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The pro-survival effects of glucose-dependent insulinotropic

polypeptide receptor signalling on beta cells

Scott B Widenmaier

B.Sc. (High Honours) University of Regina, 2005

A Thesis Submitted in Partial Fulfillment of the

Requirements for the Degree of

Doctor of Philosophy

The Faculty of Graduate Studies

(Physiology)

The University of British Columbia

(Vancouver)

July, 2010

Abstract Gastrointestinal incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) have been targeted for the treatment of diabetes due, in large part, to their physiological actions on pancreatic β-cells in potentiating glucose-stimulated insulin secretion and increasing insulin biosynthesis. In recent years, incretins have also been revealed to exert potent actions on β-cell proliferation and survival. However, most studies have focused on GLP-1, and so there is a relative paucity of evidence regarding GIP. In the current thesis, it was hypothesized that GIP receptor (GIPR) activation in β-cells enhances critical anti- apoptotic signalling networks and promotes β-cell survival, which in rodent models of type 2 diabetes mellitus results in an elevation in β-cell mass and improvement in glycaemic control. This hypothesis was initially tested by examining the effects of GIPR activation on the protein- serine/threonine kinase Akt, which is known to exert potent pro-survival actions in β-cells. It was revealed that GIPR stimulation activates Akt signalling through both canonical and non- canonical mechanisms. Next, the effects of GIPR activation on pro- and anti-apoptotic Bcl-2 family proteins were examined on β-cells under pro-apoptotic stress. GIPR activation was found to suppress the onset of stress-induced apoptosis by preventing mitochondrial translocation of pro-apoptotic proteins Bad and Bim as well as activation of the key pro-apoptotic protein Bax, and this effect was due to Akt-mediated inhibition of the apoptosis signal regulating kinase-1. Lastly, the effects of GIPR activation on β-cell survival were examined in vivo by administering a long acting GIP analogue to multiple rodent models of diabetes that exhibit elevated β-cell apoptosis. In all models the GIP analogue promoted β-cell survival and improved glycaemic control. Overall, these studies supported the hypothesis proposed. In conclusion, similar to the signalling actions of the GLP-1 receptor, GIPR signalling exerts potent actions on β-cell survival, and therefore, therapeutics that enhance GIPR signalling in β-cells merit consideration for the treatment of type 2 diabetes mellitus.

Preface Studies in this thesis have been published in the following: 1) Widenmaier SB, Sampaio AV, Underhill TM & McIntosh CHS (2009) Noncanonical Activation of Akt/Protein kinase B in β-cells by the Incretin Hormone Glucose-dependent Insulinotropic Polypeptide. Journal of Biological Chemistry. Volume 284 (16): 10764- 10773. Studies in this publication are described in Chapters 3 and 4. 2) Widenmaier SB, Ao Z, Kim SJ, Warnock G & McIntosh CHS (2009) Suppression of p38 MAPK and JNK via Akt-mediated Inhibition of Apoptosis Signal-regulating Kinase 1 Constitutes a Core Component of the β-cell Pro-survival Effects of Glucose-dependent Insulinotropic Polypeptide. Journal of Biological Chemistry. Volume 284 (44): 30372- 30382. Studies in this publication are described in Chapter 4. 3) Widenmaier SB, Kim SJ, Yang GK, De Los Reyes T, Nian C, Asadi A, Seino Y, Kieffer TJ, Kwok YN & McIntosh CHS (2010) A GIP Receptor Agonist Exhibits β-cell Anti-Apoptotic Actions in Rat Models of Diabetes Resulting in Improved β-cell Function and Glycaemic Control. PLoS One. Volume 5 (3): e9590. Studies in this publication are described in Chapter 5.

Contributions of authors: Drs. AV Sampaio, TM Underhill, Z Ao, G Warnock, Y Seino, TJ Kieffer and YN Kwok provided materials and analysis tools. Mr. A Asadi and Mrs. C Nian provided training and assistance. Dr. SJ Kim contributed Appendix B and provided intellectual insight. Mr. GK Yang performed surgical procedures and provided intellectual input for the pancreas perfusion study (Fig. 26). Mr. T De Los Reyes assisted in studies with streptozotocin treated rats (Fig. 27) and Akita mice (Fig. 29). All studies were conceived and designed by SB Widenmaier and Dr. CHS McIntosh. All studies described in Chapters 3, 4 and 5 were performed by SB Widenmaier. The writing of this thesis and all publications were performed by SB Widenmaier with editing provided by Dr. CHS McIntosh.

Certificates of Approval (scanned copies provided in Appendix C): Biohazard Approval Certificate: #B07-0060 Animal Care Certificate: #A04-0322

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Table of Contents

Abstract ...... ii

Preface ...... iii

Table of Contents ...... iv

List of Figures ...... vii

List of Abbreviations ...... ix

Acknowledgments ...... xiii

Chapter 1 Introduction to GIP, Incretins and Type 2 Diabetes ...... 1 1.1 Overview ...... 1 1.2 Discovery of the Incretins ...... 1 1.3 Nature of Glucose-dependent Insulinotropic Polypeptide (GIP) ...... 3 1.3.1 Origin ...... 3 1.3.2 Gene and Peptide ...... 4 1.3.3 Biosynthesis and Localization ...... 4 1.3.4 Secretion ...... 5 1.3.5 Metabolism ...... 7 1.4 Nature of the GIP Receptor ...... 8 1.4.1 Gene and Protein ...... 8 1.4.2 Localization ...... 9 1.4.3 Receptor Interaction with GIP ...... 9 1.5 Biological Actions of GIP ...... 10 1.5.1 Effects of GIP on the Pancreatic Islet ...... 10 1.5.2 Effects of GIP on Adipose Tissue ...... 11 1.5.3 Effects of GIP on the Gastrointestinal System ...... 12 1.5.4 Effects of GIP on the Cardiovascular System ...... 13 1.5.5 Effects of GIP on Bone ...... 14 1.5.6 Effects of GIP on the Brain ...... 14 1.6 Type 2 Diabetes Mellitus and a Therapeutic Role for GIP ...... 15 1.6.1 Significance to Society and Natural History ...... 15 1.6.2 Role of Insulin Resistance ...... 16 1.6.3 Role of the β-cell ...... 18 1.6.4 The Incretin Axis and GIP in Type 2 Diabetes ...... 20 1.6.5 GIP and Type 2 Diabetes Therapeutics ...... 21 1.7 Mechanisms of GIP Action in the β-cell ...... 24 1.7.1 Regulation of Insulin Secretion ...... 24

1.7.2 Regulation of Insulin Biosynthesis ...... 27 1.7.3 Regulation of β-cell Mass ...... 28 1.8 Thesis Investigation ...... 31 1.8.1 Rationale ...... 31 1.8.2 Hypothesis ...... 32 1.8.3 Objectives ...... 32

Chapter 2 Materials and Methods ...... 33 2.1 Sources of Materials ...... 33 2.2 Cell Culture ...... 33 2.3 Experimental Treatment of Cell Cultures ...... 34 2.4 Cell Death Assays ...... 35 2.5 Plasmid Constructs, siRNA and Transfection Protocol ...... 35 2.6 Cell Lysis and in vitro Kinase Assays ...... 36 2.7 Western Blots ...... 37 2.8 Mitochondrial/Cytosolic Fractionation and Bax Crosslinking ...... 38 2.9 Animal Studies ...... 38 2.10 Pancreatic Perfusions ...... 40 2.11 Histological Analysis ...... 40 2.12 Statistical Analysis ...... 41

Chapter 3 Mechanisms by Which GIP Activates Akt in β-cells ...... 42 3.1 Introduction ...... 42 3.2 Results ...... 43 3.2.1 GIP Activates Akt Without Increasing the Phosphorylation of Thr-308 ...... 43 3.2.2 Neither PI3K Signalling nor Phosphorylation of Thr-308 or Ser-473 are Required for GIP-mediated Activation of Akt ...... 43 3.2.3 GLP-1 and Adenylate Cyclase Activation Mimic the Stimulatory Effects of GIP ...... 49 3.2.4 Stimulation of Akt by GIP Appears to Involve EPAC2 and not PKA ...... 49 3.3 Discussion ...... 54

Chapter 4 Effects of GIP on the Mitochondrial Apoptotic Pathway ...... 57 4.1 Introduction ...... 57 4.2 Results ...... 58 4.2.1 GIP Promotes the Survival of β-cells Exposed to Glucolipotoxic Stress ...... 58 4.2.2 GIP Signalling Inhibits the Mitochondria-mediated Apoptotic Pathway...... 58 4.2.3 GIP Anti-apoptotic Signalling Require cAMP Production but not Insulin‟s Actions ...59 4.2.4 GIP Inhibits Mitochondrial Bad and Bim Translocation and Bax Activation ...... 59 4.2.5 GIP Anti-apoptotic Signalling Does Not Require Transcriptional Changes ...... 67 v

4.2.6 GIP Anti-apoptotic Actions Involves Akt Signalling ...... 67 4.2.7 GIP Anti-apoptotic Actions are Mediated via Akt-dependent Suppression of p38 MAPK and JNK ...... 73 4.2.8 GIP Inhibits p38 MAPK and JNK via Akt-mediated Phosphorylation of ASK1 ...... 80 4.2.9 Inhibiting p38 MAPK and JNK is a Core Anti-apoptotic Mechanism of GIP Action ..81 4.3 Discussion ...... 85

Chapter 5 Effects of GIPR Activation on β-cells in Diabetic Rodents ...... 88 5.1 Introduction ...... 88 5.2 Results ...... 88 2 5.2.1 D-Ala GIP1-30 (D-GIP1-30) is a DPP-IV Resistant GIP Analogue with Equivalent Islet Actions to Native GIP ...... 88

5.2.2 Effects of D-GIP1-30 in Streptozotocin Treated Rats ...... 93 5.2.3 Effects of D-GIP1-30 in Vancouver Diabetic Fatty (VDF) Rats ...... 93 5.2.4 Effects of D-GIP1-30 in Akita Mice, a Mouse Model of β-cell ER Stress ...... 94 5.2.5 Effects of D-GIP1-30 in Zucker Diabetic Fatty (ZDF) Rats ...... 103 5.2.6 Cultured Adipocytes Differentially Respond to D-GIP1-30 and GIP1-42 ...... 104 5.3 Discussion ...... 109

Chapter 6 Further Thoughts and Conclusions ...... 113 6.1 On the Regulation of Akt in β-cells by GIP ...... 113 6.2 On the Anti-apoptotic Actions of GIPR Signalling in β-cells ...... 118 6.3 On the Proposed Hypothesis and Future Directions ...... 125

References ...... 129

Appendices ...... 174 Appendix A. An Illustration of the Canonical Mechanism of Akt Activation...... 174

Appendix B. Cultured 3T3-L1 Adipocytes Differentially Respond to D-GIP1-30 and GIP1-42 175 Appendix C. Certificates of Approval ...... 176

List of Figures

Figure 1: GIP Activates Akt Without Increasing Phosphorylation of Akt at Thr-308 ...... 45

Figure 2: PI3K is Not Required for GIP-mediated Activation of Akt ...... 46

Figure 3: Phosphorylation of Thr-308 or Ser-473 is Not Required for GIP-mediated Activation of Akt...... 47

Figure 4: Akt Inhibitor IV (Akt IV) Inhibits GIP-mediated Activation of Akt Without Affecting the Phosphorylation of Akt at Thr-308 or Ser-473 ...... 48

Figure 5: GLP-1 and Forskolin but Neither High Glucose nor Insulin Mimics the Akt Kinase Stimulatory Effects of GIP...... 50

Figure 6: Stimulation of Akt by GIP Does Not Involve Protein Kinase A (PKA) ...... 51

Figure 7: Stimulation of Akt by Forskolin Involves EPAC and Not PKA ...... 52

Figure 8: Stimulation of Akt by GIP is Mimicked by EPAC but is Not Modulated by Glucose 2+ Metabolism or Involve Ca , MEK 1/2 or iPLA2 signalling ...... 53

Figure 9: GIP Promotes the Survival of INS-1 Cells and Dispersed Mouse and Human Islets Exposed to Glucolipotoxic Stress ...... 61

Figure 10: GIP Inhibits the Mitochondria-mediated Apoptotic Pathway in Staurosporine (STS) Treated INS-1 Cells ...... 63

Figure 11: GIP Mediated Anti-apoptotic Signalling Requires the Production of cAMP but Does Not Involve Insulin Autocrine Signalling ...... 64

Figure 12: GIP Dynamically Regulates Mitochondrial Levels of Bad and BimEL ...... 65

Figure 13: GIP Promotes Survival of STS-treated INS-1 Cells by Suppressing the Activation of Mitochondrial Bax ...... 66

Figure 14: GIP-mediated Anti-apoptotic Signalling in STS-treated Cells Does Not Require Transcriptional Changes ...... 69

Figure 15: GIP Stimulation of Survival in STS-treated INS-1 Cells Involves Akt ...... 70

Figure 16: GIP Enhances Akt Signalling in STS-treated INS-1 Cells...... 72

Figure 17: GIP Anti-apoptotic Signalling Requires Akt and Involves Suppression of p38 MAPK and JNK ...... 75

Figure 18: Inhibitors of p38 MAPK and JNK Suppress STS-induced INS-1 Cell Death in a Concentration Dependent Manner ...... 77

Figure 19: GIP Promotes Survival of STS-treated MIN6 Cells via Suppression of p38 MAPK and JNK ...... 78 vii

Figure 20: Inhibition of p38 MAPK and JNK Signalling Mimics the Survival Actions of GIP ...79

Figure 21: GIP Inhibits the Activity of Apoptosis Signal Regulating Kinase-1 (ASK1) in STS- treated INS-1 Cells ...... 82

Figure 22: GIP Suppresses p38 MAPK and JNK Activity via Inhibition of ASK1 in INS-1 cells and Human Islets...... 83

Figure 23: GIP Mediated Suppression of p38 MAPK and JNK Promotes the Survival of INS-1 Cells Exposed to ER and Genotoxic Stress ...... 84

Figure 24: A DPP-IV Resistant GIP Analogue (D-GIP1-30) Exhibits Potent Insulinotropic Actions in Lean and Obese Vancouver Diabetic Fatty (VDF) Rats...... 90

Figure 25: The GIP Receptor (GIPR) is Required for the Insulinotropic Effects of D-GIP1-30 .....91

Figure 26: D-GIP1-30 Demonstrates Equivalent Islet Actions to GIP1-42 ...... 92

Figure 27: D-GIP1-30 Protects β-cells in Streptozotocin (STZ) Treated Rats ...... 96

Figure 28: D-GIP1-30 Improves Islet Function and Diminishes Islet Pro-apoptotic Protein Levels in Obese VDF Rats, While Not Promoting Weight Gain ...... 98

Figure 29: D-GIP1-30 Improves β-cell Function and Survival and Glucose Tolerance in Akita Mice ...... 100

Figure 30: The Mechanism by Which D-GIP1-30 Promotes the Survival of Cultured β-cells Under ER Stress Does Not Involve Suppression of CHOP Protein Levels ...... 102

Figure 31: D-GIP1-30 Improves Glycaemic Control in Obese ZDF Rats ...... 105

Figure 32: D-GIP1-30 Improves β-cell Function and Survival and Glucose Tolerance in Obese ZDF Rats ...... 106

Figure 33: Proposed Mechanism by Which GIP Activates Akt in β-cells ...... 117

Figure 34: Proposed Anti-apoptotic Actions of GIPR Signalling in β-cells ...... 124

Figure 35: The Pleiotropic Actions of Incretin Hormones ...... 128

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List of Abbreviations °C Celsius 8-cpt cAMP adenosine 3‟, 5‟-cyclic monophosphate, 8-(4-chlorophenylthio)-2‟-O-methyl AC adenylate cyclase AKAP A-kinase anchoring protein Akt / PKB protein kinase B ANOVA analysis of variance APAF1 apoptosis protease-activating factor 1 ASK1 apoptosis and signal regulating kinase-1 ATP adenosine triphosphate AUC area under the curve Bad Bcl-2 associated death promoter Bak Bcl-2 antagonist killer Bax Bcl-2 associated X protein Bcl-2 B-cell lymphoma protein Bcl-XL B-cell lymphoma-extra large protein BH Bcl-2 homology domain Bid BH3 interacting domain death agonist Bim Bcl-2 interacting mediator of cell death BMH bismaleimidohexane BSA bovine serum albumin BW body weight Ca2+ calcium cAMP cyclic-adenosine monophosphate caspase cysteine/aspartate protease

CaV voltage-dependent calcium channel cDNA complementary deoxyribonucleic acid CHOP C/EBP homologous protein CHX cycloheximide CNS central nervous system

CO2 carbon dioxide COX cyclooxygenase CRE cAMP response element CREB CRE binding protein DAB 3,3'-diaminobenzidine DAPI 4',6-diamidino-2-phenylindole DM diabetes mellitus

DMEM Dulbecco‟s modified Eagle medium DPP-IV dipeptidyl peptidase IV DMSO dimethyl sulfoxide

EC50 half maximal effective concentration EDTA ethylenediaminetetraacetic acid eIF2α eukaryotic initiation factor 2α ELISA enzyme-linked immunosorbent assay EPAC exchange protein activated by cAMP ER endoplasmic reticulum ERK extracellular-regulated kinase FACS fluorescence-activated cell sorting FBS fetal bovine serum FFA free fatty acid FoxO1 forkhead O1 transcription factor GFP green fluorescent protein GIP glucose-dependent insulinotropic polypeptide GIPR GIP receptor GLP-1 glucagon-like peptide-1 GLP-1R GLP-1 receptor GLP-2 glucagon-like peptide-2 GLUT glucose transporter protein GSIS glucose-stimulated insulin secretion GSK3 glycogen synthase kinase 3 GST glutathione-S-transferase h hour HA haemagglutinin HBSS Hank‟s balanced salt solution H&E hematoxylin and eosin HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HOMA SI homeostatic model of insulin sensitivity IGF-I insulin-like growth factor-I IGF-II insulin-like growth factor-II IGF-IR IGF-I receptor IGT impaired glucose tolerance IKK IĸB kinase IRE-1 inositol-requiring enzyme 1 INS-1 rat insulinoma β-cell line (clone 832/13) i.p. intraperitoneal x

iPLA2 group VIA phospholipase A2 IR insulin receptor IRS insulin receptor substrate i.v. intravenous IPGTT intraperitoneal glucose tolerance test JNK jun N-terminal kinase KA kinase activity

KATP ATP-sensitive potassium channels

KV voltage-dependent potassium channel LPL lipoprotein lipase MDM2 murine double minute 2 MEK MAPK kinase min minute MIN6 mouse insulinoma β-cell line mRNA messenger ribonucleic acid mTOR mammalian target of rapamycin mTORC1 mTOR/raptor protein complex mTORC2 mTOR/rictor protein complex NFĸB nuclear factor ĸB NTS nucleus tractus solitarius

O2 oxygen OGTT oral glucose tolerance test p38 MAPK p38 mitogen-activated protein kinase PBS phosphate-buffered saline PC prohormone convertase PCNA proliferating cellular nuclear antigen PDE phosphodiesterase PDK-1 3-phosphoinositide dependent kinase-1 PDX-1 pancreatic and duodenal homeobox-1 pen/strep penicillin G-sodium and streptomycin sulphate pH potential for hydrogen ion concentration PH pleckstrin homology domain PI propidium iodide PI3K phosphatidylinositol 3-kinase

PIP3 phosphatidylinositol (3,4,5)-trisphosphate PKA protein kinase A PKC protein kinase C PKR double stranded RNA dependent protein kinase xi

PMSF phenylmethylsulfonyl fluoride PPAR peroxisome-proliferator activated receptor PTB protein tyrosine binding domain Puma p53-upregulated modulator of apoptosis RIA radioimmunoassay ROS reactive oxygen species RP-cAMP adenosine 3‟, 5‟-cyclic monophosphorothioate Rp-isomer RPMI Roswell Park Memorial Institute s.c. subcutaneous SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis SEM standard error of the mean SH2 src homology domain 2 siRNA small interfering ribonucleic acid SNP single nucleotide polymorphism Src sarcoma protein STS staurosporine STZ streptozotocin T2D type 2 diabetes mellitus TCF7L2 transcription factor-7 like 2 TG triglyceride TNFα tumour necrosis factor-α TNF-R TNF-α receptor Trx thioredoxin TSC tuberous sclerosis complex TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling TZD thiazolidinedione UBC University of British Columbia UPR unfolded protein response UTR untranslated region VDF Vancouver diabetic fatty rat WT wild type ZDF Zucker diabetic fatty rat

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Acknowledgements Earning a PhD is a remarkable journey that comes with numerous moments of joy and discovery as well as difficultly. I have been blessed by such a journey, and it is soon coming to a finish, but before it does, there are many deserving of mention. The bulk of my gratitude goes to Chris McIntosh. None of this would have been possible without you. Thanks for the opportunities, patience and effort. You were great in providing me with new ideas and a strong foundation of the literature, and have a natural talent for encouragement. I thank you as well for your friendship and allowing me the freedom to pursue my own interests. I would also like to thank Harold Weger and Richard Manzon who took a chance on me during my undergraduate program at the University of Regina, providing my first spark in research, and Ed Moore, Bruce Verchere, Steven Pelech, Eric Accili, Tim Kieffer and Jim Johnson for your comments and insights. Many offered intellectual insights and brought a great deal of joy during the last five years. Thanks to Dan, Rob, Rhonda, Emilyn, Gary, Jasna, Thomas, Su-Jin and all the others from the Kieffer, Johnson, Verchere, Proud and Accili labs as well as those from St. John‟s College who were involved in this process. A special thanks to Cuilan who has been a good friend and the one who taught me the basics of the lab. I hope all the best to each of you in your future. My family, especially my parents, plays an important role in my life and I will always be grateful. My work ethic and character derives from you and I hope that you can be proud. I must also recognize the role of God in my PhD studies. About 10 years ago, God became very involved in my life and shifted my focus towards the needs of others and the amazing beauty of biology, resulting in my interests in diabetes and biomedical sciences, which, to me, is a mix of both. Moreover, God‟s sustenance has enabled me to get through the rough patches of my „PhD years‟ and, for that, I am exceedingly grateful and pray that I can bless others in turn. It was not long ago that I thought I would never find someone to share my life with. Now, a few years later, I have my wonderful wife Jaclyn and we will soon be adding a baby to our family. Honey, you have been so wonderful in encouraging me, especially in the time leading up to my comprehensive exam and while writing this thesis. You always inspire me and I only wish I could be the husband that you truly deserve. Although my PhD is finishing, in the grand scheme it is only a turn of the page. Now, it is time to begin a new journey…

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Chapter 1 Introduction to GIP, Incretins and Type 2 Diabetes 1.1 Overview Bayliss and Starling‟s seminal discovery of secretin revealed a mechanism of inter-organ communication via a blood-borne chemical messenger that they termed a hormone (Bayliss and Starling, 1902). We now know of numerous hormones that regulate virtually every aspect of physiology and development; one clear example being the regulation of glucose homeostasis by insulin. The effects of insulin are tightly synchronized with caloric intake, circulating glucose levels and tissue sensitivity to its actions, and disruptions in these relationships results in the debilitating conditions of diabetes. Gastrointestinal hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are major modulators of insulin levels, and in recent years, they have been targeted for treating diabetes. However, there is uncertainty over the application of GIP-based therapies since, paradoxically, arguments have been made for benefits of treatment with both GIP agonists and antagonists. A deeper understanding of the physiological roles of GIP and the mechanistic basis underlying its actions is critical for both understanding its biology and establishing its potential therapeutic application. The studies described in this thesis focus on the mode of GIP action with emphasis on its pro-survival effects on β-cells in the context of type 2 diabetes mellitus. Discussions of GLP-1 are also included in areas where there is much greater knowledge regarding its biology and when it is reasonable to assume that GIP could have similar properties.

