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Regulation of glucose-dependent insulinotropic polypeptide (GIP) receptor expression in the pancreatic… Lynn, Francis Christopher 2002

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R E G U L A T I O N OF G L U C O S E - D E P E N D E N T INSULINOTROPIC POLYPEPTIDE (GIP) R E C E P T O R E X P R E S S I O N I N T H E P A N C R E A T I C B - C E L L by FRANCIS CHRISTOPHER L Y N N B . S c , The University of British Columbia, 1997 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T FOR T H E D E G R E E OF D O C T O R OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department o f Physiology) We accept this thesis as conforming to the required standard  U N I V E R S I T Y OF BRITISH C O L U M B I A December 2002 © Francis Christopher Lynn, 2002  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head of my department or by h i s o r her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  Department The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada  11  Abstract Glucose-dependent insulinotropic polypeptide (GIP) is a peptide hormone that is released postprandially from the small intestine and acts to potentiate glucose induced insulin secretion from the pancreatic (3-cell. In type 2 diabetes (T2D) there is a decreased responsiveness of the pancreas to G I P . The literature suggests that the ineffectiveness of G I P in T 2 D may be a result of chronic homologous desensitization of the G I P receptor (GIPR); however, there has been no conclusive evidence suggesting that G I P levels are elevated in diabetes. The first hypothesis of this thesis is that one cause of decreased responsiveness to G I P in T 2 D is an inappropriate expression of the G I P R on the pancreatic islet. This hypothesis was tested using the Vancouver Diabetic Fatty ( V D F ) strain of Zucker rats. The V D F rats were unresponsive to a G I P infusion during an intraperitoneal glucose tolerance test (IPGTT). G I P did not alter insulin* secretion or c A M P production in isolated V D F islets, nor did it stimulate insulin secretion from perfused V D F pancreata. The expression of G I P R m R N A and protein in islets from V D F rats was significantly reduced. The second hypothesis is that hyperglycemia and hyperlipidemia are able to regulate 6-cell expression of the G I P receptor. High glucose was able to significantly reduce G I P R m R N A levels in INS(832/.1.3) cells after only 6 hours. Palmitic acid and the P P A R a activator, W Y 14643, produced an approximate doubling of G I P R expression in INS(832/13) cells under 5.5 m M but not 25 m M glucose conditions, suggesting that free fatty acids can regulate G I P R expression via P P A R a in a glucose-dependent manner. A dominant negative form of P P A R a transfected into  * Circulating hormone levels determined by radioimmunoassay are most accurately described as immunoreaclive (IR) peptides (eg IR-GIP or IR-insulin).  For the sake of brevity, the prefix IR- has been omitted from "insulin" in this text;  although, in most cases insulin levels are measured by radioimmunoassay.  Ul INS(832/13) cells caused a significant reduction in G I P R expression in 5.5 but not 25 m M glucose. In hyperglycemic clamped rats, there were reductions in G I P R expression in the islets and in GIP-stimulated insulin secretion. Thus, evidence is presented that the G I P R is controlled at normoglycemia by the fatty acid load on the islet; however, when exposed to hyperglycemic conditions the G I P R is down-regulated. The final hypothesis of this thesis is that glycosylation of the G I P R is able to control receptor expression on the cell surface. Here we demonstrate that cell surface expression of the G I P R and G I P stimulated insulin secretion are dependent on glycosylation of the G I P R . Furthermore, the asparagine-linked glycosylation sites on the G I P R include Asn-59, Asn-69, and A s n 200 and alteration of any of these sites decreased total cell surface G I P R expression. Overall, this thesis presents evidence that the G I P R is regulated negatively by glucose and positively by free fatty acids. Additionally, it is demonstrated that hyperglycemia leads to downregulation of the G I P R in models of T 2 D . This glucose-induced downregulation is a result of a decrease in transcription of the receptor as well as a glucose-induced defect in glycosylation of the receptor.  IV  Table of Contents Abstract  ii  Table of Contents  iv  List of Tables  ix  List of Figures  x  Abbreviations  xiii  Acknowledgements  xvi  Chapter 1 - Introduction  1  1.1 Overview  1  1.2 The Incretin Concept  2  1.3 Glucose-Dependent Insulinotropic Polypeptide  4  1.3.1 1.3.2 1.3.3 1.3.4  The Discovery of GIP GIP Sequence and Homology GIP Gene Structure and Posttranslational Processing Tissue Distribution, Release and Gene Expression  1.4 The GIP Receptor  1.4.1 Discovery of Specific Binding Sites for GIP 1.4.2 Gene Structure and Homology 1.4.3 Binding of GIP and Signaling Pathways 1.4.4 Structure-Function Relationships 1.4.5 Tissue Distribution 1.5 Biological Actions of GIP  1.5.1 Gastric Secretion 1.5.2 Adipose Tissue and Fat Metabolism 1.5.3 GIP and Islet Hormone Secretion 1.5.4 Other Biological Effects 1.6 Evidence for Other Incretins  4 5 6 7 10  10 11 12 14 15 16  16 17 19 22 23  V  1.7 Glucagon-Like Peptide-1  23  1.8 GIP and GLP-1 Metabolism  25  1.9 Pathophysiology o f GIP release and Actions  26  1.10 Nutrient Regulation o f Gene Expression 1.10.1 Glucose Regulation of Gene Expression 1.10.2 Fat Regulation of Gene Expression - Peroxisome Proliferator Activated Receptors 1.10.3 Other Fat-Activated Transcription Factors  29 29  1.11 Rationale  37  C h a p t e r 2 - Methods  31 36  38  2.1 Chemicals  38  2.2 Animals  39  2.3 Intraperitoneal Glucose Tolerance Test (IPGTT)  39  2.4 Measurement of Immunoreactive GIP  40  2.5 In Vitro Pancreatic Perfusion  40  2.6 Isolation and Culture o f Rat Pancreatic Islets  41  2.7 Perifusion of Pancreatic Islets  41  2.8 Measurement of Insulin and cyclic A M P Production by Islets  42  2.9 Isolation and Measurement of Islet GIP Receptor messenger R N A by Real-Time Reverse Transcription Polymerase Chain Reaction 43 2.10 Western Blot Analysis of Islet GIP Receptor Protein  44  2.11 Culture o f B R I N - D l l and INS(832/13) Cells  45  2.12 Transfection of INS(832/13) Cells  45  2.13 Isolation and Measurement o f GIP Receptor m R N A from Isolated Islets and Cultured Cells  46  2.14 m R N A Degradation and Half-Life Analyses  47  2.15 Iodination o f GIP and Saturation Binding Studies  48  2.16 Cloning of the Rat 5' GIP Receptor Promoter  48  VI  2.17 GIP Receptor 5'-Promoter Stimulated Gene Transcription and Luciferase Assay 49 2.18 7/7 Vivo Hyperglycemic Clamp Experiments  51  2.19 Pancreatic Perfusions of Hyperglycemic-Clamped Rat Pancreata  51  2.20 Site-Directed Mutagensis  51  2.21 Transfection, Affinity Purification of GIP Receptor Protein and Western Analyses 53 2.22 Competitive Binding and c A M P Production Analyses in H E K 293 and INS(832/13) Cells  55  2.23 Insulin Release from 1NS(832/13) cells  56  2.24 Fatty A c i d Oxidation in BRIN-D11 Cells  56  2.25 Tunicamycin Treatment of INS(832/13) Cells  57  2.26 Data Analysis  57  C h a p t e r 3 - Development of Competitive R T - P C R and T a q M a n Real T i m e R T P C R Methodologies  60  3.1 Competitive R T - P C R  60  3.2 Real Time R T - P C R - The Taqman System  62  3.3 A Comparison of the Two Methodologies  68  C h a p t e r 4 - G I P and the Vancouver Diabetic Fatty Z u c k e r V D F R a t M o d e l of T y p e 2 Diabetes  71  4.1 Background  71  4.2 Effect of GIP on glucose tolerance in the V D F rat  73  4.3 Effect of GIP on insulin secretion in the Zucker rat  77  4.4 Effect of GTP on insulin release from the pancreas of the Zucker rat  81  4.5 GIP receptor m R N A expression in the Zucker rat islets  86  4.6 GIP receptor protein expression in the Zucker rat islets  89  Vll 4.7 Glucose Tolerance, Insulin Secretion, and GIP Receptor Expression in Prediabetic V D F Rats 89 4.8 D I S C U S S I O N  93  Chapter 5: The Regulation of GIP Receptor Expression in Rat Clonal 6-Cell Lines 102 5.1 Background  102  5.2 Characterization of GIP Binding, GIP-stimulated c A M P Production and G I P stimulated Insulin secretion in the INS(832/13) Clonal (3-Cell Line  104  5.3 The Effect of GIP on Insulin Secretion from INS(832/13) Cells  104  5.4 GIP Stimulates Palmitate Oxidation in B R I N - D 1 1 Clonal B-Cells  107  5.5 The effects of glucose on GIP receptor m R N A expression in INS(832/13) cells. 107 5.6 The Effect of Free Fatty Acids and P P A R a Activation on G I P R Expression in Islets, B R I N - D 1 1 , and INS(832/13) Cells  115  5.7 The Interaction Between Fat and Glucose and the Effect on GIP Receptor Expression  120  5.8 Glucose, Palmitate , W Y 14643 and Gene Transcription  122  5.9 The Effect of .Osmolality on GIP Receptor Expression in INS(832/13) Cells  126  5.10 The Effect of Activation of P P A R y on GIP Receptor Expression in INS(832/13) Cells 126 5.11 Discussion  130  Chapter 6: Glucose-Induced GIP Receptor Downregulation in the Lean VDF Rat 136 6.1 Background  136  6.2 Glucose-Induced Downregulation o f the GIP Receptor in Hyperglycemic Clamped Rats 137 6.3 Discussion  137  Vlll  Chapter 7: Glycosylation of the GIP Receptor, the Effect of Glycosylation on Cell Surface Expression and Insulin Secretion 7.1 Background  142 142  7.2 The I - G I P Competitive Binding and Signaling Properties of the Glycosylation Site GIP Receptor Mutants 145 125  7.4 The Effect of Treatment of INS(832/13) Cells with Tunicamycin on Cell Surface GIP Receptor Expression 152 7.5 The Effect of Tunicamycin on GlP-Stimulated Insulin Secretion from INS(832/13) Cells 153 7.6 Discussion  156  Chapter 8: Discussion and Future Directions  161  Bibliography  168  IX  L i s t of Tables  Table 1: Megaprimers used for glycosylation site mutation  53  X List of Figures Figure 1: A typical gel obtained during competitive R T - P C R of G I P receptor R N A  63  Figure 2: Standard curve derived from G I P receptor competitive P C R band density data. 64 Figure 3: Raw, standard curve data obtained from the P C R amplification of synthetic standard G I P receptor c D N A  66  Figure 4: Real time R T - P C R standard curve  67  Figure 5: Dose related effects of G I P on plasma glucose during an I P G T T  74  Figure 6: Glucose response to infused G I P (A) and saline ( • ) in control, Fa/? rats  75  Figure 7: Glucose response to infused G I P (A) and saline ( • ) in V D F (fa/fa) rats  76  Figure 8: The integrated G I P response of saline infused control (Fa/?,lean) and V D F (fa/fa,Fat) rats during the I P G T T  78  Figure 9: Insulin responses to infused G I P (A) and saline ( • ) in control, Fa/? rats  79  Figure 10: Insulin responses to infused G I P (A) and saline ( • ) in V D F (fa/fa) rats  80  Figure 11: Insulin responses from the perfused pancreata of control rats  82  Figure 12: Insulin responses from the perfused pancreas of V D F (fa/fa) rats  83  Figure 1.3: Insulin release from perifused islets isolated from control (Fa/?) rats  84  Figure 14: Insulin release from perifused islets isolated from V D F (fa/fa) rats  85  Figure 15: Islet c A M P responses to G I P and forskolin from control and V D F rat islets. 87 Figure 16: G I P receptor m R N A levels in the islets of control (Fa/?) and V D F (fa/fa) rats measured by ( A ) real time R T - P C R or (B) competitive R T - P C R 88 Figure 17: G I P receptor protein expression in the islets of control (Fa/?) and V D F (fa/fa) rats 90 Figure 18: Oral glucose tolerance test from 4 week old control (Fa/?) and V D F (fa/fa) rats 91 Figure 19: Insulin release from isolated islets from 4 week old control (lean, Fa/?) and V D F (fat, fa/fa) rats 92 Figure 20: Perfusion of 4 week old control and V D F Zucker rat pancreata with saline or with a 0-50 p M gradient of G I P 94 Figure 21: G I P receptor m R N A levels in the islets of control (lean, Fa/?) and V D F (fat, fa/fa) rats 95 Figure 22: G I P receptor binding (A) and c A M P signaling (B) in INS(832/13) clonal 6cells 105 Figure 23: Insulin secretion from INS(832/13) cells in response to increasing glucose concentrations in the presence of G I P 106  XI  Figure 24: GIP-stimulated palmitate oxidation in B R I N - D 1 1 clonal 6-cells  108  Figure 25: The effect of time of exposure of INS(832/13) cells to 25 m M glucose on G I P receptor m R N A expression 109 Figure 26: The effect of glucose on G I P receptor m R N A expression in 1NS(832/13) cells: GIP receptor m R N A downregulation in response to graded glucose concentrations. 1 10 Figure 27: Saturation binding analysis of INS(832/13) cells treated with high glucose. 112 Figure: 28: Total cell surface G I P receptor numbers at 5.5 m M and 25 m M glucose.... 1 13 Figure 29: The effect of various inhibitors of cell growth and proliferation on glucoseinduced G I P receptor m R N A downregulation in INS(832/13) cells 114 Figure 30: G I P receptor expression in islets following incubation with the P P A R a activator W Y 14643 (100 u M ) or 2 m M palmitate  116  Figure 31: G I P receptor expression in B R I N - D 1 1 cells following incubation with P P A R a activator, 100 u M W Y 14643 or 2 m M palmitate 117 Figure 32: A time-course for palmitate-stimulated induction of G I P receptor expression in INS(832/13) clonal 6-cells 118 Figure 33: Saturation binding analysis of INS(832/13) cells treated with W Y 14643 and 2 m M palmitate 119 Figure 34: G I P receptor m R N A expression following culture of INS(832/13) cells for 24 hours in various glucose concentrations with 2 m M palmitate 121 Figure 35: The effect of a specific P P A R a antagonist on glucose induced G I P receptor downregulation 123 Figure 36: The effect of stimulating or blocking P P A R a activity in INS(832/13) cells. 124 Figure 37: G I P receptor m R N A degradation curves in INS(832/13) cells  •.  125  Figure 38: G I P receptor 5' promoter driven luciferase activity in response to W Y 14643 and 2 m M Palmitate (Fat) in INS(832/13) cells 127 Figure 39: The effect of osmolarity on G I P receptor expression in INS(832/13) clonal 6cells 128 Figure 40: The effect of activation of P P A R y on G I P receptor expression at increasing glucose concentrations in INS(832/13) clonal 6-cells 129 Figure 41: The effect of hyperglycemic clamping on G I P receptor expression in islets of lean Zucker rats 138 Figure 42: The effect of hyperglycemic clamp on G I P stimulated insulin release from the perfused lean Zucker rat pancreas 139 Figure 43: G I P binding (A) and c A M P production (B) by single site glycosylation mutants transfected into H E K cells  146  Xll  Figure 44: G I P binding (A) and c A M P production (B) by multiple site glycosylation site mutants transfected into H E K 293 cells 149 Figure 45: A representative electromobility shift assay using affinity purified G I P receptor extracted from transfected H E K 293 cells  151  Figure 46: G I P saturation binding analysis from INS(832/13) cells treated with tunicamycin  154  Figure 47: Tunicamycin decreases GIP-stimulated insulin secretion from INS(832/13) cells 155  Xlll  Abbreviations AA  arachidonic acid  1-Oct  octamer-1  ACC  acetyl C o A carboxylase  ACS  a c y l - C o A synthase  AOX  a c y l - C o A oxidase  aP  adipocyte fatty acid binding protein  ATP  adenosine triphosphate  AUC  area under the curve  BCA  bicinchoninic A c i d  Bis  bisindolylmaleimide  bp  base pairs  BSA  bovine serum albumin  cAMP  cyclic adenosine monophosphate  CCK  cholecystokinin  cDNA  complementary deoxyribonucleic acid  cPCR  competitive R T - P C R  cpm  counts per minute  CPT-1  carnitine palmitoyl transferase-1  CRE  c A M P response element  Ct  cycle threshold  C-terminal  carboxy-terminal  DMEM  Delbucco's modified eagle media  dNTP  deoxyribonucleotide triphosphates  DP I V  dipeptidyl peptidase 4  EC50  effective concentration where a 50 % maximal response occurs  ECL  enhanced chemi-luminescence  EMS A  electromobility shift assay  ERK  extracellular regulated protein kinase  FAM  l-Dimethoxytrityloxy-3-[0-(N-carboxy-(di-0-pivaloyl-fluorescein)-3aminopropyI)]-propyl-2-0-succinoyl-long chain alkylamino  FAS  fatty acid synthase  FAT/CD36  fatty acid translocase  FATP  fatty acid transport protein  FFA  free fatty acid  GAPDH  glyceraldehyde-3-phosphate dehydrogenase  GFP  green fluorescent protein  GHRH  growth hormone releasing hormone  GIP  gastric inhibitory polypeptide/Glucose-dependent insulinotropic polypeptide  GIPR  G I P receptor  GLP  glucagon-like peptide  xiv  GPCR  G-protein coupled receptor  GRE HBSS+  glucose response element Hank's Balanced Salt Solution supplemented with 10 m M H E P E S , 2 m M L-glutamine and 0.2 % B S A  HEK  human endothelial kidney  HPLC  high performance liquid chromatography  IBMX  3-isobutyl-l-methylxanthine  IC50  inhibitory concentration where 50 % maximal binding occurs  IJ  intrajejunal  IP  intraperitoneal  IP3  inositol trisphosphate  IPGTT  intraperitoneal glucose tolerance test  IR  immunoreactive  IV  intravenous  KATP  inwardly rectifying potassium channel (Kir) 6.2  Kb  kilobase  Kd  dissociation constant  kDa  kilodaltons  KRBH  Krebs-Ringer bicarbonate H E P E S buffer  LACS  long-chain acyl-CoA synthetase  LC-CoA  long chain acyl-CoA esters  L-FABP  liver fatty acid binding protein  LPL  lipoprotein lipase  MAP  mitogen activate protein  mRNA  messenger ribonucleic acid  NPY  neuropeptide Y  N-terminal  amino-terminal  OGTT  oral glucose tolerance test  ORF  open reading frame  PCR  polymerase chain reaction  PEPCK  phophoenolpyruvate carboxy-kinase  PK  pyruvate kinase  PKA  protein kinase A  PKC  protein kinase C  PLA2  phospholipase A  PLC  phospholipase C  PMSF  phenylmethylsulfonylfluoride  PNGase  peptide:/V-glycosidase F  PPAR  peroxisome proliferator activated receptor  PPRE  peroxisome proliferator response element  PUFA  polyunsaturated fatty acid  RIA  radioimmunoassay  2  XV  rPCR  real-time R T - P C R  RT  reverse transcription  RT-PCR  reverse transcription polymerase chain reaction  RXR  retinoid X receptor  SDS-PAGE  sodium dodecylsulphate-polyacrylamide gel electrophoresis  sh  synthetic human  sp  synthetic porcine  SRE  sterol regulatory element sterol regulatory element binding protein/adipocyte determination differentiationS R E B P / A D D 1 dependent factor 1 type 2 diabetes T2D l-Dimethoxytrityloxy-3-[0-(N-carboxy-(Tetramethyl-rhodamine)-3TAMRA aminopropyl)]-propyl-2-0-succinoyl-long chain alkylamino Taq T. aquaticus D N A polymerase TBST  tris-buffered saline with 0.5 % Tween 20  TF  transcription factor  UCP1 USF/MLTF  uncoupling protein 1 uracil N-glycosylase upstream stimulatory factor/major late transcription factor  UTR  untranslated region  VDCC VDF  voltage-dependent calcium channels Vancouver diabetic fatty  VLP  vasoactive intestinal polypeptide  WT  wild-type  ZDF  Zucker diabetic fatty  UNG  XVI  Acknowledgements To start out, I'd like to thank my parents, Denis and Portia for all the support and inspiration over the years. Their curiosity in the natural world was the driving force for my development of an interest in biological sciences and continues to motivate my research today. Over the last 7 years, Ray Pederson and Chris Mcintosh have also provided a source of inspiration. Their dedication to Physiology and enthusiasm for new techniques and projects has kept life interesting. Ray and Chris always allowed a great amount of intellectual freedom and encouraged the investigation of projects that were of direct interest to me. The nurturing environment that was provided by them has definitely made life in the lab fun. Outside of work, I ' l l always be appreciative of the "Grad retreat" weekends and other times spent with Ray and Margaret Pederson on Mayne Island and at W . 2 3 Ave. These times were at least important in my personal development as the time spent doing benchwork, and these times definitely made the department a more congenial place to work and fostered friendships that I'm sure will last lifetimes. I'd also like to acknowledge all the people that have worked in the lab and department and made life interesting. Heather White and her strange view of science made the first years of life in the lab interesting. Gord Rintoul, R i c k Gelling, and Chris Brett were always ready to make the trek to the Gallery for a pint or two, a necessary therapeutic tool when, yet again, the experiments failed. Simon Hinke and his contrary ways of trying to get under your skin were always appreciated. Jan Ehses for all the " y o ' momma" days, Andrew Pospisilik (pops) for putting up with me, and Nathalie Pamir for raising her voice when at first 1 didn't understand. In all seriousness though, these four individuals have had a profound effect on this thesis by providing me with: techniques, advice on techniques, experiments that I could carry out or by doing some of the experiments. I am most grateful to all of you. I am also grateful to the 4' year students, Eddy N g and Stephen Thompson, which I have had the pleasure of supervising over the years. W e learned together and your work was always appreciated. The lab definitely would not have continued to function without the meticulous work of Irene Bremsak, Cuilan Nian, and Madeleine Speck, these individuals and their contributions were greatly appreciated. I'd also like to acknowledge the familial environment that the Department of Physiology at U B C provided. I don't think that there are many departments left that are this special; hopefully, this atmosphere will not change in the future. Finally, I'd like to acknowledge the love and support of Sophika Kostyniuk, who kept me on track during the preparation of this manuscript and during the defense process. Thanks! rd  h  XVII  Portions of this work are published in: LYNN, F. C , PAMIR, N . , NG, E . H . C , MCINTOSI-I, C. H . S., KIEFFER, T. J. & PEDERSON, R. A . (2001). Defective glucose-dependent insulinotropic polypeptide receptor expression in diabetic fatty Zucker rats. Diabetes 50, 1004-1011. LYNN, F.C., THOMPSON, S.A., POSPISILIK, J.A., EHSES, J.A., HINKE, S.A., PAMIR, N . , MCINTOSI-I, C . H . S . & PEDERSON, R . A . (2002). A Novel Pathway for the Regulation of Glucose-Dependent Insulinotropic Polypeptide (GIP) Receptor Expression. FASEB J Nov 15, 2002: 10.1096/fj.02-0243fje.  1  Chapter 1 - Introduction 1.1 Overview Gastric inhibitory polypeptide/glucose-dependent insulinotropic polypeptide (GIP) is a 42 amino acid polypeptide that is synthesized and released by the K-cells o f the duodenal and jejunal mucosa (Brown et al., 1981; Buchan et al., 1978; Jornvall et al., 1981). GIP is released following ingestion of a meal by direct stimulation of the K-cell with the digestive products of glucose, fat, and protein (Dupre et al., 1973; Falko et al, 1975; Pederson et al., 1975; Thomas et al, 1978; Wolfe & McGuigan, 1982; Wolfe et al, 2000). One of the main physiological actions o f GIP following its release is potentiation of glucose-induced insulin secretion from the endocrine pancreas (Beck, 1989; Pederson, 1994; Pederson & Brown, 1976; Pederson et al, 1975). For this reason it is termed an incretin and, along with its partner incretin, glucagon-like peptide-1, comprises the endocrine axis of the physiological connection between the gut and the endocrine pancreas, known as the enteroinsular axis (Kieffer & Habener, 1999). Type 2 diabetes (T2D) is characterized by impaired glucose tolerance and thereby, an inability to properly secrete insulin following a glucose load. Because both GIP and GLP-1 are insulin secretagogues, there is considerable interest in using these polypeptides in the treatment o f diabetes. One characteristic o f T 2 D is the apparent loss of a GIP stimulated insulin response; however G L P - 1 , which signals through similar transduction pathways, seems to retain full potency (Elahi et al., 1994; Krarup et al., 1987; Meneilly etal., 1993; Nauck etal,  1993b).  Early studies attempted to link the lack o f potency o f GIP in T 2 D to a defective GIP receptor. T w o studies, in Japanese and Danish populations, demonstrated several  2  mutations in the GIP receptor (A207 V , E354Q and C198G) that did not have prevalence in T 2 D populations (Almind et al, 1998; Kubota et al, 1996). The Japanese group, but not the Danish group, showed that there was a decreased ability o f GIP (~ 70-fold rightshift o f the dose-response relationship) to stimulate c A M P accumulation in C H O K J cells transfected with the E354Q form of the receptor when compared to wild-type conditions. However, since none of these mutations seem to be linked directly to the development of overt diabetes, it appears as i f a mutation in the GIP receptor is probably not responsible for the decreased insulinotropic potency of GIP in T 2 D . Another possible cause for a loss o f potency o f GIP in T 2 D is a decrease in cell surface expression o f the receptor in this disease which could occur via desensitization and internalization followed by downregulation (Hinke et al, 2000a; Tseng et al, 1996a; Tseng & Zhang, 1998a, b). It has been hypothesized that receptor downregulation is a major pathway by which GIP actions become attenuated (Hoist et al, 1997; Livak & Egan, 2002).  1.2 The Incretin Concept The field o f endocrinology began in 1902 when Bayliss and Starling reported that a substance from the gut could influence secretion of pancreatic juice. These initial studies demonstrated that hydrochloric acid, when introduced into the duodenum of dogs with denervated small intestines produced an increase in the volume of secretion from the exocrine pancreas into the small intestine. Furthermore, when these same investigators infused a duodenal extract into these dogs, there was a similar increase in pancreatic secretion. They called this substance secretin (Bayliss & Starling, 1903).  3  A t around the same time, investigators were postulating that the "internal secretion" from the pancreas could control blood glucose. In 1906 Moore et al. hypothesized a role for gut secretions in the stimulation of the "internal secretion" from the pancreas. They were unable to show any effect of porcine gut extracts on the hyperglycemia o f diabetic individuals, probably because of a total absence o f B-cells. It was not until 1921 that Banting and Best isolated insulin and proved that it was the elusive "internal secretion." This discovery led to a revival of interest in the effects of duodenal extracts on hyperglycemia. To this end, La Barre and colleagues showed that a crude extract of secretin, when injected intravenously, could lower blood glucose levels in some dogs (LaBarre & Still, 1930; Zunz & LaBarre, 1929). They concluded from these studies that the secretin extract contained another substance which they termed incretin for its ability to stimulate release of the "internal  secretion" i.e. insulin from the  endocrine pancreas (LaBarre, 1932). In the ensuing years, studies by Loew, Gray and Ivy demonstrated that an incretin secreted from the gut did not have a blood glucose lowering effect (Loew et al, 1939, 1940a, b). It was not until development o f the radioimmunoassay ( R I A ) in (1960) that the insulinotropic effects o f duodenal extracts were studied. Mclntyre et al. (1964) reported that intrajejunal (IJ) administration o f glucose in two healthy subjects resulted in a more profound insulin response and more rapid return to basal glycemia than an equal intravenous (IV) dose. They hypothesized that this was a result of a substance that was released from the small intestine in response to glucose that stimulated insulin secretion from the endocrine pancreas (Mclntyre et al., 1964). The following year the same group ruled out liver as a potential site for the release of an insulintropic substance by carrying  4  out similar I V vs IJ experiments and obtaining similar results in healthy control patients and those with end-to-side portacaval shunts (Mclntyre et al., 1965). These studies supported the hypothesis o f LaBarre (1932) that an incretin substance was released from the intestinal mucosa, not the liver. Perley and Kipnis (1967) quantified insulin responses in diabetic, non-diabetic, obese and normal individuals and demonstrated that the response to oral glucose was 60-70 % greater than the response to I V glucose. In a seminal review, Unger and Eisentraut (1969) brought together the ample physiological evidence to coin the term enteroinsular axis to describe the endocrine connection between the gut and the endocrine pancreas. The definition was later broadened by Creutzfeldt (1979) to include both neural and substrate stimulants o f insulin secretion.  1.3 Glucose-Dependent Insulinotropic Polypeptide 1.3.1 The Discovery o f GIP GIP was initially isolated for its ability to inhibit gastric acid secretion. This followed a long search for enterogastrone: an inhibitory messenger that was secreted from the small intestine in response to intraluminal fat and acted via the blood to decrease gastric secretion (Gray et al., 1937; Greengard et al., 1946; Kosaka & L i m , 1930). Studies by Brown and Pederson (1970) suggested that different preparations of C C K , when given in doses that stimulated equal gallbladder contractile activity, had differing inhibitory effects on pentagastrin-induced acid secretion from canine stomach pouches. In these studies, the 40 % pure C C K preparation was not able to inhibit pentagastrininduced gastric acid secretion to the same degree as an equimolar dose o f the 10 % preparation. The authors proposed that this was due to the presence o f an inhibitor of gastric acid secretion that was in greater concentration within the less pure preparation.  5  Concomitant to these studies in dogs, GIP was chemically isolated from porcine duodeno-jejunal mucosa by standard biochemical methods. GIP and C C K were then separated using Sephadex G50, and amino acid composition was determined (Brown et al, 1969).  1.3.2 GIP Sequence and Homology The amino acid sequence o f porcine GIP was initially described by Brown and Dryburgh (1971) who reported that GIP was a 43 amino acid polypeptide. A n error in the intial sequence o f porcine GIP was later corrected by removal o f a glutamine residue at amino acid 30 (Jornvall et al, 1981), leaving a 42 amino acid polypeptide with an apparent molecular weight of approximately 5 k D a . Sequence identity analysis indicated that GIP was highly conserved between species; human GIP having 95 % sequence identity with the porcine and rat forms of GIP and 91 % sequence identity with the bovine and mouse sequences. This high conservation of sequence identity may indicate that GIP is an important regulatory hormone. Additionally Jornvall et al. (1981) demonstrated that there was a minor component o f the porcine GIP preparation with a 2 amino acid deletion at the amino-terminus producing GIP3-42The structure o f the GIP gene puts it in the growth hormone releasing hormone ( G H R H ) superfamily of genes, which is thought to have evolved during a gene duplication event in invertebrates between 500 million and 1 billion years ago. This gene duplication event resulted in the formation of the G H R H / V I P gene family and the glucagon gene families (Campbell & Scanes, 1992; Inagaki et al., 1989). Sequence similarity indicates that the GIP gene was then a result of a further series of gene  6  duplication events early within the existence of the glucagon family of genes (Irwin, 2002).  1.3.3 GIP Gene Structure and Posttranslational Processing The human GIP gene spans approximately 10 kb and contains 6 exons encoding a 153 amino acid prepro-form of GIP. Similar to other members of the G H R H and glucagon families o f genes, each exon in the GIP gene codes for a specific region of the peptide: exon 1 encoding the majority o f the 5' untranslated region of the m R N A ( U T R ) , exon 2 encoding the remainder of the 5' U T R and the signal peptide; exon 3 encoding the majority of GIP, exon 4 and 5 encoding the remainder of GIP and exon 6 encoding the 3' U T R (Inagaki et al., 1989). The rat gene structure is similar to that o f the human gene; although, the rat gene product is a 144 amino acid peptide that is primarily an aminoterminal deletion of the human ortholog (the result of a splice site shift) (Higashimoto & Liddle, 1993; Tseng etal., 1993). Exon .1 o f the GIP gene contains putative T A T A and C C A A T boxes, sites that are often necessary for the initiation o f transcription, although it has been reported that the T A T A box in the rat GIP gene is not active in the adult animal (Higashimoto & Liddle, 1993; Inagaki et al., 1989). Human preproGIP is posttranslationally processed by removal o f the 21 amino acid signal peptide (at glycine-21), and the intervening 30 amino acid N-terminal peptide, as well as the removal o f the 60 amino acid C-terminal peptide by proteolytic cleavage at single arginine residues (Arg51, Arg94) flanking the mature 42 amino acid peptide (Inagaki et al., 1989; Takeda et al., 1987). The posttranslational processing o f rat preproGIP is similar to that o f the human peptide (Higashimoto & Liddle, 1993).  