of GIP related research. GLP-1 has been shown to inhibit gastric acid secretion (enterogastrone effects) (Schjoldager et al. 1989; O'Halloran et al. 1990) and gastric emptying (Schirra et al. 1996; Wil lms et al. 1996; Nauck et al 1997) in humans. As with GIP, the insulinotropic actions of GLP-1 require glucose (Kreymann et al 1987; Holz et al. 1993), but it is not yet known if a glucose-dependence is a requirement of the proliferative or survival actions of GLP-1 (Buteau et al. 1999, 2001, 2003; L i et al. 2003; Hu et al. 2003). GLP-1 can inhibit glucagon secretion (Suzuki et al. 1989) and stimulate somatostatin secretion (Heller and Aponte 1995). In contrast to GIP, there are reports that GLP-1 regulates fasting glycemia and nonenteral glucose clearance in mice. (Baggio et al. 2000). The actions of GLP-1 on other insulin-sensitive tissues (liver, skeletal muscle, and adipose tissue) are not well elucidated. GLP-1 elicits an overall anabolic effect on these tissues, resulting in stimulation of glycogenesis and lipogenesis. Most surprisingly, these actions occur without a well-established demonstration of expression of the G L P - 1 receptor in these tissues (reviewed by Kieffer and Habener 1999). One of the more recently identified biological actions of GLP-1 is its contribution to regulating feeding behavior via anorexigenic actions in the brain (Turton et al. 1996). GLP-1 receptors exist in the brain (as with GIPR m R N A ) , and GLP-1 administrated into 8 the third intracerebral ventricle in rats causes a reduction in food consumption (Turton et al. 1996). However, it is unclear whether this occurs under physiological conditions, since GLP-1 receptor knockout mice (GLP-1 R'~) exhibit normal feeding behaviour (Scrocchi et al. 1996). Human studies do, however, support a satiety effect of GLP-1 during infusion of the peptide (Flint et al. 1998; Toft-Nielsen et al. 1998) and the lack of change in feeding behaviour in GLP-1R' mice could be due to compensation by other components of the satiety system. There is also preliminary evidence for the involvement of GLP-1 in the hypothalamus-pituitary-adrenal (HPA) axis. GLP-1 can promote thyroid stimulating hormone (TSH), luteinizing hormone releasing hormone (LHRH), and adrenocorticotropic hormone (ACTH) release (Kieffer and Habener 1999; Nussdorfer et al. 2000). As suggested above, the most heavily pursued interest in GLP-1 physiology currently is its effects on (3-cell fate. While many of these studies have used the long acting GLP-1 receptor agonist, exendin-4, data also support the claim that the GLP-1 receptor can regulate (3-cell proliferation (Buteau et al. 1999, 2001; Stoffers et al. 2000), differentiation/neogenesis (Xu et al. 1999; Hui et al. 2001; Abraham et al. 2002; Zhou et al. 2002) and survival (Li et al. 2003; Wang et al. 2004, Farilla et al. 2003). Intriguingly, trophic actions of GLP-1 have also been extended to 'neuronal' (PC-12) cells in culture (Perry et al. 2002A) and anti-apoptotic GLP-1 actions have been documented in hippocampal neurons (Perry et al. 2002B). 1.1.6 GIP and GLP-1 Receptors and Signal Transduction GIP belongs to the GPCR Family B subgroup and stimulation of its receptor activates adenylate cylase resulting in the production of cAMP. Wide support for this property of GIP exists as evidence has arisen from studies in pancreatic tumour cell lines (Amiranoff et al. 1984; Maletti et al. 1987; Lu et al. 1993B; Hinke et al. 2000), a gastric cancer cell line (Gespach et al. 1984), isolated islets (Siegel and Creutzfeldt 1985; Lynn et al. 2001), FACS sorted a- and (3- cells (Moens et al. 1996), heterologous expression models (Wheeler et al. 1995; Gelling et al. 1997), endothelial cells (Ahong et al. 2000), 9 and osteoblast-like cell lines (Bollag et al. 2000). Cyclic AMP responses to GIP have been reported with EC^ values ranging from approximately 200pM (Moens et al. 1996) to 30nM (Amiranoff et al. 1984) depending on the cell type. Insulin secretory responses to GIP are associated with increases in intracellular Ca 2 + (|;Ca2+li) (Wahl et al. 1992; Lu et al. 1993B; Usdin et al. 1993; Wheeler et al. 1995; Ding and Gromada 1997). Based on the effects of EGTA or nifedipine (a voltage-dependent L-type channel antagonist) it has been concluded that increased influx of extracellular Ca 2 + is involved in GIP-mediated (3-cell actions in mouse islets (Wahl et al. 1992), HIT-T15 (Lu et al. 1993B) and RINm5F (Usdin et al. 1993) insulinoma cells and COS cells (Wheeler et al. 1995). However, there is also evidence for mobilization of intracellular stores of Ca 2 + by GIP, although it was found to have no effect on IP3 (inositol- 1,4,5-triphosphate) levels in HIT-T15 cells (Lu et al. 1993B), pointing to an alternative to phospholipase C (PLC)-induced mobilization. GLP-1 also failed to induce inositol turnover in either HIT-T15 cells (Lu et al. 1993B), fibroblasts or COS cells (Widemann et al. 1994). However, some evidence does exist that GLP-1 can elevate IP3 in COS cells (Wheeler et al. 1993) and (3-cells (Zawalich et al. 1993; Zawalich and Zawalich 1996; MacDonald et al. 2002). Thus, there may be differences between the pathways by which GIP and GLP-1 signal in the (3-cell. GIP stimulated mobilization of intracellular Ca 2 + was also evident in other cell systems. GIP increased |Ca 2 +|; in a-cells (Ding et al. 1997), endothelial cells (Zhong et al. 2000), and osteoblast-like cell lines (Bollag et al. 2000). How the GIP receptor couples to these Ca 2 + fluxes has not been established. Current theories include cAMP/Protein Kinase A (PKA) effects on Ca 2 + or non-specific cation channels, cAMP stimulated PKA-independent effects on ryanodine receptors involved in calcium induced calcium release (CICR), nicotinic acid adenine dinucleotide phosphate (NAADP) effects (Masgrau et al. 2003) or arachidonic acid effects on Ca 2 + fluxes (Ehses et al. 2001). To understand the physiological relevance of these cAMP/PKA and |Ca2+1; signals, investigations using individual mouse (3-cells were conducted (Ding and Gromada 1997). By pharmacologically inhibiting PKA, support was generated for its role in regulating GIP-stimulated insulin secretion, but no immediate connection was made between cAMP/PKA signaling and rises in [Ca2+|;. It was assumed that GIP potentiated 10 insulin secretion via cAMP/PKA signaling is downstream to the increase in |Ca2+]j. To confuse the matter further, experiments have shown that cAMP can cause both Ca 2 + -dependent and Ca2+-independent insulin exocytosis (Ammala et al. 1993)! Therefore, the current paradigm is that both cAMP and PKA can have independent effects on insulin exocytosis, the former may affect late stages of the exocytotic process and the latter can affect mobilization of the readily releasable pool of insulin granules (Gromada et al. 1998). Newer studies on GIP-mediated signaling have focused on alternative second messengers to cAMP and Ca 2 + and the regulation of other protein kinases in addition to PKA. One kinase in particular, phosphatidylinositol 3-kinase (P13K), has been implicated in GIP receptor mediated signal transduction (Straub and Sharp 1996; Kubota et al. 1997; Trumper et al. 2001). Straub and Sharp first reported that the effects of GIP on insulin secretion from HIT-T15 cells were severely blunted by the PI3K inhibitor wortmannin (Straub and Sharp 1996). Kubota and colleagues (Kubota et al. 1997), demonstrated that GIP activation of mitogen-activated protein (MAP) kinase (also known as extracellular regulated kinases 1 and 2; ERK 1/2) was influenced by wortmannin suggesting involvement of PI3K. However, this pathway is not fully established since recent studies from our laboratory reported GIP stimulated ERK 1/2 activity is wortmannin insensitive (Ehses et al. 2002). Trumper has provided direct evidence for PI3K activation by GIP in INS-1 cells (Trumper et al. 2001). Despite these initial studies, throughout the 1990s there was very little information available about GIP receptor signaling and its impact on protein kinase cascades relative to GLP-1 and other GPCRs. Given the expansion in the signal transduction field throughout this period, there was much to be elucidated and the potential for identifying novel physiological actions based on these biochemical networks is an attractive possibility. The main focus of the present Thesis was to identify novel signaling mechanisms of the GIP receptor and functionally relate them to alternative GIP actions. Since cloning of the GLP-1 receptor by Thorens (1992), mRNA transcripts have been detected in the brain, heart, hypothalamus, intestine, kidney, lung, panncreatic islets and stomach. Controversy remains as to whether the GLP-1 receptor is also expressed in 11 adipose tissue, liver, and skeletal muscle. However, reports continue to highlight in vivo GLP-1 effects on these tissues, suggesting either secondary actions or an as yet, undiscovered novel GLP-1 receptor isoform present in these tissues (reviewed in Kieffer and Habener 1999)). Following its discovery, the GLP-1 receptor was shown to stimulate adenylate cyclase in (3-cell lines (Drucker et al. 1987; Lu et al. 1993b; Widemann et al. 1994), isolated islets and (3-cells (Ahren et al. 1996; Moens et al. 1996) and some other tissues (Dhillon etal. 1993; Thorens et al. 1993; Wheeler et al. 1993). The EC 5 0 for this response is similar to that reported for GIP (0.5 to 3nM) (Fehmann et al 1995). Not surprisingly, GLP-1 has been reported to activate PKA using both pharmacological inhibition (Gromada et al. 1998) and direct activity assays (Fehmann et al. 1994; Gao et al. 2002). Furthermore, |Ca 2 +|; has been reported to increase in response to GLP-1 in several (3-cell models (Wheeler et al. 1993; Holz et al. 1995; Ahren et al. 1996) and isolated (3-cells (Ding etal. 1997). GLP-1 signaling has been investigated in terms of insulin secretion, insulin gene transcription, and growth/survival effects in (3-cells. GLP-1 induced insulin secretion has been shown to increase cAMP/PKA, which can affect both K + A T P channel closure (Holz et al. 1993; Gromada et al. 1997) and C1CR via ryanodine-sensitive stores (Wheeler et al. 1993; Holz et al. 1995; 1999; Ahren et al. 1996; Ding et al. 1997). Effects on Ca 2 + influx via effects on VDCCs (voltage dependent calcium channels) and Ca2+-activated non-selective cation channels (NSCC) can additionally be exerted by GLP-1 (Leech and Habener 1997; Gromada et al 1998; Leech and Habener 1998). Several recent studies have shown cAMP-GEF-dependent, PKA-independent effects on CICR and insulin secretion calling into question the idea of PKA dependence (Kang et al. 2001, 2003; Kashima et al 2001; Tsuboi et al. 2003). It is now thought that GLP-1-induced PLC/IP3 mediated Ca 2 + signaling only provides a minor contribution to stimulation of insulin exocytosis (MacDonald et al. 2002). Similar to GIP, cAMP/PKA have been shown to potentiate insulin secretion at a site distal to elevations in |Ca2+jj (Ammala et al. 1993; Yajimaetal. 1999). GLP-1 has been implicated in insulin gene transciption in both a cAMP/PKA-dependent (Lawrence et al. 2002) and -independent manner (Skoglund et al. 2000; 12 Chepurny et al. 2002). GLP-1 has been reported to exert these effects via a CREB (cAMP response element (CRE) binding protein) family transcription factor acting on CRE elements of the rat insulin gene, however, the identity of this transcription factor remains elusive (Chepurny et al. 2002). Finally, the roles of cAMP/PKA in the proliferative and survival effects of GLP-1 are not completely understood. GLP-1 mitogenic actions on the (3-cell have been linked to PI3K, PKC (protein kinase C) and p38 MAP kinase signaling (Buteau et al.1999, 2001) and transactivation of the EGF (epidermal growth factor) receptor (Buteau et al. 2003). In addition, the pancreatic developmental transcription factor, PDX-1, has been implicated in the mitogenic actions of GLP-1, where GLP-1 has been shown to increase its DNA binding activity and translocation to the nucleus in a cAMP/PKA-dependent manner (Buteau et al. 1999; Wang et al. 2001). GLP-1 survival effects on the (3-cell were only discovered recently and have been attributed to caspase-3 inhibition in a cAMP and PI3K- (Li et al. 2003) and PKB-dependent manner (Buteau et al. 2004). Activation of the ERK 1/2 (Frodin et al. 1995; Buteau et al. 2001; Gomex et al. 2002) and p38 MAP kinases (Montrose-Rafizadeh et al. 1999; Buteau et al 2001) have also been shown to be coupled to GLP-I receptor activation in (3-cell lines and islets. Gomez et al (2002) demonstrated that GLP-1 activation of ERK 1/2 involves Ca 2 +- and PKA-dependent phosphorylation of Mek 1/2. However, input was found to be independent from Ras, Rap and Raf isoforms (A-Raf, B-Raf, C-Raf) (Gomez et al. 2002). Activation of p38 MAP kinase by the GLP-1 receptor is dependent on MKK3/6 in CHO cells, and thought to be downstream of PI3K in INS-1 cells (Buteau et al. 1999). 1.1.7 Regulation of (3-cell Growth and Survival It has been suggested that two main branches regulate adult pancreatic (3-cell growth: replication from existing (3-cells and neogenesis of new (3-cells from precursor stem cells (Bonner-Weir 2000). Overall (3-cell mass can be regulated by variations in cell number (hyperplasia), cell size (hypertrophy), neogenesis/differentiation, and cellular apoptosis. However, a recent report has refuted the idea of cell differentiation forming new (3-cells in the adult mouse, instead showing evidence of (3-cell self-replication 13 contributing to (3-cell neogenesis (Dor et al. 2004). Reports indicate that (3-cell mass exists in a fluctuating state, and that large variations in size and function allow for maintenance of plasma glucose levels within a very narrow range (Bonner-Weir 2001). The changes in (3-cell mass have been well characterized in an animal model of type 2 diabetes, the obese diabetic Zucker fa/fa (ZDF) rat (Lingohr et al. 2002). Initially during diabetes development, an increased (3-cell mass attempts to compensate for early insulin resistance. However, as obesity and glucose intolerance worsen with age, the (3-cell population can no longer meet the requirements of worsening insulin resistance (Lingohr et al. 2002). A similar decrease in (3-cell mass in another rodent model of type 2 diabetes, the sand rat Psammoninys obesus, is correlated with onset of type 2 diabetes, and has been attributed to an increase in (3-cell apoptosis, likely due to glucotoxity and/or elevated fatty acids (Donath et al. 1999), subsequently leading to a reduction in (3-cell mass. Recently, the notion of increased apoptosis correlated with decreased (3-cell mass has also been confirmed in histological studies on human pancreatic sections from type 2 diabetics (Butler et al. 2003). Thus, the factors regulating (3-cell mass are now recognized as an important determinant in the pathophysiology and the treatment of diabetes. Numerous growth factors, for both precursor and adult (3-cells, have been identified, however the best characterized include growth hormone (GH) and insulin-like growth factor 1 (IGF-1) (Linghor et al. 2002). These hormones both act on tyrosine kinase receptors, signaling via JAK2 (Janus Kinase )/STAT5a/b (signal transducers and activators of transcription) and IRS-2 (insulin receptor substrate-2) respectively, to regulate (3-cell mitogenesis. IGF-1 can also increase phosphorylation of ERK 1/2 MAP kinases, however, the biological implications of this in terms of (3-cell proliferation