1.2 Discovery of the Incretins Soon after the discovery of secretin it was predicted that gastrointestinal hormones could regulate “internal secretions” of the pancreas and have important effects on glucose homeostasis (Moore, 1906). Using intestinal extracts, La Barre and colleagues provided evidence for such a factor that they named “incrétine” (La Barre, 1932) but inconsistencies arose in the literature, mainly due to a lack of understanding of the chemistry of peptide hormones, and the concept became dormant (Creutzfeldt, 2005). However, following the development of an insulin immunoassay (Yalow and Berson, 1961) compelling evidence was obtained for a regulatory link between the gut and pancreas when it was shown that glucose infused via intajejunal (McIntyre et al., 1964) or intragastric (Elrick et al., 1964) routes exerted greater insulinotropic effects than glucose delivered intravenously. It was estimated that ~50% of insulin secreted after an oral glucose load was due to gastrointestinal factors (Perley and Kipnis, 1967), and soon thereafter, 1

this became known as the entero-insular axis (Unger and Eisentraut, 1969). While the enteroinsular axis is comprised of hormonal, nutritional and neural components, the incretin concept is a specific reference to its hormonal arm. Creutzfeldt re-introduced the term “incrétine” in anglicized form (incretin) and established a set of criteria considered to be critical for an incretin to promote nutrient disposal without hypoglycaemic effects (Creutzfeldt, 1979). First, it should be secreted in response and proportion to enteric nutrient absorption, particularly carbohydrates. Second, it should stimulate the secretion of insulin from the pancreas at physiological levels and only when blood glucose levels elevate above fasting. Lastly, secretion should cease during the post-absorptive phase (ie. between meals). Around the time that Unger had defined the entero-insular axis, Brown and colleagues were searching for gastrointestinal factors that regulate gastric motility and acid secretion (Pederson, 1994). In physiological studies, they identified a gastric inhibitory factor within crude intestinal extracts (Brown and Pederson, 1970), and in collaboration with Viktor Mutt at the Karolinska Institute in Stockholm (Brown et al., 1970), a polypeptide was isolated, purified and named gastric inhibitory polypeptide (GIP). In parallel with these studies, Dupre and colleagues were searching for the mysterious incretin(s) and found that a similar intestinal extract to Brown‟s had insulinotropic activity (Dupre and Beck, 1966). In collaborative studies, they showed that intravenous infusion of GIP stimulated insulin secretion and improved glucose disposal in humans (Dupre et al., 1973). GIP was subsequently renamed glucose-dependent insulinotropic polypeptide, allowing retention of the acronym (Pederson and Brown, 1976). Further studies provided clear evidence that GIP fulfills the criteria of an incretin hormone (Creutzfeldt, 1979). However, the incretin effect on insulin responses to intraduodenal glucose were only partially reduced in rats receiving co-administration of GIP anti-serum (Ebert and Creutzfeldt, 1982; Lauritsen et al., 1981) and significant insulinotropic activity was found to remain in rat intestinal extracts following immuno-affinity removal of GIP (Ebert et al., 1983), indicating the possibility of an additional incretin hormone. In studies using Brockman bodies from anglerfish as an enriched source of islet cells, Habener and colleagues (Lund et al., 1982) discovered a coding sequence for a glucagon-related peptide arranged 3ʹ to the sequence encoding glucagon on preproglucagon cDNA. They had speculated that the product was structurally similar to that of GIP (Lund, 2005), and by that time, several studies had uncovered glucagon-like immuno-reactive products in the mammalian gut

(Holst, 1994). Bell and colleagues (Bell et al., 1983) were the first to isolate mammalian preproglucagon cDNA and they discovered two glucagon-related peptides arranged in tandem, one of which was structurally similar to that of the anglerfish, and they named them glucagon- like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2). The selective production of glucagon in α-cells and GLP-1 in intestinal L-cells was found to result from differential post- translational processing by tissue specific expression of prohormone convertase 2 (PC2) in pancreatic α-cells and PC1/3 in gastrointestinal L cells. In a series of subsequent studies (Holst, 1994), the processed and functionally active form of GLP-1 was identified as an N-terminally truncated version (GLP-17-37) and (GLP-17-36amide), collectively referred to herein as GLP-1, which was identified as having insulinotropic actions (Mojsov et al., 1987; Schmidt et al., 1985) and recognized as an incretin hormone (Holst, 2007; Kreymann et al., 1987). In combination, GIP and GLP-1 account for virtually the entire, if not all, of the incretin effect, amounting to ~50-65% of post-prandial insulin responses (Muscelli et al., 2006; Nauck et al., 1986b). GIP and GLP-1 contribute relatively equally to the incretin effect (Vilsboll et al., 2003a) and thus both incretins play important roles in regulating glucose homeostasis.

1.3 Nature of Glucose-dependent Insulinotropic Polypeptide (GIP) 1.3.1 Origin GIP is a member of the secretin/glucagon superfamily of hormones, currently consisting of nine bioactive peptide members, including GLP-1 (Sherwood et al., 2000). The gene for GIP appeared during whole genome duplications in early vertebrate evolution (Irwin, 2002) and gene sequences can be identified in mammals, birds, reptiles and fish (Irwin, 2009), indicating that GIP originated more than 300 million years ago. Though there is high divergence in distant relatives (Irwin, 2009), primary sequences of GIP differ by only 1-2 amino acids in mammals, including human, porcine, rat, mouse, canine and bovine species (McIntosh et al., 2009). This highly conserved feature reveals that GIP has important function(s) in mammals, but due to the lack of comparative studies, especially in lower species, there is little known regarding the functional evolution of GIP. However, based on actions identified in mammalian studies, it is arguable that GIP serves mainly anabolic functions by coordinating the post-prandial disposal of carbohydrates and fats as well as in bone deposition, while also regulating growth and development of pancreatic β-cells.

1.3.2. Gene and Peptide The human GIP gene, located on chromosome 17q (Inagaki et al., 1989), consists of 6 exons that include the preproGIP precursor encoded in exons 2-5, a 5ʹ-untranslated region in exon 1 and a 3ʹ-polyadenosine tail in exon 6. The functional peptide, GIP1-42, is encoded in exons 3 and 4. The 5ʹ-upstream region of the promoter contains a TATA motif and consensus sites for Sp1, Ap-1, Ap-2 and two cis-acting regulatory (CRE) elements (Inagaki et al., 1989; Someya et al., 1993). The promoter also contains a binding site for pancreatic and duodenal homeobox-1 (PDX-1), a transcription factor critical for development of GIP-producing K-cells (Jepeal et al., 2005). Human cDNA clones revealed a 459 base pair open reading frame encoding a 153 amino acid preproGIP (Takeda et al., 1987), predicted to consist of a 21 amino acid signal peptide, a 30 amino acid NH2-terminal peptide, GIP1-42 and a 60 residue C-terminal peptide. The rat GIP gene, preproGIP structure and 5ʹ promoter region are similar to those of human (Higashimoto and Liddle, 1993).

1.3.3 Biosynthesis and Localization GIP is mainly produced in enteroendocrine K-cells, which are highly concentrated in the duodenum (McIntosh et al., 2009), and its production is coupled to dietary intake with glucose ingestion increasing both duodenal mRNA and peptide levels (Higashimoto et al., 1995; Tseng et al., 1994). In contrast, GLP-1 is mainly produced in L-cells of the distal portion of the small intestine and colon (Eissele et al., 1992). However, recent studies on porcine, rat and human small intestine indicated that >60% of GLP-1 positive cells co-localize with GIP in mid-jejunal to mid-ileal regions (Mortensen et al., 2003), and co-localization was additionally detected in the duodenum of humans (Theodorakis et al., 2006). There also appears to be a sub-population of GIP positive cells that co-localize with a 25 amino acid peptide called xenin, but the underlying relationship to GIP at this point is uncertain (McIntosh et al., 2009). Expression of GIP has also been identified in the pancreatic α-cell (Fujita et al., 2010b), hippocampus (Nyberg et al., 2005), stomach (Yeung et al., 1999) and salivary glands (Tseng et al., 1995). Interestingly, although processed in the K-cell mainly to GIP1-42 via PC1/3 (Ugleholdt et al., 2006), in α-cells, which preferentially express PC2, GIP was found to be differentially processed to a C-terminally truncated form predicted to be GIP1-30amide (Fujita et al., 2010b). Furthermore, a subset of K-cells in mice were recently identified that express PC2 and also appeared to produce GIP1-30amide (Fujita et al., 2010a).

1.3.4 Secretion Plasma GIP increases ~6-fold in response to a meal, with carbohydrates and fats as the major stimuli (McIntosh et al., 2009). Depending on the assay, human GIP levels can range from 12-92 pM during fasting to 35-235 pM during the post-prandial phase (Jorde et al., 1983). The level of GIP secreted is proportional to the calorific value of the ingested nutrients (Vilsboll et al., 2003b) and requires nutrient absorption in the intestine (Creutzfeldt, 1979). Interestingly, both GIP and GLP-1 are secreted in high levels into the intestinal lymph (D'Alessio et al., 2007; Lu et al., 2008), although the physiological significance is unclear. Oral or intraduodenal glucose administration elicits rapid GIP release in humans (Andersen et al., 1978; Krarup et al., 1985; Lavin et al., 1998; Lucey et al., 1984; McIntosh et al., 2009), rats (Bryer-Ash et al., 1994), dogs (Greenberg and Pokol-Daniel, 1994) and pigs (Knapper et al., 1995a), with levels peaking within 15-30 min and returning to fasting levels in the post-absorptive phase. Consistent with K-cell distribution, glucose-stimulated GIP secretion decreases from the duodenum to the ileum (Thomas et al., 1977), and the rate of intestinal glucose absorption correlates with circulating GIP levels (Wachters-Hagedoorn et al., 2006). Studies in rats have shown that the sodium-dependent glucose transporter is involved in glucose- stimulated GIP secretion (Sykes et al., 1980) and blockade of glucose absorption reduces GIP secretion (Fushiki et al., 1992). Secretion is also stimulated by galactose, sucrose, and maltose but not fructose, sorbitol, mannose, or lactose (Creutzfeldt, 1979). In comparison to carbohydrates, triglycerides elicit a slow, prolonged and potent GIP response in humans (Cleator and Gourlay, 1975; Falko et al., 1975; Krarup et al., 1985), dogs (Pederson et al., 1975) and rats (Ebert et al., 1991; Hampton et al., 1983), and fat has been found to be a more potent secretagogue than isocaloric glucose in humans (Cleator and Gourlay, 1975; Krarup et al., 1985). Secretion is dependent on triglyceride metabolism (Creutzfeldt, 1979) with long chain, but not medium chain, fatty acids being mainly responsible for stimulating GIP secretion (O'Dorisio et al., 1976; Ohneda et al., 1984; Ross and Shaffer, 1981). Amino acids and duodenal acidification also stimulate GIP secretion (Ebert et al., 1979; LeRoith et al., 1980; O'Dorisio et al., 1976; Schulz et al., 1982; Wolfe et al., 2000). Thus, the overall GIP response to nutrients is dependent upon meal size, type of nutrient and rate of absorption in the intestine (Chaikomin et al., 2005; Schirra et al., 1996). Inhibitory systems also appear to be involved in regulating GIP secretion. Insulin infusion reduced GIP secretion stimulated by intraduodenal glucose in humans (Sirinek et al.,

1978) or oral gavage in rats (Bryer-Ash et al., 1994), and glucose and insulin were found to inhibit GIP secretion in response to fat, but not glucose absorption, in patients with type 1 diabetes (Creutzfeldt et al., 1980). Intravenous infusion of C-peptide was also found to reduce fat-induced GIP secretion (Dryburgh et al., 1980) and glucagon reduced GIP responses to carbohydrates (Ranganath et al., 1999). Moreover, inhibition of incretin degradation resulted in a reduction in total circulating GIP and GLP-1 levels in dogs (Deacon et al., 2002) and humans (El-Ouaghlidi et al., 2007; Herman et al., 2006), although whether the effect was due to potentiation of insulin secretion is uncertain. Similarly, it is unclear whether regulatory inputs from the nervous system play a role (McIntosh et al., 2009). Little is known regarding the cellular mechanisms of GIP secretion, mainly because it has proven difficult to isolate the diffusely dispersed cells of the enteroendocrine system for in vitro studies. However, in studies on partially purified K-cells prepared from canine intestinal mucosa, elevating glucose or gastrin releasing peptide, or immuno-neutralization of somatostatin, were found to stimulate GIP secretion (Kieffer et al., 1994), and a sub-clone of the intestinal STC-1 endocrine tumour cell line (STC6-14), containing ~30% immuno-reactive GIP positive cells, also responded to glucose (Kieffer et al., 1995a). The ATP-dependent K+ channel subunits, SUR1 and Kir6.2, are localized in K- and L- cells in the human small intestine (Nielsen et al., 2007) and K-cells also contain glucokinase (Cheung et al., 2000), both of which are critical components in β-cells for glucose-dependent insulin secretion, indicating that K-cells may have a similar mechanism of secretion. The G protein-coupled receptor, GPR119, has been localized selectively in K- and L-cells (Chu et al., 2008), and the GPR119 ligand, oleoylethanolamide, was found to stimulate GLP-1 secretion (Lauffer et al., 2009) and may also stimulate GIP secretion. GLP-1 is also secreted in response to a meal (Orskov et al., 1996), especially carbohydrates and lipids (Dubé and Brubaker, 2004; Elliott et al., 1993; Herrmann et al., 1995), in a manner that is dependent on meal size (Vilsboll et al., 2003b). Secretion from the canine ileum (Sugiyama et al., 1994) and the L-cell line, GLUTag (Gribble et al., 2003), have been shown to involve sodium-dependent glucose transporters, indicating similarities to GIP secretion. Recently, glucose-induced GLP-1 secretion was reported to involve regulation by sweet taste receptors and activation of the taste-associated G-protein α-gustducin (Jang et al., 2007; Rozengurt et al., 2006). In the study from Jang et al. (2007), it was reported that <50% of K-cells and 100% of GIP/GLP-1 co-positive cells contained α-gustducin, and that GLP-1 and GIP secretion in response to orally delivered glucose were reduced in α-gustducin knockout mice.

Moreover, treatment of the human L-cell line, NCI-H716, with the artificial non-caloric sweetener, sucralose, was found to stimulate GLP-1 secretion. By contrast, sucralose was not found to stimulate GIP or GLP-1 secretion in healthy humans (Ma et al., 2009) and an assortment of sweeteners, including sucralose, exerted no effect in rats (Fujita et al., 2009). Transgenic mouse-lines expressing fluorescently labelled proteins either in L-cells (Reimann et al., 2008) or K-cells (Parker et al., 2009) were recently generated, enabling studies on highly purified primary cells. Both K-cells and L-cells were found to express SUR1, Kir6.2, sodium- dependent glucose transporters and glucokinase. Elevating glucose on cultured cells increased incretin secretion, but again, sucralose had no effect (Parker et al., 2009; Reimann et al., 2008). Further studies are clearly needed to clarify the mechanisms underlying incretin hormone secretion and the role of sweet taste receptors.

1.3.5 Metabolism Measurements of canine renal arteriovenous differences in immuno-reactive GIP levels and the finding that patients with renal failure have elevated levels of circulating GIP (O'Dorisio et al., 1977) indicated that GIP is cleared via renal filtration. The circulating half-life of GIP in humans has been reported to be ~20 min (Vilsboll et al., 2006). However, infusion studies of both GIP and GLP-1 identified much shorter biological half-lives for either hormone (Deacon, 2005), revealing an additional regulatory mechanism for the incretin system. Endogenous or exogenously administered GIP1-42 and GLP-17-36 undergo rapid N-terminal truncation to non- insulinotropic forms GIP3-42 and GLP-19-36 (Deacon, 2005; Kieffer et al., 1995b; Mentlein et al., 1993), and the enzyme responsible was identified as dipeptidyl peptidase IV (DPP-IV). A member of the S9 family of prolyl oligopeptidases (McIntosh, 2008), DPP-IV is ubiquitously expressed and found in both circulating and membrane bound forms, including the kidney and intestinal brush-border membranes and throughout the entire vascular bed (Deacon, 2005; Drucker, 2007a; Holst and Deacon, 1998; McIntosh et al., 2006). DPP-IV selectively removes N-terminal dipeptides from oligopeptides, with preference for peptides having proline or alanine as the penultimate amino acid. In the case of GIP and GLP-1, the N-termini are Tyr-Ala and His- Ala, respectively, and are physiological substrates of DPP-IV. As expected, incretin bioactivity was found to be elevated in DPP-IV negative rats (Pederson et al., 1996) and DPP-IV knockout mice (Marguet et al., 2000). Due to the actions of DPP-IV, the biological half-lives of GIP and GLP-1 have been determined to be ~5-7 and ~2-3 min, respectively (Deacon, 2004; Deacon et

al., 2000; Deacon et al., 1995; Meier et al., 2004). An important result of studies on GIP and GLP-1 metabolism has been the development of DPP-IV inhibitors and DPP-IV resistant incretin analogues as diabetes therapeutics. Of additional interest, DPP-IV has been identified in close proximity to the site of GLP-1 release and in the surrounding capillary beds, and as a consequence, only 10-15% of GLP-1 (7- 36) reaches the systemic circulation (Hansen et al., 1999; Holst, 2007). This finding raised questions regarding the pathway by which GLP-1 acts. An extensive series of studies, discussion of which is beyond the scope of this thesis, have established that GLP-1 acts not only as an endocrine hormone, but also via activation of afferent sensory nerves and as a neurotransmitter in the central nervous system (Holst, 2007). Furthermore, non-insulinotropic biological effects of the DPP-IV metabolised GLP-1 (9-36) have also been identified. In contrast, although expression has been reported in the brain (Section 1.5.6), the general consensus is that GIP acts mainly in an endocrine manner.

1.4 Nature of the GIP Receptor 1.4.1 Gene and Protein The human GIP receptor (GIPR) gene is located on chromosome 19q13.3 (Gremlich et al., 1995) and contains 14 exons with a protein coding region of 12.5 kb (Yamada et al., 1995). The promoter contains six consensus sequences for Sp1 and Sp3 transcription factor binding (Baldacchino et al., 2005). The GIPR gene in rat has 13 exons and spans 10.2 kb and the promoter 5ʹ-flanking sequences contain several transcription factor binding motifs, including a cAMP responsive element, an octamer binding site, three Sp1 binding sites and an initiator element (Boylan et al., 1999). There has been suggestive evidence for GIPR mRNA splice variants (Gremlich et al., 1995; McIntosh et al., 2009), and a dominant negative variant was recently identified in mouse β-cells that interestingly was down-regulated in mice on a high fat diet, resulting in enhanced GIP sensitivity (Harada et al., 2008). The GIPR is a member of the class B G-protein coupled receptor family, along with receptors for GLP-1, GLP-2, secretin and glucagon (Sherwood et al., 2000). The human GIPR shares 99.4% homology with chimpanzee (Pan troglodytes), 86.2% homology with rhesus monkey (Macca mulatto) and ~80% homology with rodents. Sequence lengths range from 440- 492 amino acids, apart from the fox receptor (felix catus) that has 388 amino acids. The

sequences of the rat, human and hamster GIPRs share 40-47% identity with GLP-1 and glucagon receptors (McIntosh et al., 2009). The protein has an estimated molecular mass of ~59 kDa and exhibits a typical seven transmembrane structure with N-terminal glycosylation sites that are important for cell surface expression (Amiranoff et al., 1985; McIntosh et al., 2009). As with other class B family members (Hoare, 2005), the GIPR contains a large extracellular N-terminus. The third intracellular loop and C-terminal tail are rich in serine/threonine residues, which offer phosphorylation sites for functional coupling and internalization (Tseng and Zhang, 1998a, b; Usdin et al., 1993; Wheeler et al., 1999; Wheeler et al., 1995). Interestingly, GIP can induce homologous desensitization of the GIPR, through mechanisms apparently involving G-protein receptor kinase 2 and β-arrestin-1 (Tseng and Zhang, 1998a, 2000). Chronic hyperglycaemia also results in desensitization and down-regulation of the GIPR in β-cells (Hinke et al., 2000; Lynn et al., 2003) and this is reversible by normalizing glycaemia in diabetic rats (Piteau et al., 2007) and patients with T2D (Hojberg et al., 2009).

1.4.2 Localization Protein and mRNA expression studies have identified the GIPR in multiple rodent tissues including pancreatic islet α and β-cells, adipose tissue, stomach, brain, pituitary, heart, lung, vascular endothelium and bone (Bollag et al., 2000; McIntosh et al., 1999; Moens et al., 1996; Nyberg et al., 2005; Usdin et al., 1993; Wheeler et al., 1995; Yip and Wolfe, 2000; Zhong et al., 2000). The roles of GIPR activity in many of these tissues are unclear, but functions that have been identified are discussed in Section 1.5.

1.4.3 Receptor Interaction with GIP Studies using native and cloned human and rat receptors (Gremlich et al., 1995; Maletti et al., 1986; Wheeler et al., 1995) as well as chimeric GIP and GLP-1 receptors (Gelling et al.,

1997b) revealed kd values of 0.2-7 nM. The GIPR N-terminus and first transmembrane domain are important for receptor activation (Gelling et al., 1997b). A recent crystal structure of the human GIPR extracellular domain bound to GIP1-42 indicated that GIP binds in an α-helical conformation with the C-terminal region binding in a surface groove of the receptor and N- terminus remaining free to interact with other parts of the receptor, and that both GIP and the receptor undergo conformational changes during binding (Parthier et al., 2007). The N-terminus

of GIP1-42 is critical for bioactivity (Hinke et al., 2004b) and the high affinity binding region resides in amino acids 6-30 (Gelling et al., 1997a). Interestingly, in the β-cell C-terminally truncated GIP1-30 exhibits almost identical GIPR binding affinity and bioactivity to GIP1-42 (Hinke et al., 2004b; Pederson, 1990), whereas its effects on somatostatin secretion from the perfused rat stomach are reduced (Pederson, 1990; Rossowski et al., 1992), indicating potential cell-selective differences in GIP-receptor interactions and signalling.