7  1.3.4 Tissue Distribution, Release and Gene Expression GIP m R N A has been localized primarily to cells in the gastrointestinal tract such as the duodenum (Inagaki et al, 1989) and the stomach (Cheung et al., 2000; Yeung et al., 1999). A recent study has demonstrated GIP m R N A to be present in the duct cells o f the submandibular glands, although the physiological role of GIP in these cells is unknown (Tseng et al., 1995). GIP-like immunoreactivity has been localized to the K cells of the duodenum and jejunum in humans but immunoreactive GIP (IR-GIP) has been observed in the ileum in rats and dogs but not in the colon (Buchan et al., 1982; 1978; Polak etal,  1973).  GIP is released from cells that have been "defined by the characteristic appearance of the intracellular secretory granules having a small electron dense core surrounded by a concentric electron-lucent halo (Pederson, 1994)." These putative K cells of the duodenum and ileum are located within the intestinal mucosa, and respond to stimulation by luminal nutrients (Buchan et al, 1982; 1978; Dupre et al, 1973; Falko et al, 1975; Pederson et al, 1975; Thomas et al, 1978; Wolfe & McGuigan, 1982; Wolfe et al, 2000). GIP levels have been reported to increase from 12-92 p M basal ly, to 35235 p M postprandially: with the great degree of variability coming about as a result o f the affinities o f different antibodies for human GIP (Alam & Buchanan, 1993). In any case, most o f the literature agrees that GIP levels increase 5-6 fold basal following a mixed meal (Pederson, 1994). A more recent study by Hoffmann et al (2002), reported that fasting bioactive GIP levels in normal humans were in the low p M range and that the peak bioactive GIP level of 45 p M occurred 30 min following initiation o f an O G T T .  8  Total G I P levels (inactive +active) rose to approximately 150 p M at the 30 min timepoint. The literature reports that many o f the constituents of a mixed meal stimulate GIP release to varying degrees. One potent stimulus o f GIP release, and appropriate for its role as an incretin, is glucose. IR-GIP has been reported to increase in response to an oral glucose load in humans, dogs, rats, and mice (Cataland et al, 1974; Pamir et al, 2002; Pederson et al., 1982; Pederson et al., 1975) as well as isolated canine K-cells and from a mouse intestinal cell line (Kieffer et al., 1994; 1995a). IR-GIP is not released in response to I V glucose, indicating that luminal stimulation of the K - c e l l is necessary for release. The exact mechanism for this release involves uptake o f glucose into the enterocytes, as phloridzin an inhibitor of sodium-dependent glucose transport abolished the GIP secretory response to glucose in the perfused rat intestine (Sykes et al., 1980). Probably the most potent stimulant o f GIP release is ingestion o f triglycerides. The G I P response to oral triglycerides is more prolonged and often greater in magnitude, which may be a result of the decreased rate of gastric emptying caused by G I P and related to its enterogastrone activities (Brown & Otte, 1978; Pederson et al., 1975). Furthermore, the chain length o f fatty acids is directly related to the potency o f GIP release; long chain/highly saturated fatty acids stimulated a more profound GIP secretory profile than either medium or short chain fatty acids (Lardinois et al., 1988; Ross & Shaffer, 1981). The exact mechanism for triglyceride-stimulated IR-GIP release and differences in potency are thought to result from stimulation o f the K-cell with/and possibly by metabolism o f free fatty acids ( F F A ) that have been released by the prior action of gastric lipase (Wolfe et al, 1999). A s its name suggests, the insulinotropic  9  activity o f GIP is strictly dependent on elevation of blood glucose, and GIP released by F F A has not been shown to be insulinotropic (Pederson & Brown, 1978; Pederson et al, 1975). A m i n o acids, peptone and proteins may also cause physiologic GIP release. It has been reported that a mixture of basic amino acids (I, L , K , T, R, H) but not a mixture of aromatic amino ( M , P, Y , V ) acids stimulates GIP release (Thomas et al, 1978). However, protein meals consisting o f cod or steak did not stimulate GIP release (Cleator & Gourlay, 1975; Sarson et al, 1980). It was recently demonstrated that GIP release may be stimulated by protein, and this release was partially inhibited by omeprazole indicating that protein stimulated GIP release may be dependent on gastric acid secretion and subsequent acidification of the duodenum (Wolfe et al, 2000). The mechanism for protein stimulated GIP release has been linked to sodium-dependent amino acid transport and/or K-cell membrane potential (Schulz et al, 1982). GIP release may be inhibited by hyperinsulinemia. Bryer-Ash et al (1994), demonstrated that under euglycemic conditions, hyperinsulinemia inhibited GIP release; however, at high glucose levels the effects of hyperinsulinemia were attenuated. GIP gene expression is regulated in a parallel manner to that of GIP secretion. A number o f groups have demonstrated that glucose increases the m R N A levels of GIP in both rat intestine and intestinal cell models (Higashimoto et al., 1994; Schieldrop et al., 1996; Tseng et al., 1995; 1994). Additionally, triglycerides or F F A may increase GIP expression in rats; although the fat induced effect is very short-lived (Wolfe et al, 1999). Fasting has been shown to decrease both GIP m R N A and intestinal IR-GIP levels, (Higashimoto et al, 1994) as well as to increase GIP m R N A expression (Sharma et al,  10  1992). The effect of overall nutrition on GIP gene expression is unclear. The GIP promoter contains AP-1 and A P - 2 consensus elements for gene regulation by P K A and P K C . In addition the GIP promoter contains 3 sequence elements which share similarity to c A M P response elements although, the exact roles of any o f these sequences in controlling GIP expression have not been elucidated (Inagaki et al., 1989).  1.4 T h e G I P Receptor  1.4.1 Discovery o f Specific Binding Sites for GIP The first demonstrations of specific binding sites for GIP were carried out using GIP radiolabelled with  125  I and bound to hamster In 111 cells, human insulinomas or  mouse (3-TC3 cells (Amiranoff  al., 1984, 1985; Kieffer el al., 1993; Maletti et al.,  1987; 1983). These studies all indicated that GIP binds to its receptor with an equilibrium dissociation constant (Kd) in the low n M range. Early studies used crosslinking techniques to irreversibly bind radiolabelled GIP to hamster (3-cell membranes. These proteins were then run out on acrylamide gels with the majority o f radiation running with an apparent molecular weight of 64 kDa: indicating that the receptor was approximately 59 k D a in size. Furthermore, these studies demonstrated that treatment with dithiothreitol reduced the electrophoretic mobility of the protein, indicating the presence of a disulfide bond (Amiranoff ef al., 1986). This group was the first to demonstrate that the GIP receptor was a glycoprotein containing N-acetylglucosamine, mannose and sialic acid: moieties often associated with asparagine-linked glycosylation.  11  1.4.2 Gene Structure and Homology The GIP receptor (GIPR) was initially cloned from the rat insulinoma cell line, RTNm5F (Usdin et al., 1993). Following this, there were a number o f studies reporting the cloning from other human, hamster and rat sources (Gremlich et al., 1995; V o l z et al., 1995; Wheeler et al., 1995; Yasuda et al, 1994). Sequence analysis o f the G I P R c D N A s isolated indicates that the human gene contains a 1389 base pair (bp) open reading frame (ORF) coding a 466 amino acid protein with a predicted molecular weight o f approximately 50 kDa. The rat and hamster gene products are 455 and 462 amino acids respectively. Both the human and rat GIP receptor genes have been characterized. The human gene is composed of 14 exons spanning 13.8 kb: 13 of which encode protein sequences and the other encodes the 5' U T R ; while the rat gene is comprised o f 15 exons spanning 10.2 kb: with the extra exon encoding a 3' U T R (Boylan et al, 1999; Wolfe et al, 1999; Yamada et al., 1995). Aside from the 3' U T R found in the rat gene, the human and rat genes are identical in structure. These studies collectively demonstrated that the GIP receptor had sufficient sequence identity (25-49 %) to be considered a member of the secretin/VIP family of serpentine, seven transmembrane domain G-protein-coupled receptors (GPCRs). The rat GIP receptor has the highest homology with members o f the glucagon family o f G P C R s : sharing 44 % sequence identity with the glucagon receptor and 40 % with the glucagon-like peptide-1 receptor. The transmembrane domains are the most highly conserved sequence elements within the G I P , G L P - 1 and glucagon receptors, followed by the N-termini. The least conserved regions o f the receptors are the C-termini with only 3 common amino acids between all three receptors (Gremlich et al, 1995; Usdin et al, 1993; Wheeler et al, 1995).  12 The 5'-flanking promoter region of the rat GIPR gene has been sequenced and contains a number of transcription factor binding sites; including 3 SP-1 binding motifs, an octamer-1 (OCT-1) binding site, and a cAMP response element (CRE). However, binding of transcription factors (TF) to these sites has not been verified. The rat GIPR promoter does not contain a T A T A box directly upstream of the transcription initiation site; although, there are T A T A and C A A T motifs approximately lkb upstream from the transcription start site. There is, however, an initiator element 10 bp upstream of the transcriptional start site that is identical to the Inr sequence in other genes. Inr elements are important for the binding of R N A polymerase II. Deletion analyses indicated that the first 100 bp upstream from the transcriptional initiation site are necessary for efficient transcription. Furthermore, deletion between -100 and -2500 bp upstream did not effect the ability of the promoter to stimulate luciferase transcription in RIN38 cells (Boylan et al, 1999).  1.4.3 Binding of GIP and Signaling Pathways Wheeler et al (1995) examined the affinity of different orthologs of GIP for the rat receptor following transfection in Chinese hamster ovary K l (CHO) cells or COS-7 cells. They found that both synthetic porcine (sp) GIP and synthetic human (sh) GIP had comparable IC50 values for displacing radiolabeled spGIP from the GIP receptor. These IC50  values were approximately 3 nM and 8 nM in CHO cells and COS-7 cells  respectively and were similar to those obtained for the hamster GIP receptor (Wheeler et al, 1995; Yasuda et al, 1994). Due to the sequence similarity in the N-terminus of the GIP receptor (the postulated binding site for GIP) with other members of the glucagon receptor family, it was hypothesized that the glucagon family of peptides, which share  13  homology with G I P , may be able to activate the GIP receptor (Mcintosh et al, 1996). However, when this was tested, only 1 u M e x e n d i n ^ g or exendin-4i_39, (GLP-1 receptor antagonist and agonist respectively), were able to displace  l 2 5  I labeled G I P from the  receptor. Secretin, V I P , glucagon, GLP-1 and G L P - 2 had no effect (Wheeler et al, 1995). Prior to the cloning o f the G I P receptor it was demonstrated that G I P in the low n M range stimulated adenylyl cyclase in a hamster pancreatic tumor cell line (Amiranoff et al, 1984; L u et al, 1993), as well as isolated islets (Siegel & Creutzfeldt, 1985) and in H G T - 1 cells (Gespach et al, 1984). Whether expressed in Chinese hamster ovary ( C H O ) , lung, L V I P cells or C O S cells, the human, rat and mouse forms o f the receptor all respond to G I P by activation of adenylyl cyclase and subsequent elevation of cyclic adenosine monophosphate ( c A M P ) . However, each group of authors reported slightly different EC50 values ranging from 0.1 p M to approximately 15 n M (Gremlich et al, 1995; V o l z et al, 1995; Wheeler et al, 1995; Yasuda et al, 1994). Studies indicate that there is not a glucose dependence for GIP-stimulated c A M P production in (3-TC3 cells and in INS(832/13) cells; thus, glucose metabolism does not seem to affect this signal transduction module and the glucose dependence must come about at later steps in the exocytotic process (Ehses et al, 2001; 2002; Hinke et al, 2000a). GIP has also been demonstrated to increase C a ' levels in isolated islets at 2  elevated glucose levels (Wahl et al, 1992), as well as in HIT-T15 insulinoma cells ( L u et al, 1993) via influx through L-type voltage dependent calcium channels. Wheeler et al. (1995) demonstrated that activation of the G I P receptor in C O S - 7 cells led to an increase in intracellular calcium in a nifedipine-independent manner that could be inhibited by  14  thapsigargin: indicating that G I P was able to couple to other voltage-independent calcium channels. G I P , however, has never been shown to couple to phospholipase C ( P L C ) and stimulate the release o f inositol trisphosphate (IP3) (Lu et al, 1993; Yasuda et al, 1994). Furthermore, it was suggested by Ehses et al. (2001) that G I P could stimulate C a release from intracellular stores via activation of  PLA2  2 t  and the consequent release of  arachidonic acid ( A A ) . However, the exact pathway by which G I P increases intracellular Ca  2 +  has yet to be elucidated. It has been previously speculated that G I P may exert its effects through both A A  (Lardinois ei al, 1990) and through activation of M A P kinases (Kubota et al, 1997). Recently, Ehses et al. (2001) demonstrated that G I P liberates A A via activation o f a calcium independent form of  PLA2,  suggesting that GIP may potentiate insulin secretion  via this pathway. Furthermore, they demonstrated that  PLA2  is activated in these (3-cell  models ((3TC-3 cells) by G|3y dimers and that this activation is dependent on elevated c A M P . In another recent study, Ehses et al. (2002) demonstrated that G I P activates the E R K module in a c A M P and P K A dependent manner probably via activation o f B - R a f in INS(832/13) (3-cells. They hypothesized that GIP receptor activation could lead to proliferation/differentiation or gene transcription within the (3-cell in response to activation of the E R K module. Thus, G I P signaling pathways in the (3-cell are much more complicated than previously thought, and at present are not completely elucidated.  1.4.4 Structure-Function Relationships The amino terminus of the receptor contains consensus sequences for N-type glycosylation; in addition, the third intracellular loop and the C-terminus of the receptor are rich in serine residues that could serve as potential phosphorylation sites (Usdin et al,  15  1993; Wheeler et al, 1999; 1995). Recent studies have begun to characterize the regions of the G I P receptor which are important for binding of GIP, G-protein coupling, desensitization and internalization. Studies utilising G I P / G L P - 1 receptor chimeras have indicated that the amino-terminal tail of the G I P receptor is important for high affinity ligand binding and that the first transmembrane helix is important for coupling of the receptor to the intracellular signal transduction machinery (Gelling et al., 1997). Further studies in which the carboxy-terminal tail of the receptor was truncated, demonstrated that the C-terminus is not essential for binding or signaling but necessary for proper expression and possibly orientation of the receptor within the cytoplasmic membrane (Wheeler et al., 1999). Also, it was recently shown that C-terminal receptor truncation (at amino acid 425) did not greatly affect GIP-induced desensitization but may have slowed initial receptor uptake (Wheeler et al., 1999).  1.4.5 Tissue Distribution GIP receptor m R N A is expressed in the pancreas, stomach, intestine, adipose tissue, adrenal cortex, heart, lung, endothelium, telencephalon, diencephalon, brain stem, cerebellum and the pituitary (Usdin et al, 1993; Yasuda et al, 1994; Zhong et al, 2000); although, the function o f the receptor in some of these tissues is not known. Radiolabelled GIP binding in the rat brain has been characterized using autoradiography. Most o f the brain sections that expressed G I P R m R N A also bound  125  I - G I P , with the  exception o f the pituitary. High affinity binding sites were noted in the olfactory bulb (Kaplan & Vigna, 1994). One enigmatic point is that GIP has never been detected in brain extracts and G I P m R N A has never been detected in the brain. Therefore, it is  16  possible that an alternate molecule exists in the brain that activates the G I P R (Mcintosh etal,  1996). Expression of the G I P R in the adrenal cortex may result in increased  glucocorticoid metabolism in response to GIP release and has been shown to play a role in food-induced Cushing's syndrome (Croughs et al, 2000; Lacroix et al, 1992). Recently, it was shown that the G I P R was expressed in bone, and stimulation o f SaOS2 cells (an osteoblast cell line) by GIP led to increased expression o f collagen type I m R N A and increased alkaline phosphatase activity. Both of these effects are osteotrophic, and led Bollag et al. (2001; 2000) to propose the existence o f an enteroosseous axis; whereby GIP could control.bone density in response to nutrient intake.  1.5 Biological Actions of  GIP  1.5.1 Gastric Secretion GIP was initially isolated for its inhibitory effect on gastrin-stimulated gastric acid secretion in dogs (Pederson & Brown, 1972) and subsequent studies supported the role o f GIP as an enterogastrone (Arnold et al, 1978b; V i l l a r et al, 1976). However, some studies questioned the enterogastrone activity of GIP because they observed rather weak inhibition of gastric acid secretion and only with supraphysiological doses (Andersen et al, 1978; Arnold et al, 1978a; E l Munshid et al, 1980; M a x w e l l et al, 1980; Soon-Shiong et al, 1979; Yamagishi & Debas, 1980). However, during this time Mcintosh et al. (1979) suggested that since the onset o f acid inhibitory effects o f GIP was slow, it was possible that GIP was causing the release o f another substance that was inhibiting gastric acid secretion. Furthermore, there was ample evidence at this time that somatostatin secreting D-cells abutted on gastrin secreting G-cells (Larsson et al, 1979)  17 and that somatostatin was capable of inhibiting acid secretion (Bloom et al, 1974). Mcintosh et al. (1981b) demonstrated that IR-somatostatin was released from D-cells in response to GIP in the perfused rat stomach and that the release of somatostatin was inhibited by vagal activation or acetylcholine administration. This vagally mediated inhibition of GIP-stimulated somatostatin release was only partially blocked by atropine (Mcintosh et al, 1981b), indicating that other neurotransmitters may be involved (Mcintosh et al, 1983). The authors hypothesized that the putative processes from the D-cells in the stomach were in direct contact with gastrin releasing G-cells; with release of somatostatin having an inhibitory effect on gastrin secretion and a decreased acid output from the parietal cells (Mcintosh et al, 1981b). Another series of experiments demonstrated that sympathetic activation may also modulate GIP stimulated somatostatin secretion and thereby gastric acid secretion (Mcintosh et al, 1981 a). Subsequently, Soon-Shiong et al. (1984) reported that GIP had no effect on acid secretion if it was coadministered with Bethanechol, a cholinergic agonist. This observation suggested that the parasympathetic nervous system also controlled the enterogastrone properties of GIP. Overall, the mechanism by which GIP exerts enterogastrone action is via stimulation of somatostatin secretion in the stomach with modulation from the autonomic nervous system.  1.5.2 Adipose Tissue and Fat Metabolism Triglycerides are digested in the stomach and small intestine and the resultant FFA are absorbed by the K-cell. As previously described, these FFA are possibly the strongest stimulant of GIP release postprandially (Ebert & Creutzfeldt, 1980; Pederson, 1994; Ross & Shaffer, 1981; Yoshidome et al, 1995). Additionally, GIP receptor mRNA  18  was found in adipose tissue as well as in differentiated 3T3-L1 cells and it has been demonstrated that G I P may be involved in the subsequent clearance o f circulating triglycerides (Mcintosh et al, 1999). G I P has been shown to cause an increase in triglyceride clearance from the blood of dogs and rats (Ebert et al., 1991; Wasada et al., 1981), possibly by activation o f lipoprotein lipase (Eckel et al., 1979). G I P has also been shown to have discrete effects on lipid metabolism within adipose tissue. Although no systematic studies have been carried out, it has been demonstrated that GIP is capable o f augmenting synthesis of fatty acids from both glucose and lipid sources (Hauner et al., 1988). Furthermore, these authors and others (Dupre et al., 1973) demonstrated that GIP also strongly inhibited glucagon-stimulated c A M P production and lypolysis and may have improved insulin binding affinity in adipose tissue; concluding that G I P has insulinlike effects in this tissue (Hauner et al., 1988). In this vein, M i y a w a k i et al. (2002) recently demonstrated that G I P R -/- mice were protected from high fat induced obesity, while wild-type mice demonstrated "extreme visceral and subcutaneous fat deposition and insulin resistance." These authors also demonstrated that the ob/ob phenotype was partially rescued by crossing ob/ob mice (morbidly obese) with G I P R -/- mice. Thus, this group hypothesizes that the G I P R expressed on adipose tissue could be a potential target for anti-obesity therapy (Miyawaki et al., 2002). In contrast, G I P has also been shown to be lipolytic in some studies. Hauner et al. (1988) showed that GIP was weakly lipolytic and more recently a study demonstrated that the GIP receptor was expressed and signaled via c A M P in the differentiated 3T3-L1 adipocyte model (Mcintosh et al., 1999). Furthermore, this study demonstrated that GIP was able to stimulate glycerol release from these adipocytes in the physiological dose  19  range in a cAMP-dependent fashion, and that this could be inhibited by preincubation with insulin through a wortmannin-dependent pathway (Mcintosh et al, 1999). This paper concluded that GIP-induced lipolysis may be responsible for increasing F F A levels sufficiently, to optimize the insulin secretory response of the (3-cell. In conclusion, it is clear that further studies need to be carried out to determine the effect o f GIP on lipid metabolism; although, it could be the case that the exact effect of GIP on lipid metabolism is dependent on the ambient lipid levels and prevailing metabolic state o f the organism.  1.5.3 GIP and Islet Hormone Secretion Most o f the evidence to date supports the fact that GIP is a potent incretin and acts via the enteroinsular axis to stimulate insulin secretion from the (3-cell. The first studies indirectly showed that impure preparations o f C C K stimulated insulin secretion, and that i f the preparations o f C C K were purified, the insulinotropic potency decreased (Rabinovitch & Dupre, 1972). These observations were similar to those made by Brown et al. (1970) on the effect of C C K preparations on gastric acid secretion and once isolated, provided the impetus for examining the role of GIP on insulin secretion. Later it was demonstrated that GIP stimulated insulin secretion in humans (Dupre et al, 1973), dogs (Pederson et al, 1975) and in rats (Ebert & Creutzfeldt, 1982; Pederson & Brown, 1976; 1978). Furthermore, GIP has been shown to be insulinotropic in isolated islets (Hinke et al, 2000a; Lynn et al, 2001) as wells as in many (3-cell lines (Ehses et al, 2001; 2002; Kieffer et al, 1993; O'Harte etal,  1998).  In vivo, GIP stimulates insulin secretion in the rat (Pederson & Brown, 1976; Tseng et al, 1996b), in the human (Dupre et al, 1973; Elahi et al, 1979), and in dog  20  (Pederson et al, 1975) only when glucose levels are elevated above approximately 5 m M . In the rat perfused pancreas model, the maximum co-stimulatory glucose concentration was determined to be around 16 m M ; thus, many o f the later experiments were carried out at this glucose concentration (Pederson & Brown, 1976). This property prompted Pederson et al (1976) to suggest an alternate name for GIP: Glucose-dependent insulinotropic polypeptide. Aside from the glucose-dependence, the insulin secretory response to G I P is also dose dependent. It was demonstrated that G I P concentrations reached postprandial ly are able to stimulate insulin secretion in normal rats (Pederson & Brown, 1976; Pederson etal,  1982).  The exact pathway by which G I P stimulates secretion of insulin has begun to be elucidated and it is believed that the hormone exerts the majority o f its physiological effects on the (3-cell via activation o f adenylyl cyclase and stimulation o f c A M P production. However, as previously mentioned, other signaling pathways have been implicated (Ehses et al, 2001; Ehses et al, 2002; Trumper et al, 2002; 2001). Most of the studies carried out to date have demonstrated that glucose metabolism is a necessary prerequisite for GIP-stimulated insulin secretion. When D-glyceraldehyde was included in the perfusate, G I P was able to stimulate insulin secretion from the perfused pancreas in the absence o f glucose (Dahl, 1983). Furthermore, mannoheptalose, a glycolysis inhibitor, abolished G I P stimulated insulin secretion in the perfused rat pancreas (Mueller et al, 1982). A series of recent studies by our laboratory have indicated that G I P may also cause insulin secretion, in a  K  +  A  T  P  independent manner as well. These studies  demonstrated that G I P was able to stimulate both c A M P production and insulin secretion  21 in clonal 6-cells that had been depolarized with high external potassium and diazoxide; albeit in a C a  2 +  dependent manner.  Recently, Beguin etal. (1999) demonstrated that stimulation of the 6-cell by G I P caused phosphorylation of the Kir6.2 ( K  A I T  ) channel on serine 372 via protein kinase A .  Phosphorylation of this serine residue led to an increased open probability of the channel. This paper was the first demonstration that G I P stimulation of the 6-cell leads to protein phosphorylation. However, the physiological basis for this phosphorylation event is still unclear since Beguin and colleagues believe that Kir6.2 is maximally phosphorylated in the basal state. Additionally, GIP may have effects on 6-cell proliferation and cell survival, and recent studies in our lab and others (Trumper et al., 2001) have demonstrated that GIP is an extremely potent anti-apoptotic agent; and that these effects are manifested via inhibition of the p38 stress activated kinase signaling module. It has also been demonstrated that GIP has actions on the other cell-types within the islet. GIP-stimulated glucagon release from isolated, cultured islets (Fujimoto et al, 1978; Verchere, 1991), and from the perfused rat pancreas (Pederson & Brown, 1978). In addition, secretion of glucagon in response to GIP only occurs at glucose levels below a threshold o f 5.5 m M in humans and rats (Elahi et al., 1979; Pederson & Brown, 1978); however, GIP is able to increase glucagon secretion in the face o f high glucose in mice (Opara & G o , 1991). Thus, the effect of GIP on glucagon secretion is probably species dependent and may depend on the overall metabolic state of the organism. Finally, GIP has been demonstrated to stimulate somatostatin release from 6-cells in pancreatic islets; though, the physiological relevance of this is not clear because the direction o f blood  22  flow is believed to be from 6-cell to 5-cell and there is only a weak stimulation o f somatostatin release produced by GIP (Schmid et al, 1990; Verchere, 1991).  1.5.4 Other Biological Effects There are many other examples of the effect of GIP on other tissues; however, none o f these actions have been very well characterized. GIP has been shown to affect blood flow in the vascular beds of dogs: some beds are more highly perfused in the presence of GIP e.g. the superior mesenteric artery and portal vein while others are not affected e.g. the celiac artery and hepatic artery (Kogire et al, 1988; Kogire et al, 1992). Recently splice variants of the GIP receptor have been demonstrated in endothelial tissue that could be responsible for the disparities in GIP action in different vascular beds (Zhong et al, 2000). Zhong et al. (2000) reported preliminary data that indicated that GIP can signal to different degrees via either increases in C a  2 +  or P K A activation in  different endothelial cell types. A s previously mentioned, GIP has been implicated in bone metabolism, where it is believed to have an anabolic role (Bollag et al, 2001; 2000). Thirdly, GIP receptors have been localized to various regions in the brain; however, GIP has never been localized to any areas o f the brain. Interestingly, pharmacological doses of GIP injected into the 3 ventricle reduced plasma follicleld  stimulating hormone, and increased growth hormone levels but had no effect on luteinizing hormone, thyroid-stimulating hormone, or prolactin levels (Ottlecz et al, 1985). Presently it has not been determined whether GIP is able to cross the blood-brain barrier or i f another hormone or substance is able to activate GIP binding sites in the brain. GIP is also able to decrease lower esophageal sphincter pressure (Sinar et al, 1978), decrease intestinal motility (Fara & Salazar, 1978), decrease water and electrolyte  23  uptake across the small intestine (Helman & Barbezat, 1977) and may play a role in skeletal muscle glucose utilization (Kahle et al., 1986).  1.6 Evidence for Other Incretins Early studies reported that infusion of GIP antibodies into rats did not completely block the differential insulin response between oral or intraduodenal (Ebert & Creutzfeldt, 1982) and I V glucose. More recently, Tseng et al. (1996b) infused GIP 7. 30NI-12,  a specific antagonist at the GIP receptor, into rats and determined that the insulin  reponse to oral glucose was decreased by 72 %. Taken together, these studies indicate that GIP contributes significantly to the enteroinsular axis: release o f GIP causes 20-70 % of the response to oral vs I V glucose.  1.7 Glucagon-Like Peptide-1 G L P - 1 is genetically encoded within the proglucagon gene. Posttranslational processing cleaves G L P - 1 , G L P - 2 and glicentin from proglucagon in the L-cells of the intestine; whereas, different processing in the a-cells of the pancreas primarily produces glucagon (Fehmann et al., 1995). G L P - 1 is released from the L-cells of the ileum in response to ingested nutrients, primarily glucose and amino acids such as arginine (Elliott et al., 1993). However, the mechanisms that control the postprandial secretion of G L P - 1 are thought to be different from those that control G I P secretion. This is indicated by the fact that the majority of the L-cells are located in the distal small intestine, a site that is not directly stimulated by food prior to the rise in postprandial G L P - 1 secretion (Elliott et al., 1993; Fehmann et al., 1995). A number of studies have indicated that G I P may exert a feed forward effect on the L-cells to stimulate postprandial G L P - 1 secretion (Damholt  24  et al., 1999; Elliott et al., 1993; Herrmann-Rinke et al., 1995; Roberge & Brubaker, 1993). The G L P - 1 receptor has a wide tissue distribution including: brain, lung, stomach pancreatic islet, hypothalamus, heart, intestine, and kidney. Upon binding to its receptor, G L P - 1 activates a seven transmembrane domain G-protein-coupled receptor that has sufficient identity to be considered a member of the secretin/VIP family of G P C R s . Activation of the GLP-1 receptor involves many signaling pathways that appear to be similar to those activated by GIP, at least in the proximal, or receptor associated events. These include activation of adenylyl cyclase and an increase in c A M P , increase in 2_]_  intracellular C a  via extrusion from intracellular stores as well as opening o f V D C C , and  activation o f P L C (Kieffer & Habener, 1999). The primary biological action o f G L P - 1 is believed to be potentiation o f insulin secretion from the (3-cell. Schmidt et al. (1985) demonstrated that G L P - 1 , but not G L P - 2 , was capable o f potently stimulating insulin secretion from the perfused rat pancreas. More than 80 % o f circulating G L P - 1 is in the 7-36NH2 form, and it was hypothesized that due to the shared sequence identity between glucagon and G L P - 1 , this was probably the biologically active, highly insulinotropic form (Fehmann et al., 1995). G L P - 1 is insulinotropic in the presence o f high glucose in human (Kreymann et al, 1987), pig (Hoist et al, 1987), and rat (Mojsov et al, 1987), as well as in a number of (3-cell lines (Lu et al, 1993; Montrose-Rafizadeh et al, 1994; Susini et al, 1998), and isolated islets (Siegel etal, 1992; Suzuki etal, 1992). GLP-1 also inhibits pancreatic glucagon secretion in humans (Nauck et al, 1993b), the rat (Matsuyama et al, 1988), the dog (Kawai et al, 1989) and in isolated  25  islets (Fehmann et al, 1995) thus enhancing its glucose lowering effects. Additionally, G L P - 1 stimulates pancreatic 6-cells causing profound somatostatin release (Fehmann et al, 1995). It is still unclear whether GLP-1 inhibits glucagon secretion by causing somatostatin release or i f there are GLP-1 receptors located on a-cells (Heller et al, 1997; Moens et al, 1996). One o f the other physiological actions o f GLP-1 that may contribute to its glucose lowering effect is its inhibitory effect on gastric emptying (Nauck et al, 1997). Since fats and chyme are potent stimulators of G L P - 1 release, it has been proposed that G L P - 1 might be the major hormone acting as an 'ileal brake' and have a more minor role as an incretin (Kieffer & Habener, 1999). In support of this hypothesis, G L P - 1 does inhibit both gastric acid secretion and gastric emptying when infused in physiological concentrations in many models (Nauck et al, 1997; O'Halloran et al, 1990; Schjoldager et al, 1989; W i l l m s et al, 1996) Glucagon-like peptides and G L P - l receptors are expressed in the hypothalamus where it is believed that binding of agonist can exert anorexic effects. In fact, GLP-1 injection into the 3 ventricle leads to large decreases in food and water intake that can ld  be inhibited with exendin .3 ; indicating specificity for GLP-1 receptors (Turton et al, 9  9  1996). It is still not clear whether GLP-1 leads to satiety or food aversion and whether or not G L P - 1 is able to cross the blood-brain barrier or i f locally produced G L P - 1 acts on these neurons (Kieffer & Habener, 1999).  