or survival are still unclear. The known survival actions of GH are mediated via increased expression of the anti-apoptotic protein Bcl-xL and members of the SOCS (suppressors of cytokine signaling) family. Survival actions of IGF-1 are mediated via a PI3K-dependent PKB signaling pathway that modulates the activity of numerous effectors important for cell survival, including GSK-3 (glycogen synthase kinase-3), (3-catenin, Bad, MdM2, procaspase-9, Rb (Retinoblastoma) protein, and FKHR-1 (Forkhead family transcription factor). If a central role is established for PKB in regulating the activity of 14 these downstream targets in |3-cells, it will define a key player in the regulation of cell proliferation and survival in these cells (reviewed in Lingohr et al. 2002). Pituitary adenylate cyclase activating protein (PACAP) and GLP-2, both members of the VIP-secretin-glucagon family of peptides, have been convincingly shown to act as anti-apoptotic agents in neurons and baby hamster kidney cells, respectively (Yusta et al. 2000, 2002; Vaudry et al. 2002). These actions are accomplished by cAMP-mediated inhibition of caspase activity that involves downstream kinase cascades promoting cell survival: Mekl/2 in the case of PACAP and PKB and GSK-2|3 mediating GLP-2 effects (Vaudry et al. 2002; Yusta et al. 2002). However, the mode of coupling between class II receptors and anti-apoptotic effects has been largely unexplored, and relatively little is known about anti-apoptotic signaling in the pancreatic (3-cell. Given the pleiotropic actions of Gas and G(3y coupled receptors, their effects on MAP kinase pathways, and the established actions of GLP-1, it was hypothesized that GIP could also regulate cell fate. Roles for GLP-1 and GIP in the regulation of (3-cell fate has only become recently apparent. GLP-1 was first shown to have mitogenic effects in INS-1 cells, acting via PI3K and p38 MAP kinase signaling (Buteau et al. 1999). This has since been confirmed in vivo (Xu et al. 1999; Stoffers et al. 2000) and further studies have implicated roles for PKC and transactivation of the EGF receptor (Buteau et al. 2001, 2003). Recently, anti-apoptotic actions mediated via the GLP-1 receptor have been confirmed in vivo with exendin-4 administration in STZ (streptozotocin)-induced diabetic mice (Li et al. 2003). However, little was clarified in terms of mechanism of action. GIP growth and anti-apoptotic actions were suggested (Triimper et al. 2001, 2002) supporting our notion of the importance of GIP in (3-cell survival. 1.1.8 Pathophysiology of the Incretins The role of the incretins in regulating glucose homeostasis is well documented and begs the question as to whether they contribute to the pathophysiology of diabetes or other diseases. No clear consensus has been reached regarding the roles of GIP or GLP-1 in the pathophyiology of diabetes mellitus or obesity, however, novel insights are beginning to emerge. In type 2 diabetics, IR-GIP levels have been reported to be 15 increased (Ross et al. 1977; Elahi et al. 1984; Jones et al., 1989), normal (levitt et al,. 1980; Service et al. 1984) or blunted (Groop 1989) following meal ingestion. In obese subjects, reports include elevated fasting IR-GIP levels (Salera et al. 1982, Mazzaferri et al. 1985) and either exaggerated (Cruetzfeldt et al. 1978; Elahi et al, 1979; Jones et al. 1989; Fukase et al. 1993), normal (lauritsen et al. 1980; Jorde et al. 1983; Amland et al. 1984), or blunted (Groop 1989) responses to oral meal ingestion. These inconsistencies are likely due to inappropriate RIAs that quantify C-terminal peptide levels (both active GIP,_42 and inactive GIP3.42). Recent evidence using N-terminal specific antibodies for GIP and GLP-1 suggest no defect in meal-regulated active GIP secretion, with an attenuation in late phase active GLP-1 secretion in type 2 diabetics (Vilsboll et al. 2001). However, despite this, the evidence that abnormalities in circulating GIP and GLP-1 levels are associated with type 2 diabetes is not strong. Perley and Kipnis (1967) first demonstrated a reduced or absent incretin effect in type 2 diabetics and Nauck et al. (1993b) later reported that this may be due to the ablation of GIP responses in these patients. In contrast, numerous studies have shown that type 2 diabetics are fully responsive to exogenous GLP-1, highlighting a role for this incretin as a therapeutic agent (Nauck et al. 1993b; Elahi et al. 1994). The evidence for blunted GIP actions has been extended to first-degree relatives of type 2 diabetics and a subgroup of type 1 diabetics (Meier er al. 2001; Greenbaum et al. 2002). The underlying mechanism resulting in blunted GIP actions in diabetes mellitus has been an area of interest in our laboratory and others. In 1998, two missense mutations in the GIP receptor were identified in humans, however, these mutations were not associated with type 2 diabetes in a Japanese study group (Kubota et al. 1996; Almind et al. 1998). More recently, our laboratory reported a reduction in islet GIP effects in the Vancouver fatty Zucker rat model of type 2 diabetes (Lynn et al. 2001). This is in agreement with an earlier proposal that the underlying pathogenesis of type 2 diabetes mellitus might involve defective GIP receptor expression (Hoist et al. 1997). Lynn et al. (2003) went on to show that expression of the GIP receptor is influenced by ambient glucose and fatty acid levels at the level of the (3-cell. Fatty acids were found to up-regulate GIP receptor mRNA levels under normoglycemic conditions, however hyperglycemia resulted in a 70 % reduction in receptor mRNA which was no longer 16 regulated by fat. Thus, nutrients are clearly able to regulate the GIP receptor and in this manner may contribute to the decreased responsiveness of GIP in type 2 diabetics. 1.2 D I P E P T I D Y L P E T I D A S E I V 1.2.1 Structure, Distribution and Function Dipeptidyl peptidase IV (DP IV, CD 26, EC 3.4.14.5) is a proline specific peptidase originally discovered by Hopsu-Havu and Glenner (1966) in the rat liver and later isolated from a variety of bacterial, insect and mammalian sources. DP IV belongs to the SC clan of serine proteases and is a highly-glycosylated, multifunctional ectopeptidase. Human DP IV cDNA encodes a 766 amino acid protein with 9 potential glycosylation sites and a molecular mass of approximately 88 kDa (Darmoul et al. 1992; Misumi et a. 1992). However, purified DP IV is found to be homodimeric, the glycosylated form yielding molecular weights in the range of 200-240 kDa depending on species, age, tissue and pathophysiology. Expression of DP IV has been found in many tissues of the body, with particularily high levels on the intestinal and renal brush borders and on endothelial cells of the circulatory system. Also varying levels of enzyme have been reported to be present throughout the haematopetic system (Shon et al. 1984, Hegaen et al 1990, Buhling et al. 1994, 1995), on the surface of hepatocytes (Hopsu-Havu and Glenner 1966), within pancreatic islets of Langerhans (Mentzel et al 1996) and in particular, co-localized with glucagon within secretory vesicles of the a-cell (Poulsen et al. 1993). Dipeptidyl peptidase IV has been classified as a non-classical serine-protease since the linear arrangement of the catalytic triad (Ser 630, Asp 709, His 741) is inverted with respect to the trypsin-like serine-proteases (His-Asp-Ser). The concensus sequence proposed for serine proteases (G-X-S-X-G), however, is centered to the catalytic S residue (GWSYG). The unique substrate specificity of this enzyme is probably attributed to the inverted triad arrangement. Mammalian DP IV prefers polypeptides less than 70-80 amino acids in length with prolyl, alanyl or seryl residues at the penultimate (P,) position, though longer peptides and those with alternate P, residues can serve as lower rate substrates (Pro Ala Ser/Gly Val/Leu). Recently, this generally accepted substrate 17 selectivity, whose definition is based on cleavage of short synthetic para-nitroanilide-containing peptides, has been shown to be less stringent when dealing with natural peptide substrates, with a number of seryl containing peptides displaying more favourable cleavage kinetics than the alanyl containing glucagon-like peptides 1 and 2 (Pospisilik et al. 2001, Lambeir et al. 2002). Bulky, hydrophobic or basic amino acids are preferred at P2 while proline residues are not accepted at P,. Though seemingly restrictive, the substrate specificity for DP IV includes a large number of neuropeptides, metabolic hormones and cytokines/chemokines (reviewed by Mentlein 1999). 1.2.2 DP IV regulation of Incretins All members of the glucagon superfamily of polypeptides satisfy the requirements of substrate specificity for DP IV due to their His-Ala, Tyr-Ala, or His-Ser N-termini. It is accepted the DP IV-mediated processing of several glucagon superfamily members constitutes a crucial determinant of their activity, whereas the physiological relevance of DP IV activity in the context of neuropeptides remains unclear. Since the incretins have a requirement for an intact N-terminus for receptor activation, rapid N-terminal truncation by DP IV in vivo represents a significant clearance pathway for active (full length) GIP and GLP-1. In fact, DP IV-mediated metabolism of these peptides reduces their clearance half-lives from 5-6 minutes (largely hepatic and renal clearance) to 1-2 minutes, a process reduced by addition of specific DP I V-inhibitors (reviewed by Drucker 2003). 1.2.3 DP IV Inhibition in Type II Diabetes In 1993, Mentlein and colleagues described GIP and GLP-1 as substrates for DP IV (Mentlein et al. 1993b), an idea postulated over ten years earlier after the purification of GIP3.42 from intestinal extracts (Brown et al. 1981; Jornvall et al. 1981). However it was the subsequent demonstration of the physiological importance of this enzymatic process that provided the basis for the development of a new therapeutic paradigm for type-II diabetes (reviewed by Drucker 2003). Kieffer and colleagues first demonstrated that GIP and GLP-1 are substrates of DP IV in vivo, and showed the rapidity of the 18 process, yielding a circulating half-life of 1-2 minutes for the parent peptides (Kieffer et al. 1995B). Considering that the N-terminally truncated products of GIP 3 4 2 and GLP-19. 36amide were previously shown to be inactive at the receptor level and thus non-insulinotropic (Schmidt et al. 1986, Suzuki et al. 1989; Hinke et al. 2002), a theory was postulated that DP IV-mediated truncation of the incretins served as the primary mechanism for GIP and GLP-1 inactivation (Pauly et al. 1996a). Several groups confirmed this hypothesis and went on to demonstrate the therapeutic potential of DP IV-inhibitors in augmenting circulating incretin levels (Pauly et al. 1996b; Hansen et al. 1999; Deacon et al. 2000). By taking advantage of the insulinotropic actions of the incretins, in addition to their ability to promote (3-cell glucose competence (Huypens et al. 2000), insulin gene transcription and biosynthesis (Drucker et al. 1987; Fehmann and Habener 1992), differentiation and growth (Hui et al. 2001; Buteau et al.2001) and ability to restore islet-cell glucose responsiveness (Zawalich et al. 1993), DP IV-inhibitor mediated-enhancement of their actions presents as a unique pleiotropic approach to the treatment of type-2 diabetes. Studies in rats using the specific, reversible DP IV-inhibitor P32/98 showed enhancement of insulin secretion and glucose tolerance after a single intraduodenal glucose bolus (Pauly et al. 1996a). Further studies by Pederson et al. (1986b) on the application of DP IV inhibitor during oral glucose tolerance testing in the obese Zucker rat model of type-II diabetes showed that these improvements were much more profound in diabetic "fatty" animals than in their lean littermates. Balkan et al. (1999) confirmed these findings using the DP IV inhibitor NVP-DPP728, and went on to provide direct evidence for the previously postulated stabilization of, and rise in, plasma active-GLP7_36 (GLP-la) after inhibitor treatment. Clinical trials are currently underway. 1.3 P R O T E I N K I N A S E B (PKB/Akt) 1.3.1 Introduction Protein kinase Ba (PKBa) was initially identified by homology cloning (Jones et al. 1991). The kinase domain is similar to that within protein kinase A (PKA) and protein kinase C (PKC), therefore it was named delated to A and C-Protein /kinase (RAC-PK); later changed to PKB (Jones et al. 1991; Coffer and Woodgett 1991). Soon after, the 19 product of a murine oncogene, v-Akt (AKT8 retrovirus), turned out to be a cellular homologue of PKB, termed c-Akt (Bellacosa et al. 1991). Two additional PKB family members have also been identified, PKBp7c-Akt2 and PKBy/c-Akt3 (Cheng et al. 1992; Brodbeck et al. 1999). The tissue distribution of PKB isoforms was recently determined using quantitative RT-PCR (Yang et al. 2003). In mouse tissues, both the a and |3 isoforms are ubiquitously expressed, whereas the y isoform is not detected in several tissues in which a and (3 isoforms are highly expressed, but is relatively highly expressed in brain and testis. PKB is highly expressed in insulin target tissues, such as fat cells, liver and skeletal muscle. The PKB family of kinases is evolutionarily conserved in eukaryotes ranging from C. elegans to man (except yeast). The amino acid identity between C. elegans and human PKB is around 60%, whereas that between mouse, rat and human is more than 95%. The three PKB isoforms share a similarity in their catalytic domain with a group of kinases from the AGC family that consists of more than 80 kinases (reviewed by Hanada et al. 2004). Most of these protein kinases are regulated by second messengers such as cyclic mononucleotides, Ca 2 + or phosphoinositides and many of them are thought to be transducers of cell growth or survival signaling (reviewed by Hanada et al. 2004). 1.3.2 Phosphorylation and Regulation of PKB Full activation of PKB is a multi-step process and several proteins responsible for each step have been identified and characterized (reviewed by Brazil and Hemmings 2001). A number of stimuli can promote activation of PKB through various activation mechanisms. The classical activator of PKB, PI3K was shown to be the major kinase responsible for PKB activation (Franke et al. 1995; Burgering and Coffer 1995). Growth factors facilitate the interaction of tyrosine-phosphorylated insulin receptor substrate (IRS) with the p85 regulatory subunit of PI3K leading to activation of the catalytic subunit, pi 10, which in turn phosphorylates phosphoinositides at the 3' position of the inositol ring, generating phosphotidylinsitol phosphate (PIP3). This increase in phosphorylated phosphoinositides leads to the localization of protein kinase B (PKB) to the plasma membrane through interaction of PIP3 and PIP2 with the pleckstrin homology 20 (PH) domain of PKB. Full activation of PKB appears to be dependent on the subsequent phosphorylation of two residues: Thr-308 in the activation loop of the kinase domain and Ser-473 in the carboxyl-terminal tail (Alessi et al. 1996). The protein kinase shown to phosphorylate Thr-308 is 3-phosphoinositide-dependent kinase-1 (PDK1) (Alessi et al. 1997; Stephens et al. 1998), whereas the kinase responsible for phosphorylation of the Ser-473 residue has not yet been identified. Once active, PKB regulates multiple biological processes, such as cell proliferation and apoptosis, suggesting that it may phosphorylate a number of target proteins (reviewed in Hanada et al. 2004) 1.3.3 PKB substrates and functions PKB isoforms contribute to a variety of cellular responses, including cell growth, cell survival and metabolism. PKB has been implicated in the regulation of apoptosis, or programmed cell death, through the phosphorylation of several key targets. One of these substrates, BAD (Bcl-2/Bcl-X antagonist) is a member of the Bcl-2 family of proteins that binds Bcl-2 and Bcl-X and inhibits their anti-apoptotic potential (Downward 1999). When BAD is phosphorylated on Serl36 by PKB, it does not exhibit proapoptotic activity in cells (del Peso et al. 1997; Datta 1997). The ability of phosphorylated BAD to inhibit apoptosis is thought to be regulated by its formation of complexes with other proteins. Once phosphorylated, BAD is released from a complex with Bcl-2/Bcl-X that is localized on the mitochondrial membrane, and forms a complex with 14-3-3 proteins (Stokoeetal. 