1.5 Biological Actions of GIP 1.5.1 Effects of GIP on the Pancreatic Islet The most established physiological effect of GIP is to potentiate glucose-stimulated insulin secretion (GSIS). After initial demonstrations in humans (Dupre et al., 1973), insulinotropic effects of GIP were shown in rat, mouse, baboon and dog (Pederson et al., 1998; Pederson et al., 1975; Turner et al., 1974a; Turner et al., 1974b). Potentiation of insulin secretion has been demonstrated in isolated rat islets (Schauder et al., 1975), perfused rat pancreas (Pederson and Brown, 1976) and INS-1 β-cells (Kim et al., 2005a), revealing that GIP promotes this effect via direct β-cell stimulation. Studies in humans (Elahi et al., 1979) and in the perfused rat pancreas (Jia et al., 1995) demonstrated that GIP stimulates insulin secretion at physiological concentrations and only under hyperglycaemic conditions, consistent with the criteria proposed for an incretin (Creutzfeldt, 1979). In rats, a threshold of ~4.5 mM glucose was observed, with maximal responses at 16.7 mM (Jia et al., 1995; Pederson and Brown, 1976). However, clamp studies in humans identified small insulinotropic effects for GIP at fasting glucose levels, although pronounced stimulation required elevated glucose levels (Vilsboll et al., 2003a). The important contribution of GIP to the incretin response was initially shown in studies on rats showing that administration of GIP anti-serum reduced the insulin response to orally delivered glucose (Ebert and Creutzfeldt, 1982; Lauritsen et al., 1981). Studies using immunoadsorption of GIP from intestinal extracts (Ebert et al., 1983), intravenous administration of antibodies against the GIP receptor (Lewis et al., 2000) or GIPR antagonists (Gault et al., 2002; Tseng et al., 1996; Tseng et al., 1999), provided estimates of 50-70% for the contribution to the overall incretin effect. Confirmation of an incretin role was obtained in GIPR knockout (GIPR-/-) mice, which exhibited reduced insulin secretion and glucose intolerance during an oral glucose load (Hansotia et al., 2004; Miyawaki et al., 1999; Preitner et al., 2004). More recent advances in understanding the molecular mechanisms of GIP-induced insulin secretion and the potential

roles for GIP in promoting β-cell proliferation and survival are discussed further in Sections 1.6 & 1.7. The effects of GIP on the islet are not limited to the β-cell. GIP has been found to promote glucagon secretion from the rat (Pederson and Brown, 1978) and dog (Adrian et al., 1978) with maximal effects at low glucose concentrations. GIP exhibited glucagonotropic effects in perfused pancreata from human cadavers (Brunicardi et al., 1990), patients with liver cirrhosis and hyperglucagonemia (Dupre et al., 1991) as well as fasted healthy human subjects (Meier et al., 2003). The physiological significance of this effect is uncertain, but one argument has been that since GIP potentiates leucine and arginine-induced insulin secretion (Mazzaferri et al., 1983; Schauder et al., 1977), GIP-induced glucagon release may prevent hypoglycaemia during a high protein meal (McIntosh et al., 2009). Effects of GIP on somatostatin and pancreatic polypeptide release have also been reported, but the significance is uncertain (McIntosh et al., 2009). It is important to note that, despite strong similarities to GIP with respect to insulin secretion, the effects of GLP-1 on glucagon secretion are opposite. GLP-1 strongly inhibited glucagon secretion in humans, including patients with type 1 and type 2 diabetes mellitus (Creutzfeldt et al., 1996; Gutniak et al., 1992) as well as all other examined species (Drucker, 2007b; Dunning et al., 2005; Fridolf et al., 1991; Orskov et al., 1988), and this is one of the major anti-diabetic benefits of GLP-1 based therapeutics. However, the underlying mechanism by which GLP-1 promotes this effect remains controversial and beyond the scope of this thesis.

1.5.2 Effects of GIP on Adipose Tissue The robust effects of fat ingestion on GIP secretion and the presence of GIPRs on adipocytes indicated that GIP plays a role in lipid metabolism. GIP administration promotes chylomicron-associated triglyceride (TG) clearance from blood in dogs (Wasada et al., 1981), and lowers plasma TG levels following an intraduodenal fat load in rats (Ebert et al., 1991). However, GIP did not exert an effect when TG was administered intravenously in humans (Jorde et al., 1984) or dogs (Ohneda et al., 1983), and it thus requires chylomicron transport. While part of its actions on the adipocyte occur via stimulation of insulin secretion, GIP has also been shown to stimulate fatty acid synthesis from acetate in adipose tissue explants (Oben et al., 1991) as well as increase glucose uptake and incorporation into lipids (Hauner et al., 1988). In humans, the primary source of adipose-tissue triglyceride is dietary fat (Morgan, 1996), and in adipose tissue, the liberation of free fatty acids from circulating triglycerides is

mediated by lipoprotein lipase (LPL). GIP has been shown to enhance LPL activity in cultured 3T3-L1 and human adipocytes (Kim et al., 2007b, c) and rat adipose explants (Knapper et al., 1995b). Interestingly, in the absence of insulin, GIP exhibits lipolytic actions, whereas its lipogenic effects are permissively-dependent on insulin (McIntosh et al., 2009). The role of a dual lipolytic/lipogenic action is unclear but it has been argued that GIP may, under fasting conditions, maintain levels of circulating free fatty acids that are required for β-cell priming (McIntosh et al., 2009), whereas post-prandially, GIP would enhance lipid storage. An alternative explanation has been proposed in which GIP-induced lipolysis contributes to lipogenesis by releasing fatty acids for re-esterification (Getty-Kaushik et al., 2006). The effects of GIP on adipose tissue have been suggested to contribute to obesity. GIPR expression increases during differentiation of preadipocytes to adipocytes and GIPR signalling promotes the differentiation process (Song et al., 2007; Weaver et al., 2008), indicating that GIP may contribute to the development and, possibly, also functional maturation of adipocytes. In longitudinal studies, GIPR-/- mice on a high fat diet (Hansotia et al., 2007) or crossed with leptin (ob/ob) knockout mice (Miyawaki et al., 2002) exhibited a reduction in adiposity and elevated energy expenditure, and similar findings occurred in mice with targeted K-cell ablation (Althage et al., 2008) or chronic administration of a GIPR antagonist (Irwin and Flatt, 2009). However, relevance of these studies to humans is uncertain and the underlying mechanism in mice remains unclear. Of particular importance is the need to determine whether the loss of GIP action reduces adiposity because of GIPR signalling removal in adipocytes or indirectly via reductions in nutrient-induced insulin responses.

1.5.3 Effects of GIP on the Gastrointestinal System The discovery of GIP was based on its ability to inhibit gastric acid secretion in denervated dog stomachs, but these effects were less pronounced in the innervated stomach and physiological levels of GIP exerted minimal effects in humans (McIntosh et al., 2009; Pederson, 1994). Perfusion studies showed that GIP stimulates somatostatin secretion from the rat stomach, a process that is inhibited by acetylcholine and vagal stimulation (McIntosh et al., 1981), indicating that GIP probably inhibits acid secretion via paracrine actions on parietal cells. This conclusion was supported by studies using a selective somatostatin receptor antagonist (Rossowski et al., 1998). It therefore appears that GIP is capable of inhibiting gastric acid secretion but in a manner sensitive to parasympathetic tone (McIntosh et al., 2009).

GIP has been shown to inhibit ductal sodium transport in mandibular glands (Denniss and Young, 1978), presumably reflecting responses to locally produced peptide. Moreover, GIP has been found to stimulate intestinal water and electrolyte transport (Helman and Barbezat, 1977) and may regulate mucosal alkalinization in the proximal duodenum (Konturek et al., 1985) as well as intestinal motility (Thor et al., 1987). Studies on rodent intestine showed that GIP increases intestinal glucose uptake through a mechanism involving increased trafficking of the sodium-dependent glucose transporter into the brush-border membrane and glucose-transporter 2 (GLUT2) trafficking into the basolateral membrane (Cheeseman and O'Neill, 1998; Cheeseman and Tsang, 1996). In mice (Singh et al., 2008), GIP was found to increase cAMP levels in isolated enterocytes as well as increase short-circuit current and mucosa-to-serosa glucose flux, supporting a role for GIP in the regulation of transepithelial glucose transport. In contrast to the mild effects of GIP, GLP-1 exerts profound effects on gastrointestinal secretion and motility. Administering GLP-1 via infusion or subcutaneous injection was found to inhibit gastric emptying of a liquid meal in healthy, obese and type 2 diabetes patients (Naslund et al., 1998; Nauck et al., 1997; Nauck et al., 1996; Wettergren et al., 1993; Willms et al., 1996). The GLP-1 induced reduction in gastric emptying, through actions on vagal afferent pathways, is thought to contribute significantly to the anti-diabetic effects of incretin mimetics or enhancers (Drucker and Nauck, 2006; Holst, 2007; McIntosh, 2008; Schirra and Goke, 2005).

1.5.4 Effects of GIP on the Cardiovascular System Despite the presence of GIPR expression (Usdin et al., 1993), there have not been any significant effects of GIP identified in the heart. However, GIP may have a physiological role in regulating splanchnic blood flow since intravenous infusion of GIP increased superior mesenteric blood flow in cats (Fara and Salazar, 1978) and dogs (Kogire et al., 1988). GIP has also been shown to increase portal venous, but decrease hepatic arterial, blood flow in dogs (Kogire et al., 1988; Kogire et al., 1992), and increase pancreatic islet blood flow in rats (Svensson et al., 1997). In cell culture studies, GIP induced secretion of endothelin-1 from canine hepatic arterial cells and nitric oxide secretion from portal vein endothelial cells (Ding et al., 2004). These findings indicate that GIP probably plays a role in optimizing post-prandial nutrient delivery to the liver. In contrast, GLP-1 receptors (GLP-1R) have been localized to cardiomyocytes, coronary and vascular endothelial cells, and smooth muscle in mice (Ban et al., 2008), and GLP-1 has

been shown to exhibit inotropic and chronotropic activity as well as cardioprotective effects and improvements to cardiovascular performance (Abu-Hamdah et al., 2009). The underlying mechanisms resulting in these benefits remain uncertain and are currently under intense investigation. Of specific interest is the finding that the DPP-IV metabolite, GLP-19-36, also exhibited cardiovascular benefits in dogs (Nikolaidis et al., 2005) and it was suggested to act via a separate receptor. Similar findings were obtained in mice, although GLP-1 receptor-dependent actions were also involved (Ban et al., 2010; Ban et al., 2008; Noyan-Ashraf et al., 2009). A two pathway schema has been suggested in which the GLP-1 receptor promotes inotropic actions, glucose uptake, ischemic preconditioning and mild vasodilation, whereas GLP-19-36, in a GLP-1 receptor-independent manner, promotes glucose uptake and vasodilation through a nitric oxide/cGMP-dependent pathway (Ban et al., 2008). An interesting hypothesis recently proposed that GLP-19-36 is transported into target cells and further metabolized to form peptides that contain consensus mitochondrial target sequences and exert metabolic and antioxidative actions

(Tomas and Habener, 2010). Biological actions of the DPP-IV metabolite GIP3-42 have not been identified to date, but a similar uptake pathway is possible.

1.5.5 Effects of GIP on Bone It has been proposed that GIP is involved in an “entero-osseous axis” that promotes mineral deposition and bone formation following a meal (Bollag et al., 2001). The receptor is present on bone as well as osteoblast and osteoclast cell lines (Bollag et al., 2001; Bollag et al., 2000; Zhong et al., 2007). GIP stimulation increased alkaline phosphatase activity and collagen type I formation in osteoblasts (Bollag et al., 2000) and inhibited the resorptive activity of osteoclasts (Zhong et al., 2007). Moreover, ovarectomized rats treated with GIP had increased bone mass (Bollag et al., 2001), and studies on GIPR-/- and transgenic GIP over-expressing mice confirmed a role for GIP in enhancing bone mass, length and strength as well as reducing bone turnover (Ding et al., 2008; Tsukiyama et al., 2006; Xie et al., 2005; Xie et al., 2007).

1.5.6 Effects of GIP on the Brain GIP and the receptor have been found in the brain (Nyberg et al., 2005; Usdin et al., 1993). GIP expression was identified in multiple sites in the brain with immunoreactivity found exclusively in neurons, and it was proposed that GIP could function as a neurotransmitter (Nyberg et al., 2007). Although no distinct functional roles for GIP in the brain have been

identified, intracerebroventricular administration of GIP to rats induced proliferation of hippocampal neurons, whereas proliferation was reduced in GIPR-/- mice (Nyberg et al., 2005). Administration of GIP was also found to enhance exploratory behavior and improve motor function performance in mice (Ding et al., 2006). Clearly more studies are needed to clarify the role of GIP in the brain. Expression of GLP-1 and the GLP-1R has also been identified in the brain (Jin et al., 1988). The brainstem contains the highest concentration of GLP-1 expressing neurons, with cell bodies localized in the nucleus tractus solitarius (NTS) and reticular nucleus of the medulla (Huo et al., 2009; Jin et al., 1988). Nerve fibres expressing GLP-1 innervate numerous brain sites, particularly the hypothalamus. Many components of the complex circuitry mediating responses to NTS-derived GLP-1 are associated with regulation of feeding, and intracerebroventricular administration of GLP-1 in rats was shown to reduce appetite and food intake (Tang-Christensen et al., 1996; Turton et al., 1996). Furthermore, subcutaneous administration of a GLP-1R agonist was found to reduce food intake (DeFronzo et al., 2008) and intravenous or subcutaneous injections of GLP-1 resulted in profound reductions in appetite and caloric intake in humans (Flint et al., 1998; Verdich et al., 2001). Vagal reflex circuits, including the CNS, are now considered to also play major roles in GLP-1 mediated stimulation of insulin secretion and glucose homeostasis as well as in the inhibition of gastric emptying (Holst, 2007). Similar roles for GIP have not been identified.

1.6 Type 2 Diabetes Mellitus and a Therapeutic Role for GIP 1.6.1 Significance to Society and Natural History Diabetes mellitus (DM) is a metabolic disorder characterized by chronically elevated blood glucose. This results in increased production of reactive oxygen species in tissues such as the retina, mesangial cells and Schwann cells, promoting blindness, renal disease and peripheral neuropathy (Brownlee, 2005). Additionally, DM is commonly linked to insulin resistance, a major contributor to cardiovascular disease. With the prevalence of DM projected to surpass 320 million people by 2025 (Zimmet et al., 2003), significant attention has focused on its pathogenesis and treatment in order to ameliorate forthcoming health and economic burdens. Of all diabetes cases, the vast majority have type 2 diabetes mellitus (T2D), a form typically identified by the development of obesity-associated insulin resistance and β-cell dysfunction. Although T2D was once thought of as a disease acquired in middle-aged and older

adults, there has recently been a large rise in incidence in school-aged children, and this seems in large part reflective of the growing obesity epidemic (Permutt et al., 2005). However, there is also an underlying polygenic heritability that is critical for susceptibility to diabetes (Doria et al., 2008; Permutt et al., 2005). Thus the pathophysiological basis of T2D derives from the product of gene-environment interactions. The natural history of T2D was examined in large cohorts by the United Kingdom Prospective Diabetes Study (UKPDS, 1998) and Diabetes Prevention Program (Knowler et al., 2002). These studies demonstrated that T2D develops because of gradual deteriorations in glycaemic control, improvements in which reduce or prevent disease complications. Furthermore, during the progression of T2D the degree of insulin resistance was found to remain relatively stable, whereas β-cell function exhibited continuous declines, thus accounting for the loss of glycaemic control. Therefore, while insulin resistance is a contributing factor, β-cell dysfunction is essential for the development of T2D.

1.6.2 Role of Insulin Resistance Insulin plays a critical role in glucose metabolism (Taniguchi et al., 2006). Circulating insulin binds its membrane-bound receptor, a tetrameric protein consisting of α- and β-subunits and member of a subfamily of receptor tyrosine kinases as is the insulin-like growth factor (IGF)-I receptor (Saltiel and Kahn, 2001). The α-subunits allosterically inhibit β-subunits, but upon insulin binding, β-subunits are derepressed and transphosphorylate, further increasing their kinase activity. The key metabolic actions of insulin, as well as IGF-I, are coupled through insulin receptor substrate (IRS) proteins. IRS-1 is essential for promoting glucose-uptake in muscle, lipogenesis in adipocytes and inhibiting gluconeogenesis in liver, whereas IRS-2 is essential for β-cell growth and function (Taniguchi et al., 2006). The N-terminus of IRS proteins contain phosphotyrosine binding domains (PTB), enabling interactions with insulin receptors (IR) as well as IGF-I receptors (IGF-IR). These receptors phosphorylate IRS proteins at tyrosine residues in its mid-region, enabling binding by intracellular proteins through src homology (SH) domains, and one such protein critical for insulin‟s metabolic actions is phosphatidylinositol 3- kinase (PI3K). Conversely, serine phosphorylation of IRS proteins in the PTB domain by kinases such as jun N-terminal kinase (JNK) uncouples these interactions. Overall, these different IRS protein states play critical roles in the metabolic effects of insulin and can be greatly influenced by factors such as age, diet, fitness and genetics (Kahn, 2003; Taniguchi et al., 2006).

Insulin resistance is defined as a state of decreased responsiveness to its glucose lowering effects. It is a common feature of obesity that consistently precedes and strongly predicts the development of T2D (Lillioja et al., 1993; Warram et al., 1990). The important role of insulin resistance in T2D has been supported by the finding that therapies improving insulin sensitivity reduce the onset of T2D in humans (Gerstein et al., 2006; Savage et al., 2007). Skeletal muscle and liver are key gluco-regulatory tissues that demonstrate severe insulin resistance in patients with T2D. Resistance in skeletal muscle accounts for ~90% of the impairment in glucose disposal (DeFronzo et al., 1979; Shulman et al., 1990), whereas in liver, resistance causes elevated glucose production and thus fasting hyperglycaemia (Defronzo, 2009). Studies by Bergman and coworkers showed that insulin resistance can result from delayed interstitial insulin dispersion, especially in obesity (Bergman, 2007; Kolka et al., 2010), but the majority of evidence indicates that the key defect occurs downstream of the insulin receptor (Defronzo, 2009; Schenk et al., 2008). Studies by Shulman and coworkers showed that insulin resistance in patients with T2D results from defects in insulin-mediated plasma membrane trafficking of GLUT4 in skeletal muscle (Cline et al., 1999; Rothman et al., 1992) and suppression of gluconeogenesis in liver (Magnusson et al., 1992). Supporting evidence is found in the responses to the insulin sensitizers, thiazolidinediones (TZDs) and metformin, since they improve insulin‟s effects on skeletal muscle GLUT4 and liver gluconeogenesis (Defronzo, 2009; Petersen and Shulman, 2006). Extensive evidence has linked the origins of skeletal muscle and liver insulin resistance to derangements in adipocyte function and metabolism. In obesity, adipocytes are susceptible to hypoxic and endoplasmic reticulum (ER) stress, which causes inflammation and insulin resistance in adipose tissue, ultimately resulting in chronic low-grade systemic inflammation, elevated free fatty acid (FFA) flux, and ectopic lipid deposition in tissues such as liver and muscle. Among the effects on skeletal muscle and liver are the activation of pro-inflammatory and nutrient sensing protein-serine/threonine kinase modules such as JNK, double stranded RNA dependent protein kinase (PKR), IĸB kinase (IKK), mammalian target of rapamycin (mTOR) and atypical protein kinase C (aPKC), which promote insulin resistance via uncoupling signalling through IRS-1. Discussion of the complex series of events underlying insulin resistance is beyond the scope of the current thesis, but has been comprehensively reviewed (Hotamisligil, 2006; Hotamisligil and Erbay, 2008; Kusminski et al., 2009; Nakamura et al., 2010; Savage et al., 2007; Schenk et al., 2008; Shoelson et al., 2006; Taubes, 2009; Unger,

2003). Interestingly, TZDs decrease skeletal muscle and liver insulin resistance by reversing adipocyte derangements and increasing adipose storage capacity (Defronzo, 2009; Leahy, 2009; Sugii et al., 2009). Moreover, skeletal muscle and liver insulin resistance were reduced in obese mice with enhanced adipose tissue expansion (Kim et al., 2007a) or receiving fat transplants (Tran et al., 2008). Conversely, severe insulin resistance results from lipoatrophy in humans and mice (Pajvani et al., 2005; Savage et al., 2007). Thus, obesity in itself does not cause insulin resistance, but instead there appears to be a natural limitation to adipose storage capacity which, when surpassed, stresses adipocytes and causes insulin resistance.