1.8 GIP and GLP-1 Metabolism Upon release into the circulation G I P  M 2  is rapidly (1-2 min) degraded to G I P .  which renders the peptide biologically inactive (Jornvall et al, 1981; Kieffer et al,  3  42  26  1995b; Pederson et al., 1996; Suzuki et al., 1989). This degradative process is catalysed by the aminopeptidase, dipeptidyl peptidase I V (DP I V ) , which preferentially cleaves peptides containing a penultimate N-terminal proline or alanine residue (e.g. G I P , G L P - 1 , G H R H , N P Y ) but can also degrade peptides containing a serine in the penultimate position, such as glucagon (Hinke et al., 2000b; Pospisilik et al., 2001; Yaron & Naider, 1993). D P I V is ubiquitously distributed; however, the highest concentrations are found in the brush borders of both the kidney and the intestinal epithelia (Yaron & Naider, 1993). Recent studies have shown that inhibition of circulating D P I V , by unhydrolyzable analogue substrates, such as isoleucine thiazolidide, improves the glucose tolerance in the V D F model of T 2 D (Pederson et al., 1998b) and further that these inhibitors can alleviate the hyperglycemia associated with T 2 D (Pospisilik et al., 2002). These findings suggest that inhibition of D P I V is effective in increasing the biological half-life of G I P (and G L P - 1 ) within the circulation and thereby, augmenting the role of the incretins within the entero-insular axis and that D P I V is the primary means of modulating incretin bioactivity In vivo (Deacon et al., 2000; Hansen et al., 1999; Pauly et al, 1996).  1.9 Pathophysiology of GIP release and Actions Because G I P is an important incretin, the role of this hormone in T 2 D has been extensively studied. N o consensus exists regarding changes in circulating G I P levels in T 2 D . It has been reported that G I P levels are increased (Elahi et al, 1984; Jones et al, 1989b; Ross et al, 1977); although, there has been some research indicating G I P levels decrease (Groop, 1989) or remain unchanged (Levitt et al, 1980; Service et al, 1984) in T 2 D . Another defect in T 2 D patients is a reduced incretin effect; consequently, oral  27  glucose does not produce a markedly greater insulin response than an isoglycaemic intravenous infusion as described by Perley and Kipnis (1967). Furthermore, studies indicate that the pancreas is responsive to G I P in T 2 D (Jones et al., 1989b; 1987); however, there is a marked attenuation of G I P induced insulin secretion (Meneilly et al., 1993). Nauck et al. (1993b) and others have shown that there is little or no pancreatic response to natural or synthetic human or porcine G I P in some type 2 diabetic groups (Elahi et al., 1994; Krarup etal., 1987). In contrast, numerous investigations have shown that T 2 D patients are fully responsive to exogenous G L P - 1 and additionally, the pancreata of those patients that are unresponsive to G I P are responsive to GLP-1 (Elahi et al., 1994; Nauck et al., 1993b). Both hormones signal via seven transmembrane domain G-protein coupled receptors (of the same family) to increase adenylyl cyclase activity and intracellular c A M P concentrations (Thorens, 1995; Usdin et al, 1993; Wheeler et al., 1995) and therefore, it is interesting that the sensitivity of the diabetic 6-cell to the two hormones is so distinct (Hoist et al., 1997). A complicating factor in assessing the glucose lowering actions of G L P - 1 is that this hormone has physiologically important insulin-independent glucose lowering actions such as decreasing hepatic glucose output, increasing muscle and adipose glucose uptake, decreasing gastric emptying, and suppressing glucagon secretion (Drucker, 1998). One explanation that could be given for the lack of G I P effect on the diabetic 6cell is that these cells either do not express a G I P receptor or express a defective form. In fact, Kubota et al. (1996) identified two missense mutations in the G I P receptor gene (G198C, Q354E) in Japanese T 2 D subjects. One of these mutations (G198C) was shown to dramatically affect G I P stimulated c A M P production; however, association studies  28  were unable to conclusively provide a relationship between T 2 D and either of these mutations. Thus, it does not seem probable that a mutant G I P receptor is a causative factor in T 2 D ; however, it is possible that a mutation in the 5' flanking/promoter sequence of the GTP receptor gene could cause inefficient receptor transcription (Hoist et al., 1997). This in turn, could decrease receptor expression level and potentially predispose an individual to T 2 D . Furthermore, it has been shown by Tseng et al. (1996a) that rats rendered diabetic by streptozotocin treatment had markedly increased G I P m R N A levels. Furthermore, these experiments demonstrated that when G I P was infused over 6 hours in anaesthetized animals there was a lack of insulinotropic activity at approximately 4 hours, indicating G I P receptor desensitization. Additional studies in the L G I P R 2 cell line indicated that the G I P receptor was desensitized in a ligand specific manner, as the c A M P response to other substances was unaffected (Tseng et al., 1996a). Thus, it is also possible that the insensitivity of the islet to G I P in T 2 D is a result of chronic desensitization of the G I P receptor by the high ambient GTP levels.' Chan et al. (1984) found that the insulin secretory response to G I P was enhanced in fatty Zucker rats and additionally, that the glucose threshold for G I P actions was lower than fasting glucose levels. It has also been shown that postprandial G I P levels in obese subjects are much higher than in normal subjects (Brown & Otte, 1978). Thus, it appears that in the obese state the insulinotropic activity of G I P may become uncontrolled. A substrain of the Zucker (fa/fa) rat, the Zucker Diabetic Fatty rat ( Z D F ) has recently been described (Friedman et al, 1991). In this strain, obese animals (males more pronounced) develop severe glucose intolerance and an impaired ability of the 6cell to respond to glucose. This decreased responsiveness to glucose including the loss of  29  the first phase insulin secretory response to glucose is characteristic of T 2 D in humans (Sturis et al., 1994). A s noted above, this is in contrast to the Zucker fa/fa rat which remains hyperresponsive to all insulin secretagogues (Chan et al., 1984). The Zucker fa/fa colony maintained by our laboratory (Vancouver Zucker Fatty, V D F ) has developed a milder form of the glucose intolerance and insulin secretory defects exhibited by the Z D F rat, including fasting hyperglycaemia, and glucose intolerance as well as the loss of first phase of insulin secretion. Preliminary results also indicate a decreased responsiveness of the isolated perfused pancreas to G I P compared to lean littermates. Thus, these animals provide a model to investigate possible changes in G I P and the G I P receptor at the time of onset of the diabetic state (10-12 weeks).  1.10 Nutrient Regulation of Gene Expression 1.10.1 Glucose Regulation o f Gene Expression The regulation o f gene expression by glucose allows organisms to adapt to their internal nutritional load usually by regulation o f genes involved in lipid or glucose metabolism. Genes that are regulated by glucose can fall into two categories; those that are regulated by glucose levels greater than 5 m M and are regulated to improve the response during nutritional abundance or those which are strongly regulated in the 0-5 m M range and offer protection/adaptation to energy/glucose starvation (Foufelle et al., 1998). Most of the genes identified to date have been of the first category and are those that are induced at the transcriptional level by high glucose, for example glucose induces expression o f fatty acid sythase ( F A S ) in adipose tissue as well as pyruvate kinase ( P K ) in the liver and pancreatic (3-cell (Towle, 1995; Vaulont & Kahn, 1994). It is believed  30  that phosphorylation o f glucose is a prerequisite for its regulatory effects on gene transcription. Studies in both adipose tissue and INS-1 cells have demonstrated that 2deoxyglucose (which is phosphorylated to 2-deoxyglucose-6-phosphate but then not further metabolized) is able to regulate expression o f P K in a manner similar to glucose. Furthermore, the cellular concentrations of glucose-6-phosphate are regulated in a manner similar to the P K gene and finally the kinetics o f the upregulation match those of glucose phosphorylation (Foufelle et al., 1998). Some groups have also proposed that this regulation could occur via xylulose-5-phosphate, which is found in some cells and is an intermediate in the pentose-phosphate pathway o f non-oxidative glucose metabolism (Doiron etal., 1996) There have been two glucose response elements ( G R E ) identified: the first from the P K gene promoter (Thompson & Towle, 1991) and the second from the S14 gene promoter (Shih & Towle, 1992). Sequence comparison between these two G R E s revealed that the canonical sequence for a G R E is two E-box-like sequences o f C A N N T G separated by 5 nucleotides. Two trans-acting factors have been proposed to have roles in the control o f gene expression by glucose. First, the upstream stimulatory factor/major late transcription factor ( U S F / M L T F ) family has been implicated in binding to the P K G R E in (3-cells (Kennedy et al, 1997). Secondly, the sterol regulatory element binding protein/adipocyte determination differentiation-dependent factor 1 ( S R E B P / A D D 1 ) has been shown to activate the S14 G R E in response to glucose ( K i m et al, 1995) and in another study found to bind an E-box motif in the F A S promoter and activates transcription o f F A S ( K i m et al, 1998). The exact pathway by which g l u c o s e s phosphate stimulates activation or repression o f either S R E B P / A D D 1 or U S F is not clear  31  but involves regulating the amount of active transcription factor within the cell by any o f a number o f mechanisms, for example phosphorylation, allosteric modification by binding o f glucose-6-phosphate, or a combination of the two (Foufelle et al, 1998). There have been two reports o f genes that are suppressed by high glucose. The phosphoenolpyruvate carboxykinase gene is downregulated in hepatocytes (Cournarie et al, 1999) and the peroxisome proliferator activated receptor a gene is downregulated in the pancreatic 6-cell by high glucose (Roduit et al, 2000). Both of these studies reported that glucose phosphorylation was necessary for the downregulation to occur.  1.10.2 Fat Regulation o f Gene Expression - Peroxisome Proliferator Activated Receptors F F A regulation o f gene transcription is also a relatively new field; however, the last 10 years have yielded significant developments in understanding the effects o f fatty acids on gene transcription. One o f the most studied pathways by which polyunsaturated fatty acids ( P U F A ) negatively regulate gene expression is that o f the hepatic lipogenic enzymes including: F A S , acetyl C o A carboxylase ( A C C ) , Liver P K , A T P citrate-lyase, malic enzyme, stearoyl C o A desaturase (SCD1), apolipoprotein A - l ( a p o - A l ) , the S14 protein (S14), and A5- and A6-desaturases (Duplus et al, 2000). Most of these genes are negatively regulated by decreasing m R N A transcription; however, as yet it is unclear whether this is a direct effect o f P U F A or of their peroxidative products on gene promoters (Foretz et al, 1999). Fatty acids also regulate genes in a positive manner, mostly in adipocytes. This was first demonstrated by A m r i et al when they demonstrated F A induced adipocyte lipid-binding protein (aP2) gene transcription in pre-adipocytes through a cycloheximide-dependent mechanism; indicating that F F A are not having a  32  direct effect on gene transcription but may be acting via a transcription factor (Amri et al, 1991a; 1991b). Phosphoenolpyruvate carboxykinase (PE'PCK) is also regulated positively by F F A in adipocytes (Antras-Ferry et al., 1994; 1995) and many hepatic genes are upregulated including: acyl-CoA oxidase ( A O X ) , carnitine palmitoyl transferase-1 (CPT-1), the liver fatty acid binding protein ( L - F A B P ) , cytochrome P4504A1, acyl-CoA synthase, 3-hydroxy-3-methylglutaryl-CoA synthase, and cholesterol 7a-hydroxylase. CPT-1 expression is regulated at both the transcriptional and translational level and it appears that oxidation of fatty acids are not necessary for induction (Duplus et al., 2000). There are a number o f proposed mechanisms by which fatty acids can affect gene transcription, including: phosphorylation of T F via a kinase cascade, direct binding of F A to T F , change o f transcription rate o f target gene or T F , change in m R N A stability of target gene or T F (Duplus et al, 2000). One of the only families o f T F that fulfill the requirement o f being F A activated receptors are the peroxisome proliferator activated receptors (PPARs). The first member o f this family o f TFs was cloned in the 1990 on the basis that it was activated by 'hepatocarcinogens' that caused proliferation of peroxisomes in the hepatocytes of mice and subsequent hypolipidemia due to an increase in peroxisomal 6-oxidation (Issemann & Green, 1990). Subsequently, P P A R s were cloned from other species including, hamsters (Aperlo et al, 1995), humans (Sher et al, 1993) and. Xenopus (Dreyer et al, 1992). The study done in Xenopus indicated that there are at least 3 P P A R isoforms: they were designated P P A R a , PP A R B / 5 and P P A R y ; subsequently the nomenclature was revised for the superfamily and now they are all included as members o f group C in the subfamily 1 o f nuclear receptors or N R 1 C 1 ,  33  N R 1 C 2 and N R 1 C 3 respectively (Escher & Wahli, 2000). Furthermore, the three paralogs have now been cloned from rodents and humans and have been found to share considerable sequence identity. The P P A R genes that have been analyzed show considerable conservation in exon structure. Six exons are common to all o f the P P A R genes: one exon encodes the N-terminal A / B domain, two exons encode the D N A binding domain (one exon for each o f the two zinc fingers), one exon encodes the hinge region and two exons encode the ligand binding domain (Beamer et al., 1997; Gearing et al., 1994; Krey et al., 1993; Zhu et al., 1995). However, there is some variation in the 5' U T R structure in some of the P P A R s , specificially with P P A R y which, in humans can have 3 different splice variants (Fajas et al., 1998; Zhu et al., 1995). Using transactivation assays, it was demonstrated that PPARct could activate transcription of the A O X promoter in cooperation with the retinoid X receptor ( R X R ) in response to P U F A as well as saturated F A with chain lengths > 6 carbons (Gottlicher et al., 1992; Keller et al, 1993). Using similar techniques it has been demonstrated that P U F A and thiazolidiones are higher affinity ligands for PPAR(3 and P P A R y while saturated F A and fibrate drugs bind PPARct with high affinity (Desvergne & Wahli, 1999). Additionally, it has been demonstrated that various fatty acid metabolites, particularly leukotrienes (B4), prostaglandins (15-deoxy-A ' -prostaglandin J2) and 12 14  arachidonate (8S-hydroxeicosatetraenoic acid) derivatives, are able to bind to and potently activate P P A R s . These molecules could act as second messengers in the F A control o f gene transcription; however, the transduction cascades by which these molecules are produced are still unclear (Duplus et al, 2000). Upon binding to ligands, P P A R s heterodimerize with the R X R via the  34  ' D - b o x ' domain and are transported to the nucleus where they influence gene transcription by binding to specific sequence elements, known as peroxisome proliferator response elements (PPREs), within gene promoters. Using transactivation assays for a number of genes that are responsive to P P A R activation, the exact sequence element that makes up the P P R E has been determined (Tugwood et al., 1992). The canonical sequence is a direct repeat of the sequence A G G T C A separated by one nucleotide. O n the 5' end of the sequence there is an extended A A C T that is important for specificity and polarity o f P P A R binding with the P P A R and R X R moieties binding to the up and downstream repeats respectively (Desvergne & Wahli, 1999). Genes that are either regulated in a positive or negative manner by P P A R / P P R E s include: A O X , apoA-1, aP2, C P T - 1 , L - F A B P , S C D 1 , S I 4 , malic enzyme, uncoupling protein 1 (UCP1), and acyl-CoA synthase ( A C S ) (Desvergne & Wahli, 1999). The three P P A R paralogs are expressed in a distinct pattern within various tissues. In general, P P A R a is expressed in tissues where catabolism of fatty acids usually occurs. In the mature rodent, m R N A for P P A R a has been found in hepatocytes, cardiomyocytes, proximal tubules o f the nephron, intestinal mucosa, brown adipose tissue and in pancreatic (3-cells (Braissant et al., 1996; Ouali et al, 1998; Zhou et al., 1998). PPAR(3 is ubiquitously expressed and in most tissues has a higher expression level than either of the other paralogs (Braissant et al., 1996; Kliewer et al., 1994). P P A R y is highly expressed in adipose tissue with some expression in the large intestine, jejunum and spleen (Braissant et al, 1996; Kliewer et al, 1994). The expression and tissue distribution o f the P P A R s in humans is similar to that in rodents with the exception that  35  P P A R a may not be as highly expressed in hepatocytes (Auboeuf et al, 1997; Mukherjee etal., 1997; Palmer etal., 1998). The physiological roles o f P P A R s can be divided into three broad categories. First, the main role of P P A R a seems to be upregulation of enzymes involved in lipid oxidation in tissues of the body where this is important, primarily the liver but including the pancreatic 6-cell and the cardiomyocyte (Desvergne & Wahli, 1999). P P A R a stimulates expression of lipoprotein lipase ( L P L ) (Schoonjans etal,  1996), as well as  proteins involved in the translocation of fatty acids across the cell membrane e.g. fatty acid transport protein ( F A T P ) and fatty acid translocase ( F A T / C D 3 6 ) (Motojima et al, 1998) and L - F A B P (Issemann et al, 1992). The physiological role o f the upregulation o f these proteins is to aid in the absorption o f fatty acids into cells. Once in the cells, F A become activated as acyl-CoA thioesters by A C S (Schoonjans et al, 1995) and then may be catabolized in the peroxisomal 6-oxidation pathway by A O X (Tugwood et al, 1992), bifunctional enzyme (Zhang et al, 1992), and thiolase (Lee et al, 1995), all of which are target genes for P P A R a . A c y l - C o A esters may also be shuttled into the mitochondrial oxidative pathway. P P A R a is important in regulating mitochondrial 6-oxidation by controlling the expression o f CPT-1 (Brandt et al, 1998), acyl-CoA dehydrogenase (Gulick et al, 1994), and 3-hydroxy-3-methylglutaryl-CoA synthase (Rodriguez et al, 1994). Furthermore, P P A R a controls the expression of the C Y P 4 A family o f genes that are involved in microsomal oo-hydroxylation (Kroetz et al, 1998), the lipogenic malic enzyme (Castelein et al, 1994), and apolipoproteins ( A p o A - 2 , and ApoC-3) (Staels et al, 1995; Vu-Dac et al, 1995) as well as U C P 1 (Sears et al, 1996). In addition, the P P A R a knockout mouse has outlined the importance of P P A R a expression in the control of fatty  36  acid metabolism and lipid homeostasis (Lee et al, 1995). These animals have defective fatty acid catabolism and thus mice fed a high fat diet develop lipid accumulation in liver and heart tissue (Aoyama et al, 1998; Djouadi et al, 1998). Secondly, the main functions of P P A R p is believed to be the control of gene expression during development, particularly in the central nervous system where it is believed to have a positive effect on cell proliferation/differentiation (Braissant & Wahli, 1998). Because o f its ubiquitous expression pattern, PPAR(3 may also be involved in the control of basic cellular functions including lipid synthesis and turnover (Braissant & Wahli, 1998). Overall, PPAR|3 has not been highly studied because of the lack of specific agonists for this isoform (Desvergne & Wahli, 1999). Thirdly, P P A R y is highly expressed in adipose tissue and is an important transcription factor for differentiation of white and brown adipose tissue from preadipocytes (Dreyer et al, 1992; Tontonoz etal, 1994b). In the adipocyte, P P A R y regulates expression of: the adipocyte fatty acid binding protein 2 (aP2) (Tontonoz et al, 1994a), P E P C K , L P L , F A T P and F A T / C D 3 6 (Motojima et al, 1998; Schoonjans et al, 1996). A l l of these proteins are involved in fat storage and movement within adipocytes.  1.10.3 Other Fat-Activated Transcription Factors There is a substantial body o f evidence proving the important role o f P P A R s in gene regulation in response to fat. However, recently it has become apparent that other T F are able to bind fatty acids and as a consequence modulate gene expression. The hepatic receptor H N F 4 binds F A - C o A and is able to regulate the human A p o C 3 gene promoter (Hertz et al, 1998). Fatty acids may also be able to regulate the synthesis o f some T F . One example o f this is the fatty acid regulation of genes with a sterol  37  regulatory element in their promoters. It has been shown that P U F A can inhibit transcription of genes with a S R E in their promoter (Worgall et al, 1998). This has been attributed to downregulation of S R E binding protein 1 ( S R E B P 1 ) by P U F A , which is a post-transcriptional event (Mater et al., 1999; Shimano et al., 1999; Yahagi et al., 1999). Finally, it has been demonstrated that palmitate and oleate can positively regulate expression of the immediate-early response genes nur-77 and c-fos in pancreatic (3-cells (INS-1) by a mechanism that changes the transcription rate and is dependent on C a , 2 +  P K C and metabolism o f the fatty acid (Roche et al., 1999). Thus, in these ( S R E B P , cfos, nur-77) and probably other cases, F A can regulate the expression of T F themselves and do not seem to affect the transactivation properties o f the T F .  1.11 Rationale M u c h of the present research in the incretin field is driven by the ultimate goal of developing treatments for T 2 D (Hoist et al., 1997). However, at present it is not understood why G I P is unable to adequately stimulate insulin secretion in T 2 D , considering the effectiveness of the "partner" incretin G L P - 1 . A s previously discussed there are a number of factors that could cause the diabetic (3-cell to be unresponsive to GIP; specifically, it could be hypothesized that three pathways are involved. First, but not foremost as previously discussed, there could be a defect in the receptor; which could result from either a mutation in the promoter or within the gene itself (Almind et al., 1998; Hoist et al., 1997; Kubota et al., 1996). Secondly, there could be a defect in G I P mediated signal transduction. Thirdly, there could be a defect in G I P receptor expression, leading to desensitization and/or down regulation of the G I P receptor within the (3-cell and elsewhere (Tokuyama et al., 1995; Tseng et al., 1996a). A l l of these pathways could  38  contribute to a decreased incretin effect, much like that observed in T 2 D . Here we set out to determine i f the G I P receptor is downregulated and if so, how this downregulation might occur. We hypothesized that a defect in receptor expression was responsible for the decreased effectiveness of G I P in T 2 D . Furthermore, we believed that this downregulation may be a result of hyperglycemia, hyperlipidemia or hyperinsulinemia. One consequence of the hyperglycemia associated with T 2 D is abnormal glycosylation of proteins. Furthermore, it has been demonstrated that the expression of many G-protein coupled receptors relies on correct glycosylation. These observations led us to hypothesize that correct glycosylation of the G I P receptor may affect cell surface expression and thereby G I P responsiveness of the 6-cell. B y elucidating if these pathways alter the insulinotropic effects of G I P in T 2 D , it would become much easier to test i f alteration of these pathways in a normal animal yields glucose intolerance and 6 cell defects that would predispose it to a T 2 D -like condition.  Chapter 2 - Methods 2.1 Chemicals Synthetic human GIP (shGIP) and G L P - 1 were purchased from Bachem California, Inc (Torrance, C A , U S A ) and 3-isobutyl-l-methylxanthine ( I B M X ) was purchased from Research Biochemicals International (Natick, M A , U S A ) . A l l chemicals, of reagent or molecular biology grade were from Sigma (Oakville, O N , Canada) or Fisher Scientific International (Pittsburgh, P A , U S A ) . A l l tissue culture disposables were from B D Falcon (San Jose, C A , U S A ) .  39  2.2 Animals Diabetic fatty Zucker ( V D F ) rats spontaneously developed from a Zucker strain maintained by our laboratory. These diabetic rats are homozygous recessive for a mutation in the leptin receptor gene, /a, (Gln269Pro). Rats carrying one normal Fa allele are phenotypically lean and display normal glucose tolerance. Male animals age of 4 and 14-16 weeks of age were used in these studies. A l l animals tested displayed glucose intolerance and decreased first phase insulin response, characteristic o f V D F rats. Lean littermates were used as control animals in these experiments.  2.3 Intraperitoneal Glucose Tolerance Test (IPGTT) Zucker rats were anesthetized with an intraperitoneal (IP) injection of sodium pentobarbital (65 mg/kg) (Somnotol®, M T C Pharmaceuticals, Cambridge, O N , Canada). The right jugular vein was then exposed and cannulated with heparinized polyethylene tubing (PE50, Becton-Dickinson C o , Sparks M D , U S A ) . GIP (4 pmol/min/kg) or physiological saline was infused (30 ul/min) via the cannula using an infusion pump (Harvard Apparatus, South Natick, M A , U S A ) , for five minutes prior to an IP glucose injection (40 %, lg/kg). Blood samples (0.5ml) were collected from the tail vein into heparinized Caraway/Natelson collecting tubes (Fisher Scientific, Pittsburgh, P A , U S A ) 5 minutes prior to (basal) and 10, 20, 30 and 60 minutes following glucose injection. Concomitantly, blood glucose measurements were taken before the infusion started (basal) and then every 10 minutes following glucose administration, using a handheld blood glucose meter (SureStep®, Lifescan Inc., Burnaby B C , Canada). Plasma was then separated from red cells by centrifugation at 10 000 xg for 20 minutes at 4 °C, and then  40 stored at - 2 0 °C until G I P and insulin radioimmunoassays could be carried out (Pederson etal., 1982).  2.4 Measurement of Immunoreactive GIP Samples were diluted in assay buffer containing 5% charcoal extracted human plasma, 2 % Trasylol and 0.04 M P 0 buffer (pH 6.5). shGTP standards were diluted 4  from 7.8 pg to 2000 pg and used for the standard curve. Samples and standards were incubated at 4 °C with GIP antiserum R K 3 4 3 F (1:30 000; Linda Morgan, University of Surrey, Guilford, Surrey) for 24 hours before radiolabelled  125  I - G I P (5000 cpm/tube,  >350 mCi/mg) was added. The samples were then allowed to equilibrate for a further 24 hours before the antisera-bound G I P was separated from the unbound GIP with 25 % polyethylene glycol 8000 ( P E G 8000). Antisera bound  125  I - G I P was then counted for  radiolabel using a gamma counter ( L K B / Wallac 1277).  2.5 In Vitro Pancreatic Perfusion Anesthesia was established using Somnotol® and pancreata were isolated as previously described (Pederson & Brown, 1976). The perfusate consisted o f a modified Krebs-Ringer bicarbonate buffer containing 3 % dextran and 0.2 % bovine serum albumin ( B S A , Fraction V , R I A grade, Sigma) gassed with 95 % O2 / 5 % CO2 to achieve p H 7.4. The abdominal aorta was perfused at a rate o f 4 ml/min and portal venous outflow was collected at one minute intervals. Following a 10 minute equilibration period, the pancreatic perfusion continued with 4.4 m M glucose for 4 minutes  followed  by 8.8 m M glucose for the remainder of the experiment. GIP (10 p M ) , GLP-1 (50 p M ) or saline were introduced into the perfusion system from 20-40 minutes.  41  2.6 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 X I , Sigma) in Hank's Balanced Salt Solution supplemented with 10 m M H E P E S , 2 m M L-glutamine and 0.2 % B S A (HBSS+) (Invitrogen, Burlington, O N Canada). The pancreas was then removed from the rat and macerated with scissors prior to collagenase digestion. The pancreatic tissue was initially digested in a shaking 37 °C water bath for 20 minutes and 10 minutes for pancreata from lean and fat rats respectively; a second digestion was then carried out for 10 and 7 minutes for the lean and fat rats respectively. Following collagenase digestions, the islets were passed through a 1 mm nylon mesh and separated from exocrine tissue via centrifugation (1000 xg/ 4 °C) through a discontinuous dextran gradient. Finally, islets were picked under a dissecting microscope, washed in H B S S + and used for m R N A isolation or cultured in R P M I 1640 with 8.8 m M glucose, 10 % fetal calf serum (Cansera, Rexdale Ontario, Canada), antibiotics (50 U / m l each penicillin G and streptomycin), 0.07 % human serum albumin, 0.0025 % human apotransferrin, 25 p M sodium selenite and 20 u M ethanolamine hydrochloride for 20-24 hours in 10 cm plastic culture dishes (Falcon, Beckton Dickinson, Sparks, M D , U S A ) in a humidified, 5 % C O 2 environment.  2.7 Perifusion of Pancreatic Islets After the culture period, 40 healthy islets (healthy refers to islets that retained a characteristic pink colour when viewed with a dissecting microscope) were selected and sandwiched between two layers of Cytodex-3 beads (Amersham-Pharmacia, Baie d'Urfe,  PQ, Canada) in 0.2 m l polyethylene perifusion chambers (Endotronics Inc., Coon Rapids, M N , U S A ) . The chambers were then perifused in an Acusyst-s perifusion apparatus (Endotronics) under a humid 37 °C, 5 % CO2 environment at a flow-rate of 0.5 ml/min with 10 m M HEPES-buffered Krebs-Ringer bicarbonate buffer ( K R B H ) supplemented with 0.2 % B S A . Perifusion experiments were carried out for 80 minutes following a 60 minute equilibration period in 2.8 m M glucose (low glucose) perifusate. After 20 minutes, the perifusate glucose concentration was switched to 16 m M with or without GIP.  Samples were collected every 2 minutes and insulin levels determined by  radioimmunoassay as previously described (Pederson et al, 1982).  2.8 Measurement of Insulin and cyclic A M P Production by Islets After overnight culture, 40 healthy islets were selected, washed twice with 0.5 ml of K R B H supplemented with 0.2 % B S A and allowed to equilibrate for 30 minutes in a humidified, 5 % CO2 environment. The islets were then stimulated with either vehicle, 10 u M forskolin, or 10 n M GIP for 30 minutes in the presence o f 0.5 m M I B M X . The islets were then lysed by boiling for 5 minutes in 0.05 N hydrochloric acid. Samples were then dried by vacuum centrifugation (Speed-Vac, Sorvall, Farmingdale, N Y , U S A ) and stored at - 2 0 °C for c A M P radioimmunoassay (Biomedical Technologies, Stoughton, M A , USA).  