1997). Glycogen synthase kinase 3a/(3 (GSK3a/|3) are PKB substrates that have been linked both to metabolism in the insulin signaling pathway and to cell survival. PKB phosphorylates GSK3a on Ser 21 and GSK3(3 on Ser 9 rendering these kinases inactive and probably improving cell survival (Pap and Cooper 1998). The mechanism for GSK3a/(3-induced cell death involves phosphorylation of eukaryotic initiation factor 2B (eIF2B) causing inhibition of protein synthesis (Pap and Cooper 2002). These factors have broader actions, as exemplified by the fact that GSK3(3 knockout mice die shortly after birth from TNF-a induced hepatic degeneration (Hoeflich et al. 2000). Taken together, it is evident that maintenance of appropriate levels of GSK3 activity is crucial. 21 Caspase-9 acts as an initiator and an effecter of apoptosis (Donepudi and Grutter 2002). In human cells, PKB can phosphorylate pro-caspase 9 on Serl96 in a Ras-dependent manner thus inhibiting the cytochrome c-mediated cleavage that is required for expression of its enzymatic activity. Mutation of Serl96 to Ala reduces the apoptosis-inducing activity of pro-caspase 9 transfected into fibroblasts (Cardone et al. 1998). However, it should be remembered that the PKB phosphorylation motif is not seen in caspase-9 from lower species such as mouse or rat. There could be two reasons for this: phosphorylation of caspase-9 by PKB is not a general phenomenon, i.e. is not a major pathway for caspase-9 activation, or phosphorylation of this site is specific for higher species, for example in the development of the central nervous system. A recent study has shown that PKB regulates apoptosis through transcription factors that are responsible for pro- as well as anti-apoptotic genes. PKB can regulate the Forkhead (FH or FoxO) family of transcription factors. To date, four isoforms of FH proteins (FKHR/FoxOl, Fox02, FKHRLl/Fox03 and AFX/Fox04) have been shown to be phosphorylated by PKB directly (Rena et al. 1999; Biggs et al. 1999; Wolfrum et al. 2003; Brunet et al. 1999; Kops et al. 1999). These phosphorylation sites, that are conserved throughout the FHKR family include one threonine site in the 14-3-3 protein binding domain and two serine sites located in the nuclear localization and export sequence. Phosphorylation of FH by PKB results in the exclusion of FH from the nucleus, leading to a decrease in the transcriptional activity that is required for promoting apoptosis. The target genes for the FH family are thought to be extracellular ligands, including the Fas ligand, TRAIL (TNF-related apoptosis-inducing ligand) and TRADD (TNF receptor type 1 associated death domain), and intracellular components for apoptosis like Bim (bcl-2 interacting mediator of cell death), a proapoptotic Bcl-2 family member and Bcl-6 (Burgering and Medema 2003). The transcription factor nuclear factor kappa-B (NFKB)/Rel family is a key regulator of the immune response, as well as other genetic programmes that are central to cell growth and survival (Li and Verma 2000). In most cases, activation of N F K B is dependent on the phosphorylation and degradation of IB, an inhibitor of N F K B , by the IB kinase (IKK) complex. PKB has been shown to regulate IKK activity in both a direct and indirect manner. It has been reported that PKB interacts with and phosphorylates IKK on 22 Thr23 in a PI3K-dependent manner that is required for N F K B activation in response to TNF stimulation (Ozes et al. 1999). PKB also phosphorylates Ser/Thr kinase Tpl-2 (or Cot) on Ser400, resulting in IKK complex activation (Kane et al. 2002). This N F K B activation by PKB might result in the inhibition of apoptosis by survival genes activated by N F K B (Li and Verma 2000). However, considering that N F K B activation can bring about the opposite effect in cellular responses that PKB contributes to, such as apoptosis, the physiological consequences of positive regulation of N F K B by PKB remain to be elucidated. 1.4 THESIS INVESTIGATION There exists a potential role for the incretin hormone, GIP, in the pathophysiology of diabetes mellitus and obesity. GIP acts on its GPCR in target tissues to promote specific physiological responses such as insulin secretion and cell survival. However, the intracellular mechanisms involved in the actions of GIP are not well understood. Considering the limited knowledge available on GIP receptor stimulated intracellular signal transduction, it is of great importance to understand these mechanisms and apply them to further biomedical research. Eludication of these intracellular signals will form a greater basic science knowledge base from which to apply strategies for therapeutic intervention in the treatment of diabetes, obesity, and food-dependent Cushing's syndrome. The present thesis investigation was undertaken to further eludicate intracellular signaling cascades mediated by the GIP receptor in a (3-cell line and human islets, with the hope of highlighting novel functional roles for the hormones. The following hypotheses were tested: Hypothesis I: Stimulation of the GIP receptor with GIP results in the activation of PKB in a PI3K dependent manner. Hypothesis 2: Stimulation of the GIP receptor with GIP regulates (3-cell fate via regulation of PKB signaling. 23 CHAPTER 2: METHODOLOGY 2.1 REAGENTS All chemicals, of reagent or molecular biology grade, were from Amersham Pharmacia Biotech (Mississauga, ON), BDH Inc. (Toronto, ON), Fisher Scientific International (Pittsburgh, PA, USA), Gibco Life Technologies Inc. (now Invitrogen Canada, Burlington, ON), Merck (Darmstadt, Germany), Perkin-EImer/Mandel Scientific/NEN Life Scientific Co. (Guelph, ON), Sigma (Oakville, ON) or VWR Canlab (Mississauga, ON). All tissue culture disposables were from BD Falcon (San Jose, CA, USA) and serum was from Cansera (Rexdale, ON). The DP IV inhibitor P32/98 (di-[2S,3S|-2-amino-3-methyl-pentanoic-1,3-thiazolidine fumarate) was synthesized as previously described (Demuth. 1990) and provided by Probiodrug, Halle, Germany. Specific sources for other chemicals are indicated in brackets in the following sections describing experimental methodology. 2.2 PLASMID DNA CONSTRUCTS The empty mammalian expression plasmid pRK5 and the plasmid pRK-|3ARKTct (encoding the C-terminus of (3-adrenergic receptor kinase I bp 495-689) were kindly provided by Dr. R. J. Lefkowitz for experiments targeted at investigating a role for G(3y signaling (Koch et al. 1994). PI3K constructs, Ap85 and pi 10CAAX (dominant negative and constitutively active), were donated by Dr. G. Rutter (Bristol UK). PKB constructs, murine Akt K179M mutant (PKB-HA), murine mys-Akt which contains a src myristroylation signal sequence (myr-PKB) (dominant negative and constitutively active) and pCMV5 vector were kind gifts from Dr. A. Toker (Harvard, MA). PTEN constructs, pCIS2 expression vector and PTEN-C129R (dominant negative) were generously donated by Dr. M. J. Quon (NTH, ML). 24 2.3 CELL CULTURE AND TRANSFECTION P-INS-I cells (clone 832/13) were provided by Dr. C. B. Newgard (Duke University). Cells were cultured in 11 mM glucose RPMI (Sigma) supplemented with 2 mM glutamine, 50 (3-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, and 10% fetal bovine serum (Cansera). Prior to the protein kinase analyses and glucolipotoxicity studies, cells were harvested into 6-well plates (at 2 x 106 cells/well) in 3 mM glucose media. Passages 45-70 were used in the described studies. Transfections in all subsequent experiments employed the reagent Lipofect2000IM (Invitrogen). In experiments targeted at investigating a role for PI3Kinase signaling in glucolipotoxicity, (3-INS-l cells were transiently transfected with plasmid DNA targeting PI3K, PKB and PTEN signaling. Briefly, 80-90% confluent monolayers in 6-well culture plates (BD Biosciences) were transfected with a 2:1 ratio of Lipofect2000™ to plasmid DNA ( L i L : [ i g DNA) according to the manufactures' protocol. Single wells in 6-well paltes were generally transfected with 5 [ig of DNA. Transfections were allowed to proceed for 5 hr in DMEM media in the absence of serum, and therafter regular growth medium was added to the cells overnight. All transfections were performed concurrently with green fluorescent protein to ensure transfection and approximate efficiency (25-40% for INS-1 cells). When increasing amounts of construct DNA were transfected, empty vector was added to ensure the same amount of total DNA was used in all cases. 2.4 CHARACTERIZATION OF SIGNAL-TRANSDUCTION PATHWAYS BY WESTERN BLOT ANALYSIS For protein kinase determinations, INS-1 (832/13) cells were harvested and plated into 6-well plates 2 days prior to overnight serum starvation to establish metabolic quiescence, and subsequent stimulation by GIP or other agonists was performed on day 3. Cells were preincubated for 1 h at 37 °C in modified RPMI medium containing 3 mM 25 glucose prior to the addition of agonists or pharmacological inhibitors. Following the elapsed stimulation period, cells were washed once with ice-cold PBS and lysed on ice. Pharmacological inhibitors were added for 15 min prior to agonist. addition and maintained in the presence of agonists. These included inhibitors of: PKA (H89, Rp-cAMP), PKC (Bis; GF109203x), Mek 1/2 (U0126), PI3K (wortmannin), CaM Kinases (KN-63, KN-62), Ca2+-independent PLA 2 (HELSS), receptor tyrosine kinases (genistein), voltage-dependent Ca 2 + channels (nifidipine) and Ca 2 + release from intracellular stores (thapsigargin) (Calbiochem, La Jolla, CA), and the Ca 2 + chelator EGTA. Forskolin, IGF-1, 8-pCPT-2'-0-Me-cAMP, 8-Cpt-cAMP, N6,2'-0-Dibutyryladenosine 3\5'-cyclic monophosphate and arachidonic acid (Calbiochem, La Jolla, CA) were added at time zero following addition of inhibitors. At the completion of incubation, cells were washed with RPMI medium (2 mM glutamine, 50 / Y M |3-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, 3mM Glucose and 0.1% BSA), and protein was extracted with cellular lysis buffer (0.5% Triton X-100, 60 mM (3-glycerophosphate, 20 mM MOPS, pH 7.2, 5 mM EDTA, 5 mM EGTA, 1 mM Na 3V0 4, 20 mM NaF, 1% Trasylol, and 1 mM phenylmethylsulfonyl fluoride). Thereafter, samples were sonicated (30 s) and centrifuged (12,000 rpm for 30 min), and protein content was quantified using the BCA reagent (Pierce) in order to ensure equal loading of gels for subsequent Western blotting. Protein samples (50 ug of protein/well) were separated on a 13% SDS-polyacrylamide gel and transferred onto nitrocellulose (Bio-Rad) membranes. Probing of the membranes was performed with antibodies against phospho-Thr 308 and phospho-Ser 473 in PKB, phospho-GSK3a/|3 (Ser-21/Ser-9), phospho-FHRK/AFX (Ser-256/Ser-196), phospho-BAD (Ser-136) and total PKB/Akt (Cell Signaling Technology; New England Biolabs). Bands were visualized by enhanced chemiluminescence (Amersham Biosciences) using horseradish peroxidase-conjugated IgG secondary antibodies. For quantification of band density, indicative of phosphorylation, films were analyzed using densitometric software (Eagle Eye (Stratagene, La Jolla, CA)). 2.5 ARACHIDONIC ACID RELEASE 26 Arachidonic acid release was determined by a method adapted from Shuttleworth and Thompson. Cells were harvested and passaged into 24-well culture plates at 2 x 105 cells/well for INS-1 cells. The respective media were replaced with media containing 0.125 /^ Ci/ml | 3 H|AA (PerkinElmer Life Sciences) for 18-24 h following passaging,and the plates were incubated for an additional 36-48 h. Prior to the addition of experimental agents, the wells were washed twice with 0.5 ml of serum free RPMI +0.1 % BSA and allowed to equilibrate for 1 h. Experiments were carried out in serum free RPMI containing 2 mM glutamine, 50 uM p-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, 3mM Glucose and 0.1% BSA. The agonists were dissolved in RPMI buffer, added in triplicate (0.5 ml total volume/well), and incubated for the length of time shown in the figure legends. As a positive control, ATP was added at a final concentration of 5 pM. In experiments investigating a role for G(3y in arachidonic acid release, cells were transfected as detailed above before being harvested from 10cm plates 18-24h. post-transfection and passaged into 24-well plates. After incubation, 0.4-ml aliquots were placed into scintillation vials followed by the addition of 10 ml of Econo 2 scintillation fluid (Fisher), and the radioactivity was determined by liquid scintillation spectrometry. AA released from cells was generally between 2-6% of total | 3 H|AA incorporated into cells. 2.6 HUMAN ISLET ISOLATION AND CULTURE Human islets were kindly provided by Dr. Garth Warnock, Head of the Ike Barber Islet Transplant Unit, Department of Surgery,Vancouver General Hospital. Islets were isolated from 5 organ donors between 20 and 65 years old, none of whom had a history of diabetes or metabolic disorders. Islets were separated from the surrounding exocrine tissue by enzymatic digestion, and then cultured overnight in regular RPMI 1640 medium containing 10% FBS, 10 mmol/l HEPES, 2 mmol/l L-glutamine, 1 mmol/l sodium pyruvate, 100 IU/ml penicillin and 100 /^ g/ml streptomycin. This was done in a humidified (5% C0 2 , 95% air) atmosphere at 37 °C. The next day, intact islets were directly used in experiments involving incubation under the conditions described in 27 Section 2.8 and Section 2.10. Cell survival was determined by two different methods: measurment of LDH release to the surrounding medium and caspase-3 activation. 2.7 DETERMINATION OF CELL SURVIVAL Cell survival was assessed in the presence of prolonged exposure to glucolipotoxicity (0.4mM palmitate and 20mM glucose). Cells were initially treated with increasing concentrations of palmitate bound to 0.5% BSA and glucose for 24 h. in serum free medium. Thereafter, increasing concentrations of GIP or GLP-1 were at the start of each experiment and again, 12 h. later. Following the 24 h. glucolipotoxic incubation, cell death was quantified by counting trypan blue stained cells. Maximum cell death was measured by counting trypan blue positive cells following Triton X-100 treatment for 1 hr in control wells. Final cell numbers were always less than the initial number plated (5 x 104 cells/well) in assessing cell survival. 