1.6.3 Role of the β-cell Impairments in glycaemic control resulting from insulin resistance escalate when there is a coexisting decline in β-cell function. In patients with T2D, β-cell dysfunction is manifest in several ways including decreased insulin responses to oral and intravenous glucose and other secretagogues (Halter and Porte, 1978; Perley and Kipnis, 1967; Varsano-Aharon et al., 1970; Ward et al., 1984), altered pulsatile insulin secretion (O'Rahilly et al., 1988; Porksen, 2002) and inefficient proinsulin processing (Kahn and Halban, 1997; Ward et al., 1987). Although β-cell dysfunction was once thought to occur late in the natural history of T2D, as a result of deeper understanding of β-cell function, this viewpoint has been challenged. Insulin secretion has long been known to be modulated by circulating glucose and a range of secretagogues, but recently it has become clear that secretion is also tightly linked to insulin sensitivity, such that insulin resistant individuals maintain euglycaemia through proportional increases in insulin secretion (Kahn, 2003). As such, β-cell function can be expressed as the incremental rise in circulating insulin in response to intravenously infused glucose, divided by the degree of insulin resistance (Defronzo, 2009). Similarly, the product of insulin sensitivity and the acute insulin response to intravenous glucose is described as the disposition index (Bergman et al., 1981). Using assessments of β-cell function and the disposition index, cross-sectional and longitudinal studies have clearly shown that β-cell dysfunction commonly precedes and strongly predicts T2D and worsens in correlation with elevations in glycaemia (Kahn, 2003). A major concern regarding the nature of β-cell dysfunction has been the relative contributions of diminished β-cell mass and β-cell secretory dysfunction. Post-mortem studies have shown that pancreatic β-cell mass and/or volume is significantly reduced in individuals with impaired glucose tolerance (IGT) and even more so in patients with T2D (Butler et al.,

2003a; Rahier et al., 2008; Sakuraba et al., 2002; Yoon et al., 2003). The underlying mechanism responsible in humans (Butler et al., 2003a) and Zucker diabetic fatty (ZDF) rats (Finegood et al., 2001) is a large increase in β-cell apoptosis. Conversely, secretion studies show that acute (first phase) insulin responses to intravenous glucose infusions decline in correlation with elevating fasting glycaemia and that responses can be almost absent in those having glycaemic values that are still below „clinical diabetes‟ (Brunzell et al., 1976). Moreover, individuals with a high risk for developing T2D exhibit disturbed insulin pulsatility, oscillatory patterns and proinsulin processing (Kahn et al., 1995; Mykkanen et al., 1995; O'Meara et al., 1993; O'Rahilly et al., 1988). Thus, diminished β-cell mass and secretory dysfunctions are present early in the pathogenesis of T2D, and while it is unclear whether either is more important, based on studies in humans and various animal models, it has recently been argued that neither defect alone is sufficient and their concerted contribution is required for T2D progression (Kahn et al., 2009). An intriguing possibility is that β-cell dysfunction may share common origins with skeletal muscle and liver insulin resistance. There is extensive evidence that chronic exposure to elevated glucose, FFA and/or inflammatory cytokines promotes defects in insulin production and secretion as well as β-cell apoptosis. The underlying mechanisms involve β-cell mitochondrial and ER stress, resulting from elevated protein synthesis, increases in reactive oxygen species, production of islet amyloid polypeptide toxic fibrils and increased stress signalling via JNK, p38 mitogen activated protein kinase (p38 MAPK) and nuclear factor ĸB (NFĸB). For comprehensive discussions, refer to: (Donath et al., 2010; Donath et al., 2008; Eizirik et al., 2008; Evans et al., 2002; Haataja et al., 2008; Muoio and Newgard, 2008; Poitout and Robertson, 2008; Scheuner and Kaufman, 2008). Supporting clinical evidence that reductions in circulating glucose, FFA and cytokines improve β-cell function has been found in T2D patients treated with TZDs (Gastaldelli et al., 2007) or with an interleukin 1 receptor antagonist (Larsen et al., 2007). Thus, as with insulin resistance, defects in adipocyte function and metabolism appear to also contribute to β-cell dysfunction in T2D. Collectively, the evidence reveals that the common natural history of β-cell dysfunction in T2D ensues as follows (Poitout and Robertson, 2008; Rhodes, 2005). In obese individuals, normoglycaemia is maintained via compensatory increases in β-cell mass, insulin biosynthesis and insulin secretion, but in those with genetic or acquired predispositions, compensation is insufficient because of elevations in β-cell apoptosis and secretory dysfunction. Initially, this manifests as transient episodes of post-prandial hyperglycaemia, which combines with the FFA

and inflammatory milieu to further exacerbate the hostile environment for β-cells, ultimately causing the continuous declines in β-cell function that occurs in the pathogenesis of T2D. As such, increasing insulin biosynthesis and secretion and preventing β-cell apoptosis should improve β-cell function and glycaemic control in T2D patients.

1.6.4 The Incretin Axis and GIP in Type 2 Diabetes Incretins modulate β-cell function by enhancing post-prandial insulin secretion. However, T2D patients exhibit insulin profiles indicative of a defective incretin axis (Nauck et al., 1986a), resulting from a reduction in GLP-1 secretion, as well as mild β-cell resistance to the insulinotropic effects of GLP-1 and marked β-cell resistance to the insulinotropic effects of GIP (Meier and Nauck, 2004, 2008; Nauck et al., 1993). Although the basis for reduced GLP-1 secretion is currently unknown, mechanisms underlying defective insulin responses to GIP are beginning to be unveiled in studies on humans as well as cultured β-cells and rodent models. The disturbed incretin axis in T2D probably involves genetic components. Single nucleotide polymorphisms (SNP) in the transcription factor-7 like 2 (TCF7L2) gene are highly associated with T2D and the SNP variant rs7903146 is associated with reduced β-cell responsiveness to incretins (Lyssenko et al., 2007; Villareal et al., 2010). Moreover, a recent study showed that TCF7L2 protein is reduced in β-cells of T2D patients and that this appears to contribute to β-cell dysfunction by promoting a reduction in both GIPR and GLP-1R expression and signalling (Shu et al., 2009). Further evidence implicating a genetic component to the defective incretin axis in at least some T2D patients includes reports that insulin responses to GIP are reduced in ~50% of normoglycaemic first degree relatives of T2D patients (Meier et al., 2001), and the recent identification of a SNP in the GIPR gene that is associated with diminished glucose tolerance, reductions in insulin responses and a diminished incretin effect (Saxena et al., 2010). In a subsequent study involving more in depth physiological analyses, this same GIPR variant as well as the TCF7L2 variant were found to associate with elevations in proinsulin levels with respect to the insulinogenic index (Ingelsson et al., 2010), a finding interpreted as indicating either reduced β-cell mass, an impairment in proinsulin processing or defective insulin vesicle trafficking. Aside from genetic predispositions, acquired factors also contribute to defective incretin responses. Stimulation of the GIPR in β-cells promotes homologous desensitization, which could be an important component for GIP resistance in people exhibiting elevated GIP secretion

(McIntosh et al., 2009). However, probably the most significant contributing factor was revealed with the discovery that expression of the GIPR is greatly reduced in the Vancouver diabetic fatty (VDF) rat (Lynn et al., 2001). Subsequently it was shown that normalization of glycaemia in diabetic rats (Piteau et al., 2007; Xu et al., 2007) or patients with T2D (Hojberg et al., 2009; Meneilly et al., 1993) improved the insulinotropic actions of GIP, demonstrating that chronic hyperglycaemia contributes significantly to GIP resistance. The underlying mechanism has been attributed to decreases in GIPR expression (Lynn et al., 2001; McIntosh et al., 2009; Piteau et al., 2007; Xu et al., 2007), but it has also been proposed that hyperglycaemia elevates ubiquitination and subsequent degradation of GIPRs in islets (Zhou et al., 2007). Studies with INS-1 β-cells indicated that elevated glucose diminishes GIPR expression indirectly through down-regulation of the transcriptional activator peroxisome-proliferator activated receptor (PPAR) α (Lynn et al., 2003; Roduit et al., 2000), whereas a more recent study identified a role for PPARγ (Gupta et al., 2010). This latter finding may have significant clinical importance, since PPARγ is the major target of the TZD class of anti-diabetic drugs. Although hyperglycaemia clearly plays a role in reduced GIP responsiveness in humans, there are still factors that need clarification. In glucose-clamp studies on healthy individuals and T2D patients (Vilsboll et al., 2002), the first-phase insulin response to an intravenous bolus of GIP was found to be similar, but the late-phase insulin response was markedly diminished in those with T2D. The authors reasoned that GIP resistance in humans with T2D does not result from diminished receptor expression but instead a post-receptor defect in the signalling mechanism by which GIP promotes insulin release. Supporting evidence was recently found in a similarly designed 4-day cross-over clamp study (Aaboe et al., 2009) in which patients received an intravenous bolus of saline, GIP, the sulfonylurea glipizide which is a blocker of ATP- sensitive potassium (KATP) channels or both. Remarkably, the dose of GIP and glipizide used stimulated relatively mild and similar insulin responses when administered alone, but when used in combination the insulin response was more than two-fold greater than would be expected from the combined individual responses, demonstrating that KATP channel closure ameliorates the impaired insulinotropic effect of GIP in patients with T2D.

1.6.5. GIP and Type 2 Diabetes Therapeutics There is now widespread use of incretin-based therapeutics for patients with T2D, the main impetus being that they exert pleiotropic actions on glycaemic control. In contrast to the

commonly prescribed sulfonylureas which act by pharmacologically stimulating insulin release, incretin-based therapies act, at least in part, by „boosting‟ a natural system and counter-acting the underlying pathophysiology of T2D. Since the insulinotropic effects of GIP were found to be reduced in patients with T2D, whereas the effects of GLP-1 remained largely preserved, most attention has focused on developing GLP-1 based therapies. There are currently two marketed GLP-1R agonists. Exendin-4 is a peptide with potent stimulatory effects on the GLP-1R that was isolated from venom of the lizard, Heloderma suspectum (Eng et al., 1992), and it is marketed as exenatide (ByettaTM) by Amylin Pharmaceuticals and Eli Lilly and Company. It is resistant to DPP-IV degradation with a circulating half-life of 60-90 min and shares ~50% homology with GLP-1 (Drucker and Nauck, 2006). Liraglutide (Vicotoza) is a long-acting human GLP-1 analogue developed by Novo Nordisk that contains an Arg34Lys substitution and a 16 carbon free fatty acid moiety attached to Lys26 (Knudsen et al., 2000). Extensive pre-clinical and clinical studies have demonstrated that administering Byetta or Liraglutide to T2D patients improves glycaemic control, enhances GSIS and β-cell function, reduces glucagon secretion and gastric emptying, suppresses appetite and promotes weight loss (Amori et al., 2007; Drucker and Nauck, 2006; Nauck and Meier, 2005). These agents also increase β-cell mass in rodent models of diabetes but it is too early to determine if a similar effect occurs in humans. However, these peptides require frequent injection and commonly induce nausea, likely due to effects on gastric emptying. More recently, chronic use of incretin mimetics was linked to higher incidences of pancreatitis and the possible promotion of pancreatic cancer (Butler et al., 2010), and in the future, these endpoints will clearly be closely monitored in patients treated with GLP-1R agonists. An alternative approach in incretin-based therapies is to increase the biological half-lives of the secreted incretins with DPP-IV inhibitors and this class of therapeutics is also in wide use. Sitagliptin was developed by Merck and Vildagliptin by Novartis. As with GLP-1R agonists, extensive pre-clinical and clinical studies have shown that these agents also improve glycaemic control, enhance GSIS and β-cell function and suppress glucagon secretion, but they have milder effects on gastric emptying and no effect on appetite and weight loss (Amori et al., 2007; Drucker and Nauck, 2006). However, these agents also increase β-cell mass in rodent models of diabetes (Drucker and Nauck, 2006; McIntosh, 2008; Mu et al., 2006), but effects in humans remain uncertain. Importantly, a major benefit of DPP-IV inhibitors is that they are orally administered and significant side-effects have not been reported to date (Amori et al., 2007;

Drucker and Nauck, 2006), although concern has been raised that these agents could also promote pancreatitis via enhancing GLP-1 actions (Butler et al., 2010). Since levels of biologically active GIP are also elevated with DPP-IV inhibitors, and active GIP exerts similar effects on β-cells as GLP-1, it is likely that GIP contributes to the effects of DPP-IV inhibitors on β-cell function in T2D, though this has been generally unstudied due to the reductions in insulinotropic effects commonly found in patients. However, as previously discussed, other actions of GIP on β-cells may still remain functional and the resistance declines as glycaemia improves. Responsiveness to GIP would therefore likely be enhanced with prolonged use of DPP-IV inhibitors in patients with T2D. In regard to GIP-based therapies, roles have been suggested for both GIPR agonists and antagonists (Green and Flatt, 2007; Irwin and Flatt, 2009). Administering pharmacological levels of GIP to „GIP-resistant‟ rodents exerts significant insulinotropic effects (Hinke et al., 2002), and several approaches have been taken to develop GIP analogues that are resistant to DPP-IV removal of the Tyr1-Ala2 dipeptide such as chemical modifications to the N-terminus with N- glucitol-, N-acetyl-, or N-pyroglutamyl-GIP (Green et al., 2004; O'Harte et al., 2002; O'Harte et al., 1999), substitution of amino acid position 2 with D-alanine (D-Ala2-GIP), phosphorylated serine or glycine, as well as acetylation with long-chain or short-chain fatty acids or attaching palmitate residues to intrinsic lysine residues at position 16 or 37 (Green et al., 2004; Irwin et al., 2005). Peptides containing linkers that enhance helix formation in the C-terminal (19-30) portion of GIP have also been tested (Manhart et al., 2003). Though studies have not been performed in humans, several analogues have demonstrated prolonged biological activity, strong insulinotropic actions and improvements in glycaemic excursions in glucose tolerance tests in rodent models of T2D (Green and Flatt, 2007; Hinke et al., 2002; Irwin and Flatt, 2009). In contrast, since mice lacking functional GIPRs were resistant to obesity (Miyawaki et al., 2002), approaches have been taken to develop GIP antagonists, and substitution of amino acid position 3 with proline (Pro3-GIP) has been found, in some hands, to act as a GIP antagonist and reduce insulin secretion to a glucose load in obese mice (Gault et al., 2002; Gault et al., 2003). Intraperitoneal administration for 11 days impaired glucose tolerance and insulin sensitivity in normal mice (Irwin et al., 2004), but a similar treatment in obese mice improved glucose handling and reduced insulin resistance and the onset of diabetes (Gault et al., 2005; Irwin et al., 2007). For unknown reasons, extending the half-life of Pro3-GIP by acylation of an internal lysine did not improve its efficacy (Gault et al., 2007).

Although these results are intriguing, translation to humans is uncertain since the roles of GIP in human adipocyte metabolism remains to be defined and inhibition of the GIPR in all body sites could have significant deleterious effects on tissues such as β-cells, bone and the central nervous system (Section 1.5). Indeed, it remains possible that enhancing storage of triglycerides in adipose tissue may result in overall improvements in insulin sensitivity as well as β-cell function (Section 1.6.2 & 1.6.3). It also remains uncertain whether GIP‟s actions could promote significant therapeutic improvements to β-cells in T2D. It is therefore important to understand the mechanisms of GIP actions in β-cells in order to define potential responses to GIP therapy.

1.7 Mechanisms of GIP Action in the β-cell 1.7.1 Regulation of Insulin Secretion Incretins have been proposed to play a critical role in modulating the insulin secretory competence of β-cells in order to prepare them for forthcoming meals (Hinke et al., 2004a). Incretin-secreting cells are known to sense forthcoming nutrient loads in the gut, correspondingly secreting their „messengers‟ into the circulation, which then „inform‟ β-cells of the insulin levels that will be required to maintain circulating glucose within strict levels. An additionally intriguing feature of incretins is the requirement for elevated glucose levels to stimulate insulin secretion (Drucker, 2006; Kim and Egan, 2008; McIntosh et al., 2009), and extensive investigations have focused on the molecular mechanisms of these actions. Glucose is the primary regulator of insulin secretion (MacDonald et al., 2005; Remedi and Nichols, 2009). Circulating glucose rapidly equilibrates across the β-cell plasma membrane due to high capacity, low affinity glucose transporter proteins, with GLUT1 in humans and GLUT2 in rodents being the major type (De Vos et al., 1995). Glucose is then phosphorylated by glucokinase, considered to be the β-cell „glucose sensor‟ (Matschinsky, 1996), thus shunting glucose into glycolysis and mitochondrial oxidative metabolism. The increased ATP/ADP ratio closes ATP-sensitive potassium (KATP) channels, resulting in membrane depolarization and opening of L-type voltage-dependent calcium (Ca2+) channels and Ca2+ influx (Ashcroft and Rorsman, 2004; Hinke et al., 2004a; Hiriart and Aguilar-Bryan, 2008). Elevated intracellular Ca2+ interacts with insulin granules and the exocytotic machinery, causing insulin secretion. 2+ Membrane repolarization is mediated by voltage-dependent (KV) and Ca -sensitive potassium channels (Hiriart and Aguilar-Bryan, 2008; MacDonald and Wheeler, 2003), which shuts off

Ca2+ entry and insulin secretion. It has become apparent that incretins modulate insulin release through acting at multiple levels within the glucose-induced pathway. Activation of the GIPR and GLP-1R in β-cell lines, cell-lines transfected with GIPR or GLP-1R cDNA, cultured islets and FACS purified β-cells results in stimulation of adenylate cyclase (AC) through Gαs coupling and the production of cAMP (Amiranoff et al., 1984; Baggio and Drucker, 2007; Ehses et al., 2001; Holz et al., 2008; Lu et al., 1993; Moens et al., 1996), and this is essential for incretins to potentiate GSIS. Multiple types of AC are expressed in β-cells (Leech et al., 1999) but interestingly the Type VIII AC, which is dually activated by Ca2+- calmodulin and Gαs, was shown in β-cells to act as a „coincidence detector‟ for signals deriving from glucose and GLP-1 (Delmeire et al., 2003). The importance of such a dual signalling network is evident from studies on the „glucose competence‟ of β-cells, from which it was suggested (Pipeleers, 1987) that purified β-cells exhibit poor glucose-responsiveness due to the necessity for synergistic interaction with cAMP that normally results from glucagon secreted from α-cells and nutrient induced messengers, such as incretins. Supporting evidence was found in electrophysiological studies in which treatment with GLP-1 rendered competence to isolated β-cells that were previously unresponsive to glucose (Holz et al., 1993). Glucagon has been found to exhibit a similar effect with human islets (Huypens et al., 2000), and so it is likely that, in vivo, the combined effects of glucagon, GLP-1 and GIP modulate β-cell glucose responsiveness. In studies using real-time measurements on β-cells, synergistic interactions 2+ between GLP-1/Gαs and Ca -calmodulin promoted synchronous, in-phase, oscillations in intracellular levels of cAMP and Ca2+ (Fridlyand et al., 2007). The oscillations were thought to involve coordinated interactions with Ca2+-calmodulin activated phosphodiesterase (PDE) 1C (Holz et al., 2008), but a role for PDE 3B has also been suggested (Doyle and Egan, 2007). Incretin mediated production of cAMP promotes insulin secretion through mechanisms involving activation of protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) 2 (Ding and Gromada, 1997; Holz, 2004; Holz et al., 2006; Kashima et al., 2001; Seino and Shibasaki, 2005). Whether GIPR and GLP-1R localize in microdomains of the β-cell is not yet known, but β-arrestin-1 and scaffolding proteins such as A-kinase anchoring protein (AKAP)-18 and 79/150 may have regulatory roles (Fraser et al., 1998; Lester et al., 2001; Sonoda et al., 2008).

The KATP channel is critical for coupling glucose metabolism to insulin secretion and

GLP-1 and GIP have been shown to inhibit KATP channel activity in a glucose-dependent manner (Gromada et al., 1998). In electrophysiological studies on INS-1 β-cells, GLP-1 was found to

inhibit the KATP channel by promoting PKA-dependent phosphorylation of Ser-1448 in the SUR1 subunit (Light et al., 2002). Conversely, EPAC2 interacts with the nucleotide-binding fold-1 of

SUR1, and this has been shown to sensitize the KATP channel to inhibition by ATP (Holz et al., 2006; Kang et al., 2006; Kang et al., 2008b). Whether GIP has similar actions or EPAC1 is also involved is unknown. Furthermore, GLP-1 has also been reported to stimulate ATP production through increasing ER Ca2+ release (Rutter et al., 2006; Tsuboi et al., 2003), which would likely also contribute to inhibiting the KATP channel. However, a similar effect was not observed in rodent islets (Peyot et al., 2009). Reconciling this discrepancy will be of interest. Modulation of β-cell Ca2+ flux by incretins involves uptake and release from intracellular stores (Doyle and Egan, 2007; Gromada et al., 1998). Both GLP-1 and GIP increase Ca2+ influx through CaV1.3 channels (Gromada et al., 1998; Lu et al., 1993; Wheeler et al., 1995), and at least with GLP-1, this has been shown to involve PKA and PKC (Jacobo et al., 2009). Incretins also act on non-selective ion channels and stimulate Ca2+ release from intracellular stores (Gromada et al., 1998; Lu et al., 1993; Wheeler et al., 1995). Though complex, the mechanisms for increasing Ca2+ release from intracellular stores involves interactions with release channels such as the ryanodine and inositol 1,4,5-trisphosphate receptors, likely through EPAC2- and PKA-dependent actions (Gromada et al., 1998; Holz et al., 2006; Kang et al., 2005; Tsuboi et al., 2003).

Repolarization of the β-cell involves KV channels. Numerous subtypes are expressed in β-cells and the two major classes identified are the delayed rectifier and A-type currents (MacDonald et al., 2001; MacDonald et al., 2002b; MacDonald and Wheeler, 2003). Both GIP and GLP-1 have been shown to reduce KV channel currents, resulting in more prolonged β-cell action potentials and thus potentiation of Ca2+ signals on insulin release. Through a mechanism involving PKA and PI3K / Protein kinase Cδ (PKCδ), GLP-1 has been shown to reduce delayed rectifier channels, and Kv2.1 plays a dominant role (MacDonald et al., 2001; MacDonald et al., 2002a; MacDonald et al., 2002b; MacDonald and Wheeler, 2003). GIP has also been found to inhibit delayed rectifier currents in INS-1 cells and human β-cells (Kim, Choi, and McIntosh; unpublished observations) as well as reduce A-type currents by increasing endocytosis of Kv1.4 channels through PKA-dependent actions (Kim et al., 2005a). In addition to actions on membrane depolarization, incretins also exert distal effects on exocytosis in β-cells and this, again, involves PKA- and EPAC2- dependent pathways (Ding and Gromada, 1997; Holz et al., 2006; Seino and Shibasaki, 2005). PKA phosphorylates proteins

associated with the exocytotic machinery, whereas two models have been proposed for EPAC2 in which it either increases the probability of readily releasable pools of insulin to undergo exocytosis or interacts with the SUR1 subunit located in the plasma membrane and secretory granules (Eliasson et al., 2003; Holz et al., 2006; Seino and Shibasaki, 2005). In the latter model, EPAC2 activates secretory granule chloride channels, resulting in granule acidification and priming via H+-ATPases (Eliasson et al., 2003). In the former model, an EPAC2–Rim2 complex plays a central role by promoting Rab3A activity, a core component of the exocytotic system (Ding and Gromada, 1997; Holz et al., 2006). Additional signalling pathways have been found that contribute to incretin-mediated stimulation of insulin secretion. GLP-1 has been shown to promote activation of ADP ribosyl cyclase/CD38, resulting in elevated production of adenine dinucleotide phosphate and cyclic ADP-ribose, and this was reportedly essential for the induction of insulin secretion in mouse islets (Kim et al., 2008a). Conversely, GIP has been shown to potentiate insulin secretion by activating the group VIA phospholipase A2 (iPLA2), causing production of arachidonic acid 2+ (Ehses et al., 2001). Activating iPLA2 has been shown to promote Ca entry by increasing the rate of Kv2.1 inactivation (Jacobson et al., 2007), indicating that GIP-activation of iPLA2 may be linked to Kv channel regulation. A physiological role for the iPLA2 pathway has been supported by the findings that inhibition (Song et al., 2005) or siRNA suppression (Bao et al., 2006a) of iPLA2 in INS-1 β-cells reduces insulin secretion and iPLA2 knockout mice exhibit greater impairments in glucose tolerance than wild type mice on a high fat diet (Bao et al., 2006b).