For insulin secretion experiments, 40 islets were selected, washed with  K R B H containing 2 m M glucose and allowed to equilibrate for 60 minutes. Following equilibration, islets were incubated with 10 n M GIP for 30 minutes in 16 m M glucose K R B H . The supernatant was then collected and analyzed for insulin content by radioimmunoassay as previously described. Total insulin was measured by lysing the  43  islets in 0.2 M acetic acid followed by boiling, centrifugation, dilution and radioimmunoassay (Pederson et al., 1982).  2.9 Isolation and Measurement of Islet GIP Receptor messenger RNA by Real-Time Reverse Transcription Polymerase Chain Reaction Rat islet R N A was isolated immediately following islet isolation using Trizol® and the standard protocol supplied by the manufacturer (Invitrogen). Specifically, 1 ml of Trizol® reagent was utilized per 100 islets and the  A260/A280  ratios of isolated R N A  were > 1.80. Following R N A isolation, 1 u.g o f islet R N A was subjected to reversetranscription (RT). Total R N A was reverse transcribed in a volume o f 10 u.1 containing, 0.5 m M deoxynucleotide triphosphates, 15 pmol gene specific primer targeted at the carboxy terminus o f the rat GIP receptor (5'- G T T C T G G A G T A G A G G T C C G T G T A 3'), 75 pmol o f random hexamers (Amersham-Pharmacia), 100 U Superscript II® R N A s e FT Reverse Transcriptase (Invitrogen), 10 U R N A s e inhibitor ( R N A G u a r d ® ; AmershamPharmacia), 1 m M dithiothreitol, 50 m M T r i s - H C l , p H 8.3, 75 m M KC1 and 3 mM: M g C b . Following R T , 100 ng o f rat islet tissue c D N A was used in the real-time P C R reaction to measure GIP receptor expression; whereas, 10 ng c D N A was used in the glyceraldehyde-3-phosphate dehydrogenase ( G A P D H ) control P C R reaction. The P C R reaction mix consisted of l x TaqMan Buffer A ® (PE Applied Biosystems, Foster City, C A , U S A ) , l O m M M g C l , 200 u M d A T P , d C T P , d G T P and 400 u M d U T P , 200 n M rat 2  GIP receptor 5' forward primer (5'- C C G C G C T T T T C G T C A T C C -3'), 200 n M rat GIP receptor 3' reverse primer (5'- C C A C C A A A T G G C T T T G A C T T -3'), 200 n M GIP receptor probe co-labelled with the fluorescent dyes I ' A M and T A M R A (5 - C C C A G C f  A C T G C G T G T T C T C G T A C A G G -3'), 0.01 U / u l A m p E r a s e ® uracil N-glycosylase  44 (UNG, PE-Applied Biosystems), and 0.025 U/uI of AmpliTaq Gold® (PE Applied Biosystems). The GAPDH reactions included the above reaction conditions with the exception of the primers and probe which were purchased from PE Applied Biosystems and were directed towards rodent GAPDH. PCR reactions were carried out in triplicate in the PE Applied Biosystems 7700 sequence detection system. The reaction profile included a 10 minute preincubation at 50 °C to allow the UNG to degrade any uracil containing nucleic acids and a further 10 minute incubation at 94 °C to activate the AmpliTaq Gold®. Following these preincubations, a two-step PCR protocol was carried out, which included a denaturation step at 94 °C for 15s followed by a 1 minute annealing/extension step at 60 °C. Fluorescence was measured during the annealing/extension steps over 40 cycles and used to calculate a cycle threshold (Ct), i.e. the point at which the reaction is in the exponential phase and is detectable by the hardware. All reactions followed the typical sigmoidal reaction profile, and Ct was used as a measure of amplicon abundance (Freeman et al, 1999). 2.10 Western Blot Analysis of Islet GIP Receptor Protein Islets were isolated as previously described. Following isolation, islet GIP receptor protein was analyzed as previously described (Lewis et al, 2000). Briefly, islets were lysed in ice cold RIPA buffer (150 mM NaCI, 20 mM Tris-CI pH 7.5, 1 mM EDTA, 1 % Nonidet P-40, 1 % deoxycholate, 0.1 % SDS, 5 mM NaF, 1 mM phenymethylsulfonyl fluoride, 1 mM DTT, 10 jig/ml leupeptin, 10 u.g/ml pepstatin A, 10 u.g/ml bestatin and 1 % Trasylol (Bayer Pharmaceuticals, Etobicoke, ON, Canada)) for 30 minutes on ice. Protein concentration was determined using Bicinchoninic Acid (BCA) kit (Pierce, Rockford, IL, USA). Fifty micrograms of total islet protein were denatured  45  under reducing conditions (100 m M D T T ) at 100 °C for 5 minutes and run by S D S P A G E . Proteins were then transferred to nitrocellulose membrane, blocked with 5 % skim milk (in tris-buffered saline with 0.5 % Tween 20 (TBST)) and then incubated with a well-characterized polyclonal anti-GIP receptor antibody (Lewis et al., 2000). Membranes were then washed three times in T B S T and then incubated with horseradish peroxidase conjugated goat anti-rabbit IgG secondary antibody (Jackson Immunoresearch Laboratories, West Grove, P A , U S A ) . Following further washing, the immunoreactive bands were visualized using enhanced chemi-luminescence ( E C L ) (AmershamPharmacia). Finally, bands were subjected to densitometry using Eagle Eye II software (Stratagene, L a Jolla, C A , U S A ) and molecular weight was determined using R f analysis.  2.11 Culture of BRIN-D11 and INS(832/13) Cells B R I N - D 1 1 cells were obtained from Dr. P . B . Flatt (University of Ulster, Belfast, N . Ireland), and INS(832/13) cells were obtained from Dr. C . B . Newgard (University of Texas, U S A ) (Hohmeier et al, 2000; McClenaghan et al, 1996). C e l l lines were maintained in a humidified atmosphere containing 5 % C 0 a t 37 °C. Both cell lines were 2  grown in RPMI-1640 medium containing 11 m M glucose, supplemented with 10 % fetal bovine serum (Cansera; Rexdale ON), and penicillin/streptomycin. The media in which the INS(832/13) cells were grown was supplemented with 10 m M H E P E S (pH 7.4), 1 m M sodium pyruvate, 2 m M glutamine and 50 u M 6-mercaptoethanol.  2.12 Transfection of INS(832/13) Cells. The m P P A R a - G (a mutant (G282E) form of mouse P P A R a with low intrinsic transactivation properties but a higher affinity for W Y 14643 and other fibrates than the  46  wild-type form) construct was obtained from Dr. E . F . Johnson (Scripps Research Institute, L a Jolla, C A , U S A ) and a dominant negative form of human P P A R a (hPPARa,,.) was obtained from Dr. B . Staels (Institut Pasteur de L i l l e , France) (Gervois et al, 1999; Hsu et al, 1995). Cells were seeded at 6 x 10 cells/plate in 10 cm dishes. 6  After two days of growth or when the cells were 90 % confluent, transfection was carried out using Lipofectamine 2000™ (Invitrogen) using the manufacturer's protocol. The day after transfection the cells were transferred to 12 well plates with a seeding density of 1 x 10 cells/well and then allowed to grow for 24 hours before the media was replaced and 6  the experiment was started. Transfection efficiencies were determined by co-transfection with the jellyfish green fluorescent protein containing plasmid ( p G F P N 2 ; Invitrogen): typically the transfection efficiency was 40 %.  2.13 Isolation and Measurement of GIP Receptor mRNA from Isolated Islets and Cultured Cells For m R N A experiments, cells were seeded into 12 well plates at a density of 1 x 10 cells/well in R P M I or INS(832/13) media containing 5.5 m M glucose. Cells were 6  grown in these media for 24 hours before media were changed and experimental agents were applied. These included 100 u M W Y 14643, a specific P P A R a activator, 10 u M M K - 8 8 6 , a P P A R a antagonist (Biomol Research Laboratories, Plymouth Meeting, P A , U S A ) , 5 u M H89, a specific P K A inhibitor, 100 u M P D 98059, a M E K inhibitor, 2 u M bisindolylmaleimide, a general P K C inhibitor (Bis) (Calbiochem, L a Jolla, C A , U S A ) , 100 n M wortmannin, a PI-3 kinase inhibitor (RBI/Sigma, Natick, M A , U S A ) , 1 u M insulin or 2 m M palmitate (Sigma). After a further 24 hours incubation, messenger R N A  47 was isolated using 0.5 ml/well Trizol® and the standard protocol supplied by the manufacturer (Invitrogen). Palmitate solution was made by complexing sodium palmitate to B S A . This was accomplished by first emulsifying the sodium palmitate in water at 60 °C. This palmitate mixture was then complexed to a solution of R P M I (0 m M glucose) containing 20 % fatty acid free B S A . This mixture was then diluted in growth medium to a final concentration of 2 m M palmitate and 2 % B S A and filter sterilized. Control conditions for experiments in which palmitate was used contained the 2 % B S A without the palmitate. Islets were isolated from lean Zucker rats as previously described and grown overnight in supplemented RPMI-1640 media as described in section 2.6. Groups of 50 islets were then incubated for 8 hours with either 2 m M palmitate or 100 U.M W Y 14643. Following stimulation, R N A was isolated by addition of 1 ml of Trizol as described. R N A was then quantified using the fluorescent Ribogreen reagent (Molecular Probes; Eugene O R ) . Following R N A isolation and quantification, 125 ng of R N A was subjected to reverse-transcription.  2.14 mRNA Degradation and Half-Life Analyses These studies were carried out by applying actinomycin D (5 u-g/ml) to cells at various times following the beginning of the experiment and then measuring the amount of G I P R m R N A remaining using real-time R T - P C R (Roduit et al., 2000).  48  2.15 Iodination of G I P and Saturation B i n d i n g Studies A s previously described, synthetic porcine G I P (5 jig) was iodinated by the chloramine-T method and the  l25  I - G I P was purified using reverse phase H P L C to a  specific activity of 350 u,Ci/p,g (Kieffer et al., 1995b). Aliquots of the tracer were lyophilized and stored at - 2 0 "C until needed. Cells were seeded into 24 well plates at a density of 5 x 10 cells/well in 5.5 m M glucose containing medium. Following 24 hours 5  of culture the medium was changed and experimental conditions were applied. After a further 24 hours of culture the cells were washed twice with ice-cold K R B H containing 0.2 % B S A . The saturation binding experiment was carried out at 4 °C in K R B H containing 5.5 m M glucose and 1 % Trasylol (aprotinin: Bayer, Etobicoke, O N , Canada) and varying amounts of radiolabeled  l25  I - G I P (12.5-112 fmol). Cells were washed twice  with ice cold K R B H and radioactivity bound to cells was measured using a gamma counter. Non-specific binding was defined as that measured in the presence of 1 u M non-labeled shGIP. A l l binding data are expressed as specific binding of  l25  I - G I P to cells.  2.16 C l o n i n g of the R a t 5' G I P Receptor Promoter The proximal 2 K b o f the rat G I P R promoter was cloned from rat liver using P C R and primers generated from the published sequence (Boylan et al, 1999). Briefly, a male Wister rat was anesthetized using Somnotol®, as previously described, and a laporotomy performed to expose the liver. The animal was then sacrificed by pneumothoraectomy and a ~ 250 mg portion o f the liver was excised. Genomic D N A was then isolated from the liver tissue by lysing the cells in a buffer containing 500 u l o f 10 m M Tris H C l , 0.5 m M E D T A , 0.2 % S D S , 0.2 M N a C l , and 0.1 mg/ml Proteinase K . Cell lysis and digestion was allowed to proceed for 3 hours at 55 °C. Following lysis, one volume o f  49  isopropanol was added to the lysate and samples were mixed on a rotator for 10 minutes. Genomic D N A was then recovered by lifting it from the microcentrifuge tube with a 200 \x\ pipette tip. D N A was placed in a fresh 1.5 ml microcentrifuge tube, washed twice with 70 % ethanol and dissolved in an equal volume of 10 m M Tris FICl overnight at 37 °C. P C R was then carried out on the genomic D N A in the following manner: 1 jxg of the genomic D N A , 100 u M each d N T P , 200 n M o f each primer ( G I P R P l 5'G A A T C C C C A G T G A G G G G C - 3 ' , G I P R P 2 5 ' - C T G T A C C G A G T C C T G C T C - 3 ' ) , 2.5 U of Expand high fidelity polymerase all in the proprietary buffer mix supplied with the enzyme. P C R was carried out by using a hot start for 5 minutes at 95 °C, followed by 35 cycles o f 95 °C for 30s, 56 °C for 1 minute, and 1 minute at 72 °C and then a 10 minute final extension at 72 °C. The P C R was then run out on an agarose gel using standard methods and the 2 kb band was excised and purified using the GeneClean kit (Q Biogene, Carlsbad, C A ) and the provided protocol. The 2 K b P C R product was then cloned into the P C R 2.1 T O P O T A cloning vector and transformed into T O P 10 F cells using the manufacturers protocol (Invitrogen). To ensure that the correct sequence had been obtained, fluorescent sequencing was carried out using the N A P S unit at the University o f British Columbia. The sequence matched the previously published sequence exactly (Boylan etal,  1999).  2.17 GIP Receptor 5'-Promoter Stimulated Gene Transcription and Luciferase Assay The cloned portion of the G I P receptor promoter corresponded to the 2 kb directly upstream of the transcriptional start site. To measure transcriptional activity of this promoter region, it was subcloned into the Eco RI site of P G L 3 (Promega, Madison W l ,  50  U S A ) and two clones containing the construct in both orientations were obtained (pGL3GP+ and p G L 3 G P - ) . P G L 3 G P constructs were then transfected into INS(832/13) cells and analyzed for promoter activity using the Bright G l o Luciferase assay (Promega). First, 5 x 10 cells were plated into 10 cm dishes and allowed to grow for 24 hours. 6  Secondly, the cells were washed two times with low glucose (11 m M ) D M E M (Invitrogen) and transfected with 2.5 | i g of either p G L 3 G P + , p G L 3 G P - , p G L 3 , or p G L 3 Control; the final two being negative and positive controls respectively. The transfection consisted of mixing 2.5 ug of p G L 3 plasmid D N A plus 1 \xg of p G F P N 2 (BD-Clonetech, Palo Alto, C A , U S A ) (a green fluorescent protein containing vector co-transfected and used as a measure of tranfection efficiency) D N A in 250 ul of low glucose D M E M . Concomitantly, 7 ul of Lipofectamine 2000 (Invitrogen) were mixed in an additional 250 pi of low glucose D M E M (Invitrogen). The two, 250 p i , portions were mixed and the cationic lipid was allowed to complex with the D N A for 30 minutes. The complexed D N A was then added to the cells in a total volume of 3 ml and the tranfection was allowed to proceed for 6 hours. Following the 6 hour incubation period, 15 m l of growth media was added to the cells and they were grown overnight. Thirdly, cells were then plated at a density of 5 x 10 cells in 96 well plates and allowed to grow for a further 24 4  hours. Fourth, the medium was changed and experimental stimuli were applied (e.g. stimulation with 2 m M palmitate or 100 u M W Y 14643) and the cells were allowed to grow for a further 24 hours. Finally, the luciferase activity of the samples was measured using the BrightGlo Luciferase reagent kit (Promega) and in a Turner Designs (Sunnyvale C A , U S A ) 96 well luminometer, using the manufacturer's suggested protocols (Promega). Cells were counted under a fluorescent microscope and the percent  51  of cells that fluoresced and therefore contained green fluorescent protein (GFP) was used as a measure of transfection efficiency.  2.18 In Vivo Hyperglycemic Clamp Experiments Lean, 16 week old Zucker rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (65 mg/kg) (Somnotol; M T C Pharmaceuticals). The right jugular vein was then exposed and cannulated with heparinized polyethylene tubing (PE50, Becton-Dickinson). Blood glucose measurements were taken every 10 minutes from the tail vein using a handheld blood glucose meter (SureStep®, Lifescan Inc.) and 50 % glucose or saline was infused (0.5-3 ml/hr) via the cannula using an infusion pump (Harvard Apparatus), and the infusion rate was adjusted to maintain blood glucose levels of 5.5, 10, or 25 m M . Following 6 hours of glucose clamp, the islets were isolated and GIP receptor m R N A levels were determined as described using real-time R T - P C R .  2.19 Pancreatic Perfusions of Hyperglycemic-CIamped Rat Pancreata Circulating glucose concentrations in rats were clamped as described previously however, following 6 hours of clamp the pancreata o f the rats were perfused as previously described with 25 p M human GIP in the presence o f 8.8 m M glucose (Pederson & Brown, 1976). Samples were collected every minute and insulin secretion was determined using radioimmunoassay as previously described (Pederson et al, 1982).  2.20 Site-Directed Mutagensis Site-directed mutagenesis was carried out using modifications on the Quickchange method that was developed by Stratagene Corp (La Jolla, Ca, U S A ) . Briefly, megaprimers were synthesized (table 1) that contained the desired mutations and  52  were complementary to one another. P C R was carried out using these primers and the W T rat GIP receptor that had previously been subcloned, in frame, into the p c D N A 1  3.1(b) V 5 - H I S vector (Invitrogen). This allowed characterization o f protein using both the poly-His tag as well as the V 5 epitope. The mutagenesis was done by combining 200 pg of the vector, 125 ng o f each o f the megaprimers, 125 u M each d N T P , 2.5 U of either Pfu polymerase (Fermentas, M a , U S A ) or Expand High fidelity D N A polymerase (Roche Diagnostics, Laval Quebec, Canada), and each of the manufacturer's P C R buffers including 2.5 m M M g C b in a final volume of 50 u l . P C R reactions were then overlayed with o i l and P C R was carried out over 16 cycles in a Robocycler (Stratagene) using the following reaction profile: 30s at 95 °C, 1 minute at 55 °C and 16 minutes at 68 °C. Following the P C R reaction the product was treated with 10 Units of Dpn I (New England Biolabs, Beverly, M A , U S A ) and restriction digestion was allowed to proceed for 1 hour at 37 °C. Dpn I digests only methylated D N A , allowing only non-mutated D N A to be restriction digested. One microliter o f the resulting mutated D N A was then transformed into competent DH5ct cells via heat shock. Colonies were picked from the plates and sequenced using both radiolabeled dideoxynucleotide sequencing followed by TBE-acrylamide gel and BigDye cycle sequencing followed by analysis on a PE310 genetic analyzer. Double and triple mutants were made by subcloning portions o f the receptor or by mutating single site mutants using the same technique. A l l mutations were subcloned out of the vector in which they were mutated and inserted into a wild-type  53  vector to ensure that mutations.to the vector sequence would not affect the phenotype. They were then fully sequenced through the areas that were mutated.  Table 1: Megaprimers used for glycosylation site mutation N59T1  5'-CGA A G G A A C C A G TAC A G G CC-3'  N59T2  5'-GGC C T G T A C TGG TTCCTT GG-3'  N69T1  5'-GGC A G C CGT GTA GGT C C A G C A GGC-3'  N69T1  5 '-GCC TGC TGG ACC TAC ACG GCT GCC-3'  N74T1  5'-CGG CTG CCA CCA C C A CTG CCC GG-3'  N74T2  5'-CCG GGC AGT GGT GGT GGC A G C CG-3'  N200T1  5'-GGG TCC C T A C A C G G G A A A C C A G A C C C C T A C CC-3'  N200T2  5'-GGG T A G GGG TCTGGT TTC CCG TGT A G G G A C CC-3'  2.21 Transfection, Affinity Purification of GIP Receptor Protein and Western Analyses Following construction of the glycosylation mutants, they were transfected in H E K 293 cells, and receptor protein was isolated by affinity purification. This was accomplished by transfecting 2.5 jig of D N A into H E K 293 cells with Lipofectamine 2000 after 24 hours o f growth from a plating density of 2 x 10 cells/10 cm dish as 6  previously described. Cells were then allowed to grow for 24 hours before protein was harvested for affinity purification. Cells were lysed in 500 u.1 of buffer consisting o f 0.5 % Triton X 1 0 0 , 60 m M (3-glycerophosphate, 20 m M M O P S p H 7.2, 1 m M N a V 0 , 20 3  4  m M NaF, 1 % Trasylol 1 m M P M S F and 2x E D T A - F r e e Protein Inhibitor cocktail  54 (Protein Inhibitor tablets, Roche Diagnostics). Cells were scraped from the 10 cm dishes and extracts were put in 1.5 m l microcentrifuge tubes and sonicated with a needle-tip sonicator for 20 s on ice. C e l l extracts were then centrifuged at 14 000 x g for 30 minutes at 4 °C and supernatent was quickly added to 50 ul of Talon resin that had been preequilibrated in lysis buffer (Clonetech). The poly-histidine tagged G I P R protein in the cell extracts was allowed to bind to the Talon resin during 15 minutes of gentle agitation. The supernatant was then removed and the resin was washed 3 times with 1 ml volumes of lysis buffer. Finally, the poly-histidine tagged protein was eluted from the Talon resin by using a 0.5 M imidazole buffer. Protein concentrations were analyzed by B C A kit (Pierce). Affinity purified protein was then analyzed for glycosylation using PNGase F and Western blotting. First, purified protein extracts were denatured and digestion was carried out on 5 \ig o f protein with peptide:7V-glycosidase F (PNGase F , N e w England Biolabs) using the suggested reaction conditions, at 37 °C for 1 hour. Following digestion, proteins were loaded onto a 12 % acrylamide gel and then Western blotted using conventional techniques. The blots were blocked overnight with 5 % skim milk in tris-buffered saline containing 0.1 % Tween 20 (TBST). Following blocking, blots were incubated with a monoclonal A n t i - V 5 antibody (Invitrogen) at a dilution o f 1:1250 for 3 hours at room temperature. Membranes were then thoroughly washed and incubated with horseradish peroxidase conjugated goat anti-mouse secondary antibody (Jackson Laboratories) for one hour at room temperature. Protein bands were visualized using the enhanced chemiluminescence ( E C L ) reagent (Amersham-Pharmacia) followed by film  55  (Kodak, Rochester N Y , U S A ) exposure for up to five minutes. Molecular weights of the proteins were determined using standard R/analysis.  2.22 Competitive Binding and cAMP Production Analyses in H E K 293 and INS(832/13) Cells Competitive binding analyses were carried out as previously described, with minor modifications (Wheeler et al, 1995). Briefly, transfected H E K 293 and INS(832/13) cells were plated in 24 well plates at a density o f 6 x 10 and 5 x 10 4  5  cells/well respectively and allowed to grow for 48 hours. Cells were then carefully washed twice with 1 ml o f ice-cold K R B H . Cells were incubated for 4 hours at 4 °C with various amounts o f unlabelled GIP (10" -10" M) in the presence of 5 x 10 cpm of 6  purified  l25  12  4  I - G I P . Then the cells were washed two more times with ice-cold K R B H and  solubilized with 0.1 M N a O H . The solubilized cells were transferred to test tubes and radioactivity was counted on a gamma counter ( L K B - W a l l a c e ) . Non-specific binding was taken as the amount of label bound to cells in the presence of 1 u M non-labelled G I P and specific binding was expressed as a percent of total binding. For c A M P studies, cells were plated in 24 well plates as above. Cells were then washed twice with 37 °C K R B H and then allowed to preincubate for one hour at 37 °C. -6  12  Following the preincubation, cells were incubated with G I P (10" -10" M ) in the presence of 0.5 m M isobutylmethylxanthine ( I B M X ) : a phosphodiesterase inhibitor. After 30 minutes o f stimulation, c A M P production was arrested by addition of 1 ml o f ice cold 70 % ethanol. Cells were scraped from the plates and transferred to 1.5 ml microcentrifuge tubes; followed by centrifugation and recovery of the supernatant to fresh tubes. The  56  supernatant was then dried by vacuum centrifugation before c A M P quantification by R I A (Biomedical Technologies, Stouton, M A , U S A ) .  2.23 Insulin Release from INS(832/13) cells 1NS(832/13) cells (3-cells were seeded into 24 well plates at a density of 5 x 10  5  cells/well. Following plating, cells were allowed to grow for 2 days. On the second day, cells were washed twice with K R B H and preincubated for 1 hour in K R B H containing 2 m M glucose. Following preincubation, cells were stimulated to release insulin in varying glucose concentrations for 30 minutes in a total volume o f 200 p i . After the stimulation period, the medium was removed and centrifuged at 12 000 xg for 5 minutes at 4 °C. Concomitantly, total insulin was extracted from the cells using 2 M acetic acid. This was accomplished by adding 200 u l o f acetic acid to the cells, scraping the surface of the plate and boiling the cells for 5 minutes. Samples were then stored at - 2 0 °C for insulin R I A as previously described (Pederson et al., 1982).  2.24 Fatty Acid Oxidation in BRIN-DH Cells. Fatty acid oxidation experiments were carried out as previously described (Shimabukuro et al., 1998). B R I N - D 1 1 cells were used because these experiments were carried out prior to the availability of the INS(832/13) cells. Cells were plated in 24 well plates at a density o f 1 x 10 cells/well and allowed to grow for 48 hours in growth 5  medium containing 2 p C i / m l 9,10-[ H]Palmitic acid (PE-Applied biosystems). Medium 3  was then removed, cells were washed 4 times to remove extracellular radioactivity and preincubated for 2 hours in 11 m M glucose K R B H . Following preincubation, medium was changed and 200 pi of fresh medium containing various concentrations o f GIP  ( I n M - l u M ) was added and incubated at 37 °C for 4 hours. M e d i u m was then removed and extracted twice with an equal volume o f 10 % trichloroacetic acid to remove any excess 9,10-[ H]Palmitic acid. The supernatant was placed in a 1.5 m l microcentrifuge 3  tube and this tube was transferred, uncapped to a 10 ml scintillation vial containing 0.5 ml o f ddHbO. The scintillation vials were then incubated for 24 hours at 60 °C to allow the H 0 to equilibrate with the non-labelled water before 10 ml o f Econo 2 scintillation 3  2  fluid (Fisher) was added, and the radioactivity was determined by liquid scintillation spectrometry. A standard H 0 solution was equilibrated along with the samples to 3  2  control for the equilibration step between different experiments.  2.25 T u n i c a m y c i n Treatment of INS(832/13) Cells Cells were plated into 24 well plates as previously described. Following 1 day o f culture, the medium was changed and tunicamycin was added at a final concentration of 1 pg/ml. Cells were then allowed to grow for a further 24 hours before insulin release studies were carried out.  2.26 D a t a Analysis Where applicable, data are expressed as mean ± standard error o f the mean, with the sample size indicated in the appropriate figure legend. In general, for animal studies an n=l is one animal, and individual measurements were done in at least duplicate. For cell culture experiments an n=l means one plate, on this plate there were individual conditions were carried out in triplicate and each well was analyzed in at least duplicate. Unpaired, two-tailed t-tests were carried out to compare groups o f animals. The means from larger groups were compared using two-tailed A N O V A and either the Dunnet or the  58  Tukey post hoc test. P values < 0.05 were considered statistically significant. Area under the curve was determined using curve analysis software (Graphpad, Prism, San Diego, CA, USA). Competitive and saturation binding data were analyzed using Prism (Graphpad) and the non-linear regression software included in this software bundle. A one site model for binding was previously determined to be sufficient for describing the binding o f GIP to its receptor (Gelling, 1998). Thus for saturation binding studies, the specific binding was determined and plotted against the concentration of radiolabelled GIP that was added to the cells. The data were then fit to a curve with the following equation: Y=B Where B 125  m a x  m a x  -X/(Kd+X)  is the maximal binding, or the binding attained at saturation of the cells with  I - G I P , and K d is the concentration o f  at equilibrium. Thus, both B  m a x  regression analysis. Once the B  125  I - G I P required to reach half-maximal binding  and K d values were determined by Prism during the m a x  value was calculated using regression, the number of  receptors on each cell could be detennined using the specific activity o f the radiolabel and Avagadro's number. Competitive binding analyses were carried using a one site competition model. In these studies the amount of non-labelled GIP was varied to compete for binding sites (GIPR) with  125  I - G I P . Non-specific binding was defined as the amount of binding  observed in the presence of 1 u M non-labelled GIP. This amount of binding was subtracted from all the other binding values to yield specific binding. The specific binding was then plotted against the concentration of non-labelled GIP and non-linear regression was carried out using the following one site competitive binding equation:  59 Y=Bottom + (Top-Bottom)/(l+10 (X-LogIC o)) A  5  Where Y is the specific binding, X is the Logio[cold GIP], Top is the top plateau, Bottom is the bottom plateau and IC50 value is the concentration of cold GIP at which half of the maximal binding is displaced.  60  Chapter 3 - Development of Competitive RT-PCR and TaqMan Real Time RTPCR Methodologies 3.1 Competitive RT-PCR Initially a competitive R T - P C R strategy was developed and utilized to measure the expression of G I P receptor message in the islets of lean and fatty Zucker rats. This methodology was utilized because the Taqman, real-time P C R methodology that has now become standard for measuring R N A abundance was not yet readily available. Furthermore, there are strong points to both of these methodologies and therefore, in retrospect it proved advantageous to employ both technologies. Competitive R T - P C R (cPCR) uses an R N A competitor, which binds both the primer used during the reverse transcription step as well as the primers used in the P C R reaction. The competitor that was utilized in these studies was constructed by inserting a portion (EcoRV-T7) of the polylinker from bluescript ( p B K S ) into the Sma I site in the carboxy (C)-terminus of the G I P receptor c D N A . This manipulation resulted in a 74 bp insertion in the G I P receptor carboxy terminus, and allowed a differentiation on size basis from the wild-type receptor D N A . Competitor R N A was synthesized from this mutant G I P receptor D N A using and a Megascript kit (Ambion Inc, Austin, Tx). The synthetic mutant G I P receptor R N A was gel purified using conventional acrylamide/urea gel electrophoresis and quantified using spectrophotometry. R N A was isolated using Trizol as described in Chapter 2. Following R N A isolation, R N A was quantified using spectrophotometry and then 1.5 ug of total R N A was reverse transcribed. This was accomplished over 1 hour at 50 °C in a 20 pi reaction  61  volume containing: 0.5 m M dNTPs, 30 pmol 3' gene specific primer ( F C L 2 3 ' : C A A G A C C T C A T C T C C A G G C A C A T ) , 200 U Superscript II Rnase FT Reverse Transcriptase (Invitrogen), 10 U R N A s e Inhibitor ( R N A Guard; Pharmacia), 1 m M dithiothreitol, 50 m M T r i s - H C l , p H 8.3, 75 m M KC1, and 3 m M M g C l . After R T , 2 uJ of the R T mix was 2  amplified in a 50 uJ P C R reaction containing 67 m M Tris HC1, 3.0 m M M g S 0 , 166 m M 4  ( N H ) S 0 , 10 m M (3-mercaptoethanol, p H 8.3, with 200 m M dNTPs, 10 pmol of each 4  2  4  primer ( F C L 5 ' : 5 ' - A C C T G T A C G A G A A C A C G C A G T G C - 3 ' and F C L 2 3 ' : C A A G A C C T C A T C T C C A G G C A C A T ) , and 1 U of Taq D N A Polymerase. The P C R reaction profile included a 5 minute intial denaturation step at 94 °C, followed by 40 cycles of 94 °C (45 s), 59 °C (60 s), 72 °C (60 s), with a final extension step at 72 °C for 5 minutes. Twenty microlitre samples were then run out on 1.5 percent agarose gels and imaged using ethidium bromide fluorescence. Trial R T - P C R runs were carried out to determine the range of G I P receptor m R N A concentrations in pancreatic islets and pancreatic (3-cell lines, from which standard G I P receptor concentrations could be derived. The standard curve values that were employed used G I P receptor competitor concentrations in the range of 0.24 to 20 amol of G I P receptor competitor R N A per u,g of total cellular R N A (figure 1). The amount of wild-type G I P receptor m R N A in the original R N A sample was determined by finding the equivalence point between competitor and G I P wild-type R N A . Figure I illustrates that the competitor concentration decreases from 2 amols to 0.25 amols as the amplification of wild-type G I P receptor increases. The point at which this amplification is equal or identical is the point at which there are equal molar amounts of wild-type and competitor R N A in the sample. T o determine this point, the density of each of the bands  was determined using the gel imager and accompanying software (Eagle Eye II, Stratagene). Then, since ethidium bromide binding and subsequent fluorescence is dependent on fragment size, the densities were divided by their fragment sizes (ie 323 bp for competitor and 249 for the W T ) . The corrected densities of the competitor were then divided by the corrected densities for the wild-type D N A and the L o g , values of these 0  numbers were calculated. This was plotted against the L o g | values of competitor R N A 0  that was added to the tubes. When the amount of competitor equals the amount of wildtype D N A the L o g |  0  i l l equal 0. Therefore, the X-intercept on the resulting curve w i l l  give the amount of wild-type G I P receptor R N A (figure 2). Overall, this method was quite labor intensive; thus, real time R T - P C R was employed in further studies.  