2.8 CELL DEATH ASSAY Freshly isolated human islets, seeded into 6-well plates, were serum starved for 12-18 h (3 mM glucose RPMI 1640 with 0.1% BSA) and treated with 0.4mM palmitate and 20 mM glucose (glucolipotoxicity, GLTX) for 24 hrs in RPMI 1640 with 0.5%BSA. In studies of glucolipotoxicity, 100 nM GIP or 100 nM GLP-1 were added concomitantly with glucolipotoxic media. Cell death was determined using a cytotoxicity detection kit (Roche, Mannheim, Germany). This assay determines the percentage of dead cells, via measurement of lactate dehydrogenase (LDH) released into the medium divided by maximal LDH released per well after addition of Triton X-100 to a final concentration of 1% (100% death). LDH was measured using a colorimetric assay according to the manufacturer's instructions. 2.9 APOPTOSIS ASSAY (3-INS-l cells (5 X104cells per well) were plated in 96 well plates at a density of 28 5 x IO4 cells/well and incubated with 0.4mlvl palmitate and 20mIVl glucose (Sigma Chemical) for 24 hr. GIP (100 nM) or GLP-1 ( 100 nM) were added at the commencement of the incubaction and again at 12 hr. Quantitative detection of apoptotic cells was performed with the APOPercentage apoptosis assay kit (Biocolor Ltd., Belfast, Northern Ireland) according to the manufacturer's instructions. Briefly, following the 24 hr incubation cells were gently washed in PBS and incubated with the APOpercentage apoptosis dye for 3 hr at 37°C. The dye was then removed and the cells were gently washed with PBS and using a microplate colorimeter read plate absorbance at 550nm. 2.10 CASPASE-3 ACTIVITY Since the initial discovery that a cysteine protease (CED-3) is involved in apoptosis in the nematode Caenorhabditis elegans, caspase activation has been identifiable with the induction of cellular apoptosis (Strasser et al. 2000). The name 'caspase' is derived from the finding that these cysteine proteases cleave after an Asp residue in their substrates (Shi 2002). At least 14 distinct mammalian caspases have now been identified, and these are generally divided into initiator caspases (caspases-2, -8, -9, -10) and effector caspases (caspases-3, -6, -7). All caspases are initially found in cells as zymogens, and must undergo proteolytic cleavage to be activated during apoptosis. While initator caspases are autoactivated, effector caspases, such as caspase-3, are cleaved by initiator caspases resulting in their activation. Effector caspases are then responsible for the broad proteolytic cleavage resulting in cell death. This includes destruction of structural components, regulatory proteins, inhibitors of deoxyribonuclease, and DNA repair enzymes such as poly (ADP-ribose) polymerase (PARP) (Reviewed by Shi 2002). In the current study, caspase-3 activity was chosen as a measurement of apoptosis due to previous reports from our own lab, that GIP can increase P-cell survival by reducing caspase-3 activity (Ehses et al. 2003). (3-INS-l 832/13 cells or freshly isolated human islets, seeded into 6-well plates, were serum starved (3 mM glucose RPMI 1640 with 0.1% BSA) for 12-18 hr to establish metabolic quiescence. Following starvation, cells were treated with 0.4 mM palmitate and 20 mM glucose (glucolipotoxicity, GLTX) for 24 hr in RPMI 1640 with 0.5%BSA. In 29 studies examining the effects of glucolipotoxicity on caspase-3 activity, 100 n M GIP or 100 nM GLP-1 were added concomitantly with glucolipotoxic media. In studies in which inhibitors were used, all inhibitors (H89, U0126, wortmannin, or Bis) were added concurrently with glucolipotoxic medium ± GIP, GLP-1. Concentrations of inhibitors used were based on previous studies, and H89 has been shown to inhibit GIP-stimulated insulin secretion, ERK 1/2 phosphorylation1 and activation of rat insulin promoter activity at 5 and 10 pM (Ehses et al. 2002). Following treatment, caspase-3 activity was determined after 6, 12, or 24 h according to the manufacturer's protocol [standard: 7-amino-4-methylcoumarin (AMC), substrate: Ac-DEVD-AMC, inhibitor: Ac-DEVD-CHO; Molecular Probes]. Caspase-3 activity per well was assessed using a microplate fluorescence reader (Bio-tek FL600, excitation/emission at 360/460 nm), and corrected for total protein content using the BCA protein assay (Pierce, Roxford, IL). Caspase-3 activity was measured after 30 min. at room temperature (Manufacturer's protocol). 2.11 STUDIES ON THE VANCOUVER DIABETIC FATTY RAT The Vancouver Diabetic Fatty rat (VDF) is a substrain of the fa/fa Zucker (Zucker fatty; obese Zucker) rat that has been maintained at the University of British Columbia, Departement of Physiology for around 12 years. The fatty (fa;LeprJ") mutation, a Gln269Pro substitution in the extracellular domain of the leptin receptor, arose spontaneously in an outbred stock in the Zucker laboratory in 1961 (Zucker and Zucker 1961). Homozygous recessive littermates, devoid of a functional leptin receptor (reduced binding and signaling) (Yamashita et al. 1997), exhibit reduced production and secretion of satiety-inducing hormones (e.g. CRH) and an associated increase in orexigenic peptides such as NPY. The resultant defect in central autonomic regulation is believed to be the major causative factor for the metabolic abnormalities seen in the fa/fa Zucker rat. Reduced sympathetic responsiveness appears to contribute to a reduction in energy metabolism (particularly in brown adipose tissue) while enhanced parasympathetic signaling to the endocrine pancreas leads to hyperinsulinemia and hyperglucagonemia. Phenotypically, VDF rats are hyperphagic and severely obese. Significantly, increases in body weight over lean littermates can be seen as early as weaning (21 days of age), at 30 which point hyperinsulinemia and hyperlipidemia are also evident. Relatively profound insulin resistance is partially compensated for, and likely exacerbated by, (3-cell hyperplasia and an exaggerated insulin secretory response (left shifted and increased in magnitude). The glucose-dependence of numerous secretagogues has similarly been reported to be left-shifted. Extensive literature exists on the Zucker fatty rat (reviewed by Mcintosh and Pederson 1999) including detailed examinations of insulin resistance by means of the euglycemic-hyperinsulinemic clamp. This technique, considered the gold-standard of insulin sensitivity measurement, may be preformed in conscious animals and allows dissection of both hepatic and peripheral contributions towards whole body insulin sensitivity (Steele 1959). The obese Zucker rat is one of the best-accepted animal models of type-2 diabetes due to the strong link between the metabolic abnormalities and obesity exhibited in the animal. The VDF rat in particular, displays a relatively mild fasting hyperglycemia, but marked glucose intolerance post prandially making it especially well-suited to modeling the disorder. Similarly, both the VDF rat and the human type-2 diabetic patient have been shown to exhibit an abnormally reduced incretin effect (Perley and Kipnis 1967, Nauck et al. 1993, Elahi et al. 1994, Lynn et al. 2001). Lynn et al. (2003) recently linked this abnormaility, in the VDF rat, to a deficit in islet GIP-receptor expression both at the mRNA and protein levels. In a subsequent study, the same group went on to propose a model of glucose-stimulated downregulation of the GIP-receptor, providing the first potential mechanism for the long-recognized defect in the diabetic state (Lynn et al. 2003). 2.12 ANIMALS Four pairs of male fatty (fa/fa) and lean VDF Zucker rat littermates were randomly assigned to a control or treatment (P32/98) group at ~600 g body wt (age 15 ± 1 weeks). Animals were housed on a 12-h light/dark cycle (lights on at 0600) and allowed access to standard rat diet and water ad libitum. The techniques used in this study were in compliance with the guidelines of the Canadian Council on Animal Care and were 31 approved by the University of British Columbia Council on Animal Care, Certificate # A99-006. 2.13 PREPARATION OF ZDF PLASMA To study the effects of glucolipotoxicity on INS-I cell fate, plasma was prepared from lean and obese rats. Four pairs of obese and lean VDF Zucker rats were sacrificed and blood was collected. Blood was centrifuged at 3500 rpm for 20 min and plasma removed from the clotted blood. Resulting plasma was used immediately or stored at -20°C. 2.14 PROTOCOL FOR DAILY MONITORING AND DRUG ADMINISTRATION The treatment group received P32/98 (10 mg/kg) by oral gavage twice daily (0800 and 1700) for 100 days, and the control animals received concurrent doses of vehicle consisting of a 1% cellulose solution. Every 3 days, body weight was assessed. Blood samples were acquired from the tail, and glucose was measured using a SureStep analyzer (Lifescan Canada, Burnaby, Canada). 2.15 PROTOCOL FOR MONTHLY ASSESSMENT OF GLUCOSE TOLERANCE Every 4 weeks from the start of the experiment, an oral glucose tolerance test (OGTT; 1 g/kg) was performed after an 18-h fast and complete drug washout (12 circulating half-lives for P32/93). No 0800 dose was administered in this case. Blood samples (250 p\) were collected from the tail using heparinized capillary tubes, centrifuged, and stored at -20°C. Plasma insulin was measured by radioimmunoassay using a guinea pig anti-insulin antibody (GP-01), as previously described (Jia et al. 1995), and blood glucose was measured as described above. Plasma DP IV activity was determined using a colorimetric assay measuring the liberation of p-nitroanilide (A405 nm) from the DP IV substrate H-gly-pro-/?NA (Sigma; Parkville, Ontario, Canada). It is 32 important to note that the assay involves a 20-fold sample dilution and therefore underestimates the actual degree of inhibition occurring in the undiluted sample when using rapidly reversible inhibitors such as P32/98. 2.16 ISOLATION AND CULTURE OF RAT PANCREATIC ISLETS Rat pancreatic islets were isolated as previously described (Van der Vliet et al. 1988). Briefly, the rat was anesthetized, and a midline incision was made. The common bile duct was cannulated, and the pancreas was inflated with collagenase (320 mg/1, Type XI; Sigma) in Hank's balanced salt solution supplemented with 10 mmol/l HEPES, 2 mmol/l L-glutamine, and 0.2% BSA (HBSS+) (Life Technologies, Burlington, ON, Canada). The pancreas was then removed from the rat and macerated with scissors before collagenase digestion. The pancreatic tissue was initially digested in a shaking 37°C water bath for 20 min and 10 min for pancreases from lean and fat rats, respectively; a second digestion was then carried out for 10 and 7 min for the lean and fat rats, respectively. After the collagenase digestions, the islets were passed through a 1-mm nylon screen and separated from exocrine tissue via centrifugation (l,000g at 4°C) through a discontinuous dextran gradient. Finally, islets were picked under a dissecting microscope, washed in HBSS+, and used for mRNA isolation or cultured in RPMI 1640 with 5.5 mmol/l glucose, 10% FBS (Cansera, Rexdale, ON, Canada), antibiotics (50 U/ml each penicillin G and streptomycin), 0.07% human serum albumin, 0.0025% human apotransferrin, 25 pmol/l sodium selenite, and 20 /^ mol/l ethanolamine hydrochloride for 20-24 h in 10-cm plastic culture dishes (Falcon; Becton Dickinson) in a humidified 5% C0 2 environment. 2.17 WESTERN IMMUNOBLOT ANALYSIS For in vitro glucolipotoxicity studies, (3-INS-l cells were harvested from 6 and 12 well plates. VDF Zucker and lean rat islets were harvested from the animals as described in Section 2.16. Cells were washed with RPMI medium (2 mM glutamine, 50 pM (3-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, 3mM glucose and 0.1% BSA), 33 and protein was extracted with cellular lysis buffer (0.5% Triton X-100, 60 mM (3-glycerophosphate, 20 mM MOPS, pH 7.2, 5 mM EDTA, 5 mM EGTA, 1 mM Na3V04, 20 mM NaF, 1% Trasylol,and 1 mM phenylmethylsulfonyl fluoride). Thereafter, samples were sonicated (30 s) and centrifuged (12,000 rpm for 30 min), and protein content was quantified using the BCA reagent (Pierce) in order to ensure equal loading of gels for subsequent Western blotting. Phospho-Ser 473 PKB, total PKB, PKBa/(3, BAD and PARP antibodies were obtained from Cell Signaling Technology (New England Biolabs) while (3-Tubulin antibody was purchased from Santa Cruz Biotechnology (San Jose, CA) and 1RS-2 antibody from Upstate Biotechnology (Boston, MA). Protein samples (30 pig of protein/well) were separated on a 13% SDS-polyacrylamide gel and transferred onto nitrocellulose (Bio-Rad) membranes. Probing of the membranes was performed with the above mentioned antibodies, and bands were visualized by enhanced chemiluminescence (Amersham Biosciences) using horseradish peroxidase-conjugated IgG secondary antibodies. For quantification of band density, indicative of phosphorylation, films were analyzed using densitometric software (Eagle Eye (Stratagene, La Jolla,CA)). 2.18 DATA ANALYSIS Data are expressed as means ± S.E.M. with the number of individual experiments presented in the figure legends. All data were analyzed using the nonlinear regression analysis program PRISM (GraphPad, San Diego, CA), and significance was tested using the Student's t test and analysis of variance (ANOVA) with the Dunnett's multiple comparison test, Tukey post-test or the Newman-Keuls post test as indicated in figure legends. Statistical significance was set at 5%. 34 CHAPTER 3: REGULATION OF PKB SIGNALING BY GIP 3.1 PROJECT RATIONALE Previous reports have suggested that GIP can activate PKB in the rat (3-INS-l cell line. The major pathway by which GIP has been shown to act is via G-protein coupled receptor mediated activation of adenylyl cyclase and cAMP production. Work from our laboratory has suggested that GIP activation of PLA2 and stimulation of arachidonic acid production is also involved in the regulation of insulin secretion. The major objective of the current studies was to establish the pathways by which GIP activates PKB using the [3 (INS-1) cell model. 3.2 RESULTS 3.2.1 GIP stimulates phosphorylation of PKB and the down-stream targets Bad (Ser 136), GSK3 ct/|3 (Ser 21/9) and FHRK/AFX (Ser 256/196) in |3 (INS-1) cells. Protein kinase B (PKB) showed significantly increased phosphorylation within 15 minutes of stimulation by GIP and maximal phosphorylation of both the Threonine (Thr) 308 and Serine (Ser) 473 phospho-sites at 60 min. Phosphorylation was sustained for 120 min (Fig. 1A). GIP-mediated phosphorylation of both PKB Thr 308 and Ser 473 demonstrated concentration-dependence, with an EC^ values of 6.59 ± 0.21 nM (PKB 308) and 3.55 ± 0.137 nM (PKB 473) and maximal phosphorylation levels reaching 4 times basal (Fig. 1B). 35 B ° o 0 . 7 H O .2 0.5CH CD i . 0.25-1 i t : Q -O.OO-1 Time (min) pPKB 308 1.2&1 • PKB Thr 308 PKB Ser 473 1.00--0.75-0.50-0.25-0.00-25 50 75 Time (min) 1 1 1 100 125 150 -12 -I— -11 —I— -10 -8 15 30 60 120 pPKB 473 PKB LogJGIP] -11 pPKB 308 L o g 1 0 [ G I P ] -10 -9 -8 -7 pPKB473 PKB Figure 1. GIP stimulates P K B phosphorylation in a time (A) and concentration (B) dependent manner in INS-1 (832/13) cells. Cells were stimulated in R M P I serum free medium for 60 min (B) or indicated times (A). Stimulation was stopped by the addition of ice-cold lysis buffer, followed by sonication before centrifugation. Protein was quantified using the B C A assay and 50u.g of protein were loaded/lane for Western blotting. Membranes were probed with anti-phospho-PKB Thr 308 and anti phospho-P K B Ser 473 antibodies, prior to densitometry analysis. Data represent mean ± S . E . M . (A, n=4;B, n=4), where * indicates p<0.05 compared with respective controls ( A N O V A , with Dunnett's multiple comparison test and Newman-Keuls post hoc). P K B has been demonstrated to phosphorylate a number of downstream targets in different cell types. In p(INS-l) cells, B A D Ser 136, G S K 3 a/(3 Ser 21/9 and F H R K / A F X Ser 256/196 were all phosphorylated with a time-course that paralleled that of P K B , peaking at around 45-60 min and sustaining phosphorylation up to 120 min (Fig 2 A - C ) . Phosphorylation of all substrates also demonstrated similar GIP concentration-dependence as P K B , with E C S ) values of 9.01 ± 0.19 n M and 78 ± 0.18 n M for GSK3a/ |3 , 10.1 ± 0 . 