1.7.2 Regulation of Insulin Biosynthesis Insulin production is a tightly regulated process in β-cells. At maximal capacity, preproinsulin mRNA accounts for ~20% of total β-cell mRNA with proinsulin translation accounting for 30-50% of the total protein synthesis load and generating ~1 million proinsulin molecules per min (Scheuner and Kaufman, 2008; Schuit et al., 1988; Van Lommel et al., 2006). As with insulin secretion, glucose is a primary regulator of insulin biosynthesis. Studies on β-cell cultures showed that when glucose is increased from 3 mM to 16.7 mM there is a brief lag period, after which translation of pre-existing mRNA increases >10-fold in correlation with translation of insulin processing enzymes, and this is followed several hours later by increased preproinsulin transcription (Uchizono et al., 2007; Wicksteed et al., 2003). Glucose metabolism is required for these effects but the mechanism is distinct from that of insulin secretion, since

2+ blocking Ca influx prevents glucose-induced insulin secretion but not biosynthesis, and KATP channel blockers promote insulin secretion but not production (Uchizono et al., 2007). The effect of glucose requires cis-elements in the preproinsulin mRNA untranslated region (UTR), with the 3ʹ UTR critical for mRNA stability and the 5ʹ UTR for the dynamic rates in translation, due to a highly conserved and unique preproinsulin response element, GGUCAUUGU (Uchizono et al., 2007; Wicksteed et al., 2001; Wicksteed et al., 2007). However, the trans-acting protein factors remain unknown. Insulin mRNA and peptide levels in GIPR-/- mice are reduced ~30-40% compared to normal mice (Hansotia et al., 2007; Pamir et al., 2003), indicating that GIP is involved in maintaining insulin stores. Studies in β-cell lines have shown that GIP enhances insulin mRNA and peptide levels as well as those for glucokinase and GLUT1 (Fehmann and Goke, 1995; Wang et al., 1996b). GLP-1R signalling has been shown to increase insulin gene transcription, stabilize preproinsulin mRNA and increase its translation, and the effects on gene expression appear to involve modulation of cAMP response element binding protein (CREB) and nuclear factor of activated T-cells (Chepurny et al., 2002; Drucker, 2007b; Drucker et al., 1987; Lawrence et al., 2002; Skoglund et al., 2000). However, in a recent study it was found that GLP- 1R signalling enhances insulin production predominately by potentiating glucose-induced proinsulin biosynthesis at the translational level (Alarcon et al., 2006). The effects of GIP on the insulin 1 promoter were found to involve a PKA / CREB family member pathway (Ehses, 2003), but further studies are needed to establish the mechanism by which GIP, as well as GLP-1, modulates insulin production.

1.7.3 Regulation of β-cell Mass The plasticity of β-cell mass is essential for maintaining glucose homeostasis (Hinke et al., 2004a). An excess could promote hypoglycaemia and lead to brain dysfunction, whereas a deficiency causes hyperglycaemia (Butler et al., 2007). The development of insulin resistance results in adaptive responses in β-cell mass to maintain euglycaemia, but due to genetic or acquired predispositions, responses are deficient in those who develop T2D (Rhodes, 2005). These findings have significant therapeutic implications and have resulted in extensive investigations in the natural regulation of β-cell mass and in identifying regulatory factors that restore or preserve β-cell mass in patients with T2D.

In adults, β-cell mass is determined by β-cell size and the rates of replication and neogenesis minus the rate of apoptosis. The contribution of each is likely variable and circumstantial and possibly also species specific. For example, studies in humans have implicated a role for neogenesis and replication in increasing β-cell number (Butler et al., 2003a), but there is strong evidence that only replication is important in adult mice (Dor et al., 2004). Nevertheless, studies in patients with T2D (Butler et al., 2003a) as well as mouse and rat models of T2D (Butler et al., 2003b; Finegood et al., 2001) have shown that β-cell replication is elevated when compared to lean euglycaemic controls, but this is significantly outstripped by a markedly elevated rate in β-cell apoptosis. Apoptosis therefore appears to be the most significant contributing factor to reduced β-cell mass in the pathogenesis of T2D. A number of factors are capable of regulating β-cell mass including prolactin, growth hormone (Lingohr et al., 2002a) and neural relays emanating from the liver (Imai et al., 2008). However, though caution is warranted in extrapolating to humans, a series of studies using β-cell lines, cultured islets and genetic mouse models have revealed that the IRS-2 / PI3K / Akt (protein kinase B) signalling module is a critical node for the natural compensatory expansion in β-cell mass that occurs in the presence of obesity and insulin resistance (Bernal-Mizrachi et al., 2001; Dickson and Rhodes, 2004; Elghazi et al., 2007; Hennige et al., 2003; Lingohr et al., 2003; Rhodes, 2005; Tuttle et al., 2001; Withers et al., 1998). An intriguing evidence-based model (Weir and Bonner-Weir, 2007) has proposed that the expansion of β-cells occurs via the coordinated effects of increased glucose metabolism and cAMP-elevating factors, such as incretins, resulting in increased Ser-133 phosphorylation of CREB (CREB pS133) and, consequently, promotion of CREB-mediated IRS-2 production. Ultimately, elevating IRS-2 amplifies insulin, IGF-I and IGF-II signalling through their receptors, resulting in the promotion of β-cell replication and survival via kinases such as Akt (Jhala et al., 2003; Lingohr et al., 2006; Lingohr et al., 2002a; Lingohr et al., 2002b; Park et al., 2006; Terauchi et al., 2007; Weir and Bonner-Weir, 2007; Withers et al., 1999). In T2D however, chronic exposure of β-cells to hyperglycaemia, hyperlipidaemia, and inflammatory cytokines promotes serine phosphorylation of IRS-2 by kinases such as p38 MAPK, JNK and IKK, which causes IRS-2 degradation and ultimately enhances β-cell susceptibility to stress-induced apoptosis (Chang-Chen et al., 2008; Rhodes, 2005). Overall, these findings reveal that factors promoting IRS-2 and Akt activity should improve β-cell mass in patients with T2D.

Incretins promote β-cell proliferation and survival (Drucker, 2006; Kim and Egan, 2008; Salehi et al., 2008), although these findings are mainly based on studies focusing on GLP-1. Activating GLP-1R signalling has been shown in several rodent models of diabetes to improve β-cell mass by increasing β-cell proliferation and decreasing β-cell apoptosis (Farilla et al., 2002; Li et al., 2003; Wang and Brubaker, 2002; Xu et al., 1999). In studies on β-cell lines and cultured islets, the proliferative effects of GLP-1 on cells under growth conditions as well as the pro-survival effects of GLP-1 on cells exposed to high glucose and palmitate (glucolipotoxicity), mitochondrial stress or pro-inflammatory cytokines revealed that this occurs in an Akt-dependent manner (Buteau et al., 2004; Cornu et al., 2009; Friedrichsen et al., 2006; Li et al., 2005a; Liu and Habener, 2008; Wang et al., 2004). Additional effects of GLP-1 on β-cells include activating PKCδ and proliferation via trans-activating the epidermal growth factor receptor (Buteau et al., 2003; Buteau et al., 2001; Buteau et al., 1999), promoting PKA-mediated modulation of the unfolded protein response (UPR) during ER stress (Cunha et al., 2009; Yusta et al., 2006) and stimulating extracellular regulated kinase (ERK) 1/2 activity in a manner potentiated by glucose metabolism (Briaud et al., 2003; Gomez et al., 2002; Trümper et al., 2005). GLP-1 elevates levels of CREB pS133, thus enhancing IRS-2 production and Akt signalling, and this is required for GLP-1 mediated increases in β-cell mass (Jhala et al., 2003; Park et al., 2006). However, GLP-1 rapidly enhances Akt phosphorylation in β-cells (Wang et al., 2004), indicating additional Akt-activating mechanisms, and it was recently shown that GLP-1 elevates IGF-I receptor expression and IGF-II secretion, which forms an autocrine loop enhancing Akt signalling and promoting β-cell proliferation and survival (Cornu et al., 2010; Cornu et al., 2009). Although Akt has multiple actions (Dickson and Rhodes, 2004; Elghazi et al., 2007), studies on mice revealed that improving β-cell mass with GLP-1 required Akt-mediated phosphorylation of the forkhead O1 (FoxO1) transcription factor, which causes FoxO1 nuclear exclusion and consequently enhances the activity of PDX-1, a transcription factor shown to be crucial for the expansion of β-cell mass in animal models of obesity and T2D (Buteau et al., 2006; Kitamura et al., 2002; Kushner et al., 2002; Li et al., 2005b; Okamoto et al., 2006). In contrast to GLP-1, regulation of β-cell mass by GIP is only beginning to be understood. It has been shown that β-cell mass is paradoxically elevated in GIPR-/- mice but with insulin content diminished and islets exhibiting altered architecture (Pamir et al., 2003). These mice also exhibit elevated energy expenditure and resist development of obesity and insulin resistance (Hansotia et al., 2007; Miyawaki et al., 2002), and they are therefore not an optimal

model for studying the role of GIP in modulating β-cell mass. As alternatives, mouse and porcine models have been generated with β-cell specific transgenic expression of a dominant negative GIPR. Interestingly, both models were found to develop diabetes and exhibit reductions in β-cell mass, which in the pig, resulted from a reduction in β-cell proliferation (Herbach et al., 2005; Renner et al., 2010). In INS-1 and islet β-cells, GIP promoted proliferation via activation of Akt and ERK 1/2, and this effect was enhanced by elevating glucose metabolism (Ehses et al., 2002; Friedrichsen et al., 2006; Trümper et al., 2002; Trümper et al., 2001). Furthermore, GIP reduced caspase-3 and caspase-8 activity as well as promoting survival of STZ-treated β-cells (Pospisilik et al., 2003), modulating the UPR in β-cells under ER-stress (Yusta et al., 2006) and promoting survival of glucose- and serum-starved β-cells by suppressing p38 MAPK activity in a cAMP- dependent manner (Ehses et al., 2003; Trümper et al., 2002). In INS-1 cells, GIP enhanced CREB pS133 levels as well as Akt-mediated phosphorylation of FoxO1 (Ehses, 2003; Kim et al., 2005b; Trümper et al., 2001), and thus may enhance the IRS-2 signalling module in a similar manner to GLP-1. Induction of β-cell apoptosis with glucolipotoxic stress was reduced by GIP, and this was associated with a reduction in pro-apoptotic Bax levels and increased anti-apoptotic Bcl-2 levels (Kim et al., 2005b). The decrease in Bax resulted from Akt-mediated phosphorylation of FoxO1 and increased Bcl-2 resulted from PKA-mediated modulation of CREB activity (Kim et al., 2008b). Despite these findings, it is unknown whether enhancing GIPR signalling results in elevated β-cell mass and improved glycaemic control in animal models of diabetes.

1.8 Thesis Investigation 1.8.1 Rationale Restoration of glycaemic control has been the major target for T2D therapeutics, and immense interest has grown in the possibility of enhancing or preserving β-cell mass in order to achieve this target. Clinical findings indicate that β-cells in T2D patients have enhanced proliferation rates (Butler et al., 2007) but that the increase in replication itself enhances β-cell susceptibility to apoptosis (Meier et al., 2006; Ritzel and Butler, 2003), thus making β-cells even more sensitive to the detrimental elevations in glucose, free fatty acids and pro-inflammatory cytokines commonly found in those with T2D. Therefore therapies that reduce β-cell apoptosis will likely have an important role in promoting elevations in β-cell mass in T2D patients.

Numerous clinical trials have clearly established that incretin-based therapies improve β- cell function and lower glycaemia in T2D patients (Amori et al., 2007) with currently marketed forms being DPP-IV inhibitors and GLP-1 receptor agonists (Wajchenberg, 2007). However, the potential impact of GIPR agonists in promoting β-cell function is unknown and has received little attention. As previously discussed, the main reasons for this have been the β-cell resistance to GIP in T2D and the reduced levels of obesity observed in GIPR-/- rodents (Irwin and Flatt, 2009; Meier and Nauck, 2004; Miyawaki et al., 2002; Nauck et al., 1993), indicating that GIPR agonists would be ineffective in restoring β-cell function and may instead exacerbate obesity. However, pharmacological doses of DPP-IV resistant GIP analogues are insulinotropic in rodents that are unresponsive to physiological levels of GIP (Hinke et al., 2002; Irwin et al., 2005) and normalization of glycaemia improves insulin responses to GIP in patients with T2D (Hojberg et al., 2009; Meneilly et al., 1993). Similarly, insulin responses to GIP are acutely improved in T2D patients when sulfonylureas are added in parallel (Aaboe et al., 2009), indicating that β-cell resistance to GIP may partially represent defects in the signalling pathway by which GIP potentiates insulin release, whereas distinct pro-survival signalling pathways stimulated by GIP may remain fully operational. Therefore the pro-survival effects of GIPR signalling in β-cells merit further investigation and form the underlying motivation for this thesis.

1.8.2 Hypothesis The working hypothesis is that activation of the GIPR enhances critical anti-apoptotic signalling networks and promotes β-cell survival, which in rodent models of T2D results in an elevation in β-cell mass and improvement in glycaemic control.

1.8.3 Objectives The main objectives are to investigate: 1) the mechanism by which GIP activates Akt; 2) the effects of GIPR signalling on the mitochondria-mediated apoptotic pathway; and 3) the effects of GIPR activation on β-cell function and survival as well as glycaemic control in rodent models of diabetes.

Chapter 2 Materials and Methods 2.1 Sources of Materials Materials were obtained from Amersham Biosciences (GE Healthcare, Baie d‟Urfe, Quebec), Biocolor (Northern Ireland), BD Biosciences and Falcon (Mississauga, Ontario), Bio- Rad (Mississauga, Ontario), Pierce (Rockford, IL, USA), Cell Signaling Technology (Beverly, MA), Invitrogen (Carlsbad, CA, USA), LifeScan (Milpitas, CA, USA; a Johnson & Johnson Company), MilliporeTM (Canada), Molecular Probes (subsidiary of Invitrogen), Qiagen (Mississauga, Ontario), Santa Cruz Biotechnology (Santa Cruz, CA, USA), Sigma (St. Louis, Missouri, USA) or Thermo Fisher Scientific Inc. (Toronto, Ontario), as indicated in the text, unless otherwise stated.

2.2 Cell Culture The rat insulinoma (INS-1) β-cell line (clone 832/13) was provided by Dr. C.B. Newgard (Duke University Medical Centre, USA), mouse insulinoma β-cell line (MIN6) by Dr. J.D. Johnson (UBC, Canada), and Akita mouse β-cell line (Akita) by Prof. Akio Koizumi (Kyoto University, Japan). MIN6 cells were developed by Miyazaki et al. (1990). The Akita cell line was one of several derived from insulinomas that developed in mice produced by crossing insulinoma IT3 transgenic mice with Ins2WT/C96Y mice (Nozaki et al., 2004). INS-1 cells were maintained in 11 mM glucose RPMI 1640 (Sigma) supplemented with 2 mM glutamine, 50 µM β-mercaptoethanol, 10 mM HEPES pH 7.4, 1 mM sodium pyruvate, 10% fetal bovine serum (FBS; Sigma), 100 units/ml penicillin G-sodium, and 100 µg/ml streptomycin sulphate (pen/strep). MIN6 and Akita cells were cultured in 25 mM glucose Dulbecco‟s modified Eagle medium (DMEM) supplemented with 0.25% HEPES pH 7.4, 10% FBS and pen/strep. Mouse islets were isolated by collagenase digestion (Salvalaggio et al., 2002) from male C57BL/6 mice (age 10-12 weeks; Jackson Laboratories). Briefly, mice were euthanized and then a midline incision was made to expose the intra-peritoneal cavity. A clamp was placed on the common bile duct and the pancreas infused with ~3 ml collagenase Type XI (Sigma) in Hanks balanced salt solution (HBSS) without Ca2+. Following surgical removal, the pancreas was placed in a 50 ml conical tube (BD Falcon) containing HBSS and incubated in a water bath at 37°C for 13 min. Digestion was stopped with the addition of HBSS plus 1 mM Ca2+. Samples were briefly centrifuged and washed twice with HBSS plus Ca2+, and then resuspended in RPMI 1640 maintenance media supplemented with 5 mM glucose, 0.25% HEPES (pH 7.4), 10% FBS, 33

and pen/strep. For islet separation, the suspension was poured through a 70 µm nylon membrane filter (BD Falcon). The filter was flushed with RPMI medium, flipped upside down and the islets rinsed into a Petri dish. Islets were then transferred into a Petri dish with fresh medium. The procedure for isolating islets from lean and obese VDF rats was similar to the mouse procedure except for the separation step, which utilized the following methods (Xu et al., 2007). Medium containing digested pancreas was slowly poured onto a Histopaque density gradient solution (Histopaque® 1077; Sigma), and then centrifuged at 500 g for 30 min and 4°C. The distinct floating layer was then poured into a Petri dish and islets picked, as described in the mouse procedure. Human islets were provided by the Centre for Human Islet Transplant and β-cell Regeneration at UBC and maintained in RPMI 1640 supplemented with 5 mM glucose, 0.25% HEPES (pH 7.4), 10% FBS, and pen/strep. Approximately 16-24 h after islets were received, they were picked into fresh medium, as described in the mouse procedure.

2.3 Experimental Treatment of Cell Cultures Experimental treatments of β-cell lines were initiated when cells were ~80-90% confluent, except in cell death studies using propidium iodide, in which case cells were ~60-70% confluent to enable more accurate image quantification. In studies on primary cells, treatments were performed on intact islets except, again, for cell death studies. In this case, islets were dispersed at 50 islets/well in a 96 well plate. Islet dispersion was performed by washing in PBS and then incubating in PBS plus 0.05% trypsin/EDTA. Following dispersion, islets were washed with fresh RPMI growth medium and then added to wells. All chemical inhibitors were acquired from Calbiochem® (now a subsidiary of Merck Chemicals). The apoptosis inducers - staurosporine, thapsigargin, etoposide, and tunicamycin - were from Sigma. Unless otherwise stated, approximately 12 h prior to treatments, cells received fresh 3 mM glucose serum-starved media containing 0.1% bovine serum albumin (BSA). For Akt kinase assays, inhibitors were applied to cultures 30 min prior to the addition of stimulus (GIP or forskolin). In apoptosis studies, inhibitors were applied in parallel with the addition of the stimulating agent. However, in studies investigating the effects of a DPP-IV resistant GIP analogue on ER stress responses (Chapter 5), cells were treated in regular serum-containing growth media.

The induction of β-cell apoptosis was examined in a subset of studies by incubating cells with 5 mM glucose (control), 25 mM glucose (glucotoxicity), 200 µM palmitate (lipotoxicity), or both (glucolipotoxicity). To generate a palmitate-medium solution, ~5 mls of water, depending on exact volume required, containing 50 µl of 1 M sodium hydroxide was added to a 15 ml tube containing ~28 µg of sodium palmitate (Sigma) to make a 20 mM solution and then incubated at 70°C for 30 min. Upon dissolution, the palmitate was added to a 5% fatty acid free BSA solution to generate a stock of 3.33 mM palmitate in 4.16% BSA. This was incubated at 37°C for at least 2 h and then added to serum-free, BSA-free RPMI, ultimately making a solution of 200 µM palmitate in RPMI solution containing 0.5% fatty acid free BSA. The non-palmitate containing media were made similarly having a BSA concentration of 0.5%, and glucose was added accordingly.

2.4 Cell Death Assays Apoptosis was determined using the APOPercentageTM apoptosis assay kit according to the manufacturer‟s protocol (Biocolor). The detection system is based on the asymmetrical composition of mammalian cell membranes in which phosphatidylserine is retained on the inner membrane. During apoptosis, phosphatidylserine can transfer to the outer membrane. When this occurs, the APOPercentage dye undergoes unidirectional uptake into the cell. In contrast, the dye does not accumulate in necrotic cells. In the current studies, cells were treated as described in Figure Legends and then incubated in medium containing dye for 30 min, after which they were washed twice in PBS and then disrupted with the reagent provided in the kit and apoptosis levels quantified via colorimetric detection at 570 nm. To determine total cell death, INS-1 cells were treated in media containing 500 ng/ml propidium iodide (PI; Invitrogen) and 250 ng/ml Hoechst 33258 (Hoechst; Sigma). Cellular uptake of PI into the nucleus occurs only during the onset of cell death whereas Hoechst labels the nucleus of all cells. PI- (PI+) and Hoechst- (Hoechst+) positive nuclei were imaged with a Cellomics Arrayscan VTI (Thermo Fisher Scientific Inc.) and % cell death was calculated as the number of PI+ cells / Hoechst+ cells multiplied by 100.

2.5 Plasmid Constructs, siRNA and Transfection Protocol The pcDNA3 construct encoding haemagglutinin tagged human Akt1 (HA-Akt) was acquired from William Sellers (Hsieh et al., 2004) via Addgene Repository (www.addgene.org).

Point mutations were generated via Quikchange (Stratagene) and oligonucleotides used were ggt gcc acc atg aag gcc ttt tgc ggc aca c and gtg tgc cgc aaa agg cct tca tgg tgg cac c to generate a Thr308Ala mutation, ctt ccc cca gtt cgc cta ctc ggc cag and ctg gcc gag tag gcg aac tgg ggg aag to generate a Ser473Ala mutation, and ccg cta cta cgc cat gat gat cct caa gaa gga ag and ctt cct tct tga gga tca tca tgg cgt agt agc gg to generate a Lys179Met mutation. The pcDNA3 construct encoding human ASK1 and a kinase dead ASK1 containing a methionine mutation at lysine-709 (Ichijo et al., 1997), used for the purpose of producing a dominant negative protein, were gifts from Dr. Hidenori Ichijo (The University of Tokyo, Japan). The same ASK1 construct, but coding for protein containing an alanine mutation at Ser- 83, thus preventing Akt-mediated phosphorylation of ASK1 (Chen et al., 2001), was a gift from Dr. Moses Chao (NYU Medical Center, USA). For plasmid transfection, DNA was incubated with LipofectamineTM LTX (Invitrogen) and PLUSTM reagent (Invitrogen) at a ratio of 1 µg DNA / 3.0 µl Lipofectamine LTXTM / 1.0 µl PLUSTM in 200 µl OPTI-MEM I (GIBCO) for 30 min at room temperature and added to 2x106 cells in 1 ml maintenance media without antibiotics. Media were replaced with media containing antibiotics after 6-16 h and experiments were performed 36 h following transfection. Transfection of siRNA was adapted from Kibbey et al. (2007). Briefly, Akt 1 siRNA (siRNA ID: SASI_Rn01_00063656; Sigma) and Akt 2 siRNA (siRNA ID: SASI_Rn01_00047688; Sigma) or scramble (control) siRNA (Cell Signaling Technology; #6568) were incubated with 10 µl RNAifect Transfection reagent (Qiagen; #301605) in 200 µl OPTI-MEM I for 30 min at room temperature and then added to 1.5x106 cells in 1 ml maintenance media without antibiotics (final concentrations: Akt 1&2 siRNA, 100 nM each; scramble siRNA, 200 nM). This was replaced with antibiotic-containing media after 6-16 h and experiments were performed ~72 h following transfection.