3.2 Real Time RT-PCR - The Taqman System Due to the labor-intensive nature of competitive P C R , high sample throughput is impossible. Thus, when the Taqman system became available it was utilized for determination of G I P receptor m R N A content of total R N A samples. This system relies on the 5'-3' exonuclease activity of Taq polymerase, as well as a dual labeled fluorescent probe for quantitation of R N A . In these studies, R N A was isolated and reverse transcription was carried out as previously described; however, the P C R reactions were different, as described in detail in chapter 2 One major difference between the P C R reactions carried out for real time P C R (rPCR) and competitive P C R is the inclusion of a fluorogenic probe. The probe used in these studies contained two dyes: l-Dimethoxytrityloxy-3-[0-(N-carboxy-(di-0pivaloyl-fluorescein)-3-aminopropyl)]-propyl-2-0-succinoyl-long chain alkylamino  63  0.5  0.25  attomoles competitor  323 bp 249 bp 1.5 ug wild-type  Figure 1: A typical gel obtained during competitive RT-PCR of GIP receptor RNA. Varying amounts of competitor GIP receptor RNA were added to the reverse transcription reactions that containing 1.5 pg of total RNA extracted from BRIN-D11 cells. The reverse transcription was carried out, followed by 40 cycles of PCR. Twenty microlitres of the PCR reaction were run on a 1.5 % agarose gel and visualized using ethidium bromide fluorescence.  64  Figure 2: Standard curve derived from GIP receptor competitive P C R band density data. The band densities for each agarose gel lane were determined using the desitometry function in the Eagle Eye II gel analysis system (Stratagene). The densities were then corrected for the oligonucleotide band size (323 and 249: W T and competitor respectively) and the L o g i o (log) of the ratio of band densities was plotted on the Y-axis. The Logio of the starting concentration o f competitor R N A was plotted on the X-axis. The two lines represent competitive P C R data for R N A collected from either the islets of a lean Zucker rat (open squares) or a fatty Zucker rat (closed triangles). The X-intercept (or when the Logio of competitor/WT R N A = 0) is the equivalence point, and thus the point at which the amount of competitor equals the amount o f GIP receptor m R N A in the sample. In this case the lean rat islets contained approximately 3 times more GIP receptor m R N A than the fatty rat islets.  65  ( F A M ) and l-Dimethoxytrityloxy-3-[0-(N-carboxy-(TetramethyI-rhodamine)-3aminopropyl)]-propyl-2-0-succinoyl-long chain alkylamino ( T A M R A ) . When these dyes are attached to the probe in its native conformation, energy is transferred from F A M to T A M R A and the F A M fluorescent signal is quenched. However, when the probe binds to D N A and Taq polymerase reads through the probe sequence, the 5'-3' exonuclease activity of Taq removes the dyes from the probe and F A M is able to fluoresce. Thus with each cycle, more F A M is released and fluoresces. This signal can be detected and measured using the PE-Applied Biosystems Sequence Detector System 7700 (figure 3). T o determine the amount of G I P receptor c D N A in a sample, a threshold is set at which point the P C R reaction is still in its exponential phase. This ensures that with each passing cycle there is an exact doubling of P C R product. The point at which any of the reactions pass through this threshold value is known as the cycle threshold and is directly proportional to the amount of starting receptor c D N A (figure 3). T o compare the amount of G I P receptor in samples between P C R runs, a standard curve was used in real-time P C R . W i l d type G I P receptor m R N A standard was synthesized in vitro (Megascript K i t ; A m b i o n Inc.), followed by electrophoretic acrylamide/urea gel purification and spectrophotometric quantification. The standard curve used in all experiments consisted of 1000, 100, 10, 1, 0.1 amol G I P receptor RNA/reaction tube (figure 4). The amount of G I P receptor that was contained in each sample was then determined automatically by the sequence detection system software from a standard curve and could then be easily plotted (figure 4). Samples were analyzed in triplicate, and each P C R run contained 24 separate R N A extractions.  66  A m p l i f i c a t i o n - t f A Z / 1 4 Kun At.  uztzv  2.000 -  1.500  |  -  1.000 -  0.500 -  0.000 - h "  - 0 5 0 0 -I  0  | I | I | | | | I I I I I I I I I I I | I I | | | | I I I I | I I I I I I I t \ 2  4  6  8  10 12  14  16  18 20 22 24 26 28 30 32 34  36 38  40  Figure 3: Raw, standard curve data obtained from the PCR amplification of synthetic standard GIP receptor cDNA. Varying amounts: 0.1 amol (red), 1 amol (green), 10 amol (yellow), 100 amol (blue) and 1000 amol (mauve) of synthetic GIP receptor mRNA were reverse transcribed and then amplified as described in Chapter 2. The fluorescence in each tube was measured at the end of the extension phase of each PCR cycle and the change of fluorescence from a baseline value was plotted against the specific cycle number. The cycle threshold is the indicated by an arrow.  67  Figure 4: Real time R T - P C R standard curve. R N A was extracted from INS(832/13) cells (unknowns) or synthetically synthesized (standard) and R T - P C R was carried out as outlined in Chapter 2. Fluorescence was measured at the end o f each extension phase and standard curve was created by the A B - P E 7700 SDS analysis software using the cycle thresholds. Linear regression was carried out and the unknowns plotted on the standard curve in red.  68  3.3 A C o m p a r i s o n of the T w o Methodologies  Competitive RT-PCR and rPCR both have strong points and the two techniques complement each other well (Freeman et al., 1999). For instance, the major drawback of rPCR is that it is not able to correct for differences in the efficiencies of specific reverse transcription reactions. On the other hand, because the competitor is within the same tube as the target WT mRNA in cPCR, the reverse transcription efficiency is always accounted for. Competitive RT-PCR also has a number of problems associated with it. The greatest problem with cPCR is that it is impossible to know if the PCR reaction is in the exponential phase of the PCR (when the reaction is not limited) or if the reaction has reached the plateau phase (where primer concentrations may be limited or where Tag Polymerase may not be as efficient). Others have demonstrated that there can be a great deal of variability between reaction tubes containing the same template (Freeman et al., 1999). Thus, using cPCR, one can optimize the protocol so that most tubes will fall within the exponential phase but it is impossible to be sure that all the tubes are in the exponential phase. Additionally, because of the nature of rPCR, a signal is obtained from each tube individually and therefore, each tube is quantified individually. This is not the case in cPCR where a series of PCR reactions is required to obtain a single quantification. This characteristic also makes quantification using rPCR more accurate. Another drawback of using cPCR is that it is extremely time consuming to carry out the actual experiments and complete the data analysis. This makes screening numerous samples rather impractical. Additionally, because there are many steps in this process, and  amplification of a large amount of message, there is often opportunity for contamination of the workspace with attendant P C R problems. This is avoided in r P C R by removing the agarose gel step altogether. Therefore, in r P C R , the P C R tubes are not opened after amplification, and the risk of contamination is low. Additionally, d U T P is used in the r P C R reactions and uracil-DNA glycosylase is added to the P C R reactions prior to amplification to degrade any nucleic acids containing uracil; elimating the risk of amplification of prior P C R products. Both methods rely on equal amounts of total R N A being used in the reverse transcription. A few different techniques for measuring R N A , or correcting for the amount of R N A added to the reverse transcription were utilized in this thesis. Initially, R N A concentrations were measured using the absorbance at 260 nm. This method of measuring nucleic acids is very prone to inaccuracies due to the fluorescence of the aromatic amino acids present in proteins at around 280 nm. Thus, a small amount of protein contamination in the R N A drastically overestimates the actual amount of R N A in the sample. This was corrected for in our experimental design by: first, only using R N A samples that had A  2 6 0  /A  2 K 0  ratios of greater than 1.8 and that were almost identical in our  c P C R reactions and later on by including glyceraldehyde phosphate dehydrogenase ( G A P D H ) external control reactions in early r P C R experiments. G A P D H is a metabolic enzyme that is expressed at a constant level within the cell. In the intial r P C R studies, most investigators were using G A P D H as an external control for their studies (Zamorano et al., 1996). Thus, we also normalized our G I P receptor expression to G A P D H expression, and this helped correct for any small differences in the amount of R N A added to our reverse transcriptions. In our intial studies that compared G I P receptor levels in  70  fatty and lean Zucker rats (Chapter 4), there was good agreement between the c P C R method using total R N A and the r P C R method using total R N A and G A P D H as an external control. However, it has been consistently demonstrated that changes in metabolic state of the cell, as well as other manipulations can alter G A P D H expression levels (Zamorano et al., 1996). These studies prompted us to investigate the possibility of using alternate means to determine the amount of total R N A added to our reverse transcription reactions. W e thus switched to the Ribogreen fluorescent method (Molecular Probes) for R N A determination because it relies only on the binding of a fluorescent probe to R N A , and completely excludes protein from the measurements. The fluorescent dye utilized in this method does not efficiently bind to either nucleotide triphosphates or to small single stranded molecules; thus, there is little chance of including degraded R N A in the determinations. This method of quantification along with the development of the R N A standard allowed efficient and accurate quantification of G I P receptor R N A levels. Since the development of these techniques, we have repeated some of our initial experiments and observed very good agreement with our previous results. Thus, in our hands both c P C R and r P C R methodologies demonstrated that the G I P receptor was downregulated in the fatty Zucker rat, and both techniques demonstrated that G I P receptor expression was downregulated by approximately 70 % (figures 2 & 16).  71  C h a p t e r 4 - G I P and the V a n c o u v e r Diabetic Fatty Z u c k e r V D F R a t M o d e l of Type 2 Diabetes  4.1 B a c k g r o u n d  A large proportion of postprandial insulin secretion is stimulated by hormones secreted from the small intestine. Glucose-dependent insulinotropic polypeptide, the proglucagon gene derived glucagon-like peptide-1-(7-37) (GLP-1) and the carboxyterminal truncated form: GLP-1-(7-36)-amide are the major incretins that act via this endocrine system to potentiate glucose induced insulin secretion (reviewed in D'Alessio, 1997). GIP and GLP-1 both signal via serpentine, seven transmembrane, G-protein coupled receptors of the secretin/VIP superfamily. Binding of the incretins to their respective receptors on the |3-cell surface activates adenylyl cyclase, increases cAMP and stimulates insulin secretion (Gremlich et al., 1995; Moens et al., 1996; Wheeler et al, 1995). Recent studies have demonstrated that the GIP receptor displays similar characteristics to other G-protein coupled receptors in terms of ligand binding, desensitization and subsequent internalization (Gelling et al, 1997; Wheeler et al, 1999). A wide range of experimental techniques have been utilized to demonstrate the physiological importance of GIP and GLP-1. In vivo administration of exendin-(9-39) and GIP-(7-30), GLP-1 and GIP receptor antagonists, resulted in decreased insulin responses to oral glucose (Schirra et al., 1998; Tseng et al., 1996b; 1999). Furthermore, both GIP and GLP-1 receptor knockout mice display compromised insulin release and, therefore, altered glucose tolerance to an oral load (Miyawaki et al., 1999; Pederson et al., 1998a; Scrocchi et al., 1996). From these studies, it has been concluded that  72  secretion o f the incretins could account for up to 70 % o f the postprandial insulin response to glucose (Nauck et al., 1993a). GIP and GLP-1 both require elevated levels o f ambient glucose to stimulate pancreatic (3-cell insulin secretion; hence, there is considerable interest in using incretin analogs of these peptides in the treatment of T 2 D (Brown et al., 1978; Jia et al., 1995; Nauck, 1998; Pederson & Brown, 1976; Rachman & Turner, 1995). One shortfall of using GIP in therapy is the controversy over its effectiveness as an incretin in T 2 D (Nauck et al., 1986; 1993b). Human studies have shown that there is a decreased incretin effect in T 2 D and this has been attributed mainly to an attenuation of GIP-stimulated insulin secretion either via a change in GIP receptor expression or a change in circulating GIP levels, although, altered signal transduction pathways could play a role (Hoist et al, 1997). Presently, there is no consensus regarding possible abnormalities in circulating levels o f GIP in type 2 diabetics; studies have demonstrated increased, decreased and unchanged GIP levels (Ahren et al., 1997; Fukase et al., 1993; Jones et al., 1989a; Vaag et al., 1996). Thus, it cannot be concluded that chronic, homologous desensitization of the GIP receptor in T 2 D causes an ineffective incretin response (Hinke et al., 2000a; Tseng et al, 1996a). In addition, studies have shown point mutations in the GIP receptor gene in human populations that affect GIP signaling in cell models; however, it has not been possible to associate these mutations with T 2 D (Almind etal., 1998; KubotaeJa/., 1996). In the current study we set out to test the hypothesis that the attenuated G I P stimulated insulin responses observed in T 2 D can result from long-term downregulation  73  of GIP receptor expression in the (3-cell plasma membrane. To test this hypothesis we utilized the Vancouver diabetic fatty Zucker ( V D F ) rat as a model o f T 2 D .  4.2 Effect of G I P on glucose tolerance in the V D F rat. In order to quantify the glucose lowering potency o f GIP in V D F obese rats, the glucose lowering actions o f GIP were first assayed in lean controls for comparative purposes. In initial experiments, the optimal GIP dose was determined by carrying out a bioassay with varying GIP concentrations ranging from 2 pmol/min/kg to 20 pmol/min/kg and monitoring the glucose lowering potency (figure 5). The optimum dose determined from this study (4 pmol/min/kg) produced a submaximal glucose lowering response in the lean animals but was still within the physiological range of doses. Thus, this dose was utilized for the remainder o f the experiments. Figure 6 shows the blood glucose response to an IP glucose tolerance test in the presence or absence o f infused GIP in lean, control animals. This figure clearly shows that GIP was able to significantly improve glucose tolerance in the lean animals as early as 30 minutes following IP glucose injection. Furthermore, this improvement in glucose tolerance was maintained as long as the GIP infusion was continued. Figure 6 (inset) shows that the integrated glucose response (over 65 minutes) for the lean animals receiving GIP was significantly smaller than those receiving saline. The fat animals displayed basal hyperglycemia, with an average o f 8.3 ± 0.4 m M , compared to 5.5 ± 0.2 m M for the lean animals (cf figures 6 and 7). The fat animals also  74  700-1  2  4  10  20  Infused d o s e of G\P^.^  2  ( p m o l / m i n / k g b o d y weight)  Figure 5: Dose related effects of GIP on plasma glucose during an IPGTT. Integrated glucose responses were determined from lean Zucker rats over 65 minutes of either GIPi. 42 or saline infusion concomitant with a lg/kg intraperitoneal glucose tolerance test as outlined in the methods (n=2).  75  16-,  i  1  1  1  1  1  1  1  1  -10  0  10  20  30  40  50  60  70  Time (minutes)  Figure 6: Glucose response to infused GIP (A) and saline ( • ) in control, Fa/? rats. Basal blood glucose samples were taken and then a 4 pmol/min/kg dose of G I P was infused into the jugular vein. Following five minutes of G I P infusion, glucose (lg/kg) was administered via an intraperitoneal injection. Blood glucose measurements were made on a handheld glucose analyser at 10 minute intervals. The inset indicates integrated area under the two curves ( A U C ) . Asterisks indicate statistical significance (n=12, P <, 0.05), values are expressed as mean ± S . E . M .  76  GIP-|.42 (4 pmol/min/kg) or Saline infusion  i  1  1  1  1  1  1  1  1  -10  0  10  20  30  40  50  60  70  Time (minutes)  Figure 7: Glucose response to infused GIP ( A ) and saline ( • ) in V D F (fa/fa) rats. Basal blood glucose samples were taken and then a 4 pmol/min/kg dose of G I P was infused into the jugular vein. Following five minutes of infusion, glucose (Ig/kg) was administered via an intraperitoneal injection. Blood glucose was measured with a handheld glucose analyser at 10 minute intervals. The inset indicates integrated area under the two curves ( A U C ) . Values are expressed as mean ± S . E . M (n=12, P <, 0.05).  77  had significantly higher peak glucose levels in response to the glucose challenge, with the control (saline) values peaking at 15 m M (compared to 11 m M in lean animals); thus the fatty animals were glucose intolerant and hyperglycemic. The GIP infusion did not result in a decrease in circulating glucose levels in the fat animals, as no difference (P>0.05) was observed between GIP and saline infusions at any time following IP glucose (figure 7). Furthermore, the integrated glucose response for the fatty animals that received GIP was not different from the integrated response o f saline infused animals (inset figure 7). Thus, GIP at an effective glucose-lowering dose in lean rats, yielded no improvement in glucose tolerance in the diabetic fatty animals.  4.3 Effect of GIP on insulin secretion in the Zucker rat. In the same I P G T T experiments, plasma was collected at - 5 , 10, 20, 30 and 60 minutes following IP glucose and assayed for insulin and IR-GIP content by R I A . There was no difference observed in basal circulating IR-GIP levels between the fat (16.7 ± 1.8 p M ) and lean (15.5 ± 1.6 p M ) animals. Additionally, there was no difference in the integrated GIP response of the saline infused animals during the IPGTT, indicating both that the circulating levels o f GIP are not different in the two phenotypes and that the I P G T T was not stimulating endogenous GIP release (figure 8). GIP yielded a significant increase in circulating insulin levels in the lean animals with a peak o f 400 p M at 20 minutes, which was prior to the glucose peak, observed in the same study (cf figures 6 & 9). Additionally, the integrated insulin response was significantly greater in the lean animals that received GIP (inset figure 9). Due to the insulin resistant state of the fatty animals, insulin levels were much higher at all times during the infusion protocol in this group (figure 10). Furthermore, there was neither a  78  5000-,  Figure 8: The integrated GIP response o f saline infused control (Fa/?,\ean) and V D F (J'a/fa,¥at) rats during the I P G T T . Basal blood glucose samples were taken and then saline was infused into the jugular vein at a rate o f 30 u,l/min. Following five minutes of infusion, glucose (lg/kg) was administered via an intraperitoneal injection. Blood samples (500 ul) were collected from the tip o f the nicked tail and plasma GIP was assayed using radioimmunoassay. Values are expressed as mean ± S . E . M (n = 9, P <; 0.05).  79  Figure 9: Insulin responses to infused GIP (A) and saline ( • ) in control, Fa/7 rats. Basal blood glucose samples were taken and then a 4 pmol/min/kg dose of G I P was infused into the jugular vein. Following five minutes of infusion, glucose (lg/kg) was administered via an intraperitoneal injection. Blood samples (500 pi) were collected from the tip of the nicked tail and plasma insulin was assayed using radioimmunoassay. The inset was obtained by taking the area under the curves ( A U C ) from the timecourse study. Asterisks indicated statistical significance (n = 4, P <, 0.05). Values are expressed as mean ± S . E . M .  80  Figure 10: Insulin responses to infused GIP ( A ) and saline ( • ) in V D F (fa/fa) rats. Basal blood glucose samples were taken and then a 4 pmol/min/kg dose of G I P was infused into the jugular vein. Following five minutes of infusion glucose (lg/kg) was administered via an intraperitoneal injection. Blood samples (500 ul) were collected from the tip of the nicked tail and plasma insulin was assayed using radioimmunoassay. The inset was obtained by taking the area under the curves ( A U C ) from the timecourse study. Values are expressed as mean ± S . E . M (n = 4, P ^ 0.05).  81  significant increase in insulin secretion elicited by GIP infusion (figure 10) nor an increase in the integrated insulin response in these animals (inset figure 10).  4.4 Effect of G I P on insulin release f r o m the pancreas of the Z u c k e r rat.  Pancreatic perfusions were carried out to determine if the defect in the secretory response to GIP in obese animals was confined to the pancreas and was not a result of extrapancreatic effects of this peptide. As indicated in figure 11, 10 p M GIP and 50 p M GLP-1 in the presence of 8.8 m M glucose evoked a significant 1.5 fold increase (~ 6-fold increase in area under the curve between 20 and 40 minutes) in insulin secretion from the lean perfused pancreas. However, this augmentation in insulin secretion was not observed in the V D F Zucker pancreas (figure 12) where insulin secretion decreased from approximately 8480 p M to around 3650 p M during the high glucose and GIP infusion period. Although GLP-1 did produce an approximate 5-fold increase (~ 10-fold increase in the area under the curve) in insulin secretion from the perfused V D F pancreas. As seen in figure 13, 10 n M GIP in the presence of 16 m M glucose was able to stimulate insulin secretion from the lean Zucker islet, with a peak level 10 times the basal level of 62 pM. High glucose (16 mM) alone only produced a 4-fold increase in insulin release from the islets. Figure 14 illustrates the effects of GIP on the islets from the fatty Zucker rat. As illustrated, there was little effect of 10 n M GIP on insulin release from these islets in the presence of 16 m M glucose. The peak GIP-stimulated insulin release from the fat islets in the perifusion system was 4.3 ±1.5 -fold basal, whereas glucose alone produced about a 3.2 ± 1 . 5 -fold increase from a basal level of 95 pM. This  82  o •*-> ns (A  3 «^ <D 0.  3.0-  8.8 mM Glucose  4.41  2.5-  T  X  -T-T  2.0-  r—•i  i  L O (A  £  0.5  0.0  J  I—I—'—<—'—I—  0  10  I—I—I—I—1—I—I—I—  20  30  40  -i—i—|—i 50  Time (minutes)  Control GIP GLP-1  Figure 11: Insulin responses from the perfused pancreata o f control rats. Pancreata were perfused at a rate of 4 ml/min with Krebs buffer. Four minutes (t=4) following equilibration, the preparations were subjected to 8.8 m M glucose (open diamonds, glucose alone). G I P ( • , 10 p M ) or GLP-1 ( • , 50 p M ) was then added via an infusion pump and a side arm at twenty minutes. Samples were collected every minute and assayed for insulin content using radioimmunoassay. The area under the curves (inset, A U C ) was determined using Graphpad software (Prism). Values are expressed as mean ± S . E . M . (n=3-6). Asterisks indicate statistical significance P<0.05.  83  o TO Ui  35J  5  30H  .E c,  20-  8.8 mM  4.4  40-i  Glucose  •  153 C  10 0-  I  0  Peptide Infusion  — i — 1 — 1 — 1 — 1 — 1 — 1 — 1 — 1 — 1 — 1 — 1 — 1 — 1 — 1 — 1 —  10  20  30  40  50  Time (minutes) 6OO-1  500-  AU  0  4003002001000-  Control GIP GLP-1  Figure 12: Insulin responses from the perfused pancreas o f V D F (fa/fa) rats. Pancreata were perfused at a rate of 4 ml/min with Krebs buffer. Four minutes (t=4) following equilibration, the preparations were subjected to 8.8 m M glucose (open diamonds, glucose alone). G I P ( • , 10 p M ) or GLP-1 ( • , 50 p M ) was then added via an infusion pump and a side arm at twenty minutes. Samples were collected every minute and assayed for insulin content using radioimmunoassay. The area under the curves (inset, A U C ) was determined using Graphpad software (Prism). Values are expressed as mean ± S . E . M . (n=3-6). Asterisks indicated statistical significance P< 0.05.  84  n & ion (D (/> (0 0)  c 0•10  -I  1  1  1  1  0  1  1  1  1~  n  10  20  30  I  f-  40  Time (minutes) Figure 13: Insulin release from perifused islets isolated from control (Fa/?) rats. Islets were isolated, cultured and perifused as described in Chapter 2. Following a 70 minute equilibration at 2.8 m M glucose, 10 n M G I P in 16.6 m M glucose ( A ) or 16.6 m M alone ( • ) was applied and continued for the remainder of the experiment. The inset depicts area under the curves ( A U C ) that were determined using the trapezoid rule (Graphpad, Prism). Fractions were collected every 2 minutes and perifusate was assayed for insulin using radioimmunoassay. Data is expressed as mean ± S . E . M . (n=3).  85  Figure 14: Insulin release from perifused islets isolated from V D F (fa/fa) rats. Islets were isolated, cultured and perifused as described in Chapter 2. Following a 70 minute equilibration at 2.8 m M glucose, 10 n M G I P in 16.6 m M glucose ( A ) or 16.6 m M alone ( • ) was applied and continued for the remainder of the experiment. The inset depicts area under the curves ( A U C ) that were determined using the trapezoid rule (Graphpad, Prism). Fractions were collected every 2 minutes and perifusate was assayed for insulin using radioimmunoassay. Data is expressed as mean ± S . E . M . (n=3).  86  response was similar to the response seen in the lean rat islets to glucose alone (cf figures 13 & 14). 4.4 Effect of GIP on c A M P production in Zucker rat islets. Islet c A M P studies were carried out to locate the defect in the GIP signaling pathway in the fatty animals Figure 15 illustrates the effect of GIP on c A M P production from fat and lean rat islets. GIP (10 n M ) produced a marked response in the lean, control islets; however, there was no observable c A M P response to GIP in the islets from obese animals. The basal values of c A M P production did not differ significantly between the two phenotypes. Forskolin was included in these experiments to control for islet size and viability. A s seen in figure 15, forskolin-stimulated c A M P production did not differ significantly between islets that were isolated from the two phenotypes.  4.5 GIP receptor mRNA expression in the Zucker rat islets. The c A M P data suggested that a decrease o f GIP receptor expression or a defect proximal to adenylyl cyclase could be responsible for the decreased effectiveness of GIP signaling in the fatty Zucker rat. This hypothesis was tested by carrying out reversetranscription, real time P C R on R N A isolated from islets o f lean and fat animals. W e observed a significant (75 ± 5 %) decrease in GIP receptor m R N A in the islets from the fatty Zucker rats, as seen in figure 16 A . Additionally, the reduction o f GIP receptor m R N A was obtained when measured with RT-competitive P C R : an alternate means o f measuring R N A expression (figure 16 B ) .  87  Figure 15: Islet c A M P responses to GIP and forskolin from control and V D F rat islets. Islets were isolated and cultured as described in Chapter 2. Forty islets were allowed to equilibrate in 8.8 m M glucose K R B H at 37 °C for 30 minutes prior to G I P stimulation (10 nM). G I P , or forskolin (10 u M ) or glucose alone was then added to the islets in K R B H supplemented with 0.5 m M I B M X . Islets were stimulated for 30 minutes prior to c A M P extraction and measurement using radioimmunoassay. Data are expressed as mean ± S . E . M . (n=4) with asterisks indicating statistical significance between control and G I P stimulated conditions (P <, 0.05).  88  L e a n (Fa/?)  O b e s e (fa/fa)  Figure 16: G I P receptor m R N A levels in the islets of control (Fa/?) and V D F (fa/fa) rats measured by ( A ) real time R T - P C R or (B) competitive R T - P C R . Islets were isolated as described in Chapter 2. Following isolation, R N A was extracted from the islets and subjected to reverse-transcription P C R . G I P receptor m R N A was normalized to glyceraldehyde-phosphate dehydrogenase m R N A content (A) and expressed as a fraction of control islet content. Data are expressed as mean ± S . E . M . (n=4) with asterisks indicating statistical significance (P <. 0.05).  89  4.6 GIP receptor protein expression in the Zucker rat islets. The Western blot shown in figure 17 was typical o f those observed when comparing fat and lean islet G I P receptor protein content.  The post-translationally  modified G I P receptor appears to run at around 65 k D a which is in agreement with previous work (Amiranoff et al., 1986). Figure 17 illustrates a marked decrease in GIP receptor protein level in islets from the V D F rats. This decrease is in accordance with that seen with m R N A levels as well as insulin release and c A M P stimulation o f islets with G I P .  4.7 Glucose Tolerance, Insulin Secretion, and GIP Receptor Expression in Prediabetic VDF Rats Experiments were carried out using 4 week old prediabetic animals to determine if G I P receptor downregulation was a result of impaired glucose tolerance or whether it was a genetic defect inherent to this animal model and present at birth. The prediabetic animals displayed significantly elevated blood glucose levels in a fasted state (time 0) as well as 10 minutes following glucose administration during an oral glucose tolerance test ( O G T T ) . However, the glucose levels in young V D F animals and control animals were superimposed for the remainder of the O G T T (figure 18). These data indicate that at four weeks of age, these animals were only mildly hyperglycemic and thus, in a prediabetic state. The small size o f these animals made it impossible to collect sufficient blood to carry out I P G T T experiments similar to those performed on older animals. Thus, islets were isolated and the insulin secretory response to G I P was assessed. A s seen in figure 19, GIP stimulated a significant amount o f insulin secretion from the islets o f both the  90  75i  Figure 17: GIP receptor protein expression in the islets o f control (Fa/?) and V D F (fa/fa) rats. Islets were isolated as described in Chapter 2. Following isolation islets were lysed in ice-cold R I P A buffer. 50 \ig of total cellular protein was run on a 13 % polyacrylamide gel. The gel was transferred to a nitrocellulose membrane and blotted with GIP receptor antibody, followed by HRP-conjugated goat anti-rabbit secondary antibody. The immunoreactive bands were visualized using E C L , and G I P receptor molecular weight (65 kDa) was determined using R/ analysis. This figure is a representative o f an n=3.  91  I  1  1  0  15  30  r 45  1  1  T  1  60  75  90  105  1—  120  T i m e (min)  Figure 18: Oral glucose tolerance test from 4 week old control (Fa/?) and V D F (fa/fa) rats. Rats were fasted overnight before an oral glucose tolerance test was carried out. Glucose (lg/kg) was administered after a basal blood glucose reading was taken from the tail vein using a Surestep handheld blood glucose analyzer. Further blood glucose readings were taken as indicated. The inset shows the integrated area under the curve for the control (white) and V D F (black) animals (n=10).  92  Figure 19: Insulin release from isolated islets from 4 week old control (lean, Fa/?) and V D F (fat, fa/fa) rats. Islets were isolated and cultured as described in Chapter 2. Forty islets were allowed to equilibrate in 2.0 m M glucose K R B H at 37 °C for 60 minutes prior to GIP stimulation (10 n M ) at 16.7 m M glucose. Islets were stimulated for 30 minutes prior to collection o f the media, extraction o f total insulin and measurement of both using radioimmunoassay. Data are expressed as mean ± S . E . M . (n=4) with asterisks indicating statistical significance between control and GIP stimulated conditions (P < 0.05).  