1 6 n M for B A D , and 6.60 ± 0 . 1 9 n M and 16.2 ± 0 . 1 7 n M for F K H R and A F X (Fig. 2D-F). 36 -pGSKli Ser 21 -pGSK3[iSer9 -m- pBad Ser 136 pBAD Ser 136-Time (mm) 75 100 125 150 Time (min) D tS 100' 0 . 2 17* | I 0.50. 1 rx 0.25. g 0.00-• 0GSK3.Ser21 l pGSK3,',9 GSK3.EC = 901*0.19nM GSK3,iEC = 8 78*0.18nM Log, 0 [GIP] pGSK3.Ser21-pGSK3)iSer9 • [£ Log [GIP] 1 1.25-O 5 8 1.0O | S 0.75-1 • s s 0.50-e 3 a s a or 0 25-< ~ GO 0 oo-pBAD Ser 136 EC 5 0 = 10 1 sO 16 nM -12 -11 -10 -9 -S -7 Log i 0(GIP] 15 30 60 120 pBAD Ser 136-Log,o(GlP) -pFKHR Ser 256 -pAFX Ser 196 50 100 150 Time (min) £ 1.25-1 |S 100-S = » £ 0?5-2 . °- = 0.50-x 2 < rx 0.25-£ — 5 o.oo--10 -9 -8 Log, 0 [GIP] • pFRHR Ser 256 A pAFX Ser 196 FKHR EG = 6 60 » 0 19 nM AFX EC. = 16 2 . 0 17 nM pFKHR Ser 256—H pAFX Ser 196 — l pFKHR Ser 25SH pAFX Ser 196 -1 Time (mini 0 5 15 30 60 120 Log [GIP] -11 -10 -9 -8 -7 -6 Total PKB —1 • — Total PKB — Figure 2. GIP stimulates phosphorylation of GSK3a/p\ BAD and FKHR. INS-l cells were stimulated with GIP in RPMI containing 3 mM glucose for specified time periods (A-C) or 60 min (D-F). Fifty u.g protein samples were separated by SDS-PAGE, and membranes were blotted with phospho-GSK3a/(3 Ser 21/9 (pGSK3a/(3) antibody, phospho-BAD Ser 136 (pBAD 136) antibody or phospho-FKHR/phospho-AFX Ser 256/196 (pFKHR/pAFX) antibody prior to densitometric analysis. Data represent mean ± S.E.M. (A-C, n = 3;D-F, n-3), where * indicates p<0.05 compared with respective controls (student's t test), and blots are representative. 37 3.2.2 GIP-mediated activation of PKB involves G-protein |3y subunit activation of PLA2 and Arachidonic Acid Production It has been previously shown that GIP signals via both cAMP and arachidonic acid (AA) pathways in rGIP-15 cells and cAMP in (3-cell lines (Ehses et al. 2001). To identify potential proximal activators of the PKB module, we tested the ability of the diterpene forskolin, and arachidonic acid (AA) to increase PKB phosphorylation. Since both agents stimulated phosphorylation of Thr 308 and Ser 473 to a similar degree as GIP it would appear that both arachidonic acid and cAMP-mediated pathways are involved (Fig. 3A). However, when the effects of two cAMP analogues on INS-1 cell PKB were tested, neither 8-(4-Chlorophenylthio)-2'-0-Me-cAMP (8-OMe), a specific activator of the EPAC pathway, nor 8-(4-Chlorophenylthio-cAMP) (8-Cpt), which specifically activates the PKA pathway (Fig. 3B), had any effect on either phospho-site. However, the cAMP analogue, N6,2'-0-Dibutyryladenosine 3',5'-cyclic monophosphate (MB-cAMP), which can also act on cAMP-dependent channels in the plasma membrane did stimulate phosphorylation of PKB supporting a role for cAMP in activating PKB (Fig. 3B). The forskolin-mediated effects on PKB phosphorylation may thereore be neither PKA nor EPAC mediated, but rather by stimulating Ca 2 + influx (See studies on CAM Kinase in Section 3.2.3). We have previously shown that GIP activates a Ca2+-independent PLA 2 (iPLA2) in (3TC-3 cells (Ehses et al 2001), and the effect of the specific iPLA2 inhibitor, HELSS, on PKB Ser 473 phosphorylation in (3(INS-1) cells was therefore examined. The presence of HELSS ablated both GIP- and forskolin-stimulated PKB Ser 473 phosphorylation (Fig. 6C), suggesting the involvement of iPLA2 activation in both pathways. In order to establish that activation of iPLA2 in INS-1 cells resulted in significant arachidonic acid production, efflux of | 3 H|AA from pre-labelled cells was studied (Fig. 4). Similar to the (3-TC-3 cell line, increases in | 3 H|AA efflux in response to GIP were found to occur in low glucose concentrations (3 mM), indicating that GIP-induced and glucose-induced increases in AA release were mediated via separate pathways. Analysis of the time dependence of AA release in INS-1 cells demonstrated maximal release at 45 min (Fig. 4B), which correlates well with that for GIP-stimulated PKB activation (maximal plateau 38 reached at lhr in INS-I cells; n = 3). AA release also demonstrated a GIP concentration-dependence with increases in AA release seen at nanomolar concentrations of GIP (Fig. 4A). ATP and forskolin also stimulated AA release from INS-1 cells after 45 minutes of stimulation (Fig. 4B and 4C). GIP-stimulated release of arachidonic acid in rGIP-15 cells (Ehses et al. 2001) was shown to be dependent upon G-protein (3y dimers, although no such involvement was found in the (3TC-3 cell line. However, as shown in Figure 5, when INS-1 cells were transiently transfected with (3-ARKct ((3-adrenergic receptor kinase C-terminal tail), a competitor peptide that effectively inhibits G(3y subunit action, there was a significant reduction in GIP-stimulated PKB Ser 473 phosphorylation. No such effect was seen when phosphorylation was stimulated with forskolin. Expression of (3-ARKct also significantly suppressed the GIP-mediated stimulation of arachidonic acid production by almost 45% (Fig. 4D, p < 0.05). Therefore, GIP activation of PKB requires G(3y subunits for signalling in |3 (INS-1) cells. A B Basal Forsk 8-OMe 8-Cpt MB-cAMP Basal Glue GIP AA IGF-1 , . Figure 3. Forskolin, arachidonic acid and cAMP regulate PKB in INS-1 (832/13) cells. Cells were stimulated in RPMI buffer for 60 min containing 3mM glucose. In A, agonist concentrations were 1 ImM glucose (Glue), lOOnM GIP (GIP), 100[iM arachidonic acid (AA) and lOnM IGF-1 (IGF-1). In B, agonist concentrations were 10)iM forskolin 39 ( F o r s k ) , IOOJXM 8 - ( 4 - C h l o r o p h e n y l t h i o ) - 2 ' - 0 - M e - c A M P ( 8 - O M e ) , l O O f x M 8 - ( 4 -C h l o r o p h e n y l t h i o - c A M P ) ( 8 - C p t ) a n d 1 0 u . M N 6 , 2 ' - 0 - D i b u t y r y l a d e n o s i n e 3 ' , 5 ' - c y c l i c m o n o p h o s p h a t e ( M B - c A M P ) . F i f t y ^xg p r o t e i n s a m p l e s w e r e s e p a r a t e d b y S D S - P A G E a n d m e m b r a n e s w e r e b l o t t e d w i t h p h o s p h o - P K B T h r 3 0 8 a n t i b o d y ( p P K B 3 0 8 ) , p h o p h o -P K B S e r 4 7 3 a n t i b o d y ( p P K B 4 7 3 ) a n d t o t a l P K B a n t i b o d y . D a t a r e p r e s e n t m e a n ± S . E . M . (n=4) , w i t h * p < 0 . 0 5 c o m p a r e d to basa l c o n t r o l s ( A N O V A ) . I I I I IUU 1 1 I 1 Basal HmMGIu 5 | iM Forsk 1 0 M M Forsk Basal 10nM GIP 100nM GIP 5 | iM ATP F i g u r e 4. T h e e f f ec t o f G I P o n a r a c h i d o n i c a c i d r e l e a s e f r o m I N S - 1 c e l l s ( A , B , C ) o r t r a n s f e c t e d I N S - 1 c e l l s ( D ) . T h e c e l l s w e r e p r e l a b e l l e d w i t h | 3 H | A A f o r 3 6 - 4 8 h r a n d p r e i n c u b a t e d i n R P M I f o r 1 h o u r p r i o r to the a d d i t i o n o f a g o n i s t s . T h e m e d i u m w a s r e m o v e d at s p e c i f i c t i m e p o i n t s ( A ) o r 6 0 m i n f o r ( B , C , D ) , a n d the r a d i o a c t i v i t y m e a s u r e d b y l i q u i d s c i n t i l l a t i o n c o u n t i n g . F o r D , I N S - 1 c e l l s w e r e t r a n s f e c t e d w i t h 10p,g o f e i t h e r v e c t o r ( p R K ) o r the C t e r m i n u s o f (3-adrenerg ic r e c e p t o r k i n a s e ( p A R K c t ) 1 d a y p r i o r to e x p e r i m e n t s u s i n g L i p o f e c t 2 0 0 0 ( I n v i t r o g e n ) . D a t a r ep re sen t m e a n ± S . E . M . ( A , n=4;B, n=4;C, n=3,D, n-3), w h e r e * i n d i c a t e s p < 0 . 0 5 c o m p a r e d w i t h r e s p e c t i v e c o n t r o l s ( A N O V A ) . 4 0 pRK (J-ARKct Forskolin GIP p P K B 4 7 3 Total PKB c o w 52 a o i/> ~ o <u J = > " I 1.25n 1.00+ 0.75H 0.50H CQ CL ib. 0.25H 0.00' C D P R K • • bARKct F i g u r e 5. GIP mediated PKB phosphorylation is G(3y dependent in INS-1 cells. INS-1 cells were transfected with 10|ig of either vector or construct 2 days prior to experiments using Lipofect2000 (Invitrogen). Fifty-p,g protein samples were separated by SDS-PAGE, and membranes were blotted with phospho-PKB Ser 473 (pPKB 473) and PKB (PKB) antibody to assess total PKB. Blots are representative of three independent experiments with similar results. 3.2.3 GIP-mediated activation of PKB involves Activation of PBKinase and is Ca 2 +-dependent In order to identify the up-stream kinase pathway(s) involved in GIP-mediated PKB activation, the effects of several pharmacological inhibitors on PKB (Ser 473) phosphorylation were tested in concentrations at which they had been previously shown to act selectively (Ehses et al. 2002; Arnette et al. 2003; Trumper et al. 2002). {J-(INS-l) cells were incubated with each inhibitor for 30 min prior to exposure to either GIP or forskolin for 60 min. PKB phosphorylation is classically associated with the up-stream activator, PBKinase, and treatment of INS-1 cells with the PBKinase inhibitor 41 wortmannin completely ablated GIP- or forskolin-induced phosphorylation of PKB Ser 473 (Fig. 6A). Neither the PKA inhibitor, H89, nor the competitive inhibitor, Rp-cAMPs, inhibited PKB (Ser 473) phosphorylation in response to either GIP or forskolin (Fig 6A). Similar results were obtained with the Mekl/2 inhibitor U0126 (Fig 6A), the PKC inhibitor GF109203x (2/^ M) or the non-specific tyrosine kinase inhibitor, Genistein. As a positive control, genistein ablated PKB Ser 473 phosphorylation when stimulated by IGF-1 (Fig6C). Although the HELSS study showed that a Ca2+-independent PLA 2 was involved in the GIP activation pathway for PKB, there is evidence for the involvement of Ca 2 + in GIP-stimulated PKB activation (Trumper et al. 2002). As shown in Figure 2B, stimulation of PKB Ser 473 phosphorylation by both GIP- and forskolin were significantly inhibited by both the Ca 2 + chelator, EGTA, and thapsigargin, an inhibitor of Ca 2 + release from intracellular stores, whereas the L-type calcium channel blocker, nifedepine, had no effect on responses. Members of the family of calmodulin-activated (CaM) kinases are potential mediators of the Ca2+-related responses and both CaM Kinase inhibitors, KN-93 and KN-62, inhibited GIP- and forskolin-stimulated PKB Ser 473 phosphorylation in p(INS-l) cells (Figs. 6B). 42 +H89 +Rp-cAMP + Wort +U0126 + Bis B GIP F B GIP F B GIP F B GIP F B GIP F B GIP F pPKB 473-PKB B pPKB 473 . PKB i-EGTA -Nif B GIP F B GIP F B GIP F + Thaps +KN-93 + KN-62 B GIP F B GIP F B GIP F + H E L S S Gen B GIP F B GIP F B GIP IGF-1 pPKB 473 • PKB x + / f f / ^ ' ^ F i g u r e 6. GIP regulation of PKB is PKA, PKC, MAPK and Tyrosine Kinase independent and PBKinase, PLA2 and Ca 2 + dependent in INS-l cells. Inhibitors (lOuM H-89, 250uM Rp-cAMP, 50nM Wortmannin, 2mM GF109203 (Bis), lOuM UOl26, ImM EGTA, 25uM Nifidepine, 50uM Thapsigargicin, lOuM KN-93, lOuM KN-62, 20uM HELSS and 25uM Gen (Genestein)) were added 30 min prior to and during 60 min stimulation under basal conditions (B) or in the presence of lOOnM GIP (GIP), 10u.M forskolin (F) or lOnM IGF-l (IGF-l). Fifty-u.g protein samples were separated by SDS-PAGE, and membranes were blotted with phospho-PKB Ser 473 (pPKB 473) antibody, prior to densitometry analysis. Data represent mean ± S.E.M. (A, n=4;B, n=4;C, n=4), where * indicates p<0.05 compared with respective controls (student's t test), and blots are representative. 43 3.2.4 GIP-induced phosphorylation of GSK3a/p\ BAD and FHRK is PI3Kinase-dependent in INS-1 cells To establish a definitive role for GIP activation of PI3kinase in the phosphorylation of the down-stream targets, GSK3a/p\ BAD and FHRK, the effect of wortmannin on INS-1 cell responses to both GIP and forskolin were determined. Wortmannin consistently inhibited GIP-induced phosphorylation of GSK3 a/(3 (Ser 21/9), BAD (Ser 136) and FHRK/AFX (Ser 256/196) (Fig. 7A). GIP-induced phosphorylation of GSK3 cc/|3 was also examined in INS-1 cells transiently transfected with kinase dead PKB (HA-PKB; PKB D/N), constitutively active PKB (myr-PKB; PKB CA) or empty vector (pCMV5), as control. GIP and forskolin induced GSK3 phosphorylation were decreased in INS-1 cells expressing the PKB-HA construct when compared to the GIP stimulated pCMV control cells (Fig. 7B), supporting a PKB dependence for GIP stimulation of GSK3a/p\ However, although basal, unstimulated GSK3 phosphorylation in INS-1 cells was increased in cells expressing the myr-PKB construct, when compared with the pCMV vector control (Fig. 7B), there was no further potentiation of GIP- or forskolin-mediated effects. This may be due to maximal stimulation by the constitutively active form. 44 + Wort B GIP B GIP + Wort F F pPKBThr308 pPKB Ser 473 pGSK3(x Ser 2 1 ^ pGSK3|', Ser 9 -> pBAD Ser 136 PKB p C M V 5 P K B - H A m y r - P K B B GIP B GIP B GIP B pGSK3u pGSK3|i PKB Figure 7. GIP activation of GSK3cc/|3 is PKB dependent. In A, INS-1 cells were serum starved overnight before 50nM wortmannin was added 30 min prior to lOOnM GIP or \0\iM forskolin (F) stimulation for 1 hr. In B, INS-1 cells were transfected with 10p,g of either vector or construct 2 days prior to experiments using Lipofect2000 (Invitrogen). Vector (pCMV5), kinase dead PKB (PKB-HA) and constitutively active PKB (myr-PKB) were kind gifts from Dr. A. Toker (Harvard University). Fifty-[ig protein samples were separated by SDS-PAGE, and membranes were blotted with phospho-PKB Thr 308 (pPKB 308) antibody, phospho-PKB Ser 473 (pPKB 473) antibody, phospho-GSK3cx/|3 Ser 21/9 (pGSK3ct/|3) antibody, phospho-BAD Ser 136 (pBAD 136) antibody and total PKB prior to densitometric analysis. Blots are representative of three independent experiments. 45 3.3 D I S C U S S I O N There is accumulating evidence supporting a major role for the incretin hormones GIP and GLP-I in the regulation of pancreatic |3-cell growth and survival (Ehses et al. 2003, Buteau et al. 2001; 2004). The signal-transduction pathways mediating these actions are complex and multiple protein kinases are involved, including Erkl/2 (Ehses et al. 2002), p38 MAP Kinase (Ehses et al. 2003) and protein kinase B/Akt (Wang et al. 2004). However, there is relatively little known about the pathways by which the MAP kinases or PKB are activated by the incretins, or the down-stream targets that mediate associated transcriptional events. Protein Kinase B plays a central role in numerous pathways related to the regulation of cell metabolism, proliferation and survival. Since GIP has been shown to exhibit powerful mitogenic (Trumper et al. 2002) and anti-apoptotic effects on pancreatic (3-cells it is therefore likely that PKB is an integral component of the signalling pathways involved. The current studies were undertaken in an attempt to establish how GIP-receptor interaction is linked to PKB activation and which down-stream targets are likely involved. Figure 8 shows a speculative model of how GIP may activate PKB, including signals deduced from the results described in the current thesis, plus potential pathways that require substantiating. 