2.6 Cell Lysis and in vitro Kinase Assays Cultured cells were lysed in 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM

Na3VO4, 1 μg/ml leupeptin, and supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and Proteinase inhibitor cocktail set III (Calbiochem; #539134). Endogenous Akt kinase activity (KA) assays were performed using the Akt Kinase Assay Kit from Cell Signaling Technology (Nonradioactive; #9840). Total endogenous Akt protein was

immunoprecipitated with an anti-Akt monoclonal antibody covalently linked with agarose beads (#9279). Beads from immunoprecipitates were washed twice in lysis buffer and twice in kinase buffer, and then resuspended in 60 µl kinase buffer containing 200 µM ATP and 0.75 µg of a GST-tagged GSK3 fusion peptide (#9237; GSK3), containing residues corresponding to GSK3α/β (Ser-21/9), that was used as a substrate for Akt and contained the optimal consensus sequence for Akt phosphorylation (Alessi et al., 1996b; Obata et al., 2000). Kinase reactions were performed at 30ºC for 30 min. Kinase activity assays for the transfected HA-Akt protein were similarly performed except protein was immunoprecipitated with protein G beads (Amersham Biosciences) conjugated to anti-HA antibody and kinase reactions were extended for an additional 30 min. Phosphorylation of GSK3 was determined via Western blotting analysis with a phospho-specific antibody, unique to the Akt Kinase Assay Kit (#9327). For ASK1 kinase assays, endogenous ASK1 protein was immunoprecipitated from untransfected INS-1 cells with an anti-ASK1 antibody (Santa Cruz) conjugated to Protein A Sepharose beads (Invitrogen). Beads from immunoprecipitates were washed twice in 500 µl lysis buffer and twice in 500 µl kinase buffer and then resuspended in 60 µl kinase buffer containing 200 µM ATP and 0.75 µg of a GST tagged MEK 6 (MEK 6) fusion peptide (Cell Signaling Technology), used as a substrate for ASK1. Kinase reactions were performed at 30ºC for 30 min. Phosphorylation of MEK 6 was determined by Western blotting analysis with anti-phospho- MEK 3/6 antibody.

2.7 Western Blots Cell lysates and samples from kinase assays were subjected to 10% (for ASK1) or 15% SDS/PAGE and electroblotted onto nitrocellulose membranes (Bio-Rad), which were probed as indicated in the Results Sections with the following antibodies. From Cell Signaling Technology: anti-Akt (#9272), anti-Akt1 (mouse mAb; 2H10; #2967), anti-Akt2 (rabbit mAb; 5B5; #2964), anti-Akt3 (#4059), anti-β-actin (#4967), anti-Bad (#9292), anti-Bax (#2772), anti-Bcl-2 (#2876), anti-Bcl-XL (#2762), anti-Bim (#2819), anti-β-tubulin (#2146), anti-caspase-3 (rabbit mAb; 8G10; recognizes full length and cleaved caspase-3), anti-CHOP (mouse mAb 2895; L63F7), anti-COX IV (#4844), anti-GST (#2622), anti-cytochrome C (rabbit mAb; 136F3), anti-HA antibody (#2367), anti-JNK (#9252), anti-p38 MAPK (#9212), anti-phospho-Akt (Thr-308 (244F9); #4056), anti-phospho-Akt (Ser-473; #9271), anti-phospho-ASK1 (Ser-83; #3761; detects human isoform but not rat), anti-phospho-ASK1 (Ser-845; #3765; detects human isoform

but not rat), anti-phospho-Bad (Ser-112; #9291), anti-phospho-Foxo1 (Ser-256; #9461), anti- phospho-JNK (Thr-183/Tyr-185; mouse mAb; G9), anti-phospho-MDM2 (Ser-166; #3521) anti- phospho-MEK 3/6 (Ser-189/207; #9231), anti-phospho-p38 MAPK (Thr-180/Tyr-182; #9211), and anti-phospho-Raf1 (Ser-259; #9421). From Sigma: anti-MDM2 (#M4308). From Santa Cruz Biotechnology: anti-ASK1 (H-300; recognizes total human and rat protein). Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Biosciences) using horseradish peroxidase-conjugated IgG secondary antibodies. For quantifying band density, films were analyzed using densitometric software (Eagle Eye; Stratagene).

2.8 Mitochondrial/Cytosolic Fractionation and Bax Crosslinking The cytosolic and mitochondrial fractions of cells were obtained using the BioVision fractionation kit (Mountain View, CA) according to the manufacturer‟s protocol. INS-1 cells were treated as described in the Figure Legends, and then scraped into ice-cold PBS and centrifuged at 500 g for 5 min. Cell pellets were resuspended in cytosol buffer provided in the kit and incubated on ice for 10 min. Cell suspension was then transferred and cell membranes sheared in a dounce homogenizer, leaving a cytoplasmic fraction and heavy membrane fraction that included nuclei. Cells were centrifuged at 500 g for 5 min and the soluble fraction transferred to a new tube, and this was repeated once more. The cytoplasmic fraction was then centrifuged at 16,000 g for 30 min at 4°C. The soluble fraction was transferred to a new tube and the remaining mitochondrial pellet was resuspended in mitochondrial disruption buffer provided in the kit. Samples were stored at -80°C. Crosslinking of Bax was performed using methods described by Kim et al. (2006b). Mitochondria were incubated with 10 mM bismaleimidohexane (BMH; Pierce) in PBS for 30 min at room temperature, and the reaction was terminated by adding SDS loading buffer. Levels of crosslinked Bax were determined via Western blotting analysis with anti-Bax antibody.

2.9 Animal Studies All studies were performed in accordance with guidelines put forth by the University of British Columbia (UBC) Committee on Animal Care and the Canadian Council on Animal Care. All studies were performed on male animals maintained on a 12 h light/dark cycle with free access to standard rodent chow and water.

Obese (400-500 g) and lean (200-250 g) Vancouver Diabetic Fatty (VDF) Zucker rats (13-15 weeks of age) and C57Bl/6 GIPR knockout (GIPR-/-) (Miyawaki et al., 1999) or wild type littermate mice (20-25 g; 10-14 weeks of age) were bred and maintained at UBC. Obese Zucker diabetic fatty (ZDF) rats (strain 370; age 4-5 weeks; 140-170 g) and lean Zucker (strain 371; age 4-5 weeks; 110-130 g) rats were from Charles River Laboratory and maintained at UBC for at least 1 week prior to study. Male heterozygous Ins2WT/C96Y Akita mice (Akita; C57BL/6 background) were obtained from The Jackson Laboratory (Bar Harbor, ME) and crossed with female C57BL/6 Ins2WT/WT wild type (WT) mice obtained from the UBC Animal Care Centre or Charles River Laboratories, Inc. (Wilmington, MA). The offspring were weaned at three weeks after birth, at which time male Akita mice were selected on the basis of random fed blood glucose levels greater than 15 mM on 3 separate and sequential occasions. 2 The truncated human GIP analogue, D-Ala GIP1-30 (D-GIP1-30), was synthesized by GenScript (Piscataway, NJ) and dissolved in 2% acetic acid plus 0.4% BSA, diluted in PBS and then pH adjusted to 7.2 for administration. Identical solvent was used for control animals. During treatment periods, peptide (D-GIP1-30) or control (PBS) was administered at 8-9 am and 4-5 pm, whereas morning blood glucose levels were determined at 7-8 am. In studies using streptozotocin (STZ), citrate buffer (pH 4.5) was added to previously unopened bottles of STZ (Sigma), which was then administered (35 mg/kg of BW) to 2-hour fasted lean Zucker rats via intraperitoneal injection within 15 min of dissolution. Animals were monitored that evening and the following morning to ensure recovery. In glucose tolerance tests on rats, animals were fasted for 16-17 h and then challenged with 1 g glucose/kg of BW via oral or i.p. delivery and glucose levels determined at 0, 10, 20, 30, 60, and 120 min time points. Serum samples were collected at the same time points for insulin determinations. In glucose tolerance tests on GIPR-/- mice and litter mate controls (GIPR+/+), animals were fasted for 6 h and then challenged with 2 g glucose/kg of BW via i.p. delivery and blood glucose levels determined at 0, 7.5, 15, 30, 60, and 120 min time points. In studies on Akita mice, glucose tolerance tests were similarly performed to those on GIPR-/- mice, except animals received glucose via oral delivery. Furthermore, serum samples were collected only at the 0, 7.5, 15, and 60 min time points for insulin determinations. For all animals, blood was collected from tail vein and glucose levels measured with a SureStep glucose analyzer (LifeScan). Insulin levels were determined from serum samples by radioimmunoassay

(RIA; MilliporeTM, Cat# RI-13K) with rats and insulin ultrasensitive ELISA kit (Cat #80- INSM5U-EO1; ALPCO Immunoassays; Salem, NH) with mice.

2.10 Pancreatic Perfusions Surgical procedures for the pancreas perfusion studies were performed by Gary Yang, a PhD candidate in the laboratories of Dr. Timothy J. Kieffer and Dr. Yin Nam Kwok. VDF rats were deprived of food for at least 12 h, anesthetized, and pancreata isolated as previously described (Pederson et al., 1982). Arterial perfusion was achieved by cannulation of the abdominal aorta at a level adjacent to the superior mesenteric artery, while venous effluent was collected via cannulation of the portal vein. Perfusate consisted of modified Krebs-Ringer bicarbonate buffer containing 3% dextran (Sigma), 0.2% BSA (Sigma) and 3 mM glucose gassed with 95% O2 / 5% CO2 and was kept at 37°C with heating units. Following a 30 min equilibration period, gradients of peptide (0-1 nM) or glucose (4.4 or 16.7 mM) were administered. Perfusion was maintained at 3 ml/min with a peristaltic pump and portal vein effluent collected in 3 min intervals and stored at -20°C. Insulin levels were determined by RIA.

2.11 Histological Analysis Pancreas samples were fixed overnight at 4°C in 4% paraformaldehyde. Paraffin embedding, sectioning (5 µm), and hematoxylin and eosin (H&E) staining of samples was performed by Wax-it services (Vancouver, Canada). For immunofluorescent staining, deparaffinized and rehydrated slides underwent heat induced epitope retrieval at 95°C for 10 min in citrate buffer (10 mM citrate, 0.05% Tween 20, pH 6.0) using an EZ-Retriever™ Microwave (BioGenex, USA), and then incubated overnight at 4°C with guinea pig anti-insulin (1:1000; Millipore), mouse anti-glucagon (1:1000; Sigma), and/or mouse anti-PCNA (1:200; BD Biosciences). Apoptotic cell staining with TUNEL was according to manufacturers protocol (Roche). Primary antibodies were visualized following 1 h incubation at room temperature with secondary antibodies conjugated to AlexaFluor 488 or 594 (1:500; Molecular Probes) and then mounted in VECTASHIELD HardsetTM mounting medium with DAPI (Vector Laboratories; Cat# H-1500). Images were captured using an Axiovert 200 microscope (Carl Zeiss, Toronto, Canada) and a Retiga 2000R camera (QImaging, Burnaby, Canada) in monochrome and pseudo- coloured (fluorescent images) or in RGB format (H&E images) using the OpenLab v5.2 software (ImproVision, Lexington, USA). Following staining with 3,3'-diaminobenzidine, pancreas

sections were digitally scanned with a ScanScope CS digital slide scanner and analyzed with ImageScope positive pixel count, version 9 algorithm (Aperio Technologies Inc., USA).

2.12 Statistical Analysis Data, expressed as mean ± standard error of the mean (SEM), were analyzed using the non-linear regression analysis program PRISM (GraphPad, San Diego, CA). Statistical significance of differences in mean value was tested using analysis of variance (ANOVA) with the Bonferroni post hoc test. A p value of < 0.05 was considered significant.

The homeostatic model of insulin sensitivity (HOMA SI) was applied using the methods specifically developed for ZDF rats (Topp et al., 2007). HOMA SI was defined as the maximal rate of hepatic glucose output (RO) minus glucose effectiveness in the absence of insulin (EO) multiplied by the level of blood glucose (mmol/L), overall divided by the product of the blood glucose and serum insulin levels (nmol/L). The values of RO and EO were considered constant as provided in the following article (Topp et al., 2000). With these defined terms therefore, incorporating the measured values of glucose and insulin levels provided HOMA SI values.

(Ro  Eo)X[G] S  I [G]X[I]



Chapter 3 Mechanisms by Which GIP Activates Akt in β-cells 3.1 Introduction Akt is a protein serine/threonine kinase that exerts several anti-apoptotic actions in β-cells and promotes the expansion of β-cell mass in response to the development of obesity and insulin resistance (Dickson and Rhodes, 2004; Elghazi et al., 2007). Interestingly, Akt is involved in multiple incretin-mediated responses (Kim et al., 2005b; Liu and Habener, 2008; Trümper et al., 2002; Wang et al., 2004), but the mechanism by which incretins actually promote Akt activation in β-cells remains unclear. Stimulation of Akt by prototypical activators such as IGF-I involves the activation of

PI3K, which generates phosphatidylinositol (3,4,5)-trisphosphate (PIP3), resulting in recruitment of Akt and 3-phosphoinositide dependent kinase-1 (PDK-1) to the plasma membrane through

PIP3 binding of their PH domains (Alessi et al., 1996a; Hanada et al., 2004). PDK-1 then phosphorylates the Thr-308 residue of Akt (pT308), an event thought to be essential for Akt activation (Alessi et al., 1996a; Hanada et al., 2004; Manning and Cantley, 2007; Stephens et al., 1998). Akt is also phosphorylated at a second site that is thought to be necessary to evoke full kinase activity, Ser-473 (pS473) in the C-terminal hydrophobic motif (Yang et al., 2002a; Yang et al., 2002b), and recent evidence (Sarbassov et al., 2005) indicates that the major kinase responsible for this event is the mammalian target of rapamycin/rictor complex (mTORC2). An illustration of this canonical Akt activation mechanism is shown in Appendix A. In comparison, activation of Akt by incretins appears more complex. Stimulation of the GLP-1R in β-cells increases IRS-2 protein levels, which potentiates PI3K signalling (Park et al., 2006). Furthermore, GLP-1 activates PI3K via trans-activation of the epidermal growth factor receptor (Buteau et al., 2003; Buteau et al., 1999), and a similar effect on PI3K was recently found to occur via enhancing an IGF-II / IGF-I receptor autocrine loop (Cornu et al., 2010; Cornu et al., 2009). GLP-1 mediated activation of Akt has also been found to occur in a PI3K- independent manner (Liu and Habener, 2008). The underlying mechanism by which GIP activates Akt is unclear but may also involve enhancing IRS-2 protein levels and the IGF-II autocrine loop. Studies with INS-1 β-cells showed that GIP increases Akt activity and the levels of Akt pS473 (Kim et al., 2005b; Trümper et al., 2001), through signalling pathways proposed to involve PI3K, Ca2+-calmodulin dependent protein kinase II and PKA (Trümper et al., 2002). However, these pathways were identified in cells incubated in high glucose for relatively long periods (Kim et al., 2005b; Trümper et al., 2001). In view of the powerful insulinotropic actions 42

of GIP on β-cells, it was therefore difficult to discriminate between direct actions of GIP and autocrine actions of released insulin and/or other factors. Additionally, the majority of studies on Akt activity have used indirect methods of measurement (i.e. phosphorylation of Ser-473 and/or Thr-308), which may not necessarily equate with Akt activity. In the current study, using activity assays on immunoprecipitated Akt, the role of PI3K in the direct and rapid activation of Akt by GIP was examined in INS-1 cells and mouse islets.

3.2 Results 3.2.1 GIP Activates Akt Without Increasing the Phosphorylation of Thr-308 To assess whether GIP directly activates Akt in β-cells, INS-1 cells were treated with GIP for 0-30 min in low glucose (3 mM) and serum starved media, thus negating the secretion of insulin and the activation of Akt by serum growth factors. In response to GIP treatment, Western blotting analysis of cell lysates showed that levels of Akt pS473 increased as early as 5 min and remained elevated for the full 30 min period of study (Fig. 1). Similar temporal responses were observed with Akt activity, as demonstrated by the levels of phosphorylated Akt substrate (P- GSK3) in Akt kinase activity (KA) assays on immunoprecipitated Akt, and those of the endogenous Akt substrate, phosphorylated MDM2 (P-MDM2), in cell lysates. Surprisingly however, the levels of Akt pT308 did not change in either cell lysates or Akt KA assays, until at least 30 min of stimulation.

3.2.2 Neither PI3K Signalling nor Phosphorylation of Thr-308 or Ser-473 are Required for GIP-mediated Activation of Akt Since phosphorylation of Akt at Thr-308 is PI3K-dependent, results in Fig. 1 indicated that PI3K was not involved in the rapid, GIP-stimulated activation of Akt. To exclude the involvement of PI3K, INS-1 cells were treated with or without the PI3K inhibitor, Ly294002, or an inhibitor, Akt VIII, that interacts with the PH domain of Akt and prevents recruitment to PIP3 in the plasma membrane as well as phosphorylation of Akt at Thr-308 and Ser-473 by upstream kinases (Barnett et al., 2005; Green et al., 2008; Logie et al., 2007), prior to a 15 min period of GIP stimulation (Fig. 2A). In addition, parallel experiments were performed with IGF-I, since PI3K-dependent phosphorylation of Akt at Thr-308 and Ser-473 is a well established component of its Akt activation mechanism (Alessi et al., 1996a; Hanada et al., 2004). In the absence of inhibitor, GIP increased Akt activity as well as levels of Akt pS473, but not pT308, whereas

IGF-I increased Akt activity and phosphorylation of both sites. In cells treated with either Ly294002 or Akt VIII, levels of Akt pT308 and pS473 were significantly reduced in both GIP and IGF-I treated cells, and this was associated with a significant reduction in Akt activity in IGF-I treated cells (Fig. 2C), demonstrating that the canonical upstream Akt activation pathway was inhibited. In GIP treated cells however, the activation of Akt was not inhibited by either Ly294002 or Akt VIII (Fig. 2B), indicating that neither PI3K nor phosphorylation of Akt at Thr- 308 and Ser-473 were required for GIP stimulated activation of Akt in INS-1 cells. To ensure that the finding in cell lysates with the Akt substrate, MDM2, was indeed Akt-dependent, the stimulatory effects of GIP were examined in INS-1 cells transfected with scramble or Akt siRNA. As shown (Fig. 2D), a reduction in Akt levels (~65%) greatly ameliorated the effects of GIP on P-MDM2 levels. To further validate these findings with primary cells, mouse islets were treated with Ly294002 prior to GIP stimulation. As found with INS-1 cells, GIP stimulated the enzyme activity of Akt irrespective of the presence or absence of Ly294002 (Fig. 2E&F). To definitively exclude a requirement for Akt pT308 and pS473, INS-1 cells were transfected with plasmid DNA (Fig. 3A) encoding either HA-tagged human Akt1 (HA-Akt1) containing alanine point mutations at Thr-308 and Ser-473 residues (HA-AktT308A,S473A) or a kinase dead form containing a methionine point mutation at the lysine-179 residue (HA-

AktK179M), and responses to GIP or IGF-I stimulation for 15 min were compared to untreated cells by Western blotting analysis (Fig. 3C). Stimulation of INS-1 cells with GIP, but not IGF-I, significantly increased kinase activity of HA-AktT308A,S473A, but not HA-AktK179M (Fig. 3D). To examine the relationship between phosphorylation status and Akt activity further, INS-1 cells were treated with or without an Akt inhibitor, Akt IV, prior to GIP stimulation for 15 min. Though not well characterized, this inhibitor was chosen because it was found to inhibit Akt signalling without affecting PI3K in 786-O cells (Kau et al., 2003) and to inhibit the activation of Akt by GLP-1 in β-cells, when the PI3K inhibitor Ly294002 was ineffective (Liu and Habener, 2008). Thus it was considered possible that it could inhibit Akt activation in response to GIP stimulation in INS-1 cells, when Ly294002 and Akt VIII were ineffective. Although treatment of cells with Akt IV did not alter the basal activity of Akt (data not shown) and had no inhibitory effect on the levels of Akt pT308 and pS473, it ablated the stimulatory effect of GIP on Akt activity, when assessed with both P-GSK3 and P-MDM2 assays (Fig. 4A&B).

Figure 1. GIP Activates Akt Without Increasing Phosphorylation of Akt at Thr-308. A) INS-1 cells were stimulated with 10 nM GIP for 0, 5, 10, 15, or 30 min and Western blotting analysis or Akt kinase activity (KA) assays performed on cell lysates with indicated antibodies. (B & C) For quantification of Akt pT308 (P-Akt 308), pS473 (P-Akt 473) and phosphorylated Akt substrate peptide (P-GSK3), levels were normalized to total Akt, whereas P-MDM2 was normalized to total MDM2. Mean ± SEM changes in phosphorylated protein relative to time 0 (n ≥ 4) for cell lysates (B) and KA assays (C). Immunoblotting with anti-β-actin and anti-GST (GSK3) antibodies were used as internal controls. Significant differences (p < 0.05) were obtained at all time points (5-30 min) in levels of Akt pS473 and P-MDM2, whereas Akt pT308 levels were significantly different only at the 30 min time point.

Figure 2. PI3K is Not Required for GIP-mediated Activation of Akt. A) INS-1 cells were pre-treated with DMSO (control) or inhibitors of PI3K/Akt signalling (15 µM Ly294002 or 5 µM Akt VIII), then treated ± 10 nM GIP or 10 nM IGF-I for 15 min. Western blotting analysis and Akt KA assays were performed on cell lysates with indicated antibodies. (B & C) Mean change ± SEM in Akt activity (P-GSK3/Akt) relative to DMSO control (n = 4) for GIP (B) and IGF-I (C) treated cells. #, p < 0.05 vs DMSO control. $, p < 0.05 vs IGF-I without inhibitor. D) INS-1 cells were transfected with scramble (200 nM) or Akt 1&2 (100 nM each) siRNA as described in the Materials and Methods Chapter and experiments performed 72 h later, in which INS-1 cells were treated ± 10 nM GIP for 15 min. Western blotting analysis were performed on total cell lysates with indicated antibodies. Shown are representative blots of three independent experiments. E) Mouse islets were pre-treated with DMSO (control) or 15 µM Ly294002, then treated ± 10 nM GIP for 15 min. Western blotting analysis and Akt KA assays were performed with indicated antibodies. F) Mean change ± SEM in Akt activity relative to DMSO control (n = 3). #, p < 0.05 vs DMSO control. Immunoblotting with anti-β-actin and anti-GST (GSK3) antibodies were used as internal controls.

Figure 3. Phosphorylation of Thr-308 or Ser-473 is Not Required for GIP-mediated Activation of Akt. (A) Illustration of HA-Akt1 constructs and the respective point mutations used for this study. (B) Western blotting showing representative level of HA-AktT308A,S473A protein in transfected cells relative to endogenous Akt in untransfected cells, as well as the purity of AktT308A,S473A immunoprecipitates. (C) INS-1 cells were transfected with HA-AktT308A,S473A or HA-AktK179M and 36 h later treated ± 10 nM GIP or 10 nM IGF-I for 15 min. Western blotting analysis was performed on Akt KA assays with indicated antibodies. (D) Mean change ± SEM in Akt activity (P-GSK3/Akt) relative to Basal (n = 4) for INS-1 cells transfected with HA- AktT308A,S473A or HA-AktK179M. #, p < 0.05 vs Basal. Immunoblotting with anti-GST (GSK3) antibodies were used as internal controls.