young V D F and young control animals. Thus, it appears as i f G I P retains insulinotropic potency in islets of young V D F rats. To determine whether the insulin secretory profile in response to G I P was similar in the 4 week old animals, the pancreata from control and prediabetic V D F animals were perfused with a 0-50 p M gradient (figure 20). G I P perfusion stimulated insulin secretion from both the control and the prediabetic pancreata when compared to saline infusions. However, it appears that the 4 week old prediabetic V D F animals are hypersensitive to GIP: as stimulation with a low G I P concentration caused a profound insulin secretory response compared to the G I P response obtained from the 4 week old control animals (figure 20). In addition, the prediabetic V D F animals were not able to increase their insulin response to higher concentrations of G I P to the same degree as the control animals. This could be a result of a rapid desensitization of the islets of the prediabetic V D F animals to G I P or because the 6-cells of these animals are already maximally secreting insulin in response to low G I P concentrations. Finally, when the G I P receptor expression levels were analyzed in the islets of young control and V D F animals (figure 21), it was determined that there was a statistically significant 32 % decrease in the expression level of the G I P R in the islets from young V D F animals. This decrease amounted to approximately one-half the level of downregulation observed in the older V D F rats.  4.8 DISCUSSION Human type 2 diabetics have been characterized by a decreased incretin response (Ahren et al, 1997; Nauck et al, 1986). This has been attributed to dysfunction in the  94  i  i  i  1  1  1  0  10  20  30  40  50  Time (minutes) Figure 20: Perfusion o f 4 week old control and V D F Zucker rat pancreata with saline or with a 0-50 p M gradient of GIP. Rats were anesthetized, pancreata isolated as described in materials and methods and perfused with a gradient of GIP in the presence of 8.8 m M glucose. Open circles denote saline infused V D F rats, filled circles denote GIP infused V D F rats. Open squares denote control saline infused animals and filled squares denote control animals infused with GIP. Perfusate was collected at one minute intervals and analyzed for insulin content using radioimmunoassay (n=4).  95  Figure 21: GIP receptor m R N A levels in the islets o f control (lean, Fa/?) and V D F (fat, fa/fa) rats. Islets were isolated as described in Chapter 2. Following isolation, R N A was extracted from the islets and subjected to real time reverse-transcription P C R . G I P receptor m R N A was normalized to glyceraldehyde-phosphate dehydrogenase m R N A content and expressed as a fraction of control islet content. Data is expressed as mean ± S . E . M . (n=4) with asterisks indicating statistical significance (P <, 0.05).  96  GIP portion o f the enteroinsular axis since G L P - 1 continues to exert relatively normal insulinotropic and blood glucose lowering actions in these individuals (Ahren et al, 1991; Lewis et al., 2000; Nauck et al., 1993b). The studies described in this chapter examined the expression and function o f the GIP receptor on the pancreatic |3-cell o f the V D F rat - an animal model of T 2 D . It was shown for the first time that there is a decreased level o f GIP receptor m R N A in the pancreatic islets o f animals exhibiting characteristics o f T 2 D : hyperglycemia, and insulin resistance. Additionally, this decrease in GIP receptor m R N A , decreased the ability of islets in these animals to respond to physiological GIP doses. The 14-16 week o l d V D F rats used in these experiments were both glucose intolerant (figure 7) and hyperinsulinemic (figures 9 and 10). The pancreas perfusions (figures 11 and 12) demonstrated that there is a blunted first phase insulin secretion in the fat animals: 125 % increase compared to a 600 % increase in lean animals in response to the introduction of 8.8 m M glucose. Furthermore, in the normal, lean animals, 10 p M GIP stimulated a characteristic, biphasic insulin response; whereas, this hormone had no effect on second phase insulin secretion in the fat animals (c/figures 11 and 12). The perifusion data demonstrate that GIP is able to significantly increase first phase insulin secretion in islets from the lean animals, but continued exposure to 10 n M GIP probably leads to desensitization o f (3-cell surface GIP receptors and no further significant effect of GIP on insulin secretion is observed (Hinke et al, 2000a). Recently, there has been considerable interest in using dipeptidylpeptidase I V (DP I V ) inhibitors in T 2 D therapy (Demuth et al., 2002). Circulating D P I V inactivates both G L P - 1 and G I P by cleaving the amino terminal dipeptide from the parent incretin  97  polypeptides rendering them biologically inactive; thereby, decreasing the circulating half-life o f the 'active' incretins. Presently, there is controversy as to whether GIP levels are elevated, normal, or lowered in T 2 D and animal models of the disease (Ahren et al, 1997; Fukase et al, 1993; Jones et al, 1989a; Kieffer et al, 1995b; Vaag et al, 1996). One possible explanation for the ineffectiveness of GIP in the V D F rat is an increase in DP I V in these animals, which could inactivate GIP prior to its actions on the (3-cell. Since it was impossible to measure differences in intact versus amino terminally truncated GIP with our radioimmunoassay, it was not possible to determine the role of D P I V in our findings, nor was it possible to determine the concentration of bioactive GIP in these animals. However, it has been demonstrated that the levels o f circulating D P I V in these fatty and lean Zucker rats are similar; therefore, this explanation for GIP ineffectiveness can be ruled out (Pederson et al, 1998b; Pospisilik et al, 2002). Interestingly, exposure o f the rat islet to GIP leads to desensitization o f the islet to GIP (Hinke et al, 2000a). Presently, it is not clear whether this is due to a reversible phosphorylation o f the GIP receptor, a decrease in GIP receptor m R N A expression or both. However, it was demonstrated that there was a rapid, homologous desensitization of the GIP stimulated insulin secretion in mouse (3-cells ((3TC3) that occurred both at the receptor level, as well as further downstream in the signaling cascade (Hinke et al, 2000a). In contrast, experiments reported here demonstrate a decreased ability of GIP to increase c A M P levels in the islets of diabetic V D F animals; suggesting there is a defect in the GIP receptor - adenylyl cyclase portion of the GIP receptor intracellular signaling pathway. Furthermore, since we were unable to measure elevated ambient GIP levels in the V D F rat (figure 8), there is no reason to believe that hyperGIPemia caused a loss o f  98  functional cell surface receptors, either by desensitization or downregulation, in these animals. The expression o f other G-protein coupled receptors (such as the glucagon receptor) in the superfamily is regulated by glucose as well as the adenylyl cyclase activator forskolin (Abrahamsen & Nishimura, 1995). Thus, it is possible that GIP receptor downregulation and dysfunction occurs in response to inappropriate stimulation of the (3-cell by abnormal levels o f glucose or c A M P elevating agents in the V D F rat. The pancreases o f the 4 week old Zucker rats were much smaller than those of the mature animals. This made it difficult to carry out experiments on these animals in the same manner as those done on the older animals. Thus, the data obtained from the 4 week old animals are not directly comparable to the data obtained from the older diabetic animals, and for that reason any direct comparisons and conclusions that are made must be considered as purely speculative. In addition, further experiments need to be conducted to further characterize the timeline for development o f defective GIP receptor expression in these animals. These prior considerations aside, the 4 week old (but not the mature) V D F animals retained an insulin response to GIP (figures 12, 19 and 20). This is o f interest because these prediabetic V D F animals were only mildly hyperglycemic and were able to clear the glucose during an O G T T as efficiently as the young control animals (figure 18). Furthermore, this normal disposal of oral glucose is the result of a near normal level of GIP receptor expression in these animals (figures 18 and 21). However, the prediabetic V D F animals do display basal hyperglycemia and do not seem to respond as quickly to an oral glucose load as the control animals (figure 18). Lewis et al (2000) administered a G I P R antibody to rats and reduced the first phase insulin response by 35 %,  99  demonstrating that GIP may be important for early phase insulin response. It is tempting to speculate that the 30 % decrease in GIP receptor expression that we observed in the islets from prediabetic V D F animals may be sufficient to significantly impair first phase insulin secretion in vivo without affected the overall glucose tolerance of the animals. Therefore, a decrease in GIP-stimulated first phase insulin secretion could explain the elevated 10 minute blood glucose values in the prediabetic V D F animals during the OGTT. The level o f GIP receptor expression in the prediabetic V D F animals was decreased 30 %; however, there is no major change in overall oral glucose tolerance in these rats i.e. they are able to adequately clear the glucose over the course of the O G T T . To speculate further, i f there were a genetic mutation in the promoter or G I P R gene in these animals that directly altered GIP receptor expression, it would be likely that GIP receptor expression would be downregulated throughout their lives. Additionally, the decrease in GIP receptor expression seems to be inversely correlated with the degree of overnutrition in these animals. Thus, GIP receptor expression is probably controlled by a metabolite that is abnormally regulated in T 2 D , such as glucose or fat. This observation provides a basis for future experiments designed to determine exactly how GIP receptor levels are modulated by nutrient status. Furthermore, this observation lends support to the concept that a genetic defect in GIP receptor expression is not a primary cause for T 2 D , but rather that downregulation o f GIP receptor levels during the development o f T 2 D may exacerbate the disease. These experiments demonstrate, for the first time, that GIP receptor m R N A expression is downregulated in the pancreatic (3-cell of the diabetic Zucker rat (figure 16).  100  This observation suggests that there is a decrease in receptor expression on the pancreatic (3-cell, and that this decrease in cell surface expression leads to the decreased potency of GIP as an insulinotropic agent. Figure 17 demonstrates that there is a decrease in total GIP receptor protein within the islets of the V D F rats. However, we have been unable to develop a method to assess cell surface GIP receptor protein expression on the islets o f rats, as we do not have an ample supply o f reliable antibody directed against the GIP receptor nor have we been able to develop a reproducible saturation radioligand binding protocol for use in islets. However, we have been able to demonstrate in clonal beta cells that changes in m R N A expression also produce similar changes in cell surface GIP receptor expression using radioligand saturation binding curves (Chapter 5). Therefore, we believe that there is a decrease in cell surface GIP receptor concomitant with the decrease in intracellular GIP receptor m R N A in these islets. Beguin et al. (1999), demonstrated that stimulation of the (3-cell by GIP caused phosphorylation o f the Kir6.2 ( K T P ) channel via protein kinase A on serine 372. A  Phosphorylation o f this serine residue led to an increased open probability o f the channel. This recent paper was the first demonstration that GIP stimulation o f the (3-cell leads to protein phosphorylation. However, the physiological basis for this phosphorylation event is still unclear since Beguin and colleagues believe that Kir6.2 is maximally phosphorylated in the basal state. It is tempting to speculate that decreased levels of GIP receptor on the (3-cell surface, would decrease the phosphorylation state of the K A T P channel and decrease the open probability. This in turn would lead to a membrane depolarization and insulin secretion. I f this receptor deficit was great enough there could  101 be uncoupling of glucose stimulated insulin secretion and 6-cell decompensation, as observed in the fatty rats. In conclusion, glucose tolerance and insulin responses were studied following GIP infusion in the diabetic V D F rat. In these animals GIP did not potentiate glucose induced insulin secretion, either in vivo or from the perfused rat pancreas and isolated perifused rat islets. Moreover, GIP failed to stimulate c A M P production in isolated fa/fa islet static incubations. Finally, GIP receptor mRNA and protein levels were shown to be downregulated in the islets of these animals, and this was hypothesized to be the basis for their insensitivity to GIP. In addition, the pancreata of young, prediabetic V D F and control animals are responsive to GIP and GIP receptor mRNA downregulation is not as severe as in the prediabetic animals. Thus, it appears that GIP receptor expression is decreased, possibly by hyperglycemia, during the development of T 2 D . As a consequence, GIP stimulated insulin secretion is greatly compromised during the development of T 2 D , and this may contribute to the etiology of this disease.  102  C h a p t e r 5: The Regulation of G I P Receptor Expression i n R a t C l o n a l B-Cell Lines  5.1 B a c k g r o u n d Postprandial insulin secretion is controlled in part by the gut derived incretin hormones G I P and G L P - 1 . These incretins stimulate pancreatic (3-cell insulin secretion by binding to a serpentine, seven transmembrane, G-protein coupled receptor and subsequently activating adnenylyl cyclase, phospholipase A ( P L A ) , and extracellular 2  2  regulated kinases ( E R K , M A P ) as well as changing cellular ion fluxes (Beguin et al., 1999; Ding & Gromada, 1997; Ehses et al, 2001; Mcintosh et al, 1996; Trumper et al, 2001; Wheeler  al, 1995).  Knockout mouse studies have demonstrated that both the G I P and G L P - 1 receptors are integral to the release of insulin from the pancreas following a meal. Both G I P and G L P - 1 receptor null mice displayed compromised insulin secretion and therefore exhibited poor glucose tolerance to an oral glucose load (Miyawaki et al, 1999; Scrocchi et al, 1996). Furthermore, in vivo administration of exendin (9-39) and G I P (7-30) antagonists at the G L P - 1 and G I P receptors respectively decreased glucose tolerance to an oral glucose load in rats (Schirra et al., 1998; 1996b; Tseng et al., 1999). From these studies it has been estimated that together G I P and G L P - 1 could account for over 50 % of the insulin secretory response to a meal. The major stimuli for GIP secretion from the gastrointestinal tract are carbohydrates and fatty acids (Pederson et al, 1975). Thus it follows that GIP may play a role in fat metabolism in the adipocyte as well as other cell types expressing its  103  receptor. Our laboratory has demonstrated that GIP is lipolytic in differentiated 3T3-L1 cells in a c A M P dependent manner (Mcintosh et al, 1999). Furthermore, Mcintosh et al. suggested that this lipolytic activity of GIP could prime the 6-cell for the ensuing meal by causing an increase in free-fatty acids in the circulation. However, other groups have shown GIP to be lipogenic in rat adipose tissue (Beck & M a x , 1983; Oben et al, 1991). The role of GIP in fat metabolism in other cell types is at present poorly defined. The peroxisome-proliferator activated receptors (PPARs) are a family of nuclear T F that are activated in vivo by fatty acids; binding of an activator of P P A R a stimulates heterodimerization with the retinoid X receptor followed by translocation to the nucleus where transcriptional regulation can occur (Desvergne & Wahli, 1999). P P A R a is expressed in the 6-cell and is activated by free fatty acids such as palmitate and oleate as well as synthetic fibrate drugs such as clofibrate and W Y 14643 (Roduit et al, 2000; Wang et al, 1999). Furthermore, P P A R a has been demonstrated to tightly control expression of genes involved in fatty acid oxidation in the pancreatic 6-cell including upregulation of acyl-CoA-synthetase and carnitine palmitoyl transferase-1 (Zhou et al, 1998). Additionally, it is believed that activation of P P A R a is the main pathway by which leptin stimulates lipolysis in the pancreatic 6-cell; thereby, protecting the 6-cell from lipotoxicity (Unger et al, 1999). Recently, it has been demonstrated that G I P may be ineffective at stimulating insulin secretion in T 2 D and the V D F animal model of T 2 D , probably because there is a decrease in the expression of the G I P receptor on the 6-cell in the disease (Hoist et al, 1997; L y n n et al, 2001; Nauck et al, 1986). However, G L P - 1 stimulated insulin secretion remains normal or even augmented in T 2 D as well as in the hyperglycemic,  104  hyperlipidemic V D F rat animal model (Lynn et al, 2001; Nauck et al., 1993b). The mechanisms governing G I P receptor downregulation in T 2 D are unclear; although, in Chapter 4 it was hypothesized that G I P R downregulation may be elicited by hyperglycemia or hyperlipidemia. Since it is difficult to manipulate glycemia and lipidemia within the whole animal without inducing widespread metabolic changes, |3cell lines and islets maintained in cultured conditions were utilized to examine the effects of glucose and fat on receptor expression.  5.2 Characterization of GIP Binding, GIP-stimulated cAMP Production and GIPstimulated Insulin secretion in the INS(832/13) Clonal |3-Cell Line G I P binds to INS(832/13) cells in a specific manner with an I C  50  of 30 n M and a  maximum specific binding of approximately 500 cpm after incubating with 50 000 cpm of label with a specific activity of 350 mCi/mg (figure 22A). This level of G I P receptor expression and G I P affinity is similar to that observed in other |3-cell lines; however, the affinity is slightly right-shifted from cells tranfected with the wild-type G I P receptor (chapter 7). c A M P production in INS(832/13) cells was stimulated by G I P with an E C  5 0  of 6.6 n M (figure 22B). The maximal c A M P production was approximately 1.5 times basal. This degree of G I P stimulated c A M P production as well as the E C  5 0  are also in  line with other (3-cell models (Hinke et al., 2000a).  5.3 The Effect of GIP on Insulin Secretion from INS(832/13) Cells. G I P stimulated insulin secretion from INS(832/13) cells in both a concentration (data not shown) and glucose dependent manner (figure 23). Figure 23 illustrates that in the presence of 0 m M glucose, 50 n M G I P was unable to stimuate insulin secretion.  105  Figure 22: G I P receptor binding ( A ) and c A M P signaling (B) in INS(832/13) clonal 6cells. Cells were plated in 24 well plates at a density o f 5 x 10 cells/well in 5.5 m M glucose and allowed to grow for 48 hours before experiments were conducted. For competitive binding analyses, various concentrations o f unlabelled G I P were incubated for 4 hours at 4 °C in presence o f I - G I P . Cells were then washed and the amount o f GIP bound was determined using a gamma counter. For c A M P studies (B), cells were preincubated in 5.5 m M glucose and then incubated for 30 minutes at 37 °C with 0.5 m M I B M X . c A M P was then extracted using 70 % ethanol and quantified using radioimmunoassay. Values are expressed as mean ± S E M of 4 independent determinations. 5  125  106  Figure 2 3 : Insulin secretion from I N S ( 8 3 2 / 1 3 ) cells in response to increasing glucose concentrations in the presence o f GIP. Cells were plated at a density o f 5 x 1 0 cells/ml in 2 4 well plates and grown in 5.5 m M glucose for 4 8 hours. The experiment consisted of a 1 hour preincubation for 6 0 minutes in 1 m M glucose. Cells were then stimulated for 3 0 minutes at 3 7 °C in the presence of 5 0 n M GIP. Media was then collected and analyzed for insulin release using radioimmunoassay, as described in Chapter 2. Asterisks indicate statistical significance from 0 m M glucose conditions as determined by A N O V A and Dunnet's post hoc test (P< 0.05, n=5). 5  107  None of the G I P stimulated conditions were significantly different from control conditions (at that glucose concentration); however, there is a trend that indicates that G I P stimulated insulin secretion at all glucose levels, and it is probable that increasing the sample size would have resulted in statistical significance. The maximal glucose and GIP-stimulated insulin secretion occurred at 11 m M glucose, there was no further increase in the ability of G I P to stimulate insulin secretion at glucose concentrations greater than this.  5.4 GIP Stimulates Palmitate Oxidation in BRIN-D11 Clonal p-Cells. G I P stimulated palmitate oxidation in the B R I N - D 1 1 (3-cell model. This stimulation of palmitate oxidation had an E C  5 0  of 27 n M and a maximum of 1.6 times  basal levels (figure 24). These values fit well with the I C  50  values for G I P binding and  insulin secretion in this cell model (data not shown)  5.5 The effects of glucose on GIP receptor mRNA expression in INS(832/13) cells. Glucose strongly downregulated expression of G I P receptor m R N A in both a time and concentration dependent manner (figures 25 & 26). A s illustrated in figure 26, there was a significant decrease in G I P receptor m R N A at glucose concentrations greater than 11 m M . Under the condition of 25 m M glucose, G I P R expression decreased to 30 % of that seen under zero glucose conditions. Furthermore, this decrease in receptor level occurred rapidly with a significant difference being observed at 6 hours following exposure to the 25 m M glucose (figure 25). N o further decrease in receptor level was observed following the 18 hour time point (cf figures 25 & 26) at which time G I P receptor expression was reduced to 28 % of the basal level. Culture of cells longer than  108  Figure 24: GIP-stimulated palmitate oxidation in B R I N - D 1 1 clonal 6-cells. Cells were grown in the presence of H-Palmitate for 48 hours, then washed and. stimulated with G I P for 4 hours. The medium was collected, and centrifuged following stimulation with GIP. H 0 was used as a measure of fatty acid oxidation, H 0 was allowed to equilibrate for 24 hours at 60 °C with non-labelled water. The radioactivity was then measured in samples and normalized to 0 G I P conditions. Values are expressed as mean ± S E M of 4 independent determinations. 3  3  3  2  2  109  0  2  4  6  8  1 0  1 2  1 8  Hours of Exposure to 25mM Glucose Figure 25: The effect of time o f exposure of INS(832/13) cells to 25 m M glucose on GIP receptor m R N A expression. Cells were incubated in regular media supplemented with 25 m M glucose for times varying between 0 and 24 hours. Following incubation R N A was isolated and quantified using real-time R T - P C R as described in Chapter 2. Data were normalized to the basal conditions: i.e. for expression level at 0 hours. Asterisks indicate statistical significance compared to basal levels P<0.05 n=4  t  0  2  5 . 5  8 . 8  11  1 6  2 0  2 5  Glucose Incubation Condition (mM) Figure 26: The effect of glucose on GIP receptor m R N A expression in INS(832/13) cells: GIP receptor m R N A downregulation in response to graded glucose concentrations. Cells were incubated for 24 hours in varying glucose concentrations between 0 and 25 m M . Following incubation, R N A was isolated and quantified using real-time R T - P C R as described in Chapter 2. Data were normalized to the basal conditions: i.e. 0 m M glucose. Asterisks indicate statistical significance compared to basal levels P<0.05 n=4  Ill  24 hours in 25 m M glucose does not allow the cells to desensitize to the high glucose conditions and the expression of G I P R m R N A remained at approximately 30 % of the basal level (data not shown). Saturation binding analyses (figures 27 & 28) showed a marked, statistically significant decrease in the amount of G I P receptor expressed on the cell surface of the INS(832/13) cells grown in high glucose conditions. The number of G I P R binding sites per cell grown at 5.5 m M glucose was 1930 ± 200, while the number of G I P R binding sites per cell grown at 25 m M was approximately 910 ± 130 (figure 28). In addition, the dissociation constant (Kd) for G I P was the same under both conditions, with K d values of 400 ± 135 and 427 ± 145 p M in 5.5 m M and 25 m M glucose respectively (figure 27). This indicates that the kinetics of binding were identical under both high and low glucose conditions. In an effort to determine how the downregulation of the G I P R m R N A was occurring we cultured INS(832/13) cells in the presence of various inhibitors of cell growth and proliferation (shown in figure 29). W e did not see a reversal of the effects of 25 m M glucose in any of the conditions that we used. However, we observed that both wortmannin, a PI-3 kinase inhibitor and H89, a P K A inhibitor significantly increased G I P R m R N A levels above basal. Furthermore, we utilized insulin to ensure that high insulin levels were not contributing to the downregulation of the G I P receptor, as high insulin levels occur during incubation of these cells in high glucose. A s seen in figure 29, insulin increased G I P receptor expression and therefore, was not contributing to the glucose-induced downregulation. Neither Bis (2 u M ) , a highly-specific, cell permeable P K C (a,61,62,Y,6,e isoforms) inhibitor, nor P D 98059 (100 p M ) , a M E K inhibitor, had  1  Figure 27: Saturation binding analysis o f INS(832/13) cells treated with high glucose. Cells were incubated for 24 hours in either 5.5 m M (squares), or 25 m M (triangles) glucose. Following incubation varying amounts of I - G I P was added to the cells and allowed to equilibrate over 4 hours at 4 °C. Cells were then washed, the amount of I G I P was counted and specific binding was calculated. The Y-asymptote at which the curve reaches a theoretical maximum denotes the number of specific G I P binding sites. l25  l25  113  5.5 25 Glucose Incubation Condition (mM) Figure: 28: Total cell surface GIP receptor numbers at 5.5 m M and 25 m M glucose. The theoretical cell surface receptor number was calculated from 4 independent saturation binding analyses carried out on INS(832/13) cells and plotted. The asterisks indicate statistical significance as determined by the two-tailed student's t-test (P<0.05).  114  Figure 29: The effect o f various inhibitors of cell growth and proliferation on glucoseinduced GIP receptor m R N A downregulation in INS(832/13) cells. Cells were grown for 24 hours in either 5.5 or 25 m M glucose in the presence o f drugs (5 u M H89, 100 u M P D 98059, 2 u M B i s , 100 n M wortmannin, 1 u M insulin) as described in Chapter 2. Following this incubation period, R N A was harvested and GIP receptor m R N A levels were measured using real-time R T - P C R . Data are expressed as a fraction basal (5.5 m M ) conditions, asterisks indicate statistical significance from basal levels P<0.05, n=3.  115  any significant affect on G I P receptor expression at either glucose concentration (figure 29).  5.6 The Effect of Free Fatty Acids and PPARa Activation on GIPR Expression in Islets, BRIN-D11, and INS(832/13) Cells. Prior to acquiring the recently developed INS(832/13) cell line, we carried out initial experiments in B R I N - D 1 1 (3-cells. A s shown in figure 31, incubation of the B R I N - D 1 1 cells in both 2 m M palmitate and with the P P A R a activator, W Y 14643, produced significant increases in G I P receptor levels. Both stimuli produced an approximate 3-fold increase in G l P receptor expression under 5.5 m M glucose conditions. Additionally, incubation of these cells in a medium containing a high fatty acid concentration upregulated the G I P receptor expression at the cell surface as determined by saturation binding analyses. In fact W Y 14643 and palmitate also significantly increased G I P receptor m R N A levels in islets isolated from lean Zucker rats as seen in figure 30. Palmitate was a stronger stimulant of receptor transcription in islets, producing an 11-fold increase in G I P receptor m R N A expression, while 100 u M W Y 14643 caused a 7-fold increase in receptor expression. Fatty acids were also capable of increasing G I P receptor expression in the INS(832/13) cells. Figure 32 demonstrates that there was significant induction of G I P receptor transcription after only 4 hours of stimulation with 2 m M palmitate. Furthermore, this upregulation of G I P receptor expression continues through 24 hours, reaching a maximum of approximately 5 times basal levels at 10 hours (figure 32). Figure 34 shows that in the presence of 5.5 m M glucose, 2 m M palmitate significantly increased receptor m R N A levels. Figure 36 illustrates that W Y 14643 can increase  116  15n  Incubation Condition Figure 30: GIP receptor expression in islets following incubation with the P P A R a activator W Y 14643 (100 u M ) or 2 m M palmitate. Islets were isolated from lean Zucker rats and then cultured overnight with 11 m M glucose. Following the recovery period, islets were incubated at 5.5 m M glucose with either W Y 14643 or 2 m M palmitate for 8 hours before R N A was harvested. GIP receptor expression was determined by carrying out real-time P C R on total islet R N A . Asterisks indicate statistical significance from control conditions P<0.05, n=4.  117  Figure 31: GIP receptor expression in B R I N - D l 1 cells following incubation with P P A R a activator, 100 u M W Y 14643 or 2 m M palmitate. B R I N - D l 1 cells were cultured for 24 hours in the presence o f W Y 14643 or 2 m M palmitate (Fat). R N A was then isolated and GIP receptor expression was quantified using real-time R T - P C R . GIP levels were normalized to G A P D H m R N A levels. Asterisks indicate statistical significance P<0.05, n=3.  118  O c  7.5-1  0  .25  .5  1  2  4  6  8  10  12  24  Time of Stimulation With 2 mM Palmitate (hours) Figure 32: A time-course for palmitate-stimulated induction o f GIP receptor expression in FNS(832/13) clonal (3-cells. Cells were grown in 12 well plates for 24 hours at 5.5 m M glucose before media was changed and cells grown for various times (0-24 hours) in 2 m M palmitate in the presence of 5.5 m M glucose. R N A was then isolated and GIP receptor expression was quantified using real-time R T - P C R . GIP levels were normalized to basal GIP receptor m R N A levels. Asterisks indicate statistical significance ( P O . 0 5 , n=4).  119  Figure 33: Saturation binding analysis o f INS(832/13) cells treated with W Y 14643 and 2 m M palmitate. Cells were incubated for 24 hours in 5.5 m M glucose with 100 u M W Y 14643 or with 2 m M palmitate. Following incubation, varying amounts o f I - G I P were added to the cells and allowed to come to equilibration over 4 hours at 4 °C. Cells were 125  125  then washed, the amount o f I-GIP was counted and specific binding was calculated. The theoretical cell surface receptor number was calculated from 4 independent saturation binding analyses as described in Chapter 2 (Data Analysis) and plotted. The data are expressed as a fraction of basal (5.5 m M ) cell surface receptors. The asterisks indicated statistical significance P<0.05.  120 receptor expression in INS(832/13) cells transfected with the mPPARa-G form of the the transcrition factor. mPPARa-G is a mutant (G282E) form of PPARa with low intrinsic transactivation properties but a higher affinity for WY 14643 and other fibrates than the wild-type form. Thus, both fatty acids and activation of PPARa were able to upregulate GIPR expression in the INS(832/13) cells. Additionally, stimulation of INS(832/13) cells with both WY 14643 and with 2 mM palmitate was able to increase cell surface GIP receptor expression approximately 3-fold (figure 33). Therefore, as in the case of glucose-stimulated downregulation of cell surface GIP receptor expression (figures 27 & 28), induction of GIP receptor mRNA expression is directly linked to an increase in cell surface expression. 5.7 T h e Interaction Between F a t and Glucose and the Effect on G I P Receptor Expression.  Recently Roduit et al. (2000) showed that glucose induced downregulation of PPARa in INS(832/13) cells. We hypothesized that, if GIP receptor expression was under the control of PPARa, then glucose may result in downregulation of the GlP receptor via a decrease in the ability of PPARa to stimulate or maintain the basal level of expression. To test this hypothesis we first incubated INS(832/13) cells in the presence of 2 mM palmitate in varying glucose concentrations. Figure 34 shows that at glucose concentrations higher than 8 mM, palmitate had no effect on GIP receptor expression. Furthermore, at high glucose levels (25 mM), fatty acids were unable to even maintain receptor levels at those seen basally and a significant decrease from basal level was observed. Furthermore, the PPARa antagonist, MK-886 (Kehrer et al, 2001), caused a  121  Figure 34: GIP receptor m R N A expression following culture o f INS(832/13) cells for 24 hours in various glucose concentrations with 2 m M palmitate. Cells were incubated overnight in 5.5, 8, 16 or 25 m M glucose in the presence or absence or 2 m M palmitate (Fat). Following this incubation, R N A was harvested and subjected to real-time P C R for quantification o f GIP message. Asterisks indicate statistical significance compared to basal, 5.5 m M conditions P O . 