46 Figure 8. Proposed coupling of the GIP receptor to PKB activation. GIP interaction with its receptor activates both the Get and G(3y subunits. Ga activates adenylyl cyclase (AC) producing cAMP causing Ca 2 + influx, possibly by opening Ca 2 + channels in the plasma membrane. Arachidonic acid is capable of increasing iCa2 + by activating plasma membrane Ca 2 + channels and releasing Ca 2 + from intracellular stores. These increases in iCa2+ may then result in calcium induced calcium release (CICR). The overall increase in iCa2+ may result in calmodulin kinase (CaMK) activation and activatation of the phosphatidylinositol 3 kinase (PI3K)/ protein kinase B (PKB) cascade. Additionally, G(3y can cause arachidonic acid (AA) accumulation via phospholipase A 2 (PLA2) activation. Arachidonic acid may also have direct effects on PI3K itself. In agreement with Trumper and co-workers (2002), GIP was found to stimulate phosphorylation of PKB at the Ser 473 site in a sustained and concentration-dependent manner (Fig 1A); threonine 308 was also shown to be phosphorylated with similar kinetics. In view of the insulinotropic actions of GIP, and the ability of insulin to activate 47 PKB, it was important to establish that phosphorylation was not mediated via an autocrine pathway. The general tyrosine kinase inhibitor genistein was therefore applied and found to have no effect on GIP-induced phosphorylation of PKB (Ser 473) (Fig 6C). Similar PKB responses have recently been reported for GLP-1 (Wang et al. 2004). Having established that PKB was a downstream target of GIP action, it was important to establish whether, as in many other cell types, activation involved the upstream kinase PI3-K in the |3(INS-1) cell line (Cousin et al. 1999). Application of the PI3-K inhibitor wortmannin completed ablated GIP-induced PKB phosphorylation, establishing that this was indeed the case (Fig 6A). Partial blockade of GIP-stimulated phosphorylation of GSK3a/|3 by the mutant dominant-negative PKB construct (PKB-HA - D/N) supported a role for GIP-activated PKB in GSK3cx/|3 regulation at low glucose levels (Fig 7B). The pancreatic (3-cell and insulinoma cell lines contain a number of phospholipase A 2 isoforms (Chen et al. 1996; Loweth et al. 1994; Ramanadham et al. 1994), including Ca2+-dependent cytosolic PLA 2 (Ma et al. 1998), ATP-stimulatable, Ca2+-independent cytosolic PLA2 (iPLA2) and secretory PLA 2 (Ramanadham et al. 1998). These enzymes catalyze the hydrolysis of the sn-2 fatty acids from glycerophospholipid substrates, resulting in the production of both free fatty acid and 2-lysophospholipid (Balsinde and Dennis 1997; Mukherjee et al. 1994), and roles for all isoenzymes in glucose-induced insulin release have been suggested (Chen et al. 1996, Loweth et al. 1994). Glucose activates PLA 2 within the (3-cell, leading to increased arachidonic acid (AA) production and amplification of insulin secretion (Turk et al. 1987; 1993). Recently arachidonic acid has also been shown to improve growth and survival in a (3-cell line (BRIN-11) (Dixon et al. 2004), suggesting it could be involved in the regulation of cell fate in addition to its acute secretory actions (Neeli et al. 2003). We previously established that, in addition to adenylyl cyclase, GIP can activate a Ca2+-independent PLA 2 (Ehses et al. 2001). In CHO-K1 cells transfected with the GIP receptor, but not the (3TC-3 tumor cell line, activation of PLA 2 was shown to occur through a G protein |3y subunit-mediated pathway (Ehses et al. 2001). In the current study, the specific iPLA 2 inhibitor HELSS was shown to ablate GIP-stimulated phosphorylation of PKB (Ser 473) in (3(INS-1) cells (Fig 6C). Additionally, since both arachidonic acid production and PKB phosphorylation were blocked by expression of the 48 C-terminal fragment of the [3-adrenergic receptor kinase ((3-ARKct), receptor coupling to PLA2 via G|3y protein subunits appears to be involved in this pathway (Fig. 4 and 5D). Endothelin-mediated activation of PKB in an endothelial cell line has also recently been shown to be mediated through a G protein |3y subunit-mediated pathway (Liu et al. 2003). What are the potential links between arachidonic acid production and PI3K activation? Arachidonic acid has been shown to cause calcium release (Alessi et al. 1997), and it may be mainly involved in release from the smooth endoplasmic reticulum (SER) in (3-cells. GIP activation resulted in the release of arachidonic acid in both a time and concentration dependent manner, mimicking temporally that of GIP-induced PKB phosphorylation (Fig 4A-C). Contradicting proposals have recently been made regarding the possible cell fate-related role played by arachidonic acid in (3-cells (Dixon et al 2004; Prasad et al. 2003). Dixon et al. (2004) reported that arachidonic acid can stimulate (3-cell growth and improve survival, whereas Prasad and co-workers proposed that activation of arachidonic acid production reduces cell viability (Prasad et al. 2003). Interestingly, in agreement with the current study, vascular smooth muscle cells also demonstrated a delayed time-dependent activation of PI3K and PKB and the authors suggested that the delay in PKB activation could be linked to the metabolic production of arachidonic acid (Neeli etal. 2003). Surprisingly, GIP, a potent stimulator of cAMP production, was not found to require PKA or EPAC activation for its effects on PI3K/PKB as displayed by the failure of the PKA inhibitors H89 or Rp-cAMP to inhibit GIP induced PKB phosphorylation (Fig 6A), and the lack of effect of the cAMP analogues 8-Cpt-cAMP (PKA specific) or 8-OMe-cAMP (EPAC specific) (Fig 3B). Similarly, effects of forkolin on PKB phosphorylation were not influenced by PKA inhibition. However, since both forskolin and dibutyryl cAMP stimulation resulted in phosphorylation of PKB there is convincing evidence for a role for the cyclic nucleotide in PKB activation. One major possibility involves a further action of cyclic AMP: the activation of plasma membrane ion channels, resulting in increased levels of iCa2+. It has been previously shown that Ca 2 + has a central role in insulinotropic and mitogenic signalling in (3-cells. Inhibition of Ca2+/calmodulin kinases (CAMK) by KN93 and KN62 or blockade of Ca 2 + influx by EGTA coupled with intracellular inhibition of Ca2+release by thapsigargin have been shown to interfere with 49 glucose-induced insulin release (Neeli et al. 2003). In the present study, we demonstrated an important regulatory role for Ca 2 + influx and CAMKs in the regulation of PKB signal transduction by GIP. By blocking Ca 2 + influx with EGTA, the phosphorylation of PKB was inhibited, contrary to a previous report on GIP signal transduction (Trumper et al. 2002). To inhibit the different CAMKs known to be expressed in (3-cells (Easorn 1999), we used KN62 and KN93. KN62, which specifically inhibits CaMK isoforms I/IV and II, caused a return to basal PKB Ser 473 phosphorylation during stimulation with GIP. KN93, which is a more selective inhibitor for CaMKII, also inhibited PKB phosphorylation demonstrating its likely importance in the PKB signaling module, although non-specific effects of CaMKII inhibitors on other CaMKs, as well as Ca 2 + channels (Sihra and Pearson 1995) have been reported. Thapsigargin causes a blockade of intracellular Ca 2 + release and displayed itself as a potent inhibitor of PKB ser 473 phosphorylation (Fig 6B). All of these findings contribute to the idea that iCa2 + is essential for PKB signaling within (3-cells. In addition to expanding upon the previously proposed role for PKB signalling in survival in other cell types, the current study also provided evidence for a role in the regulation of several deonstream targets in (3-cells, including BAD, FKHR and GSK3. FKHR is a member of the forkhead family of transcription factors and induces expression of proapoptotic genes, e.g. Fas ligand and Bim (Brunet et al. 1999; Dijkers et al. 2000). Phosphorylation of forkhead family members appears to inhibit their import into the nucleus, thus decreasing their transcriptional capability (Brownawell et al. 2001). Consistent with the findings in a limited number of other cell types, GIP stimulated FKHR phosphorylation in (3-cells. BAD is another proapoptotic member of the Bcl-2 family whose phosphorylation promotes its dissociation from other proapoptotic members of the Bcl-2 family and binding to 14-3-3 proteins, the net effect of which is anti-apoptotic (Stokoe et al. 1997). BAD is regulated by phosphorylation of two serine residues, Serl 12 and Serl36 (Zha et al. 1996), and several studies have revealed that the Serl36 site can be specifically phosphorylated by Akt/PKB (Datta et al. 1997; del Peso et al. 1997). The present study demonstrates that in (3-cells, GIP induces phosphorylation of BAD on Ser 136 (Fig 2B). Thus, marked activation of PKB by GIP in (3-cells can stimulate Ser 136 phosphorylation and could contribute to anti-apoptotic effects of GIP in (3-cells (Ehses et al. 2003) by allowing Bcl-2 and Bcl-xl to inhibit BAX and BIM, 50 preventing pro-apoptotic proteins such as BAX and BAK to aggregate and initiate cytochrome c release and subsequent caspase-activation (Wei et al. 2001; Cheng et al. 2001). Another known substrate of PKB is GSK3a/|3, protein kinases implicated in several biological and metabolic processes whose phosphorylation inhibits their activity (Wei et al. 2001). GIP stimulated GSK3a/|3 phosphorylation in a similar time scale as that of PKB (Fig 2A, D). As discussed earlier, the inhibition of GIP stimulated GSK3a/|3 phosphorylation with kinase inactive PKB (PKB-HA) (Fig 6B) displays the importance of PKB in GSK3a/|3 regulation, but it is unclear as to what lies downstream and if it relates to the control of (3-cell survival. Hence, the functional roles of many of these GIP stimulated PKB substrates needs to verified in pancreatic (3-cells. 51 CHAPTER 4: INCRETIN MEDIATED PROTECTION OF p - C E L L S A G A I N S T C E L L D E A T H : GIP P R O T E C T I O N A G A I N S T GLUCOLIPOTOXICITY-INDUCED C E L L D E A T H IN VITRO AND T H E EFFECT OF ORALLY ADMINISTERED P32/98 ON p-CELL SURVIVAL IN VDF ZUCKER RATS 4.1 RATIONALE Pancreatic (3-cell death, either through autoimmune reactions or in response to stresses such as glucolipotoxicity, is the major cause of both type 1 and type 2 diabetes mellitus. It has been previously suggested that GIP stimulates phosphorylation and activation of PKB in a rat (3 (INS-1) cell line. GLP-1 has also been shown to activate PKB in various (3-cell models and with mouse, rat and human islets. Once activated, PKB functions as a focal point for conveying a survival response in many different tissues, including the (3-cell. It was of significant interest therefore to investigate a possible role for GIP as a survival factor for improving the life of pancreatic (3-cell in vitro and in vivo. 4.2 RESULTS 4.2.1 GIP and GLP-1 reverses glucolipotoxicity in INS-1 cells It has been suggested that elevated levels of both saturated fatty acids, such as palmitate, and glucose can cause pancreatic (3 cell dysfunction and cell death (El-Assaad et al. 2003), but their combined effect is now considered to be more damaging (El-Assaad et al. 2003), and this form of (3 cell stress has been termed glucolipotoxicity. To test the effect of glucolipotoxicity on the survival of (3 cells, INS-1 cells were incubated in the presence of increasing glucose concentrations coupled with elevated levels of palmitate (Fig 9A). From the data in figure 9A, it can be seen that at higher doses of palmitate and glucose, cell death was extensive producing as high as 75% cell death. An optimal glucolipotoxic condition of 0.4 mM palmitate combined with 20 mM glucose was selected and proved to be toxic enough to produce a level of cell death that would be likely to respond to GIP and GLP-1 treatment (Figure 9A-C), based upon previous reports of the incretins survival 52 a c t i o n ( E h s e s et a l . 2 0 0 3 ; B u t e a u et a l . 2 0 0 4 ) . T o test w h e t h e r G I P a n d G L P - 1 e x h i b i t a n t i - a p o p t o t i c a c t i o n s , (3-1 N S - 1 c e l l s w e r e i n c u b a t e d f o r 2 4 h r , i n g l u c o l i p o t o x i c c o n d i t i o n s i n the p r e s e n c e o r a b s e n c e o f i n c r e t i n h r o m o n e . F i g u r e s 9 B a n d 9 C s h o w the c o n c e n t r a t i o n d e p e n d e n t p r o - s u r v i v a l a c t i o n s o f b o t h G I P a n d G L P - 1 . S i g n i f i c a n t i m p r o v e m e n t s i n c e l l s u r v i v a l o f I N S - 1 s u b j e c t e d to g l u c o l i p o t o x i c t y w e r e s e e n at c o n c e n t r a t i o n s as l o w as l O n M f o r b o t h p e p t i d e s , w i t h E C 5 0 v a l u e s o f 5 .13 ± 0 . 2 9 n M a n d 4 . 0 0 ± 0 . 2 5 n M f o r G I P a n d G L P - 1 r e s p e c t i v e l y . 8CV 60' S 40H Q O 20' 0-« • 5 mM Glucose • 11mM Glucose • 20mM Glucose 0.0 o!s B 200-rviva sal) 150-= ra if) CO — o 100-o 50 J I I 1.0 1.5 mM Palmitate c 200-1 150-1 2.0 E C 5 0 = 5.13*0.29 nM 100-1 50' E C 5 0 = 4.00 ± 0.25 nM -12 -11 -10 -9 -8 -7 Log 1 0 [GIP] -12 7i To 1! -&• T" L 0 g io[GLP-1] F i g u r e 9: T h e e f fec t o f g l u c o l i p o t o x i c i t y ( A ) c o u p l e d w i t h v a r y i n g G I P ( B ) o r G L P - l ( C ) c o n c e n t r a t i o n s o n the s u r v i v a l o f ( 3 - I N S - l c e l l s . C e l l s w e r e s e r u m s t a r v e d o v e r n i g h t b e f o r e e x p o s u r e to v a r y i n g c o n c e n t r a t i o n s o f p a l m i t a t e a n d g l u c o s e f o r 2 4 hr ( A ) . In B a n d C , c e l l s w e r e i n c u b a t e d i n 0 . 4 m M p a l m i t a t e a n d 2 0 m M g l u c o s e (he rea f t e r r e f e r r ed to as g l u c o l i p o t o x i c c o n d t i o n s , G L T X ) i n the p r e s e n c e o f v a r y i n g c o n c e n t r a t i o n s o f G I P ( B ) o r G L P - l ( C ) f o r 2 4 hr. D e a d c e l l s w e r e c o u n t e d u s i n g t r y p a n b l u e p o s i t i v e c e l l s a n d c e l l s u r v i v a l (% B a s a l ) w a s c a l c u l a t e d b y d i v i d i n g t r y p a n b l u e p o s i t i v e c e l l s b y b a s a l d e a t h . D a t a e x p r e s s e d as m e a n ± S . E . M (n=4) w h e r e * p < 0 . 0 5 . 4 . 2 . 2 G I P a n d G L P - l r e d u c e ( 3 - I N S - l c e l l g l u c o l i p o t o x i c i t y 5 3 T o e s t a b l i s h w h e t h e r the c e l l s u r v i v a l e f fec ts o f G I P a n d G L P - 1 w e r e d u e to a n t i -a p o p t o t i c a c t i o n s o f the p o l y p e p t i d e s , a c t i v a t i o n o f the e f f e c t o r c a s p a s e - 3 d u r i n g g l u c o l i p o t o x i c c o n d i t i o n i n g o f b o t h I N S - 1 c e l l s a n d h u m a n i s l e t s . A s s h o w n i n f i g u r e s 1 0 A a n d 1 0 B , c a s p a s e - 3 a c t i v i t y w a s i n c r e a s e d b y 2 4 h r i n (3 - INS-1 c e l l s e x p o s e d to g l u c o l i p o t o x i c c o n d i t i o n s a n d that t h i s a c t i v a t i o n w a s p a r t i a l l y r e v e r s e d b y the c o n c u r r e n t a d d i t i o n o f G I P o r G L P - 1 . T h e s e l e c t i v e c a s p a s e - 3 i n h i b i t o r , A c - D E V D - C H O w a s u s e d to e n s u r e that m e a s u r e d p ro tease a c i t i v i t y w a s i n f ac t d u e to c a s p a s e - 3 (da ta no t s h o w n ) . A h a l l m a r k s i g n a l o f a p o p t o s i s i s the l o s s o f the a s y m m e t r i c a l p h o s p h o l i p i d m e m b r a n e s t u c t u r e w i t h p h o s p h a t i d y l s e r i n e a p p e a r i n g o n the p l a s m a m e m b r a n e o u t e r s u r f a c e . U s i n g a p h o s p h o l i p i d c o n v e r s i o n a s s a y , G I P a n d G L P - 1 w e r e f o u n d to s i g n i f i c a n t l y r e d u c e the l e v e l s o f p h o s p h o c h o l i n e c o n v e r s i o n , a h a l l m a r k c h a r a c t e r i s t i c o f c e l l s u n d e r g o i n g a p o p t o s i s . F i g I O C a n d 1 0 D d i s p l a y the p r o t e c t i v e e f fec t s o f b o t h G I P a n d G L P - 1 t o w a r d s g l u c o l i p o t o x i c I N S - 1 c e l l s . A •o > c •2*6 -1 <D Q. 4- . 