Figure 4. Akt Inhibitor IV (Akt IV) Inhibits GIP-mediated Activation of Akt Without Affecting the Phosphorylation of Akt at Thr-308 or Ser-473. (A) INS-1 cells were pre-treated with DMSO (control) or Akt inhibitor, 500 nM Akt IV, for 1 h, then treated ± 10 nM GIP for 15 min and Western blotting analysis performed on cell lysates and Akt KA assays with indicated antibodies. (B) Mean change ± SEM in Akt activity (P-GSK3/Akt) relative to DMSO control (n = 4). #, p < 0.05 vs DMSO control. $, p < 0.05 vs GIP without inhibitor. Immunoblotting with anti-β-actin and anti-GST (GSK3) antibodies were used as internal controls.

3.2.3 GLP-1 and Adenylate Cyclase Activation Mimic the Stimulatory Effects of GIP Since both GIP and GLP-1 generally promote similar actions in β-cells through stimulation of Gαs (Drucker, 2006), it was evaluated whether GLP-1 or forskolin also stimulated Akt in a PI3K-independent manner (Fig. 5A). In the presence of 3 mM glucose, GLP-1 activated Akt in an equivalent manner to GIP irrespective of the presence of Ly294002, whereas neither high glucose nor insulin (500 pM) stimulated Akt activity (Fig. 5B), indicating that secreted insulin was not contributing to incretin-induced activation. Forskolin (1 µM) also activated Akt in INS-1 cells, whether treated with or without Ly294002 and Akt VIII (Fig. 5C&D), indicating that cAMP production was likely involved in the underlying mode of Akt activation by GIP.

3.2.4 Stimulation of Akt by GIP Appears to Involve EPAC2 and not PKA Since GIP had previously been shown to activate PKA in INS-1 cells, its involvement in Akt activation was examined by treatment with the PKA inhibitors, H89 or adenosine 3‟, 5‟- cyclic monophosphorothioate Rp-isomer (RP-cAMP), prior to a 15 min stimulation with GIP (Fig. 6A-D) or forskolin (Fig. 7A&B). It was found that neither inhibitor diminished the ability of GIP or forskolin to activate Akt. GIP has also been shown to activate the cAMP responsive protein, EPAC2, in β-cells (Kashima et al., 2001). To examine a role for EPAC2 in Akt activation, INS-1 cells were treated with 100 µM adenosine 3‟, 5‟-cyclic monophosphate, 8-(4- chlorophenylthio)-2‟-O-methyl (8-cpt cAMP), an EPAC selective agonist, and this resulted in a significant increase in Akt activity (Fig. 7C&D). To validate that primary cells responded in a similar manner, mouse islets were similarly treated, and the EPAC2 agonist also resulted in increased Akt activity (Fig. 7E). Furthermore, 8-cpt cAMP stimulation was also found to enhance P-MDM2 levels in INS-1 cells whether pre-treated with DMSO or Ly294002 in addition to GIP (Fig. 8A). Lastly, in an attempt to identify potential factors mediating the effects of GIP/EPAC signalling on Akt, INS-1 cells were pre-treated with elevated levels of glucose, 2+ inhibitors of known GIP modulated signalling pathways (Ca , ERK 1/2, and iPLA2 signalling), or Akt IV and then treated with GIP for 15 min. As shown (Fig. 8B), elevated glucose levels had no effect on GIP-mediated activation of Akt and only blockade with the Akt IV inhibitor decreased activity. Thus, neither glucose metabolism nor various signalling modules examined were found to be involved in the mechanism of Akt activation by GIP, whereas the agonist study indicated a role for EPAC2.

Figure 5. GLP-1 and Forskolin but Neither High Glucose nor Insulin Mimics the Akt Kinase Stimulatory Effects of GIP. A) INS-1 cells were pre-treated with DMSO (control) or 15 µM Ly294002 for 30 min and then treated ± 10 nM GIP, 10 nM GLP-1, or 500 pM insulin (all with 3 mM glucose) or 16 mM glucose for 15 min, and Western blotting analysis performed on Akt KA assays with indicated antibodies. (B) Mean change ± SEM in Akt activity (P-GST- GSK3/Akt) relative to DMSO (n = 4). #, p < 0.05 vs Control without Ly294002. (C) INS-1 cells were pre-treated with DMSO (control) or PI3K/Akt signalling inhibitors (15 µM Ly294002 or 5 µM Akt VIII) for 1 h, then treated ± 1 µM forskolin for 15 min., and Western blotting analysis performed on cell lysates or Akt KA assays with indicated antibodies. (D) Mean change ± SEM in Akt activity (P-GSK3/Akt) relative to DMSO control (n = 4). #, p < 0.05 vs DMSO control. Immunoblotting with anti-β-actin and anti-GST (GSK3) antibodies were used as internal controls.

Figure 6. Stimulation of Akt by GIP Does Not Involve Protein Kinase A (PKA). INS-1 cells were pre-treated with DMSO (control) or PKA inhibitor, 10 µM H89 (A) or 250 uM adenosine 3‟,5‟-cyclic monophosphorothioate, RP-isomer (RP-cAMP) (C) for 30 min, then treated ± 10 nM GIP for 15 min and Western blotting analysis performed on Akt KA assays with indicated antibodies. Mean change ± SEM in Akt activity (P-GSK3/Akt) relative to DMSO control (n = 4) in treated cells involving H89 (B) or RP-cAMP (D). #, p < 0.05 vs DMSO control. Immunoblotting with anti-GST (GSK3) antibodies were used as internal controls. Note that the concentration of H89 used herein has been shown in INS-1 cells to inhibit PKA signalling in response to forskolin as well as GIP stimulation (Kim et al., 2008b).

Figure 7. Stimulation of Akt by Forskolin Involves EPAC and Not PKA. A) INS-1 cells were pre-treated with DMSO (control) or PKA inhibitor, 10 µM H89 or 250 uM RP-cAMP for 30 min, then treated ± 1 µM forskolin for 15 min and Western blotting analysis performed on Akt KA assays with indicated antibodies. B) Mean change ± SEM in Akt activity (P-GSK3/Akt) relative to DMSO control (n = 4). #, p < 0.05 vs DMSO control. C) INS-1 cells were treated ± 100 µM of the EPAC agonist, 8-cpt-2‟-O-Me-cAMP (8-cpt cAMP) for 15 min and Western blotting analysis performed on Akt KA assays with the indicated antibodies. D) Mean change ± SEM in Akt activity relative to control (n=4). #, p < 0.05 vs DMSO control. E) Mouse islets were treated ± 100 µM 8-cpt cAMP for 15 min and Western blotting analysis performed on Akt KA assays with the indicated antibodies (n=1). Immunoblotting with anti-GST (GSK3) antibodies were used as internal controls.

Figure 8. Stimulation of Akt by GIP is Mimicked by EPAC but is Not Modulated by 2+ Glucose Metabolism or Involve Ca , MEK 1/2 or iPLA2 signalling. A) INS-1 cells were pre- treated with DMSO (control) or inhibitors of PI3K/Akt signalling (15 µM Ly294002) for 30 min, then treated ± 10 nM GIP or 8-cpt cAMP for 15 min and Western blotting analysis performed on cell lysates with indicated antibodies. (B) INS-1 cells were pre-treated with DMSO (control), elevating levels of glucose (3, 8, or 16 mM), inhibitors of Ca2+ signalling (10 µM Nifedipine, 1 mM EGTA, or 100 µM BAPTA), inhibitors of MEK 1/2 signalling to ERK 1/2 (50 µM PD985002 or 5 µM U0126), an inhibitor of iPLA2 (15 µM HELSS), or the demonstrated Akt inhibitor (500 nM Akt IV) for 30 min, then treated ± 10 nM GIP for 15 min and Western blotting analysis performed on cell lysates with indicated antibodies. Blots are representative of at least three independent experiments and immunoblotting with anti-β-actin antibodies were used as internal controls. Note: in some cases, SDS-PAGE was prolonged to achieve greater protein resolution, and this sometimes resulted in a double band appearance in Western blotting such as that for P-MDM2 shown in B, right side.

3.3 Discussion In the present study, the role of PI3K in Akt activation by GIP was evaluated in the INS-1 β-cell. Consistent with previous reports (Kim et al., 2005b; Trümper et al., 2002; Trümper et al., 2001), GIP treatment elevated Akt activity in association with increased Akt pS473 levels (Fig. 1). However, GIP-mediated stimulation of Akt activity was not found to require PI3K or phosphorylation of Akt at Thr-308, and even the phosphorylation of Akt at Ser-473 was not essential for stimulation of enzyme activity in either INS-1 cells or mouse islets (Fig. 2A&B, D- F, 3C&D). This was in contrast to IGF-I, which was unable to activate Akt in the presence of PI3K/Akt pathway inhibitors (Fig. 2A&C) or activate transfected HA-Akt1 protein that contained alanine point mutations at the Thr-308 and Ser-473 residues (Fig. 3C&D), consistent with previous studies (Alessi et al., 1996a; Hanada et al., 2004). One caveat of using inhibitors is that they often lack absolute target specificity and, indeed, Ly294002 (Davies et al., 2000) and Akt VIII (Logie et al., 2007) have been shown to inhibit off-target proteins. However, since Ly294002 and Akt VIII are potent inhibitors of PI3K/Akt signalling, yet did not affect GIP- stimulated activation of Akt, such off-target effects of either inhibitor did not contribute to the responses obtained. Based on these results, it is apparent that GIP stimulated Akt via a non- canonical pathway. Although the mechanism of action of Akt IV has not been clarified, since it blocked the activation of Akt by GIP (Fig. 4), it is proposed that it interferes somehow with this alternative mode of Akt activation. Similar to GIP, GLP-1 also stimulated Akt in a PI3K independent manner (Fig. 5A&B), a finding that is consistent with the observations of Liu and Habener (2008), who demonstrated that Akt IV but not Ly294002 prevented Akt-mediated activation of the Wnt signalling pathway under GLP-1 stimulated conditions, although they did not assess the underlying mechanism of action. Furthermore, neither high glucose nor insulin increased Akt activity, under the experimental conditions applied. This finding is consistent with studies by Rhodes and colleagues, who have shown that neither insulin (Wicksteed et al., 2003) nor high glucose (Dickson et al., 2001) has any notable effect on Akt in β-cells during short-term stimulation. Thus the effects on Akt in the present study were a result of direct incretin stimulation and not autocrine effects of secreted insulin. Moreover, incretins stimulate Gαs (Drucker, 2006) and cAMP production (Ehses et al., 2003; Leech et al., 1996; Siegel and Creutzfeldt, 1985) in β-cells, and forskolin was also capable of stimulating Akt activity in a PI3K independent manner (Fig. 5C&D), indicating a likely role for cAMP in incretin-mediated

stimulation of Akt activity. Since a role for PKA was not supported in the current study (Fig. 6&7), it was proposed that this alternative mode of Akt activation could involve exchange proteins activated by cAMP (EPAC), which have been described to be activated by both GIP and GLP-1 in β-cells (Kashima et al., 2001). Indeed, it was shown that stimulation of INS-1 cells with 8-cpt cAMP could enhance Akt activity (Fig. 7&8). The mechanism by which EPAC mediates this response is unknown but based on the current findings, it does not involve 2+ signalling by glucose metabolism, intracellular Ca , ERK 1/2 or iPLA2 (Fig. 8). There has been a widely held view that incretins activate Akt solely via the canonical PI3K-mediated pathway (Baggio and Drucker, 2007; Holz, 2004; Kim and Egan, 2008; Salehi et al., 2008). This proposal was mainly based on the fact that PI3K inhibitors prevented the phosphorylation of Akt at Ser-473. However the current studies demonstrate that GIP and GLP-1 are capable of activating Akt independent of PI3K, indicating that the role of Akt in GLP-1 and GIP mediated actions may need to be re-evaluated. For example, in the recent study by Yusta et al. (2006) it was reasoned that Akt was not essential for GLP-1 mediated survival of β-cells under conditions of ER stress. This conclusion was based on the finding that Ly294002 blocked Akt phosphorylation, but did not influence GLP-1 mediated effects on eIF2α, ATF-4 or CHOP in cells exposed to thapsigargin. However, since Ly294002 does not appear to suppress GLP-1 mediated stimulation of Akt activity, a role for Akt cannot be excluded. The determination of phosphorylation state, rather than enzyme activity, may also explain disparate results regarding the mode of action of GLP-1 reported in the literature (Tews et al., 2008; Wang et al., 2004). Wang et al. (2004) showed that GLP-1 acutely stimulated phosphorylation of Akt at Ser-473 in a PI3K-dependent manner and concluded that GLP-1 rapidly increased Akt enzyme activity. On the other hand, Tews et al. (2008) using the same cell line, reported that phosphorylation of Ser- 473 did not occur until after 24 h and concluded that GLP-1 activated Akt indirectly through elevations in IRS2 protein levels. Previous studies on GIP-mediated activation of Akt in β-cells using PI3K inhibitors have examined only the phosphorylation of Akt at Ser-473 (Trümper et al., 2002) or applied inhibitor for prolonged periods of 6 h (Kim et al., 2005b). The functional relationships between the phosphorylation status of Thr-308 and Ser-473, early GIP stimulation of Akt kinase activity and phosphorylation of endogenous Akt substrates (e.g. MDM2) have not been previously reported in -cells. Based on the current results, it is clear that their phosphorylation is not required for Akt activation.

Alternative, PI3K-independent, pathways for Akt activation have been reported that involve stimulation of cAMP production (Sable et al., 1997) and PKA (Filippa et al., 1999), activation of calcium/calmodulin dependent kinase kinase (Yano et al., 1998) as well as other undefined pathways (Moule et al., 1997), but this appears to be the first demonstration of a hormonal activation of Akt that does not depend on phosphorylation of Akt pT308. This finding is contrary to the current consensus that phosphorylation of Thr-308 is essential for Akt activation (Alessi et al., 1996a; Hanada et al., 2004; Manning and Cantley, 2007; Yang et al., 2002a). The main reason that such an alternative pathway has been previously overlooked is probably because the focus of the majority of studies on the mechanism of Akt activation was via receptor tyrosine kinases (e.g. the IGF-I receptor), rather than G-protein coupled receptors, such as those for the incretins. Earlier studies have provided evidence of alternative pathways. β- adrenergic agonists were found to activate Akt in a wortmannin insensitive manner in rat adipocytes (Moule et al., 1997) but an in-depth analysis of the Akt status was not performed, because phosphorylation-site specific antibodies were not yet available. Since forskolin stimulation led to Akt activation in INS-1 cells, similar to GIP stimulation, it is quite likely that activation of several other GPCR proteins that are present on β-cells (e.g. PACAP and glucagon) also promote similar responses. The biological significance of this finding will be an interesting avenue of future research. Three major questions arise from this study: 1) What is the signalling mechanism by which EPAC mediates Akt activation by incretins? 2) Can Akt be activated in a similar manner in vivo, and if so, can this be exploited as a therapy for diabetes? 3) Is Akt activated in a similar manner in other tissues by other hormones or growth factors? Since, in addition to its importance in β-cell survival, Akt is also crucial for insulin-mediated glucose disposal (Whiteman et al., 2002) and the coordination of a wide range of additional biological actions (Manning and Cantley, 2007); answers to these questions may uncover further aspects of Akt physiology that can be targeted for therapeutics.

Chapter 4 Effects of GIP on the Mitochondrial Apoptotic Pathway 4.1 Introduction The mitochondria-mediated apoptotic pathway is thought to play a significant role in the loss of β-cell mass in patients with T2D (Donath et al., 2008; Eizirik et al., 2008; Evans et al., 2002; Scheuner and Kaufman, 2008). Many aspects of this process are evolutionarily conserved throughout vertebrates with a critical step being the release of cytochrome C from mitochondria (Oberst et al., 2008). In the cytoplasm, cytochrome C binds to the apoptosis protease-activating factor 1 (APAF1), inducing a conformational shift that exposes the caspase activation and recruitment domain, which results in dimerization, cleavage and activation of the initiator cysteine/aspartate protease (caspase)-9 that itself then cleaves and activates executioner caspase- 3 and caspase-7, the key proteases and the so-called „point of no return‟ in apoptotic programmed cell death (Galluzzi et al., 2008; Li and Yuan, 2008). The release of cytochrome C from mitochondria is regulated by the B-cell lymphoma (Bcl-2) family proteins, which contain Bcl-2 homology domains (BH) that are critical for their functions and interactions (Chipuk et al., 2010). These proteins are functionally classified as anti-apoptotic proteins such as Bcl-2 and Bcl- XL or pro-apoptotic proteins such as Bcl-2 associated X protein (Bax), Bcl-2 antagonist killer (Bak), Bcl-2 interacting mediator of cell death (Bim), p53-upregulated modulator of apoptosis (Puma), BH3 interacting domain death agonist (Bid) and the Bcl-2 associated death promoter (Bad). Furthermore, the Bax and Bak pro-apoptotic proteins that contain BH domains 1-4 translocate and insert into mitochondria, and, upon activation, homo-oligomerize into proteolipid pores, causing the release of cytochrome C. The anti-apoptotic Bcl-2 and Bcl-XL proteins also contain multiple BH domains and prevent apoptosis by inhibiting Bax and Bak. The other set of pro-apoptotic proteins contain only the BH3 domain, which is critical for functional interactions. Some, such as Bad, sensitize cells to apoptosis by inhibiting anti-apoptotic proteins, whereas others, such as Bim, promote activation of Bax and Bak (Chipuk et al., 2010; Kim et al., 2006b; Kim et al., 2009; Reed, 2006; Shi, 2002). Ultimately the regulation of the mitochondrial apoptotic pathway is coordinated by inputs on Bcl-2 family proteins, involving transcriptional and post-translational modifications. The mechanisms by which GIP promotes β-cell survival are unclear. GIP stimulation promoted survival and inhibited activation of caspase-3 in STZ-treated INS-1 cells (Pospisilik et al., 2003), and prevented the activation of caspase-9, poly-ADP ribose polymerase and caspase- 3, thus promoting the survival of glucose- and serum-starved INS-1 cells (Ehses et al., 2003; 57

Trümper et al., 2002). Subsequently, it was shown that GIP promotes the survival of β-cells exposed to glucolipotoxic stress, and this was associated with reduced transcription of Bax and increased transcription of Bcl-2 (Kim et al., 2008b; Kim et al., 2005b). These findings revealed that GIP signalling may inhibit the mitochondrial apoptotic pathway by interacting with Bcl-2 family proteins. In the current study, the modulatory effects of GIP on the dynamic interactions between cytoplasmic Bcl-2 family members and the mitochondria were examined in cultured β- cells exposed to apoptosis-inducers.

4.2 Results 4.2.1 GIP Promotes the Survival of β-cells Exposed to Glucolipotoxic Stress Studies investigating the effects of GIP on β-cell survival have generally used INS-1 cells, but in some instances species differences in β-cell responses have been observed (Parnaud et al., 2008). Observations in rodent cell-lines or primary β-cells may therefore not necessarily translate to humans. It was therefore determined whether the survival effects of GIP were conserved among cultured INS-1 β-cells and dispersed mouse and human islets. Cells were exposed to 25 mM glucose and 200 µM palmitate (glucolipotoxicity) under serum starved conditions. Each type of β-cell exhibited some degree of cell death within 20 h of glucolipotoxic exposure. Moreover, GIP promoted the survival of INS-1 cells in a time and concentration- dependent manner (Fig. 9A-D), as determined via apoptosis and cell death assays and/or Western blotting analysis for cleaved caspase-3, and also promoted the survival of dispersed mouse islets (Fig. 9E&F) and dispersed human islets (Fig. 9G) in a similar fashion, demonstrating that the pro-survival effects exerted via GIPR activation are of relevance for human β-cells.

4.2.2 GIP Signalling Inhibits the Mitochondria-mediated Apoptotic Pathway The anti-apoptotic action of GIP on β-cells under glucolipotoxic stress was observed in studies lasting 15-24 h and likely involved pleiotropic effects on gene expression, protein action and apoptotic pathways. In order to examine whether GIPR signalling directly regulates the interactions of pro- and anti-apoptotic Bcl-2 family proteins with mitochondria, studies were performed on INS-1 β-cells exposed to staurosporine (STS), an apoptotic agent that has been shown in INS-1 cells to rapidly promote the generation of reactive oxygen species and mitochondrial lipid peroxidation, loss of mitochondrial membrane potential and onset of the mitochondrial apoptotic pathway (Seleznev et al., 2006). In studies whereby INS-1 cells were

treated with STS ± GIP, stimulation with GIP significantly delayed onset and reduced levels of cell death and apoptosis (Fig. 10A&B) in a concentration-dependent manner (EC50=515±32 pM; Fig. 10C). In parallel, effects of 0-6 h STS treatment on the release of cytochrome C from mitochondria into the cytoplasm and cleavage of caspase-3 were examined. After 4 h, STS significantly increased both parameters (Fig. 10D&E), and these responses were potently suppressed by GIP stimulation, indicating that GIP promoted cell survival via suppressing activation of the mitochondria-mediated apoptotic pathway.

4.2.3 GIP Anti-apoptotic Signalling Require cAMP Production but not Insulin’s Actions In β-cells, GIP activates adenylate cyclase and subsequently the production of cAMP (Drucker, 2006; McIntosh et al., 2009). To determine whether cAMP was required for GIP- mediated survival signalling, INS-1 cells were treated without or with STS ± GIP for 6 h in the absence or presence of the adenylate cyclase inhibitor, MDL-12,300A. The inhibitor had no additive effect on STS-induced cell death yet completely ablated the pro-survival effects of GIP (Fig. 11A), thus showing that GIP-mediated survival requires the production of cAMP. Since GIP-mediated cAMP production promotes insulin release via PKA (Ding and Gromada, 1997) and EPAC2 (Kashima et al., 2001), this raised the possibility that part of the pro-survival effects of GIP were mediated via insulin autocrine actions. Although studies were performed with low glucose conditions, under which GIP does not stimulate insulin release (Kim et al., 2005a), reports that low levels of insulin (≈200 pM) exerted growth effects on β-cells (Beith et al., 2008; Johnson et al., 2006) made it necessary to test whether GIP could have acted via insulin signalling. This was examined by treating INS-1 cells ± STS with increasing concentrations (0- 100 nM) of insulin for 6 h and comparing responses to those with GIP, forskolin and IGF-I. While significant protection of INS-1 cells was observed with GIP, forskolin and IGF-I, insulin had no effect except with an extremely high level (100 nM) that is capable of cross reacting with the IGF-IR (Fig. 11B). Based on this finding, it was considered unlikely that insulin played a significant role in promoting INS-1 cell survival under GIP-treated conditions.