0 5 , n=4.  122 small decrease in GIP receptor expression at low glucose levels; however, it had no effect at levels higher than 8 m M glucose (figure 35). Finally, transfection of INS(832/13) cells with a high affinity form of P P A R a (mPPARa-G) increased GIPR mRNA levels to 1.7 x basal levels in the presence of W Y 14643 (figure 36). Transfection of 1NS(832/13) cells with a dominant negative form of P P A R a (Gervois et ah, 1999) caused a significant decrease in the expression of the GIP receptor to levels obtained with 5.5 m M glucose, while having no effect at 25 m M glucose. Taken together, these data strongly suggest that P P A R a is able to maintain GIPR mRNA levels at low glucose but is ineffective at higher glucose levels.  5.8 Glucose, Palmitate , WY 14643 and Gene Transcription GIPR mRNA half-life was analyzed because a decrease in the GIPR mRNA degradation rate would lead to an increase in the total amount of GIPR mRNA. This was carried out by incubating the cells with actinomycin D, an agent that intercalates into double stranded D N A and inhibits further nucleic acid synthesis. Figure 37 demonstrates that the high glucose induced downregulation of GIP receptor mRNA was not due to a reduced half-life of GIPR mRNA. The half-lives of the mRNA encoding the GIP receptor were not statistically different at 5.5 and 25 m M glucose (figure 37). Thus, it does not appear that high glucose affected the R N A degradation pathway, and it is likely that there was a decrease in GIPR mRNA synthesis resulting from high glucose levels. As with the glucose studies, incubation of INS(832/13) cells in high fat or W Y 14643 (figure 37) did not affect the degradation of GIPR mRNA. The half-lives of the GIPR mRNA in the cells that were grown in high fat (data not shown) and under control  123  Figure 35: The effect of a specific P P A R a antagonist on glucose induced G I P receptor downregulation. Cells were grown in 5.5, 11, 16 or 25 m M glucose i n the presence or absence o f M K - 8 8 6 , a P P A R a antagonist, for 24 hours. R N A was then harvested and subjected to real-time R T - P C R for G I P receptor expression determination. Data are expressed as a fraction o f the 5.5 m M condition; n=4.  124  Figure 36: The effect o f stimulating or blocking P P A R a activity in INS(832/13) cells. Cells were transfected with two mutant P P A R a isoforms: either the G M U T ( m P P A R a G) form which has an increased affinity for W Y 14643 or the h P P A R a form which is a dominant negative protein as described in research design and methods (page 57). Cells were then grown for 24 hours in the presence of W Y 14643 in either high (25 m M ) or low (5.5 m M ) glucose. R N A was then harvested and G I P receptor expression was quantified using real-time R T - P C R . Asterisks indicate statistical significance of 5.5 m M groups compared to 5.5 + W Y 14643 (P<0.05; n=3). t r  125  I 0  1  1  1  1  2  3  1  4  1  1  1  5  6  7  Time Following Addition of Actinomycin D (hours) Figure 37: G I P receptor m R N A degradation curves in INS(832/13) cells. Cells were exposed to 5.5 m M glucose ( • ) , 25 m M glucose ( A ) or W Y 14643 (100 u M ) (O) for 24 hours prior to the addition o f 5 u.g/ml actinomycin D . Cells were then allowed to incubate in actinomycin D for varying times between 0 and 6 hours before R N A was harvested and G I P receptor m R N A expression was assessed by real-time R T - P C R . Data are expressed as a fraction o f that seen at basal conditions or before addition of actinomycin.  126  conditions were both approximately 30 min. Therefore, the increase in G I P R m R N A levels was probably a result of an increase in the transcription of G I P R m R N A . In addition both 100 u M W Y 14643 and 2 m M palmitate stimulated increases in G I P receptor promoter driven luciferase transcription of 1.4 and 1.7 times basal promoter activity in INS(832/13) cells (figure 38). Furthermore, basal luciferase activity was approximately 1.4 times greater than that seen with cells transfected with empty vector; indicating that the proximal 2 k B of the G I P receptor promoter can actively control gene transcription in this cell line (data not shown).  5.9 The Effect of Osmolarity on GIP Receptor Expression in INS(832/13) Cells. When INS(832/13) cells were grown in high D-mannitol, hyperosmolar conditions mimicking those of hyperglycemia, there was no effect on G I P receptor expression (figure 39). In addition, it appears as if the INS(832/13) cell response to hyperosmolarity is a slight, although non-significant, increase in G I P receptor expression (figure 39). Thus, the increase in osmlarity of high glucose culture has no downregulatory effect on G I P receptor expression.  5.10 The Effect of Activation of PPARy on GIP Receptor Expression in INS(832/13) Cells. There was no significant increase in G I P receptor expression at low glucose levels when INS(832/13) cells were cultured in ciglitazone, a P P A R y activator (figure 40). However, the glucose-stimulated downregulation of the G I P receptor was not as profound as that observed in earlier experiments (cf. figures 34 and 40). The reason for this is  127  Stimulation C o n d i t i o n  Figure 38: GIP receptor 5' promoter driven luciferase activity in response to W Y 14643 and 2 m M Palmitate (Fat) in INS(832/13) cells. The proximal 1960 bp o f the 5' GIP receptor promoter were cloned from Wistar Rat liver and inserted into the polylinker of the p G L 3 luciferase promoter. INS(832/13) cells were transfected with the promoter construct as described in Chapter 2 and allowed to grow for 24 hours before media was changed and experimental stimuli were applied. Following a further 24 hours of growth medium was removed, fresh medium was added and luciferase activity was determined using the BrightGlo kit (Promega) and a 96 well, Turner Designs luminometer. Data is expressed as arbitrary light units, with asterisks indicating statistical significance from basal activity as determined by A N O V A and Dunnet's post-hoc tests (p<0.05, n=6).  128  5.5 G r o w t h g l u c o s e Equivalent  11  16  Condition +  with  D-Mannitol  G l u c o s e  25 5.5 to  m M Give  C o n c e n t r a t i o n  (mM)  Figure 39: The effect of osmolarity on GIP receptor expression in INS(832/13) clonal (3cells. Cells were grown in 12 well plates for 24 hours in the presence o f 5.5 m M glucose supplemented to the givien osmolarity with the non-metabolizable sugar D-mannitol to the given equivalent glucose concentration. R N A was then harvested and subjected to real-time R T - P C R for GIP receptor expression determination. Data are expressed as a fraction o f the 5.5 m M condition; n=6.  129  Figure 40: The effect o f activation o f P P A R y on GIP receptor expression at increasing glucose concentrations in INS(832/13) clonal 6-cells. Cells were grown for 24 hours in varying glucose concentrations in the presence o f 10 u M ciglitazone: a thiazolidinedione that activates P P A R y . R N A was then harvested and subjected to real-time R T - P C R for GIP receptor expression determination. Data are expressed as a fraction of the 5.5 m M condition; n=4.  130 unclear but it is possible that the higher passage number (passage 65) of the cells used in these experiments contributed to this effect. 5.11  Discussion  In T2D there is a marked reduction in the insulinotropic potency of GIP. Studies outlined in Chapter 4 demonstrate that the cause of this reduction in potency may be decreased GIP receptor expression on |3-cells of the Vancouver diabetic fatty Zucker rat model of T2D. However, currently there are no data to suggest the mechanisms by which GIP receptor downregulation occurs in type 2 diabetic patients or in animal models of the disease. Here we demonstrate that elevated glucose levels are able to significantly reduce GIP receptor expression in vitro and in vivo and that this effect is not reversed by blocking any of the common cell growth and proliferation pathways. We also demonstrate a novel pathway for stimulation of GIP receptor expression at normal glucose levels through fat-stimulated PPARa activation, which is unable to reverse the GIP receptor downregulation associated with hyperglycemia. Recently, Roduit et al. (2000) and Laybutt et al. (2001) demonstrated that high glucose caused downregulation of PPARa in both INS(832/13) cells and in pancreatectomized rats. The time-course for downregulation of PPARa in 20 mM glucose was almost identical to that seen in GIP receptor downregulation studies by high glucose reported here. Where we observed a significant reduction in GIP receptor expression after only 6 hours in high glucose (figure 25), Roduit et al. reported a significant and total ablation of PPARa expression at 6 hours (Roduit et al., 2000). Their study also demonstrated that downregulation of PPARa led to a decreased expression of the mRNA for uncoupling protein 2 (UCP2), carnitine palmitoyltransferase 1 (CPT 1)  131  and acyl-CoA oxidase: genes that all have well defined P P A R response elements in their promoters. Therefore, downregulation of P P A R a by glucose can cause a downregulation of genes normally controlled by this nuclear transcription factor. Finally, their paper demonstrated that 0.4 m M oleate had no effect on P P A R a expression in INS(832/13) cells in the presence of either high or low glucose levels. Thus, our observation that F F A were unable to increase G I P R m R N A levels under high glucose conditions was likely not a result of downregulation of P P A R a by fatty acids but rather because P P A R a was downregulated by elevated glucose. The upregulatory effect of palmitate on G I P receptor expression followed kinetics similar to those observed by Sato et al. (2002) in a recent paper in which they studied the time course for induction of various P P A R a target genes in response to W Y 14643. Here we demonstrated that 2 m M palmitate caused a significant increase in G I P receptor expression with approximately a 4 hour lag time following application of high palmitate containing media (figure 32). Similarily, it was demonstrated that A O X , L - F A B P , longchain acyl-CoA synthetase ( L A C S ) , and the peroxisomal bifunctional enzyme are induced in response to W Y 14643 in rat hepatoma Fao cells with a 4-6 hour lag time (Sato et al., 2002). Thus, the induction of the G I P receptor observed here fits with the kinetics observed for well-characterized targets of P P A R a ; therefore, one could speculate that P P A R a stimulated transcription of G I P receptor expression occurs in a similar manner to other genes. To determine i f the downregulatory effects of glucose on G I P receptor expression could be attributed to the action of another common signal transduction pathway, various inhibitors of these pathways were applied to cells for 24 hours. A s can be seen in figure  132  29, none of these inhibitors reversed the glucose-induced downregulation of G I P receptor expression. However, a number of interesting observations were made. First, we observed that wortmannin, a PI-3K inhibitor, H89, a P K A inhibitor and insulin all increased receptor expression. Insulin was included as a control because it was observed that under high glucose conditions the amount of insulin in the media was 2.5 times that of basal conditions (figure 23) and it was hypothesized that insulin could be causing the downregulation of the G I P receptor. However, insulin appeared to have the opposite effect. The apparent contradiction between the insulin and wortmannin data could be explained by a desensitization of the insulin signaling pathway in these (3-cells by a prolonged, potent stimulation with insulin (Blake et al, 1987; Kulkarni et al., 1999). Thus, we expect that long-term stimulation with insulin probably had much the same functional effect as stimulation with wortmannin. Interestingly, the M A P kinase signaling module has been implicated in the activation of P P A R a ; and we did see a small but non-significant decrease in the expression of the G I P receptor at 5.5 m M glucose in the INS(832/13) cells that were incubated with the M E K inhibitor P D 98059. These data indicate that an actual decrease in P P A R a expression is probably more important than the activation (phosphorylation) state of P P A R a in the regulation of G I P receptor expression. The control of G I P receptor expression by P P A R a appears to be limited to low glucose conditions at which point it stimulates an increase in expression. The physiological significance of this is obscure since at low glucose levels G I P does not stimulate insulin secretion. However, in the presence of 0 m M glucose G I P is able to stimulate adenylyl cyclase, resulting in c A M P accumulation (Hinke et al, 2000a) as well as activation of M A P kinase (Ehses et al, 2002) and P L A (Ehses et al, 2001) . 2  133 Therefore, it is possible that G I P has functional roles in the 6-cell in addition to insulin secretion. Intraduodenal fat is probably the most potent stimulant of G I P secretion from the gut and therefore, it follows that fat should be able to regulate G I P receptor expression. In this manner, stimulation by free fatty acids or long chain acyl-CoA esters ( L C - C o A ) derived from either the adipocyte during the interdigestive period or early in the prandial process may ready the 6-cell for the ensuing glucose stimulation. In addition recent data from our laboratory (figure 24) shows that G I P stimulates fatty acid oxidation within the pancreatic 6-cell; thus, G I P may act to prime the 6-cell with A T P , using intracellular fat stores. This would allow a more rapid glucose stimulation of insulin secretion. When glucose levels are high, there is a dramatic and reproducible downregulation of the G I P receptor in vivo and in vitro; whereas, palmitate has the opposite effect (figures 25 & 26). However, fat is no longer able to induce G I P receptor expression at high glucose levels. This also makes physiological sense i f G I P is acting to cause fat oxidation within the 6-cell (figure 24) in order to prime the insulin secretory or metabolic pathways for the ensuing meal. It would thus be expected that when glucose levels are high, the 6-cell would no longer have a need for GIP-stimulated oxidation of fatty acids. Therefore, expression of the G I P receptor is downregulated and G I P becomes ineffective at high glucose levels. The downregulation occurs quickly with a significant difference seen after only 6 hours in high glucose. This time-course would allow G I P to have an incretin effect on the 6-cell, but would limit its actions in prolonged hyperglycemia. Additionally, our group has shown that G I P receptors are quickly internalized in response to G I P with a significant reduction in cell surface receptors  134  occurring after only 10 minutes of exposure to G I P (Hinke et al., 2000a). Thus, within minutes of G I P receptor activation, bioactivity is probably governed by phosphorylation events but in the hours following, G I P receptor activity is probably controlled by the level of expression of the receptor at the cell surface. Accordingly, due to the chronic hyperglycemia in type 2,diabetic individuals, G I P receptor levels are decreased. Interestingly, these data fit with the hypothesis put forward by Prentki et al. (1997) which suggest that glucose seems to positively regulate expression of genes involved in its metabolism and negatively regulate genes involved in metabolism of other fuels. In view of that, free or non-esterified fatty acids stimulate expression of genes involved in their metabolism such as CPT-1 (Assimacopoulos-Jeannet et al, 1997). Thus, G I P R expression seems to be regulated in a manner that is consistent with other metabolic genes within the 6-cell. In addition, one pathway by which G I P could stimulate 6-cell function and cytoprotection could be by decreasing fatty acid levels within the cell, thereby preventing lipotoxicity. It has been demonstrated that both palmitate and W Y 14643 stimulate transcription of the G I P receptor gene as opposed to increasing the half-life of G I P receptor m R N A within the cell (figures 37 & 38). However, it is not known whether the action of P P A R a on G I P receptor expression is a direct effect or i f it occurs via activation of other T F . For example, Schinner et al. (2002) recently demonstrated that activation of P P A R y inhibits glucagon expression in the a-cell by inhibiting Pax6 transcriptional activity. The pancreatic 6-cell also expresses Pax6 and it could conceivably interact with P P A R a to induce receptor expression. Further studies using  the recently cloned G I P receptor promoter need to be carried out to determine the exact sequence elements that control the fatty acid stimulated increase in receptor expression. In conclusion, the current studies have demonstrated a novel pathway by which glucose and fat can control G I P receptor expression in both clonal (3-cells and under in vivo conditions. W e found that free fatty acids were able to bind to and activate P P A R a at low glucose conditions and stimulate G I P receptor transcription either directly or indirectly. However under high glucose conditions, P P A R a itself is downregulated and is no longer able to maintain basal G I P receptor expression. These results may account for the downregulation of the G I P receptor that is observed in the hyperglycemic, hyperinsulinemic V D F model of T 2 D and may underlie the decreased responsiveness of type 2 diabetic patients to G I P .  136  Chapter 6: Glucose-Induced GIP Receptor Downregulation in the Lean VDF Rat 6.1 Background The conclusions of the previous two chapters were that: 1) G I P receptor expression is decreased in the V D F model of T 2 D , and 2) Hyperglycemia may result in downregulation of the G I P receptor by decreasing expression of P P A R a . Additional observations such as: the rapid time course of G I P receptor downregulation (6 hours), the degree to which G I P receptor expression was decreased (~ 75 %) and the mild hyperglycemic levels needed to stimulate a downregulation of the G I P receptor all led us to hypothesize that i f we were able induce hyperglycemia in normal animals, we should be able to mimic the conditions that we observed in the V D F animals. Hyperglycemic clamps were utilized to achieve this end. Anesthetized lean animals from the V D F colony were maintained at either a mild level of hyperglycemia (10 m M ) or a severe level of hyperglycemia (25 m M ) for 6 hours. Following this treatment, the animals were tested for either insulin secretory capacity in response to G I P using pancreas perfusions or the levels of G I P receptor m R N A were quantified using r P C R . The hypothesis to be tested was that both G I P R m R N A and GIP-stimulated insulin secretion would be attenuated in a dose-dependent manner by hyperglycemia in the clamped animals.  137  6.2 Glucose-Induced Downregulation of the G I P Receptor i n H y p e r g l y c e m i c C l a m p e d Rats  Hyperglycemic clamps were performed on lean Zucker rats to determine if hyperglycemia was able to downregulate GIPR expression in vivo. Figure 41 demonstrates that islets of rats clamped at 25 m M glucose expressed only 33 ± 7 % the GIPR mRNA level seen in 5.5 m M clamped animals. Those animals that were glucose clamped at 10 m M also showed a significant reduction in GIP receptor expression of 60 ± 15 % of levels observed in 5.5 m M clamped animals. Concomitant with reduced GIP receptor expression, there was a reduction in GIP stimulated insulin secretion from the perfused pancreata of animals clamped at 25 m M (figure 42). This is reflected as a 71 % reduction in the area under the curve in the treated animals as seen in the inset of figure 42. These data establish a causal link between GIPR levels and islet sensitivity to GIP.  6.3 Discussion  Data presented in the previous chapter demonstrated that GIPR expression is controlled reciprocally by the ambient glucose concentration in neoplastic clonal 6-cells. However, the expression of genes in cell lines is often quite different from their expression in vivo and here we set out to determine if GIPR expression was controlled in the whole animal in a similar manner. As indicated in figure 41 we found that expression of the GIP receptor was decreased in animals that were subjected to hyperglycemic clamps. The GIPR downregulation was dose-dependent and resulted in a decrease in insulin secretion from the pancreata of these animals. Interestingly, the degree of downregulation in the pancreata from animals that were clamped to 25 m M glucose was  138  1.5  n  5.5 m M G l u c o s e  10 m M Clamp  25 m M Condition  Figure 41: The effect o f hyperglycemic clamping on G I P receptor expression in islets of lean Zucker rats. Plasma glucose levels of anesthetized lean Zucker rats were clamped at either 5.5, 10 or 25 m M glucose for 6 hours. Islets were then harvested at the clamped glucose concentrations and R N A was isolated for subsequent real-time R T - P C R . The inset depicts area under the curves ( A U C ) for the perfusion time interval in which G I P was included in the perfusate (10-30 min). Asterisks indicate statistical significance compared to basal conditions (P<0.05; n=3).  139  Time (min)  Figure 42: The effect o f hyperglycemic clamp on GIP stimulated insulin release from the perfused lean Zucker rat pancreas. Lean animals were clamped at 5.5 ( • ) or 25 (A) m M glucose for 6 hours prior to pancreatic perfusion with the protocol outlined in the figure and in the research design and methods section. Insulin secretion is expressed as a fraction o f that seen in the average of the first 5 minutes o f the perfusion. Asterisks indicate statistical significance (P<0.05; n=3).  140  identical to the downregulation observed in the V D F animals (65 % decrease, figure 16). This is not surprising when the blood glucose concentrations in the V D F animals are examined. In the fasted state, a condition that would almost never occur in these obsese animals, the blood glucose levels are approximately 8 m M (Pederson et al, 1998b; Pospisilik et al., 2002). Furthermore, the genetic defect that these animals carry is a disruption in the leptin receptor. One of the results of a defect in the leptin signaling system is an inability to control nutrient intake. Thus, they are hyperphagic and the blood glucose levels in these animals rarely drop below approximately 10 m M and are often as high as 20 m M (Pederson et al, 1998b; Pospisilik et al, 2002). Therefore, a glucose clamp to 25 m M is within the range of plasma glucose expected in the V D F rat, and the degree of G I P receptor downregulation observed during these clamps should be similar to those observed in this animal model. Additionally, figure 41 illustrates that the G I P R expression is significantly downregulated (40 % of control levels) with hyperglycemic clamps of only 10 m M : 5 m M above fasting levels in these lean animals. Furthermore, there is no statistical difference between the degree of downregulation observed with the 10 m M hyperglycemic clamp and the 25 m M hyperglycemic clamp. Therefore, this lends credence to the hypothesis that glucose could account solely for the downregulation observed in the V D F animals. There was significant insulin secretion from the pancreata of control animals in response to G I P ; however, the hyperglycemic clamped animals did not respond to G I P . This produced a significant difference (3 -fold) in the area under the curve for the two conditions for the perfustion interval between 10 and 30 minutes (figure 42). In  conclusion, both the G I P receptor level and the GIP-stimulated insulin secretory profile can be blunted by a 25 m M hyperglycemic clamp in lean Zucker animals. Furthermore, the levels of downregulation observed following glucose clamping are similar to those observed in the diabetic V D F rats; thus, hyperglycemia may be the primary factor resulting in G I P receptor downregulation in T 2 D .  142  Chapter 7: Glycosylation of the GIP Receptor, the Effect of Glycosylation on Cell Surface Expression and Insulin Secretion 7.1 Background The initial biochemical characterization of the G I P receptor utilized crosslinking experiments to determine that the hamster  (3-cell G I P receptor  125  I-GIP  was a protein  with an apparent molecular weight of 59 k D a (Couvineau et al., 1984). Further studies demonstrated that dithiothreitol was able to reduce the electrophoretic mobility of the G I P receptor without effecting G I P binding indicating the presence of a disulfide bond (Amiranoff et al, 1986). These authors also demonstrated that the G I P receptor-  l25  I-GIP  complex could be adsorbed by wheat germ agglutinin and concanavalin A coupled to sepharose beads. This interaction could be specifically reversed indicating for the first time that the GIP receptor was a glycoprotein (Amiranoff  al, 1986).  More recently, cloning o f the GIP receptor has shed more light on the degree of glycosylation that occurs on the GIP receptor. Studies in the mid-1990's indicated that the mature hamster GIP receptor protein contains 462 amino acids with a predicted molecular weight o f approximately 52 k D a (Yasuda et al, 1994). Cloning studies in other animals indicate that the predicted molecular weight of the GIP receptor in all species is approximately 50 k D a (Gremlich et al, 1995; Wheeler et al, 1995). The difference between the predicted molecular weight and the actual electrophoretic size o f the protein lends support for the indirect observation by Amiranoff et al. (1986) that the protein is a glycoprotein.  143  Wheeler et al. (1995) reported four asparagine (N) consensus sites for glycosylation within the sequence of the GIP receptor: N59, N 6 9 , N 7 4 and N200. These four sites all fall on predicted extracellular regions o f the receptor: three on the aminoterminal tail o f the receptor and 1 on the first extracellular loop. However, the role o f any of these sites in actual glycosylation o f the G I P receptor has never been examined. Additionally, there has never been any direct evidence directed at proving that the receptor is glycosylated or what the effects of this glycosylation are on receptor structure, expression or function. The glycosylation of the secretin, V I P , G L P - 1 , and gastrin-releasing peptide receptors has been demonstrated to affect the cell surface expression of the proteins (Benya et al., 2000; Couvineau et a l , 1996; Goke et a l , 1994; Pang et a l , 1999). In addition, all of the receptors that have been examined to date have at least two glycosylation sites, which are located on the extracellular N-terminus or in the first extracellular loop. Removal of these sites by mutation decreases the ability of hormone to bind to the receptor because the receptor is not delivered to the plasma membrane or is incorrectly folded. For example, Couvineau et al, (1996) demonstrated that N58, N69 and N100 of the V I P receptor were all glycosylated; however, only mutation of all three sites affected delivery of V I P receptor to the cell surface and with this mutant receptor protein was retained in the perinuclear endoplasmic reticulum. In addition, they demonstrated that the 9 k D a carbohydrate residue at N58 was involved in the calnexindependent folding of the V I P receptor, but N69 was not. Others groups have demonstrated that receptor glycosylation does not seem to be necessary for delivery to the cell surface; however, it may increase the efficiency of the delivery of receptors to the  144  plasma membrane (Goke et al., 1994; Lanctot et al., 1999; Pang et al., 1999). Thus, glycosylation of G P C R s is important for optimal folding as well as delivery to the cell surface. Because there is a dysregulation of glucose homeostasis within the 6-cell of type 2 diabetics it is conceivable that there could be a defect in the glycosylation machinery within the cell. In fact, it has been demonstrated that glycation of insulin can occur in T 2 D , and that this leads to decreased biological activity of the hormone (Abdel-Wahab et al., 1997). Furthermore, there has been a recent explosion in the amount of literature exploring the effects of advanced glycosylation end product ( A G E ) accumulation via non-enzymatic glycosylation in T 2 D : much of the research pointing to pathologic effects caused by the deleterious effect of adding sugars to the extracellular matrix (Brownlee, 1995). The following studies were designed to test the hypothesis that G I P receptor glycosylation is necessary for correct cell surface receptor expression and G I P binding. In addition, it is hoped that by understanding the role of glycosylation in G I P binding, signaling and cell surface expression, the potential role of inappropriate glycosylation in T 2 D could be predicted. It is hypothesized that glycosylation is necessary for the correct expression of the G I P receptor on the cell surface, as well as in binding and signaling. To test this hypothesis four potential asparagine (N)-linked glycosylation sites ( N 59, 69, 74, 200) in the extracellular amino-terminus and the first extracellular loop of the receptor were mutated to threonine residues using site-directed mutagenesis, generating 8 mutants. These mutants were fully sequenced, and expressed in H E K - 2 9 3 cells, which have been shown to utilize complex glycosylation pathways. Furthermore, the effect of  145  glycosylation of the G I P receptor on insulin secretion from INS(832/13) cells was examined by treatment of the cells with tunicamycin (an inhibitor of glycosylation).  7.2 The  125  I-GIP Competitive Binding and Signaling Properties of the Glycosylation  Site GIP Receptor Mutants To determine whether the glycosylation sites were involved in expression of the protein at the cell surface, competitive binding and c A M P production analyses were carried out. When transfected into H E K cells, all of the mutant receptor proteins were able to bind G I P with an affinity in the near physiological range. The wild-type tagged G I P receptor (WTtag) had an I C  50  of 3.56 ± 1.1 n M . The affinity of the WTtag receptor  for G I P was found to be approximately 10-fold greater than the affinity observed in (3-cell lines (Chapter 5) but similar to affinities of the non-tagged G I P receptor transfected into other cell lines (Wheeler et al, 1995). There were minor differences in the affinities of the single glycosylation site G I P receptor mutants for GIP. The N 5 9 T mutant had an I C  5 0  of 2.57 ± 1.4 n M , the N 6 9 T mutant had an I Q of 6.16 ± 1.2 n M , the N 7 4 T mutant had 0  an I C  5 0  of 5.63 ± 2.4 n M and the N200T mutant had an I C  5 0  of 5.51 ± 1.1 n M (figure  43A). A l l of these mutants had reduced maximal binding from the WTtag receptor, which by definition had a calculated maximal binding (B/Bo) of 100 ±2%.  The N 5 9 T  mutant had a maximal binding of 81 ± 3 %, the N 6 9 T of 71 ± 2 %, the N 7 4 T of 51 ± 4 % and the N200T of 81 ±2%  (figure 43 A ) . The maximal binding is related to the amount  of receptor at the cell surface and since the transfection efficiency was equivalent in all of the transfections; thus, the WTtag mutant was most highly expressed followed by the N59T= N200T >N69T>N74T.  146  A  1.25 1.00-  2  WT  A  59 69 74  T •  0.75-  o m  •  •  N200T  0.50 0.25 0.00' -0.25-12  -11  -10  -9  -7  Log [GIP] 10  B v> 1.5-i  "35 o o o o  •  WT  •  59  1.0-  69 •  < o  0.5-  •  74 200  o E  Q. 0.0-12  -11  -10  -9  -8  Log [GIP] 10  Figure 43: G I P binding ( A ) and c A M P production (B) by single site glycosylation mutants transfected into H E K cells. Single site mutants were constructed as described in Chapter 2.19. Mutant receptor D N A (2.5 u,g) was then transfected into H E K 293 cells using Lipofectamine 2000, and cells were plated in 24 well plates. Following 48 hours o f growth, cells were washed and incubated with (A) various concentrations o f GIP at 4 °C for 4 hr in the presence o f a constant amount of I - G I P . Cells were then washed, and the amount o f radiolabel was determined. Data are expressed as percent total binding o f WTtag G I P R (mean ± S . E . M . , n=4). (B) Here cells were incubated with various amounts of GIP for 30 min at 37 °C. Cells were then solubilized with 70 % ethanol and the c A M P production was determined using radioimmunoassay. Data are expressed as mean ± S . E . M (n=4). 