00 O) JS.E = e o " 2 H E •£ Q. 0J 4' o i l * Basal GTX LTX GLTX ] Basal I GLP-1 Basal GTX LTX GLTX D Basal GIP GLTX GLTX + GIP Basal GLP-1 GLTX GLTX + GLP-1 F i g u r e 10. G I P a n d G L P - 1 r e d u c e g l u c o l i p o t o x i c i t y i n I N S - 1 ( 8 3 2 / 1 3 ) c e l l s . C e l l s w e r e s e r u m s t a r v e d b e f o r e a n d d u r i n g the e x p e r i m e n t , a n d e i t h e r l O O n M G I P ( A , C ) o r l O O n M G L P - 1 ( B , D ) w e r e a d d e d at t h e s t a r t a n d 12 h r i n t o t h e 2 4 h r e x p o s u r e t o g l u c o l i p o t o x i c i t y ( G L T X ) . F o l l o w i n g the 2 4 h r i n c u b a t i o n , c a s p a s e - 3 a c t i v i t y w a s 5 4 quantified using the substrate, Z-DEVD-AMC, over 30 min. Caspase-3 activity was corrected for total protein concentration using the BCA protein assay. Apoptotic cells were quantified using the APOpercentage apoptosis assay kit and expressed as fold change relative to control. Experiments are all n=3 were * represent p<0.05 vs. respective controls (ANOVA with Newman Keuls Multiple comparison post hoc test). 4.2.3 GIP and GLP-1 reduce glucolipotoxicity in human islets To verify the pro-survival effects of GIP and GLP-1 seen using the rat (3-ceII line INS-1 (832/13), human islets were tested. Human islets were exposed to the identical glucolipotoxic conditions of the (3-INS-l cells in serum free medium. lOOnM GIP or lOOnM GLP-1 were added at the commencement of the experiment and each 12 hours following. Fig 10D shows the cell death of the islets after 24, 48 and 72 hr. Fig 11A-C displays the caspase-3 activity measured at the same time points. GIP treatment resulted in a reduction in both glucolipotoxicity induced cell death and caspase-3 activation following 24 hr, but this reduction was lost over the subsequent 48 or 72 hr. However, GLP-1 reduced cell death and caspase-3 activity at all time points. This discrepancy in GIP mediated signaling may be attributed to the decrease of GIP receptor levels when incubated in high levels of glucose (Lynn et al. 2003). 55 A B Figure 11. GIP and GLP-1 reduce glucolipotoxicity in isolated human islets. Islets were serum starved before and during the experiment, and were exposed to glucolipotoxicity (GLTX) and caspase-3 activity was measured after (A) 24 hr, (B) 48 hr, (C) 72 hr in the presence or absence of lOOnM GIP or lOOnm GLP-1. Incretins were re-administered every 12 hr to the incubation medium. In D, incubation medium from 24, 48 and 72 hr was collected and assayed for cell death using a cytotoxic detection kit. Following the 24 hr incubation, caspase-3 activity was quantified using the substrate, Z-DEVD-AMC, over 30 min. Caspase-3 activity was corrected for total protein concentration using the BCA protein assay. The percentage cell death was determined using the Cytotoxic detection assay (Promega) which measures lactate dehydrogenase (LDH) released into the medium. All experiments are n=3, were * represent p<0.05, # represent p<0.01 vs. respective controls (ANOVA with Newman Keuls Multiple comparison post hoc test). 4.2.4 GIP and GLP-1 promote cell survival via PKA and PI3K A pharmacological approach was also taken to identify the GIP receptor mediated signaling pathway responsible for inhibition of caspase-3 activation. Inhibition of both 56 P K A ( H 8 9 , 10\IM) a n d P I 3 K ( W o r t m a n n i n , 5 0 ^ M ) r e s u l t e d i n a p a r t i a l r e v e r s a l o f the G I P a n d G L P - 1 a s s o c i a t e d r e d u c t i o n i n l i p o t o x i c i t y - i n d u c e d c a s p a s e - 3 a c t i v i t y . H o w e v e r , P I 3 K i n h i b t i o n b y w o r t m a n n i n c o m p l e t e l y a b l a t e d the r e d u c t i o n i n c a s p a s e - 3 a c t i v i t y , w h e r e a s P K A i n h i b i t i o n b y H 8 9 o n l y r e s u l t e d i n p a r t i a l r e d u c t i o n ( ~ 5 0 % ) i n c a s p a s e - 3 a c t i v i t y m e d i a t e d b y G I P a n d G L P - 1 ( F i g 1 2 A a n d I 2 B ) . T h e r e w a s n o e v i d e n c e f o r s u c h a r o l e f o r M e k 1/2 ( U 0 1 2 6 , l O f x M ) o r P K C ( G F 1 0 9 2 0 3 x ( B i s ) , l O O ^ x M ) ( F i g u r e 1 2 C a n d 1 2 D ) . T h i s s u g g e s t s that a P I 3 K m e d i a t e d c e l l s u r v i v a l a c t i o n o f G I P a n d G L P - 1 is the m a j o r p a t h w a y i n v o l v e d . A B F i g u r e 12. G I P a n d G L P - 1 r e d u c e g l u c o l i p o t o x i c i t y i n I N S - I ( 8 3 2 / 1 3 ) c e l l s . C e l l s w e r e s e r u m s t a r v e d be fo re a n d d u r i n g the e x p e r i m e n t , a n d e i t h e r l O O n M G I P o r l O O n M G L P - 1 w e r e a d d e d e v e r y 12 h r f o r the en t i r e 2 4 h r e x p o s u r e to g l u c o l i p o t o x i c i t y ( G L T X ) . P h a r m a c o l o g i c a l i n h i b i t o r s 1 0 ^ M H 8 9 ( A ) , l O n M w o r t m a n n i n ( B ) , 5 0 ; < M U 0 1 2 6 ( C ) o r IOO7 /M b i s ( D ) w e r e a d d e d f o r the d u r a t i o n o f the e x p e r i m e n t . F o l l o w i n g the 2 4 h r i n c u b a t i o n , c a s p a s e - 3 a c t i v i t y w a s q u a n t i f i e d u s i n g the subs t r a t e , Z - D E V D - A M C , o v e r 3 0 m i n . C a s p a s e - 3 a c t i v i t y w a s c o r r e c t e d f o r to ta l p r o t e i n c o n c e n t r a t i o n u s i n g the B C A p r o t e i n a s s a y . E x p e r i m e n t s are a l n = 4 w e r e * r e p r e s e n t p < 0 . 0 5 v s . r e s p e c t i v e c o n t r o l s ( A N O V A w i t h N e w m a n K e u l s M u l t i p l e c o m p a r i s o n pos t h o c test) . 5 7 To investigate further the finding that GIP mediated INS-1 cell survival is dependent upon PI3K and its main target, PKB. (3-INS-l cells were transiently transfected with the pCMV5 vector containing DNA constructs coding for mutated kinases (Fig. 13): dominant negative Ap85 PI3K, the constitutively active pllOCAAX PI3K or empty vector pCMV5 (Fig. 13A). The glucolipotoxic stimulus-induced caspase-3 activity was significantly reduced by the addition of GIP in pCMV5 vector transfected (3-(INS-l cells. However, the protective effect of GIP was lost in |3-(INS-1) cells transfected with the Ap85 PI3K (DN) construct. A reduction in caspase-3 activity was evident in |3-(INS-1) cells transfected with pi 10CAAX PI3K (CA) in the absence of GIP as compared to the pCMV5 vector control. These findings show a PI3K dependence for GIP's protective action. In the studies shown in Fig 13B, (3—INS-1 cells were transiently transfected with the pCMV5 vector containing DNA constructs coding for either the dominant negative PKB-HA, the constitutively active myr-PKB or pCMV5. Once again, GIP treatment resulted in a reduction in caspase-3 activity in (3-INS-l cells expressing the pCMV5 vector and exposed to a glucolipotoxic environment. The GIP induced reduction in caspase-3 activity was ablated in (3-INS-l cells expressing the PKB-HA (DN) construct (Fig. 13B). In (3-INS-l cells expressing the myr-PKB (CA) construct, glucolipotoxicity induced caspase-3 activity decreased in the absence of GIP, when compared to caspase-3 levels seen in the untreated pCMV5 vector. These findings demonstrate support a critical role for of PKB signaling in GIP mediated (3-INS-l cell survival. PTEN is a phosphatase involved in regulating the level of PI3K-induced phosphorylation of phospholipids in the plasma membrane. PTEN dephosphorylates PIP3 into PIP2 resulting in a reduction of PKB migration to the plasma membrane and subsequent overall PKB activity. In the studies shown in Fig 13C, INS-1 cells were transiently transfected with the pCIS2 vector containing DNA constructs coding for dominant negative PTEN-C129R or empty vector. GIP decreased caspase-3 activity in pCIS2 vector containing |3-INS-1 cells exposed to a glucolipotoxic environment. When (3-INS-l cell expressing the PTEN-C129R (DN) construct were exposed to glucolipotoxicity in the absence of GIP, a reduction in caspase-3 activity was observed. This decrease was only slightly further increased by the addition of GIP. Disruption of 58 P T E N s h o w s i t c a n r e g u l a t e the r e d u c t i o n i n c a s p a s e - 3 a c t i v i t i o n b y G I P i n d u c e d b y g l u c o l i p o t o x i c i t y . A B Vector D/N PTEN F i g u r e 1 3 . T h e P I 3 K / P K B m o d u l e i s i n v o l v e d i n G I P - m e d i a t e d r e d u c t i o n s i n g l u c o l i p o t o x i c i t y - i n d u c e d c a s p a s e - 3 a c t i v i t y i n I N S - 1 ( 8 3 2 / 1 3 ) c e l l . C e l l s w e r e t r a n s f e c t e d w i t h e i t h e r P I 3 K c o n s t r u c t s ( p C M V 5 v e c t o r , A p 8 5 P I 3 K d o m i n a n t n e g a t i v e c o n s t r u c t ( p 8 5 P I 3 K ) , o r the c o n s t i t u t i v e l y a c t i v e p i 1 0 C A A X P I 3 K ( p i 10 P I 3 K ) ) , P K B c o n s t r u c t s ( p C M V 5 v e c t o r , d o m i n a n t n e g a t i v e P K B - H A ( D N - P K B ) , the c o n s t i t u t i v e l y a c t i v e m y r - P K B ( C A - P K B ) o r P T E N c o n s t r u c t s ( p C I S 2 v e c t o r o r ( d o m i n a n t n e g a t i v e P T E N - C 1 2 9 R ( D N - P T E N ) ) o n e d a y p r i o r to the e x p e r i m e n t . C e l l s w e r e s e r u m s t a r v e d b e f o r e a n d d u r i n g the e x p e r i m e n t , a n d e i t h e r l O O n M G I P o r l O O n M G L P - 1 w a s a d d e d e v e r y 12 h r f o r the en t i r e 2 4 h r e x p o s u r e to g l u c o l i p o t o x i c i t y ( G L T X ) . F o l l o w i n g the 2 4 h r i n c u b a t i o n , c a s p a s e - 3 a c t i v i t y w a s q u a n t i f i e d u s i n g the subs t ra te , Z - D E V D - A M C , o v e r 3 0 m i n . C a s p a s e - 3 a c t i v i t y w a s c o r r e c t e d f o r to ta l p r o t e i n c o n c e n t r a t i o n u s i n g the B C A p r o t e i n a s say . E x p e r i m e n t s are ( A , B n = 5 ; C n=3) w e r e * r ep re sen t p < 0 . 0 5 , * * r ep re sen t p<0 .01 v s . r e s p e c t i v e c o n t r o l s ( A N O V A w i t h N e w m a n K e u l s M u l t i p l e c o m p a r i s o n pos t h o c test) . 4 . 2 . 5 E f f e c t s o f P 3 2 / 9 8 t r ea tmen t o n o r a l g l u c o s e t o l e r a n c e In o r d e r to e s t a b l i s h tha t e n d o g e n o u s i n c r e t i n s are c a p a b l e o f i n f l u e n c i n g (3-ce l l s u r v i v a l , D P I V i n h i b i t o r t r e a t m e n t w a s u s e d to i n c r e a s e t h e i r c i r c u l a t i n g h a l f - l i v e s 5 9 resulting in a potentiated insulinotropic effect. Long-term DP IV inhibitor treatment has been shown to improve glucose tolerance (Pospisilik et al. 2002). The metabolic status of the inhibitor- and vehicle-treated VDF Zucker rats was assessed by performing oral glucose tolerance tests (OGTTs) at 4, 8 and 12 weeks following initiation of the study, in the absence of circulating P32/98. At 4 weeks, 60 min blood glucose values in the DP IV inhibitor treated group showed significant decreases (3.6 mmol/l) despite overlapping plasma insulin excursions (Fig. 14B). At 8 weeks, the 30, 60 and 120 min blood glucose values were both significantly lower in the treated group than in the control group and the insulin profiles displayed a difference in first phase insulin secretion with the treated group displaying higher insulin peaks after 10 min compared to controls (Figs. 14C and D). The final OGTT, performed after 12 weeks of treatment, showed the greatest difference in glucose tolerance between the two groups, with significantly decreased blood glucose values observed at the 30, 60 and 120 min time points. Peak blood glucose values in the treated group averaged 13.8 ± 2.6 mmol/l, 7.9 mmol/l less than those of control animals (Fig. 14E and F), and 2-hr values in the treated group had returned to 8.9 ± 1.3 mmol/l, close to half the blood glucose concentration of the controls (Fig. 14E). The early-phase insulin response measured in the treated group exceeded that of the control animals. Figure 14G shows the effect of P32/98 treatment on the circulating DP IV activity levels over the 12 week study. DP IV activity was constant in the treated animals, while the control animals demonstrated an increase that reached a plateau at 8-12 weeks. 60 A B 0 2 4 6 8 10 12 ?4 Time (weeks) F i g u r e 1 4 . O r a l g l u c o s e t o l e r a n c e tests ( O G T T ) a d m i n i s t e r e d to b o t h D P I V i n h i b i t o r t r e a t ed ( n = 4 , f i l l e d t r i a n g l e ) a n d v e h i c l e - t r e a t e d c o n t r o l ( n = 3 , f i l l e d s q u a r e ) V D F rats a f te r f o u r ( A , B ) , e i g h t (C ,D) a n d t w e l v e (E,F) w e e k s o f t r e a t m e n t . B l o o d g l u c o s e a n d p l a s m a i n s u l i n m e a s u r e m e n t s w e r e p e r f o r m e d i n b o t h s e r i e s o f tes ts , as w e r e f a s t i n g p l a s m a D P I V a c t i v i t y m e a s u r e m e n t s ( G ) . S t a t i s t i c a l s i g n i f i c a n c e ( p < 0 . 0 5 ) is i n d i c a t e d b y an a s t e r i sk . 61 4.2.6 DP IV inhibitor treatment of VDF Zucker rats affects pro-survival protein expression Treatment of VDF rats with P32/98 increases the time of exposure of islets to intact, biologically active GIP and GLP-1 (Pospisilik et al. 2002). GIP and GLP-1 have been shown to stimulate a number of intracellular kinases involved in cellular fate, such as PKBcx/|3 (Aktl, Akt2) and IRS-2 (Trumper et al. 2002; Wang et al. 2004; Jhala et al. 2003). In the current study, protein expression was measured using immunoblotting of islet extracts from either VDF or lean animals that were treated with P32/98 or vehicle. Figure 15A and 15B show that PKBa (AKTT) and |3 (AKT2) protein expression were increased significantly in the islets from P32/98-treated fatty rats, compared to vehicle-treated animals. Additionally, the phosphorylation state of PKB at the Ser 473 was also at a higher level in the P32/98 treated animals (Fig. 15C). PKB expression and phosphorylation levels were unchanged in the lean animals regardless of treatment. IRS-2 is a protein that has recently been implicated in improving (3-cell function (Hennige et al. 2003) and has been shown to be up-regulated by GLP-1 treatment (Jhala et al. 2003), and (3-cell expression was also increased by P32/98 treatment, although to a lesser extent that PKB (Fig 15D). 62 pPKB 473 | i - Tubulin Lean VDF F i g u r e 15. P r o t e i n e x p r e s s i o n i s e n h a n c e d i n V D F rats ( n=3 ) t r ea ted f o r t w e l v e w e e k s w i t h the D P I V i n h i b i t o r P 3 2 / 9 8 ( P ) r e l a t i v e to v e h i c l e - t r e a t e d c o n t r o l s ( C ) . Is le ts w e r e i s o l a t e d f r o m b o t h t rea ted a n d un t rea ted V D F a n d l e a n l i t t e rma te s . P r o t e i n w a s q u a n t i f i e d u s i n g the B C A a s s a y a n d 3 0 fig o f p r o t e i n w e r e l o a d e d / l a n e f o r W e s t e r n b l o t t i n g . M e m b r a n e s w e r e b l o t t e d w i t h a n t i b o d i e s a g a i n s t P K B a ( A k t l ) ( A ) , P K B ( 3 ( A k t 2 ) ( B ) , p h o s p h o - A k t S e r 4 7 3 ( p P K B 4 7 3 ) ( C ) , I R S - 2 ( D ) o r ( 3 - t ubu l i n , p r i o r to d e n s i t o m e t r i c a n a l y s i s . D a t a r ep re sen t m e a n ± S . E . M . ( n = 3 ) , w h e r e * p < 0 . 0 5 , * * p<0 .01 c o m p a r e d to r e s p e c t i v e c o n t r o l s . 4 . 2 . 