125  147  The c A M P production by the single mutants was also determined (figure 43B). The half maximal concentration of G I P or E C  5 0  values for c A M P production for all the  mutant proteins were slightly right shifted from the WTtag receptor value of 0.86 ± 0.5 n M (figures 43B and 44B). Furthermore, all the mutants had significantly decreased maximal c A M P stimulating potency when compared to the WTtag receptor with the exception of the N200T mutant (figures 43B and 44B). The E C  5 0  values for c A M P  production by the single glycosylation site mutants were: N 5 9 T = 5.44 ± 1.0, N69T = 1.70 ± 1.5 n M , N 7 4 T = 3.08 ± 1.4 n M and N200T = 19.5 ± 1.2 n M . The maximal c A M P production from the WTtag receptor was 0.64 ± 0.01 pmol c A M P / 1 0 0 0 cells. The maximal c A M P production by the N59T mutant was 0.33 ± 0.01 pmol c A M P / 1 0 0 0 cells, 0.34 ± 0.02 pmol c A M P / 1 0 0 0 cells for the N 6 9 T mutant, 0.38 ± 0.02 pmol c A M P / 1 0 0 0 cells for the N 7 4 T mutant and 1.06 ± 0.04 pmol c A M P / 1 0 0 0 cells for the N200T mutant (figure 43B). These maximal c A M P levels were all lower than the WTtag receptor, with the exception of the N200T mutant. The multiple glycosylation site mutants were also tested for G I P binding and G I P stimulated c A M P production (figure 44). A l l of these mutants bound G I P with significantly decreased maximal binding than the WTtag receptor. The N59/69T double mutant displayed 22 ± 2 % WTtag binding, the N59/74T double mutant displayed 10 ± 1 % WTtag binding, the N69/74T double mutant displayed 28 ± 6% WTtag binding and the N59/69/74T (567) triple mutant displayed 25 ± 4 % WTtag maximal binding. However, the affinities of each of these mutant receptors for GIP did not differ dramatically from the WTtag receptor. These mutants had the following I C  5 0  values for  125  I - G I P displacement by unlabelled GIP: N59/69T =14.3 ± 3  148  n M , N59/74T = 2.28 ± 2.4 n M , N69/74T = 12.3 ± 4 n M and N59/69/74T = 76.7 ± 10 n M . The maximal c A M P production values for the double mutants also differed from the WTtag receptor (0.64 ± 0.01 pmol c A M P / 1 0 0 0 cells). The N59/69T mutant had a maximal c A M P production/1000 cells of 24 ± 0.2 pmol, the N59/74T mutant had a maximal c A M P production/1000 cells of 22 ± 0.8 pmol, the N69/74T mutant had a maximal c A M P production/1000 cells of 14 ± 0.4 pmol and the triple mutant had a maximal c A M P production/1000 cells of 11 ± 0 . 1 pmol. The EC50 values for GIP stimulated c A M P production for the mutiple glycosylation site mutants were right-shifted from the WTtag mutant (0.86 ± 0.5 n M ) with the N59/69T, N59/74T, N69/74T and the N59/69/74T mutants having values of 3.62 ± 0.5 n M , 3.76 ± 1.2 n M , 8.76 ± 1.4 and 86.2 ± 5 n M respectively.  7.3 THE EFFECTS OF MUTATION OF GIP RECEPTOR G L Y C O S Y L A T I O N SITES ON R E L A T I V E ELECTROPHORETIC MOBILITY. From the previous series of experiments it is clear that the glycosylation site mutants of GIP receptors were expressed on the cell surface to differing degrees. To determine i f this was due to a difference in the glycosylation state of the receptors, electromobility shift assays ( E M S A ) s were carried out with samples of affinity purified GIP receptor that had been treated with PNGase F: an amidase that cleaves between the innermost N-Acetylglucosamine  ( G l c N A c ) and the asparagine residues of high mannose,  hybrid and complex oligosaccharides in N-linked glycoproteins. When the WTtag receptor was western blotted with the mouse anti-V5 tag antibody two bands appeared in the wild-type, non PNGase F treated extract. The major band ran with a molecular mass  149  A • •  WT 59/69 0 59/74 A 69/74 V 567  1.25-  1.00-  0.75-  o CQ  2  0.50-  0.25-  0.00H -0.25 -12  -11  -9  -10  Log [GIP] 10  B w 0.75aj o o o o  < u  o E  Q.  0.50H  0.25-  • WT • 59/69 o 59/74 A  69/74  V  567  0.00I—  -12  -11  -10  -9  -8  —  i  -7  -6  Log [GIP] 10  Figure 44: G I P binding (A) and c A M P production (B) by multiple site glycosylation site mutants transfected into H E K 293 cells. Multiple glycosylation site mutants were constructed as described in Chapter 2. Mutant receptor D N A (2.5 u.g) was then transfected into H E K 293 cells using Lipofectamine 2000, and cells were plated in 24 well plates. Following 48 hours of growth, cells were washed and incubated with ( A ) various concentrations of GIP at 4 °C for 4 hr in the presence of a constant amount o f I - G I P . Cells were then washed, and the amount o f radiolabel was determined. Data are expressed as percent total binding o f WTtag G I P R (mean ± S . E . M . , n=4). (B) Here cells were incubated with various amounts of GIP for 30 min at 37 °C. Cells were then solubilized with 70 % ethanol and the c A M P production was determined using radioimmunoassay. Data are expressed as mean ± S . E . M (n=4). l25  150  of 68 ± 3 k D a , while a minor band was detected at approximately 58 ± 2 kDa. The identities of the first band was presumed to be fully glycosylated G I P receptor and the second, less dense band was thought to be incompletely glycosylated or immature G I P receptor. It is hard to distinguish the difference between these because whole cell membrane and not plasma membrane preparations were utilized. When the WTtag G I P receptor was treated with PNGase F there was a shift in the mobility of the band to approximately 51 ± 2 k D a (figure 45). There was only one band observed with no smaller bands present. The major band from the N 5 9 T mutant ran with an apparent molecular weight of approximately 63 ± 3 k D a . However, there were two other bands observed in this lane; one that ran at 56 ± 2 k D a and another that ran with a similar mobility to the digested WTtag protein. When the N 5 9 T mutant affinity purified protein was treated with PNGase, the major band also appeared at 49 ± 1 k D a . The non-digested N 6 9 T mutant protein ran with an electrophoretic mobility similar to the N 5 9 T mutant with the major band running with an apparent molecular mass of 64 ± 3 k D a , and one minor band appearing at 56 ± 2 k D a . Furthermore, when this protein was digested with PNGase F , two bands also appeared: the major one at 49 ± 1 k D a and the second faint band around 51 k D a . The N 7 4 T His-tag purified proteins ran with an apparent molecular mass of 68 ± 3 k D a , with a minor band in the non-digested extract with an apparent mobility o f 56 ± 2 k D a . When this extract was digested with PNGase F , the apparent molecular weight o f the single band was the same in all the extracts and ran at 52 ± 3 k D a (figure 45). The N 2 0 0 T mutant was also analyzed for gel shift in response to PNGase F treatment. The apparent molecular weights of this mutant were similar to that o f the  151  59/69 C  P  59/74 C  f t  P  69/74 C  P  567 C  P  —  50 kDa  —  35  Figure 45: A representative electromobility shift assay using affinity purified G I P receptor extracted from transfected H E K 293 cells. Cells were transfected with G I P receptor constructs (5 u.g) in 10 cm dishes and then protein was extracted as described in Chapter 2.20. The protein concentrations o f the extracts were determined and 5 \ig o f purified protein was digested with 200 U o f PNGase F for 1 hr at 37 °C. Control (C) and digested (P) proteins were run on a 12 % polyacrylamide gel and subjected to Western blot as described in section 2.20. Apparent molecular weights were calculated using R/ analysis.  152  N 5 9 T banding pattern except the major non-digested band ran at around 60 k D a (data not shown). The same experiments were done on the double glycosylation site mutants to substantiate the single site mutant data. The major band of the N59/69T non-digested mutant protein extract ran with an apparent molecular mass o f 59 ± 4 kDa, with minor bands running at 51 ± 2 k D a and 48 ± 2 kDa. When this extract was digested, two bands appeared at 51 k D a and 48 k D a in figure 45; however, other experiments only had the 48 ± 2 k D a band. The major band in the non-digested N59/74T protein extract had an apparent molecular mass o f 67 ± 7 k D a , with a minor bands running at 53 ± 3 kDa. The digestion o f this extract yielded a major band at 48 ± 2 kDa. The control N69/74T protein extract ran at 70 ± 9 k D a with a minor band in most extracts at 51 ± 4 k D a (however, the major band in figure 45 was at 47 kDa). When this extract was digested with PNGase F the major band ran at 49 ± 2 kDa. Finally, the triple mutant protein ran with an apparent molecular mass of 50 ± 3 k D a , when this protein extract was digested with PNGase F, the running distance did not change (figure 45). In addition, the amount of His-tagged protein purified from the triple mutant was never near the level o f protein obtained from the other mutants indicating that this triple mutant is not posttranslationally processed properly: perhaps one o f the sites for glycosylation is needed for correct targeting for expression at the cell surface.  7.4 The Effect of Treatment of INS(832/13) Cells with Tunicamycin on Cell Surface GIP Receptor Expression Figure 46 illustrates a representative saturation binding profile o f INS(832/13) cells that have been treated with tunicamycin: an antibiotic which prevents the transfer o f  153  G l c N A c - l - P from U D P - G l c N A c to dolichyl-P i.e. the first step in the glycosylation process. These experiments were carried out to determine the effect of glycosylation of proteins on G I P R expression and GIP-stimulated insulin secretion in (3-cells. In all the experiments testing the effect of tunicamycin on INS(832/13) cells, a decrease in cell surface GIP binding was observed, indicating that GIP receptors were not being delivered as efficiently to the plasma membrane when glycosylation was blocked. Control cells expressed an average of 2443 ± 400 G I P receptors on the cell surface; whereas tunicamycin treated INS(832/13) cells expressed 760 ± 70 G I P receptors on the cell surface. Additionally, the dissociation constants (Kd) of G I P from the surface of these cells did not differ between the control (455 ± 50 p M ) and tunicamycin (345 ± 100 p M ) treated cells.  7.5 The Effect of Tunicamycin on GIP-Stimulated Insulin Secretion from INS(832/13) Cells The GIP-potentiated secretion of insulin from cells treated with tunicamycin was also attenuated (figure 47). A s illustrated in figure 47, G I P caused a small increase in insulin secretion at 5.5 m M glucose in control conditions. N o difference in insulin secretion was also observed in cells treated with tunicamycin and then stimulated with G I P in 5.5 m M glucose conditions. In 11 m M glucose conditions, G I P caused a significant increase in insulin secretion from control cells; however, G I P was unable to potentiate 11 m M glucose-induced insulin secretion from cells that were treated with tunicamycin (figure 47). Additionally, growth in tunicamycin did not change the glucose stimulated insulin secretory response in these cells in response to either 5.5 m M or 11  154  7500-. Ui c c  •  WT  A  Tunicamycin  25  50  5000-  m o o  §_ 2500-  0-  0  f m o l  75  100  125  label  Figure 46: GIP saturation binding analysis from INS(832/13) cells treated with tunicamycin. Cells were plated into 24 well plates (5 x 10 cells/well) and grown for 24 hours. Cells were then treated with 1 pg/ml tunicamycin, an antibiotic that inhibits glycosylation, for 24 hours before the saturation binding analyses were carried out as described in section 2.14 and 2.26. 5  155  1 5 n  E  5.5 mM 5.5 mM + Tunicamycin  .2 1<H  11 mM 11 mM + Tunicamycin  3  o-  Control I n c u b a t i o n  50 nM GIP c o n d i t i o n  Figure 47: Tunicamycin decreases GIP-stimulated insulin secretion from INS(832/13) cells. Cells were plated into 24 well plates (5 x 10 cells/well) and grown for 24 hours. Cells were then treated with 1 p,g/ml tunicamycin, an antibiotic that inhibits glycosylation, for 24 hours before insulin release experiments were carried out (section 2.22). These were done by incubating cells in presence of either 5.5 m M or 11 m M glucose with or without 50 n M GIP for 30 minutes at 37 °C. The supernatant was then collected and assayed for insulin content using R I A . Data are expressed as mean ± S . E . M . , n=4. Asterisks indicate statistical significance compared to basal conditions (PO.05). 5  156  m M glucose; therefore, tunicamycin was probably not having adverse toxic effects on the cells.  7.6 Discussion The decrease in GIP responsiveness in type 2 diabetics may be a result of factors other than hyperglycemia. For example, glycosylation o f many G P C R s such as the G L P 1 receptor and the V I P receptor has been demonstrated to be important in the correct cell surface expression (Couvineau et al., 1996; Goke et al., 1994). Here we demonstrate that glycosylation o f the GIP receptor affects cell surface expression and subsequent function of the receptor, and it is interesting to postulate that the G I P responsiveness in T 2 D may be affected in part by abnormal glycosylation within the 6-cell. Here we verify that the wild-type G I P receptor is a glycosylated protein with a molecular mass of approximately 59 kDa. Furthermore, when this receptor is treated with PNGase F , there is a band shift to an apparent molecular mass o f 48 k D a . This indicates that TV-linked carbohydrates account for a large portion o f the G I P receptor structure (i.e. approximately 35 % by mass) (figure 45A). The literature indicates that the w i l d type G I P receptor is a 59 k D a glycoprotein (Amiranoff et al, 1986). Furthermore, molecular mass analysis o f the cloned sequence o f the G I P receptor predicts a mass o f 50 k D a (Usdin et al, 1993; Wheeler et al, 1995). Here we overestimate the molecular mass of both the intact glycoprotein by approximately 10 kDa. This is probably a result o f the reducing conditions that were utilized in our study; whereas, Amiranoff et al (1986) used non-reducing conditions in their study. In fact when they used D T T to reduce disulfide bonds they saw a shift o f the apparent molecular weight o f the G I P /  157  receptor complex to around 73 k D a , which is in the molecular weight range determined in this study. This observation indicates that the GIP receptor is "normally" glycosylated in the H E K 293 cell line. In any case, due to the structural contribution o f the glycosylation to the GIP receptor, we set out to determine the exact effect o f glycosylation on cell surface expression and function. The first series o f experiments that we carried out was to look at the effect o f tunicamycin on GIP receptor expression in the INS(832/13) rat (3-cell model. When these cells were grown in the presence of tunicamycin, glycosylation was 1 25  blocked and there was a decrease in  I-GIP binding to the surface o f the cells on  saturation: which corresponded to a 70 % decrease in cell surface GIP receptor number (figure 46). Concomitantly, the dissociation constant did not change in these experiments, indicating that the binding affinity o f label for the wild-type receptor was not affected by glycosylation o f the receptor. Furthermore, the majority o f the glycosylation-site knockouts did not display dramatic differences in IC50 values from the WTtag receptor when competitive binding analyses were carried out; the exception was the triple mutant that was not highly expressed making an accurate binding isotherm hard to obtain (figure 44A). The amount ot total binding in a competitive binding analysis is a rough determination o f total cell surface expression o f the receptor. Figure 4 4 A and 45 demonstated that there is much lower cell surface expression observed with the multiple glycosylation site mutants than with the single site mutations. Using these figures we can see that the W T receptor is most highly expressed followed by this sequence: N59T~N200T>N69T>N74T>N69/74T~N567T~N59/69T>N59/74T. These data indicate  158 that glycosylation affects GIP receptor expression, but does not change the affinity o f the receptor for GIP; an observation that is in agreement with most of the studies done to date (Benya et al, 2000; Pang et al, 1999; Walsh et al, 1998). The GIP signaling properties o f the mutants were also measured using c A M P as a marker o f receptor activation (figures 44B & 45). These studies indicated that the mutants had right-shifted EC50 values for c A M P production. However, only the N200T and N567T mutants were significantly right-shifted with respect to the WTtag receptor. The maximal c A M P stimulation levels that were obtained from each of these mutant receptors were in line with their relative cell surface expression, with the single mutants producing much greater responses than the double mutants (c.f. figures 44B & 45). The only exception to this was the N200T mutant which produced a maximal c A M P response that was 1.6 times the WTtag receptor. This mutant was not more highly expressed at the cell surface; therefore, it is believed that removal o f the carbohydrate, addition of a threonine residue, or both affects signaling. The affinity of the N 2 0 0 T receptor for G I P was found to be normal; however, EC50 for c A M P was right-shifted for this mutant meaning that coupling o f this mutant receptor to G-proteins is disturbed. Using gel shift analysis, the sites within the GIP receptor that were glycosylated were determined (figure 45). Figure 45 shows that both the N 5 9 T and N 6 9 T mutants have greater electrophoretic mobility than the wild-type tagged protein: each running approximately 5 k D a faster than the WTtag protein. Furthermore, the major band observed for the non-digested N59/69T mutant ran at approximately 59 k D a , corresponding to removal o f the the 2 single glycosylation sites. The G I P receptor does not seem to be glycosylated at asparagine 74, since none o f the mutants o f this site had  159  gel shifts from the WTtag receptor. Finally, the N 2 0 0 T mutant was gel shifted in the non-digested form and ran with an apparent molecular mass of 63 k D a , indicating that this mutant was also glycosylated. Therefore, these data indicate that asparagines 59, 69 and 200 are all glycosylated in the GIP receptor. Another interesting observation from the gel analysis was that most o f the nondigested lanes included a band that ran with a greater mobility than the major glycosylated band. The identity o f this band was not determined; however, it is absent in many of the PNGase digested lanes which indicates that it is in fact a glycosylated protein that runs with the same eletrophoretic mobility as the WTtag G I P receptor. This band was present in almost all the gels that were run, and it is believed to be GIP receptor protein that has not undergone full posttranslational processing. In these studies the GIP receptor was expressed at superphysiological levels under control o f C M V promoter. In many cell lines this leads to the concentration of the overexpressed protein within inclusion bodies that generally surround the nuclear membrane and never fully mature. It is believed that the minor band represents GIP receptor that has been shuttled into this pathway; however, additional studies need to be carried out to identify this protein. Another observation from the Western blots was that the triple mutant (N59/69/74T) was not glycosylated at all, and was generally very poorly expressed. None o f the gels that were run showed a marked expression o f this mutant, and it is believed that upon translation (if translated at all), the majority o f the protein is incorrectly folded or inserted into the membrane leading to immediate degradation. This is supported by the competitive binding and c A M P studies which demonstrated very little cell surface expression o f this mutant.  160  The final series of experiments in this study examined the role o f GIP receptor glycosylation on the ability o f GIP to stimulate insulin secretion. Figure 47 illustrates GIP was unable to potentiate insulin secretion in INS(832/13) cells that have been treated with tunicamycin. However, GIP was able to fully stimulate insulin secretion from INS(832/13) non-treated cells. There was no change in glucose-stimulated insulin secretion: indicating that insulin secretion was otherwise unaffected. A n explanation for these findings is that non-glycosylated GIP receptor was not expressed on the cell surface and therefore GIP was not able to stimulate insulin secretion (figure 46). In conclusion, the GIP receptor is glycosylated at asparagine residues 59, 69 and 200. This glycosylation is important for correct expression in the plasma membrane, although, it does not seem to be involved in modulating the binding o f GIP. Furthermore, the removal o f glycosylation at N200 seems to augment the ability o f GIP to signal via c A M P , but does not change the affinity o f GIP for the receptor. Finally, disruption o f the glycosylation o f proteins could affect cell surface GIP receptor expression. Although it seems unlikely that it could abrogate GIP binding completely, it is possible that overglycosylation could adversely affect GIP signaling. GIP seems to lose its insulin secretory ability in diseases such as T 2 D . One reason for this attenuation o f GIPstimulated insulin secretion could be the overglycosylation o f proteins that is concomitant with high ambient glucose levels in T 2 D .  161  Chapter 8: Discussion and Future Directions Previous reports have demonstrated that the insulin response to GIP is blunted in T 2 D (Elahi et al, 1994; Krarup et al, 1987; Nauck et al, 1993b). However, these studies have not demonstrated a mechanism by which this occurs (Hoist et al, 1997). The goal o f this thesis was to elucidate the mechanisms by which GIP receptor expression is controlled and to determine how this control is disturbed in T 2 D ; with the possible goal o f altering disease therapy to allow GIP to have normal effects in the disease. The data in this thesis describes a mechanism by which fat is able to upregulate GIP receptor expression in the presence of low glucose; however, at high glucose GIP receptor expression is downregulated in part by the downregulation o f P P A R a . The degree of GIP receptor downregulation was found to be dependent on the ambient level o f glucose in young and diabetic V D F animals, as demonstrated in Chapter 4. Here it is shown that the mature animals display an approximate 70 % downregulation of the GIP receptor m R N A and protein. O n the other hand, the GIP receptor levels in the 4 week old prediabetic V D F animals are only reduced by 30 %. The ambient blood glucose levels in these animals are positively correlated to their age: with old V D F animals having a fasted blood glucose of 7.5 m M and young prediabetic animals having a fasted blood glucose of 5 m M . Furthermore, older animals are glucose intolerant with peak levels o f approximately 19 m M after 60 minutes. In contrast, the prediabetic animals have near normal glucose tolerance with a difference occurring only at the 15 min time point. After 60 minutes the blood glucose level in these animals has returned to near basal levels. Based on the above discussion, one would predict that at these glucose  162  levels, there should not be any difference in GIP receptor expression in the prediabetic animals. These in vivo data compare very well with the data presented in both Chapters 5 and 6. In these studies it was demonstrated that glucose is able to downregulate GIP receptor expression in both cell lines and in hyperglycemic clamped animals. Interestingly, glucose levels in the 10 m M range caused a significant decrease in GIP receptor expression in both cell lines (30 % decrease) and in lean animals (60 % decrease) that had undergone a hyperglycemic clamp for 6 hours. Blood glucose levels of 10 m M are easily obtained during the daily cycle o f blood glucose in the same animals (Pospisilik et al, 2002). The cell and hyperglycemic clamp data also demonstrate that elevation in blood glucose is able to rapidly control GIP receptor expression. Thus, i f there is a state of prolonged hyperglycemia, there could be concurrent reduction in the expression o f the GIP receptor. The physiological basis for this downregulation is at present unclear, since most o f the G I P within the circulation is degraded to an inactive form quickly following secretion and therefore is not biologically available to stimulate the (3-cell (Kieffer et al, 1995b). One possible explanation in an acute setting is that downregulation o f the GIP receptor following a 6 hour period o f hyperglycemia would protect the |3-cell from further stress by limiting insulin secretion. A more chronic situation could occur in Western society where overnutrition results in hyperglycemia throughout the day. In this case, a glucose induced downregulation o f the GIP receptor would render the (3-cell insensitive to physiological concentrations of GIP and therefore would remove the cytoprotective/antiapoptotic and  163  mitogenic effects o f GIP that have recently been described in our laboratory. The cumulative effects o f a decreased insulin secretion and a decreased ability of the 6-cell to respond to GIP could lead to increased levels of blood glucose which hypothetically could stress the 6-cell and lead to a decompensation in the insulin response to glucose and over time to T 2 D . Another interesting observation from all of our studies was that in the 6-cell there seems to be a basal amount o f GIP receptor expression that cannot be regulated by hyperglycemia. In these studies this amounts to approximately 25 % total expression. For example, the maximal amount of downregulation observed in the V D F animal was around 75 % (Chapter 4), in the cell lines the maximal downregulation was also approximately 75% (Chapter 5), and in the hyperglycemic clamps a 75 % downregulation was also observed (Chapter 6). Therefore, a basal level o f GIP receptor transcription is maintained that cannot be inhibited by glucose or by any o f the other drugs that were tested in figure 29. One pathway through which this apparent maximum amount o f downregulation could be occurring is the P P A R a pathway. Roduit et al. (2000) demonstrated that at 20 m M glucose, a 20 % basal level o f P P A R a is maintained. It is possible that this amount of P P A R a expression is able to maintain receptor expression even in the face o f hyperglycemia. However, this explanation does not fit exactly with our data since we demonstrate in figure 36 that transfection o f 6-cells with a mutant form of P P A R a which is constitutively expressed does not elevate the GIP receptor expression level at hyperglycemia as would be expected. It is possible that the dimerization partner of P P A R a , the R X R , is also downregulated by high glucose and therefore, our G I P R  164  expression in our tranfections may have been limited by the R X R levels as well. Expression of a coactivator of P P A R s such as peroxisome proliferator gamma coactivator-1 (PGC-1) may also be regulated by glucose in the (3-cell and may provide a mechanism by which tranfection o f (3-cells with P P A R a does not lead to an increase in receptor transcription at hyperglycemia (Knutti & K r a l l i , 2001; Oberkofler et al., 2002). In these experiments we were unable to decrease the basal expression o f G I P R promoter driven luciferase activity with hyperglycemia (data not shown). However, we did see an increase in luciferase activity following incubation o f our promoter construct trasfected cells with both 2 m M palmitate and with the P P A R a activator W Y 14643 (figure 38). This is another observation that is hard to explain when we use the simplified explanation that glucose-induced P P A R a downregulation leads to G I P R downregulation. It would be expected that since P P A R a stimulates luciferase activity in the proximal 5' promoter, that a hyperglycemia induced decrease in P P A R a would also decrease receptor expression in the same portion o f the promoter. It is possible that there are multiple T F involved in binding and regulating GIP receptor transcription under hyperglycemia and that the a larger portion o f the promoter is required for a full complement o f these elements to bind and have effect at high glucose. Thus, there are probably factors (in addition to P P A R a ) involved in downregulation o f the GIP receptor at high glucose levels. Further investigation of a more complete promoter is warranted in this regard. The cloning o f the G I P R promoter was carried out, however, time constraints prevented a complete characterization o f the sequence elements within this promoter. These studies would provide important insight into the specific sequence elements that  165  are necessary for glucose and fat stimulated G I P R regulation. Gene therapy with a specific knockout of these putative sequences would allow the GIP receptor to be expressed at relatively normal levels on the 6-cells o f a model o f T 2 D , and could further elucidate the role of GIP in T 2 D . What are the possible therapeutic implications o f a downregulation of the GIP receptor in the treatment o f T2D? W e have demonstrated that GIP receptors are downregulated significantly in this disease; however, it is probably still possible to use GIP or GIP analogues for therapy o f T 2 D . Hinke et al. (2002) demonstrated that D - A l a 2  GIP stimulated insulin secretion from the pancreas of the V D F rat; however, pharmacological doses of 8 nmol/kg were required for an effect to be observed. One drawback of using high doses o f modified GIP is that the long-term effects of administration of such a peptide are unknown and may result in further dowregulation of pancreatic GIP receptors. Another possible therapeutic strategy would be to use oral D P I V inhibitors such as those used by Pospisilik et al (2002) that were demonstrated to lower blood glucose levels in these animals. Drugs such as this in combination with GIP analogs in more physiological doses, and strict control o f nutrional intake may be effective in the restoration o f 6-cell function without having any adverse affects. Another possible strategy would be to use G L P - 1 analogs in conjuction with D P I V inhibitors because G L P - 1 is much more effective at stimulating insulin secretion in T 2 D ; presumably when the blood glucose levels had been lowered sufficiently GIP receptor expression would be restored and GIP would be able to stimulate insulin secretion and have other important effects. Future studies could examine the expression of GIP receptors in V D F rats that have had their hyperglycemia ameliorated by D P I V inhibition  166  or by an insulin sensitizing agent such as metformin. These studies would help to determine i f improving glucose tolerance increases G I P R expression, and therefore, i f the beneficial effects o f GIP on (3-cells could be fully restored in T 2 D . Another potential mechanism o f improving GIP responsiveness in diabetic individuals would be gene therapy possibly using virus vectors. One method of testing the efficacy o f G I P R gene therapy would be to construct a (3-cell specific inducible G I P R knock-in mouse model. This kind of system would allow a custom tailoring o f G I P R expression to the organism's glucose intolerance by administering an oral drug such as tetracycline. A system like this would ideally lead to a restoration o f GIP-stimulated insulin secretion in T 2 D without the need for peptide therapy. Yaney et al. (2001) demonstrated GLP-1 stimulated lipolysis and F F A oxidation within the HIT-15 (3-cell model and that this effect could be mimicked by forskolin and inhibited by orlistat, a lipase inhibitor. They proposed that G L P - 1 activates hormone sensitive lipase, F F A are released from triglycerides and then L C - C o A is oxidized within the mitochondrion. Additionally, they demonstrated that this effect could be blocked by the addition of glucose, probably because glucose leads to an increase in malonyl-CoA and a subsequent inhibition o f C P T - 1 . Preliminary data are presented in this thesis, which indicate that GIP may also play a role in the oxidation of F F A within the (3-cell. However, currently the pathway by which GIP stimulates F F A oxidation in the (3-cell has not been determined, and future experiments could be directed towards this. One mechanism by which GIP could stimulate insulin secretion is by increasing the synthesis and oxidation of L C - C o A early following a meal presumably via c A M P and P K A . A s in the case o f G L P - 1 , this effect  167  would probably be inhibited by glucose. There is physiological evidence, which suggests that GIP is more important in early first phase insulin secretion (Lewis et al, 2000). This could partly be explained by an effect o f GIP on lipolysis within the 6-cell, which is inhibited once the blood glucose levels elevated. Another series o f studies could be designed to determine i f lipolysis and/or fatty acid oxidation plays a role in the antiapoptotic action o f GIP in 6-cells. In conclusion, GIP receptor expression is important for the normal stimulation of postprandial insulin secretion. In T 2 D GIP receptor expression may be decreased by hyperglycemia and the consequent downregulation of P P A R a . The glycosylation o f the GIP receptor may also be abnormal in T 2 D and may lead to a decrease in cell surface GIP receptor expression. 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