7 D P I V i n h i b i t o r t r e a tmen t o f V D F Z u c k e r rats i m p r o v e s |3 c e l l s u r v i v a l It has b e e n p r e v i o u s l y s h o w n i n o u r l a b o r a t o r y that l o n g - t e r m t r e a t m e n t o f V D F Z u c k e r rats w i t h P 3 2 / 9 8 i m p r o v e s g l u c o s e t o l e r a n c e , i n s u l i n s e c r e t i o n a n d s e n s i t i v i t y ( P o s p i s i l i k et a l . 2 0 0 2 ) . In the c u r r e n t s t u d y w e i n v e s t i g a t e d the a b i l i t y o f P 3 2 / 9 8 to i m p r o v e the a b i l i t y o f i s l e t s to s u r v i v e the g l u c o l i p o t o x i c e n v i r o n m e n t t h e y e n c o u n t e r w i t h i n V D F z u c k e r rats. C a s p a s e - 3 a c t i v i t y w a s m e a s u r e d as i n d i c a t o r s o f i s l e t a p o p t o s i s . F i g u r e 16 d i s p l a y s the c a s p a s e - 3 a c t i v i t y r e su l t s o f V D F rat i s l e t s t rea ted w i t h v e h i c l e o r P 3 2 / 9 8 f o r 12 w e e k s . C a s p a s e - 3 a c t i v i t y w a s s i g n i f i c a n t l y d e c r e a s e d i n the i s l e t s f r o m 6 3 P 3 2 / 9 8 t rea ted i s l e t s c o m p a r e d to the un t r ea t ed a n i m a l s . L e v e l s o f c a s p a s e - 3 a c t i v i t y a n d f a i l e d to a l t e r f o l l o w i n g P 3 2 / 9 8 t r e a t m e n t i n the l e a n a n i m a l s . T h e s e r e su l t s s u g g e s t D P I V i n h i b i t o r t r e a t m e n t c a n i m p r o v e (3-ce l l s u r v i v a l b y r e d u c i n g c a s p a s e m e d i a t e d a p o p t o s i s . L e a n V D F F i g u r e 1 6 . C a s p a s e - 3 a c t i v i t y w a s r e d u c e d i n V D F rats ( n=3 ) t r ea t ed f o r t w e l v e w e e k s w i t h the D P I V i n h i b i t o r P 3 2 / 9 8 ( P ) r e l a t i v e t o c o n t r o l s ( C ) . I s le t s w e r e i s o l a t e d f r o m b o t h t r ea ted a n d u n t r e a t e d V D F a n d l e a n l i t t e r m a t e s . C a s p a s e - 3 a c t i v i t y w a s q u a n t i f i e d u s i n g the subs t r a t e , Z - D E V D - A M C , o v e r 3 0 m i n . C a s p a s e - 3 a c t i v i t y w a s c o r r e c t e d f o r t o t a l p r o t e i n c o n c e n t r a t i o n u s i n g the B C A p r o t e i n a s s a y . D a t a r e p r e s e n t m e a n ± S . E . M . ( n = 3 ) , w h e r e * p < 0 . 0 5 c o m p a r e d to r e s p e c t i v e c o n t r o l s . In o r d e r to a s s e s s the g l u c o l i p o t o x i c p o t e n t i a l o f p l a s m a f r o m Z D F ra t s , w e d e t e r m i n e d the e f f ec t o f e x p o s i n g ( 3 - I N S - l c e l l s to m e d i u m s u p p l e m e n t e d w i t h p l a s m a o b t a i n e d f r o m l e a n a n d fa t a n i m a l s . B a s a l c e l l d e a t h w a s r e l a t i v e l y h i g h w i t h I N S - 1 c e l l s e x p o s e d to m e d i u m s u p p l e m e n t e d w i t h l e a n p l a s m a c o m p a r e d to p r e v i o u s s t u d i e s o n i n c u b a t i o n i n m e d i u m a l o n e . In t h o s e s t u d i e s , b a s a l c e l l d e a t h a n d c a s p a s e - 3 a c t i v a t i o n a f te r g l u c o l i p o t o x i c e x p o s u r e w e r e c l o s e r to 2 0 % o r 1.5 p m o l s u b s t r a t e c l e a v e d i n 3 0 m i n / L i g p r o t e i n ( F i g . 9 A a n d 1 0 A ) . M e a n c e l l dea th w a s r e d u c e d b y the p r e s e n c e o f e i t h e r G L P - 1 o r G I P , but o n l y the d e c r e a s e p r o d u c e d b y G L P - 1 r e a c h e d s i g n i f i c a n c e ( F i g . 1 7 A ) . I N S - 1 c e l l s e x p o s e d to m e d i u m s u p p l e m e n t e d w i t h V D F p l a s m a f o r 2 4 h r d i s p l a y e d o v e r 5 0 % c e l l d e a t h ( F i g . 1 7 A ) a n d b o t h G I P a n d G L P - 1 r e d u c e d the l e v e l o f d e a t h to that s e e n w i t h l e a n p l a s m a ( F i g . 1 6 A ) . In a d d i t i o n , G I P a n d G L P - 1 r e d u c e d c a s p a s e - 3 6 4 a c t i v a t i o n w h e n I N S - 1 c e l l s w e r e e x p o s e d to s u p p l e m e n t e d V D F rat p l a s m a o v e r a 2 4 h r p e r i o d ( F i g 1 7 B ) . F i g u r e 1 7 . G I P a n d G L P - 1 r e d u c e V D F p l a s m a - i n d u c e d g l u c o l i p o t o x i c i t y . I N S - 1 c e l l s w e r e s e r u m s t a r v e d o v e r n i g h t b e f o r e e x p o s u r e to p l a s m a f r o m e i t h e r l e a n o r fat V D F Z u c k e r rats s u p p l e m e n t e d w i t h 2 0 m M g l u c o s e , i n the p r e s e n c e o f D P I V i n h i b i t o r P 3 2 / 9 8 f o r 2 4 hr . G I P o r G L P - I w e r e a d d e d at the b e g i n n i n g a n d 12 h r o f the e x p e r i m e n t a n d c e l l d e a t h ( A ) o r c a s p a s e - 3 a c t i v i t y ( B ) w e r e m e a s u r e d as i n d i c a t o r s o f g l u c o l i p o t o x i c i t y . D e a d c e l l s w e r e c o u n t e d u s i n g t r y p a n b l u e p o s i t i v e c e l l s a n d c e l l s u r v i v a l ( % B a s a l ) w a s c a l c u l a t e d b y d i v i d i n g the n u m b e r o f t r y p a n b l u e p o s i t i v e c e l l s b y b a s a l d e a d n u m b e r s . C a s p a s e - 3 a c t i v i t y w a s q u a n t i f i e d u s i n g the s u b s t r a t e , Z - D E V D - A M C , o v e r 3 0 m i n . C a s p a s e - 3 a c t i v i t y w a s c o r r e c t e d f o r t o t a l p r o t e i n c o n c e n t r a t i o n u s i n g the B C A p r o t e i n a s s a y . D a t a r e p r e s e n t m e a n S . E . M . ( n = 4 ) , w h e r e * i n d i c a t e s p < 0 . 0 5 c o m p a r e d to r e s p e c t i v e c o n t r o l s . 6 5 4.3 D I S C U S S I O N It has been suggested that the etiology of both type 1 and 2 diabetes mellitus involves a reduction in the mass of functional pancreatic (3-cells. In order to maintain euglycemia, (3-cell mass must be held relatively constant through a dynamic process that involves neogenesis and/or differentiation, proliferation, and apoptosis (Bonner-Weir 2000, 2001). In light of recent evidence that showed adult pancreatic (3-cells are formed by self-duplication rather than stem-cell differentiation, the contribution of differentiation to (3-cell neogenesis could be relatively small (Dor et al. 2004). Only recently, have the growth factors and hormones responsible for maintaining this equilibrium been identified, and they include glucose, insulin, prolactin, growth hormone, insulin-like growth factor (IGF), and the incretin, GLP-1. From recent studies in our laboratory (Ehses et al. 2003) and others (Trumper et al. 2002), it is evident that GIP is capable of stimulating (3 (INS-1) cell proliferation, as well as promoting cell survival. The survival effects of GIP on (3 (INS-1) cells are comparable to those obtained with GLP-1 (Vaudry et al. 2000; Yusta et al. 2000). We have also recently found a dysregulation of islet size in GIPR -/- mice (Pamir et al. 2003), in addition to a protective role for incretins in STZ-induced diabetic rats (Pospisilik et al. 2003), implying a physiological role for GIP in the regulation of cell fate. Recent studies on the effect of chronic elevations of glucose and non-esterified free fatty acid (NEFA) concentrations on (3-cell function have shown that long-term exposure to high glucose induces glucose desensitisation, depletion of the readily releasable pool of insulin and may cause apoptosis (Poitout and Roberson. 2002), whereas long-term exposure to NEFA increases basal insulin release but inhibits glucose-induced insulin secretion (Segall et al. 1999). NEFA also reduces glucose-induced insulin gene expression (Gremlich et al. 1997) and induces beta cell apoptosis (Unger. 1995). According to the glucolipotoxicity concept, elevated levels of glucose and fatty acids synergise in causing beta cell malfunction, due to the fact that high glucose inhibits fat oxidation and consequently lipid detoxification (Prentki 1996; Prentki and Corkey. 1996). The present study shows that both GIP and GLP-1 can protect against beta cell glucolipotoxic cell death in human islets as well as in (3—INS-1 (832/13) cells. 66 Recent studies have shown that GLP-1, via transactivation of the EGF receptor, can activate P1-3K (Buteau et al. 2003), resulting in activation of a cascade of signal transduction that prevents apoptosis in several systems. We have been unable to establish the existence of a GIP-mediated transactivation pathway involving PI3K and PKB, although PKB does appear to play a central role in GIP signal-transduction, and evidence for a pathway involving G|3y and PLA2-mediated activation is presented in the current Thesis (Chapter 3). In the studies described in this Chapter we addressed the involvement of PKB in the anti-apoptotic actions of GIP and GLP-1. The overexpression of a dominant-negative PKB construct in |3-INS-1(832/13) cells suppressed the anti-apoptotic effects of GIP and GLP-1 on glucolipotoxicity, whereas overexpression of a constitutively active form of PKB abolished the cytotoxic effects of glucose and palmitate. These results support the view that GLP-1 prevents glucolipotoxicity (Buteau et al. 2004), at least in part, via the activation of PKB and its downstream targets. Two recent studies on transgenic mice expressing a constitutively active form of PKB demonstrating significantly greater beta cell mass are consistent with such a central role for PKB in the regulation of (3-cell mass (Bernal-Mizrachi et al. 2001; Tuttle et al. 2001). Moreover, these transgenic mice were protected from streptozotocin-induced diabetes. Both studies reported increased beta cell neogenesis and one of them showed an increase in beta cell proliferation (Bernal-Mizrachi et al. 2001). Both these transgenic studies and the current studies are supportive of an important role for PKB in the prevention of (3-cell apoptosis. Indeed, from the results of our study, we envision that the suppression of either PKB (by DN-PKB in Fig. 12B) or PI3K (by DN-PI3K in Fig. 12A) sensitizes cells to apoptosis in a context of low serum concentration, thus resulting in a increased basal level of apoptosis. In an earlier study it was shown that GIP treatment can reduce apoptosis resulting from incubation of (3-INS-(832/13) cells in medium containing zero glucose in a PI3K independent manner (Ehses et al. 2003). Clearly, therefore, there are multiple pathways by which incretins can reduce apoptosis, although the signalling mediators are not established. Conceivably, cyclic AMP production in response to GIP and GLP-1, acting in a PI3K/PKB-independent manner, could mediate their anti-apoptotic actions under relatively low stress conditions (Ehses et al. 2003; Hui et al. 2003), PI3K/PKB. signalling 6 7 would predominate under the major stress of glucolipotoxicity. This agrees with the observation that inhibition of PKA by H89 only partially reduced the protective effect of GIP, but PI3K inhibition by wortmannin completely caused complete reversal (Fig. 11A and B). Therefore we suggest that PKB may play a key role in the regulation of beta cell mass via prevention of apoptosis in addition to effects on |3-cell neogenesis and replication. Treatment of ZDF rats with DP IV inhibitor effectively reduced both prandial blood glucose levels and blood glucose responses to an OGTT (Fig 13). Although these differences were likely attributable, at least in part, to an acute increase in circulating incretin levels induced by P32/98, the pronounced early-phase insulin peak exhibited during the OGTT was not, because the OGTT was performed after complete drug washout. These data therefore support an earlier proposal (Pospisilik et al. 2002) that DP IV inhibition results not only in increased insulin sensitivity, but also in an enhanced |3-cell glucose responsiveness. Recent data suggest that GIP and GLP-1 can function as (3-cell survival factors in the stressful environment of glucolipotoxicity. VDF Zucker rats are an animal model of glucolipotoxicity and some of the survival properties of GIP and GLP-1 are evident in the data from long term DP IV inhibitor (P32/98) treatment of these animals. Caspase-3 activity is a marker of apoptosis in (3-cells and P32/98 treatment reduced this in the islets of VDF Zucker rats. This effect could be partially attributed to the greater activation of the PI3K/PKB pathway by GIP and GLP-1 due to there longer half-lives, and this proposal is supported by the increased protein expression levels of PKBa/(3 observed. IRS-2 levels were also found to be increased in the islets of P32/98 treated animals, similar to the recent study where GLP-1 was shown to increase IRS-2 expression in a CREB dependent manner and this could affect (3-cell survival (Jhala et al. 2003). In addition, exposure of INS-1 cells to serum from VDF Zucker rats in the presence of high levels of glucose, mimicked the effect of glucolipotoxicity; an effect that could be reversed by the addition of GIP and GLP-1 (Fig 16A-D). Therefore, we propose that DP IV inhibition not only increases the metabolic but also the anti-apoptotic effects of GIP and GLP-1. 68 In conclusion, our data demonstrate that GIP and GLP-1 reduces glucolipotoxicity-induced apoptosis in human islet cells, as well as in |3-INS-1(832/13) cells. Long-term treatment with the DP IV inhibitor P32/98 not only improved glucose tolerance and insulin secretion, but also reduced caspase-3 activity and in the VDF Zucker rats islets. The signal-transduction pathways responsible involve PKB activation and a myriad of downstream kinases and transcription factors. The anti-apoptotic actions of GIP and GLP-1, coupled with their incretin effects, reinforce their potential as therapeutic agents in the treatment of diabetes. Their administration may even be beneficial prior to the onset of type 2 diabetes, since they could prevent glucolipotoxicity-induced damage of beta cell that may be responsible for beta cell decompensation in the development of obesity-associated Type 2 diabetes. 69 C H A P T E R 5: C O N C L U S I O N S There has been an overwhelming amount of work dedicated to the incretin GLP-1 in the last decade, given its therapeutic application in treating type 2 diabetes. However, interest in GIP has also begun to mount due to the therapeutic potential of long-acting GIP analogues (Hinke et al. 2002) and DPIV inhibitors (Pospisilik et al. 2002) in type 2 diabetes. Furthermore, recent studies (Triimper et al. 2002; Ehses et al. 2003) and results presented in this thesis also implicate GIP in the regulation of (3-cell fate (growth and survival). Thus, the potential of both GIP and GLP-1 for therapeutic intervention in type 1 diabetes is also becoming a realistic possibility. The intracellular signaling cascades stimulated by Family B GPCRs have been largely neglected, and there is a paucity of rigorous scientific analysis with regards to the molecular mechanisms coupling receptor effectors to physiological actions. Given the recent expansion of the signal transduction field, effectors coupled to the GIP receptor clearly need to be identified. It is hoped that this thesis has provided some novel insights into the coupling of the GIP receptor to intracellular signaling of the pancreatic (3-cell and thereby identified novel elements involved in GIP-stimulated (3-cell survival. Results from this thesis can be characterized as follows: a. GIP regulates PKB in INS-1 (3-cells via a PLA 2 and PI3K-mediated pathway b. GIP promotes INS-1 (3-cell survival via a PI3K/PKB-inhibition of caspase-3 activation. c. Long term DP IV inhibitor (P32/98) treatment increases PKB expression and reduces caspase-3 activation in the VDF Zucker islet. 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