4.2.4 GIP Inhibits Mitochondrial Bad and Bim Translocation and Bax Activation Since mitochondrial release of cytochrome C and initiation of the apoptotic program is coordinated by the Bcl-2 family of proteins (Harwood et al., 2005; Reed, 2006; Shi, 2002), the effects of GIP stimulation on mitochondrial levels of the pro-apoptotic proteins, Bax, Bad and

BimEL, and two anti-apoptotic proteins, Bcl-2 and Bcl-XL, were examined in STS-treated INS-1 cells. Following treatment with STS ± GIP for 4 h, total or mitochondrial/cytoplasmic protein fractions were collected and analyzed via Western blotting analysis (Fig. 12). STS induced a significant increase in mitochondrial levels of Bcl-XL, Bad and BimEL, and there was a trend for increased Bax, but Bcl-2 levels were unaltered. In the presence of GIP, levels of mitochondrial Bax, Bad and BimEL were all similar to control levels. However, GIP had no effect on levels of mitochondria-associated Bcl-XL. STS also induced a significant elevation in the total levels of Bad protein, an effect that was ablated by GIP treatment. The elevated levels of mitochondrial Bad and BimEL and the associated release of cytochrome C indicated that Bax was activated in STS treated cells. Upon activation, Bax undergoes a conformational shift and forms homodimers, as well as heterodimers with Bak, and it has been established that this activated state can be measured using the bismaleimidohexane (BMH) crosslinking method (Kim et al., 2006b). Therefore, in parallel to the studies above, the functional state of Bax was examined in mitochondrial samples collected from INS-1 cells treated without or with STS ± GIP. Treatment with STS resulted in a dramatic increase in levels of crosslinked Bax, and this effect was significantly diminished by GIP stimulation (Fig. 13A). Moreover, the functional change in Bax activity was directly linked to INS-1 β-cell survival, since a Bax channel blocker was found to suppress the onset of STS-induced cell death in a concentration-dependent manner (Fig. 13B). Collectively, these studies indicated that the protective effects of GIP on STS treated INS-1 cells were largely mediated through inhibition of Bax activity, which likely involves a signalling pathway that inhibits translocation of pro- apoptotic Bad and BimEL proteins to the mitochondria.

Figure 9. GIP Promotes the Survival of INS-1 Cells and Dispersed Mouse and Human Islets Exposed to Glucolipotoxic Stress. A) INS-1 cells were treated with control (5 mM glucose) or glucolipotoxic (25 mM glucose + 200 µM palmitate) media ± 10 nM GIP for 0-20 h and onset of apoptosis determined. B) INS-1 cells were treated with control, 25 mM glucose, 200 µM palmitate, or glucolipotoxic (GLT) media ± 10 nM GIP for 15 h and onset of apoptosis determined. C) INS-1 cells were treated with control or GLT media ± elevating levels of GIP (0- 100 nM) for 15 h and onset of apoptosis determined. Mean ± SEM (n=6); #, p < 0.05 vs GLT without GIP or as indicated. D) INS-1 cells were treated with control or GLT media ± 10 GIP for 15 h and Western blotting analysis performed with indicated antibodies. Immunoblotting with anti-β-actin antibodies were used as an internal control. Blots are representative of at least three independent experiments.

Figure 9 continued. E) Dispersed mouse islets were treated with control or GLT media ± 10 nM GIP for 24 h and onset of apoptosis determined. Mean ± SEM (n=4); #, p < 0.05 as indicated. F) Dispersed mouse islets were treated with control or GLT media ± 10 nM GIP for 0-40 h and levels of cell death (PI+) determined. Mean ± SEM (n=4); #, p < 0.05 vs GLT without GIP. Dispersed human islets were treated with control or GLT media ± 10 nM GIP for 20 h and % cell death determined. Mean ± SEM (n=4); #, p < 0.05 as indicated.

Figure 10. GIP Inhibits the Mitochondria-mediated Apoptotic Pathway in Staurosporine (STS) Treated INS-1 Cells. A) INS-1 cells were treated without or with STS (100 nM) ± 10 nM GIP for 0-10 h and levels of cell death (PI+) determined. Mean ± SEM (n=4); significance as indicated. B) INS-1 cells were treated without or with STS (100 nM) ± 10 nM GIP for 0-8 h and onset of apoptosis determined. Mean ± SEM (n=4); $, p < 0.05 vs STS without GIP. C) INS-1 cells were treated without or with STS + increasing concentrations of GIP (0-100 nM) for 6 h and onset of apoptosis determined. Mean ± SEM (n=7); $, p < 0.05 vs DMSO control; #, p < 0.05 vs STS without GIP. In the upper right is the calculated EC50 value. D) INS-1 cells were treated without or with STS ± 10 nM GIP for 0-4 h and Western blotting analysis performed on mitochondrial or cytoplasmic protein fractions with the indicated antibodies. E) INS-1 cells were treated without or with STS ± 10 nM GIP for 0-6 h and Western blotting analysis performed on whole cell lysates with indicated antibodies. Blots shown in D and E are representative of at least three independent experiments. Immunoblotting with anti-β-actin, anti-β-tubulin and anti-COX IV antibodies were used as internal controls.

Figure 11. GIP Mediated Anti-apoptotic Signalling Requires the Production of cAMP but Does Not Involve Insulin Autocrine Signalling. A) INS-1 cells were treated without or with STS (100 nM) ± 10 nM GIP for 6 h in the presence or absence of the adenylate cyclase inhibitor, MDL-12,300A (200 µM), and cell death determined. Mean ± SEM (n=5) #, p < 0.05 vs DMSO control; $, p < 0.05 vs STS without GIP; %, p < 0.05 vs STS + GIP without MDL-12,300A. B) INS-1 cells were treated without or with STS (100 nM) + increasing concentrations of insulin (0- 100 nM), IGF-I (10 nM), GIP (10 nM) or forskolin (1 µM) for 6 h. Mean ± SEM (n=8); #, p < 0.05 vs STS without GIP.

Figure 12. GIP Dynamically Regulates Mitochondrial Levels of Bad and BimEL. A) INS-1 cells were treated without or with STS (100 nM) ± 10 nM GIP for 4 h and Western blotting analysis performed on mitochondrial and cytoplasmic protein fractions and total cell lysates with indicated antibodies. B-D) For quantification, protein levels were normalized to β-tubulin (cytoplasmic; B), COX IV (mitochondrial; C) or β-actin (cell lysates; D). Mean ± SEM (n=3-6) changes in protein levels relative to control; #, p<0.05 as indicated.

Figure 13. GIP Promotes Survival of STS-treated INS-1 Cells by Suppressing the Activation of Mitochondrial Bax. A) INS-1 cells were treated without or with STS ± 10 nM GIP for 4 h and Western blotting analysis performed with anti-Bax or anti-Cox IV antibodies on mitochondrial protein fractions that were incubated with 10 mM bismaleimidohexane (BMH) for 30 min at room temperature. Note that BMH treated fractions display 2 bands, one is the predicted molecular mass of Bax (Monomeric Bax) and the other is approximately double its mass (Dimeric Bax). Blots are representative of at least three independent experiments. Immunoblotting with anti-COX IV antibodies were used as internal controls. B) INS-1 cells were treated without or with STS + increasing concentrations of Bax channel blocker (0-5 µM) for 6 h and cell death determined. Mean ± SEM of cell death (n=5); #, p < 0.05 vs STS alone.

4.2.5 GIP Anti-apoptotic Signalling Does Not Require Transcriptional Changes Given the rapid onset of STS-induced INS-1 β-cell death, it is unlikely that the effects of STS or GIP involved changes in gene transcription, but rather pro- and anti-apoptotic signals mediated via the existing Bcl-2 protein family members. However, since there was a significant elevation in Bad protein levels in cell lysates (Fig. 12A&D), a role for transcriptional changes could not be excluded, and therefore, INS-1 cells were treated without or with STS ± GIP in the absence or presence of the mRNA translation inhibitor, cycloheximide (CHX). Treatment of cells with CHX and/or STS resulted in increased cell death in association with an elevation in Bad protein, and stimulation with GIP significantly reduced these effects (Fig. 14). Transcriptional changes were therefore not required for the activation of cell death by STS or the inhibition of cell death by GIP. Moreover, since STS ± inhibition of protein translation with CHX resulted in elevated Bad protein levels, the increase in Bad with STS treatment was likely due to a decrease in Bad degradation.

4.2.6 GIP Anti-apoptotic Actions Involves Akt Signalling In an attempt to identify signalling modules by which GIP inhibited STS-induced Bax activation and apoptosis, the effects of GIP on STS-treated INS-1 cells were examined in the absence or presence of an Akt inhibitor (Akt IV), PI3K/Akt pathway inhibitors (Ly294002, Akt VIII or wortmannin) or inhibitors of other kinases shown to be involved in GIP action: ERK 1/2 (UO126 or PD985002) or PKA (H89). Interestingly, the identified inhibitor of GIP-mediated Akt activation, Akt IV, was found to inhibit the survival effects of GIP on STS-induced INS-1 cells, whereas all other inhibitors were without effect, including PI3K/Akt pathway inhibitors (Fig. 15A), indicating that GIP promoted survival via the non-canonical mechanism of Akt activation identified in Chapter 3. As supportive evidence, IGF-I promoted survival of STS-treated INS-1 cells by a pathway that was blocked by inhibiting PI3K/Akt signalling with Akt VIII (Fig. 15D), indicating that Akt signalling was preventing apoptosis, but that the mechanism of Akt activation by IGF-I differed from GIP. Furthermore, forskolin was also found to promote the survival of STS-treated INS-1 cells in the presence or absence of H89, but not in the presence of Akt IV (Fig. 15B), and EPAC2 activation with 8-cpt cAMP promoted the survival of STS-treated INS-1 cells in a concentration-dependent manner (Fig. 15C). Both of these agents were also shown to stimulate Akt activity in a similar manner to GIP (Chapter 3).

To further investigate a role for Akt in mediating the anti-apoptotic actions of GIP, INS-1 cells were transfected with wild type HA-Akt1 or eGFP and then treated with STS ± GIP. The percent reduction and absolute reduction in cell death resulting from GIP treatment were both greater in cells transfected with wild type HA-Akt1 than those transfected with eGFP (Fig. 15E) as was the reduction in levels of cleaved caspase-3 (Fig. 15F). In addition, to validate that GIP enhanced Akt signalling under STS-treated conditions, INS-1 cells were incubated with or without Akt IV, Ly294002 or Akt VIII prior to and during a 4 h treatment with STS ± GIP. GIP stimulation resulted in Akt activation as demonstrated by enhanced phosphorylation levels of the Akt substrates, Foxo1, Raf1 and MDM2, in cell lysates, and GSK3 in Akt KA assays, despite the loss of Akt phosphorylation in Ly294002 and Akt VIII treated INS-1 cells (Fig. 16). Conversely, despite robust increases in Akt phosphorylation in Akt IV treated cells, activation of Akt by GIP was completely ablated in association with increased levels of cleaved caspase-3. Together these findings indicate that the anti-apoptotic effects of GIP on STS-treated INS-1 cells involves Akt signalling modules, and consistent with the results in Chapter 3, clearly does not require Akt activation via phosphorylation of Thr-308 and Ser-473.

Figure 14. GIP-mediated Anti-apoptotic Signalling in STS-treated Cells Does Not Require Transcriptional Changes. INS-1 cells were treated without or with STS (100 nM) ± 10 nM GIP for 4 h (A) or 6 h (B) in the presence or absence of the mRNA translation inhibitor, cycloheximide (CHX; 10 µg/ml), and Western blotting analysis performed on cell lysates with indicated antibodies (A) or cell death determined (B). Mean ± SEM of cell death (n=4); #, p < 0.05 vs DMSO control; $, p < 0.05 vs STS without GIP; %, p < 0.05 vs STS + CHX without GIP. Immunoblotting with anti-β-actin antibodies were used as internal controls. Blots are representative of at least three independent experiments.

Figure 15. GIP Stimulation of Survival in STS-treated INS-1 Cells Involves Akt. A) % cell death in INS-1 cells treated for 6 h with STS (100nM) ± 10 nM GIP without or with inhibitors of Akt (500 nM Akt IV), PI3K signalling (15 µM Ly294002, 5 µM Akt VIII, or 200 nM wortmannin), ERK 1/2 signalling (5 µM U0126 or 50 µM PD985002), or PKA signalling (10 µM H89). Mean ± SEM of dead cells (n = 6); #, p < 0.05; comparisons shown in panel. B) % cell death in INS-1 cells treated for 6 h with STS (100nM) ± 1 µM forskolin without or with inhibitors of Akt signalling (500 nM Akt IV) or PKA signalling (10 µM H89). Mean ± SEM of % cell death (n = 6); #, p < 0.05 vs DMSO control; $, p < 0.05 vs STS without inhibitor; %, p < 0.05 vs STS + forskolin. C) % cell death in INS-1 cells treated for 6 h with STS (100 nM) + increasing concentrations of the EPAC agonist, 8-cpt cAMP. Mean ± SEM of dead cells (n = 6); #, p < 0.05 vs DMSO control; $, p < 0.05 vs STS without 8-cpt cAMP. D) % Cell death in INS-1 cells treated for 6 h with STS ± 10 nM IGF-I ± PI3K signalling inhibitor, 5 µM Akt VIII. Mean ± SEM of dead cells (n = 4); #, p < 0.05; comparisons shown in panel.

Figure 15 continued. E) % Cell death in INS-1 cells transfected with eGFP or HA-Akt1 and treated for 6 h with STS ± GIP. Mean ± SEM of % cell death (n = 4); significance shown in panel. F) INS-1 cells were transfected with eGFP or HA-Akt1 and treated for 4 h with STS ± GIP and Western blotting analysis performed with indicated antibodies. Immunoblotting with anti-β-actin antibodies were used as internal controls. Blots are representative of at least three independent experiments.

Figure 16. GIP Enhances Akt Signalling in STS-treated INS-1 Cells. A) INS-1 cells treated with STS ± GIP for 4 h without or with Akt inhibitor (500 nM Akt IV) or PI3K signalling inhibitors (15 µM Ly294002 or 5 µM Akt VIII) and Western blotting analysis performed on cell lysates and Akt KA assays with indicated antibodies. Immunoblotting with anti-β-actin and anti- GST (GSK3) antibodies were used as internal controls. Blots are representative of at least three independent experiments.

4.2.7 GIP Anti-apoptotic Actions are Mediated via Akt-dependent Suppression of p38 MAPK and JNK Since treatment with STS resulted in actions on Bcl-2 family proteins that were reversed by GIP, attempts were made to identify the upstream signalling events involved. Both p38 MAPK and JNK are central pro-apoptotic proteins in β-cells (Donath et al., 2008; Eizirik et al., 2008; Evans et al., 2002; Haataja et al., 2008; Kim et al., 2005c; Poitout and Robertson, 2008; Scheuner and Kaufman, 2008; Sumara et al., 2009) that can act on Bax (Kim et al., 2006a), Bad (Grethe et al., 2006; Grethe and Porn-Ares, 2006) and BimEL (Cai et al., 2006). Since the findings described above revealed that Akt signalling mediates the anti-apoptotic actions of GIP, studies using selective inhibitors, overexpression of dominant negative Akt (Akt-DN) and Akt 1&2 siRNA were used to determine if GIP promoted INS-1 cell survival via Akt-dependent inhibition of p38 MAPK and/or JNK. INS-1 cells were treated without or with STS ± GIP for 4 h in the absence or presence of Akt-IV. Treatment with STS resulted in elevated levels of phosphorylated MEK 3/6 (upstream kinase of p38 MAPK), p38 MAPK and JNK (active forms), and this was associated with a marked dephosphorylation of Bad at Ser-112 (activated form (Yang et al., 1995; Zha et al., 1996)) and elevated levels of cleaved caspase-3 (Fig. 17A). GIP stimulation greatly reduced the effects of STS on MEK 3/6, p38 MAPK, JNK, Bad and caspase- 3 in the absence, but not in the presence, of Akt-IV. Parallel experiments were then performed with INS-1 cells transfected with GFP or Akt-DN (Fig. 15B) as well as with cells transfected with scramble or Akt 1&2 siRNA (Fig. 15C; Fig. 15D demonstrates the selective knockdown of Akt 1&2, and not Akt 3). Similar to the effects of Akt-IV, overexpression of Akt-DN and knockdown of Akt 1 and 2 clearly reduced the ability of GIP to suppress the effects of STS on p38 MAPK, JNK, Bad and caspase-3 (Fig. 17B&C). To determine if the effects of STS on phosphorylated Bad at Ser-112 and INS-1 cell survival were due to activation of p38 MAPK and/or JNK, INS-1 cells were treated without or with STS ± the p38 MAPK inhibitors, SB 203580 and SB 202190 and/or the JNK inhibitors, SP600125 and JNK inhibitor VIII (Fig. 18). Inhibition of p38 MAPK and JNK promoted the survival of STS-treated INS-1 cells in a concentration-dependent manner (Fig. 18A&C) and prevented STS-induced dephosphorylation of Bad at Ser-112 and cleavage of caspase-3 (Fig. 18B). Noteworthy was the fact that the survival of INS-1 cells treated with both inhibitors was equivalent to that achieved with GIP (Fig. 18C). To additionally validate the findings observed in INS-1 cells, the effects of STS ± GIP and STS ± inhibitors of p38 MAPK and JNK were

determined in an alternative β-cell line, mouse insulinoma (MIN6) cells. As with INS-1 cells, treatment of MIN6 cells with STS induced phosphorylation of p38 MAPK and JNK, dephosphorylation of Bad at Ser-112 and elevated levels of cleaved caspase-3 and cell death (Fig. 19A&B), and all of these effects were diminished by GIP. Similarly, inhibiting p38 MAPK and JNK with SB203580 + SP600125 suppressed STS-induced dephosphorylation of Bad at Ser- 112 as well as the increase in levels of cleaved caspase-3 and cell death (Fig. 19C&D). To determine whether the effects of GIP on Bcl-2 family proteins (Fig. 12&13) were due to inhibition of p38 MAPK and JNK, INS-1 cells were treated without or with STS ± both SB203580 + SP600125 and mitochondrial/cytoplasmic fractions collected for analysis of levels of mitochondrial BimEL, Bax and Bad. Inhibition of p38 MAPK and JNK in INS-1 cells prevented STS induced elevations in BimEL, Bax and Bad (Fig. 20A), similar to those found in cells treated with GIP. Moreover, STS-induced activation of mitochondrial Bax was also prevented by p38 MAPK and JNK inhibitors (Fig. 20B). Collectively, these studies indicated that the suppression of p38 MAPK and JNK by GIP could explain the anti-apoptotic actions of GIP in STS-treated INS-1 cells.

Figure 17. GIP Anti-apoptotic Signalling Requires Akt and Involves Suppression of p38 MAPK and JNK. A) INS-1 cells were treated without or with STS (100 nM) ± 10 nM GIP for 4 h in the absence or presence of Akt inhibitor IV (Akt IV; 500 nM) and Western blotting analysis performed on total cell lysates with indicated antibodies. B) INS-1 cells transfected with GFP or dominant negative Akt (Akt-DN) were treated without or with STS (100 nM) ± 10 nM GIP for 4 h and Western blotting analysis performed on total cell lysates with indicated antibodies. Immunoblotting with anti-β-actin antibodies were used as internal controls. Blots are representative of at least three independent experiments.

Figure 17 continued. C) INS-1 cells transfected with scramble or Akt 1&2 siRNA were treated without or with STS (100 nM) ± 10 nM GIP for 4 h and Western blotting analysis performed on total cell lysates with indicated antibodies. Of note, total protein levels of Akt were reduced 60- 70% in cells transfected with Akt 1&2 siRNA, as shown in D) in which INS-1 cells were transfected with scramble (200 nM) or Akt 1&2 (100 nM each) siRNA. Levels of Akt 1 and Akt 2 were reduced but not Akt 3. Immunoblotting with anti-β-actin antibodies were used as internal controls. Blots are representative of at least three independent experiments.

Figure 18. Inhibitors of p38 MAPK and JNK Suppress STS-induced INS-1 Cell Death in a Concentration Dependent Manner. A) INS-1 cells were treated without or with STS (100 nM) ± increasing concentrations (0.5-10 µM) of p38 MAPK inhibitors (SB 203580 or SB 202190) or JNK inhibitors (SP600125 or JNK Inhibitor VIII) for 6 h and cell death determined. Mean ± SEM of cell death (n=4); #, p < 0.05 vs STS only. B) INS-1 cells were treated without or with STS ± p38 MAPK inhibitor (5 µM SB 203580) or JNK inhibitor (5 µM SP 600125) for 4 h and Western blotting analysis performed on total cell lysates with indicated antibodies. Immunoblotting with anti-β-actin antibodies were used as internal controls. Blots are representative of at least three independent experiments. C) INS-1 cells were treated without or with STS ± GIP, SB 203580, SP600125, or SB 203580 + SP600125 for 6 h and % cell death determined. Mean ± SEM of cell death (n=6); #, p < 0.05 vs STS alone.

Figure 19. GIP Promotes Survival of STS-treated MIN6 Cells via Suppression of p38 MAPK and JNK. A) MIN6 cells were treated without or with STS (500 nM) ± 10 nM GIP for 4 h and Western blotting analysis performed on total cell lysates with indicated antibodies. B) MIN6 cells were treated without or with STS (500 nM) ± 10 nM GIP for 6 h and cell death determined. Mean ± SEM (n=4); #, p < 0.05 as indicated. C) MIN6 cells were treated without or with STS (500 nM) ± p38 MAPK inhibitor (5 µM SB 203580) and JNK inhibitor (5 µM SP600125) for 4 h and Western blotting analysis performed on total cell lysates with indicated antibodies. D) MIN6 cells were treated without or with STS ± p38 MAPK inhibitor (5 µM SB 203580) and JNK inhibitor (5 µM SP600125) for 6 h and cell death determined. Mean ± SEM of cell death (n=4); #, p < 0.05 as indicated. Immunoblotting with anti-β-actin antibodies were used as internal controls. Blots are representative of at least three independent experiments.

Figure 20. Inhibition of p38 MAPK and JNK Signalling Mimics the Survival Actions of GIP. A) INS-1 cells were treated without or with STS ± SB 203580 and SP600125 for 4 h and Western blotting analysis performed on mitochondrial and cytoplasmic fractions with indicated antibodies. B) INS-1 cells were treated without or with STS ± SB 203580 and SP600125 for 4 h and Western blotting analysis performed with anti-Bax or anti-Cox IV antibodies on mitochondrial fractions that were incubated with 10 mM bismaleimidohexane (BMH) for 30 min at room temperature. Note that BMH treated fractions display 2 bands, one is the predicted molecular mass of Bax (Monomeric Bax) and the other is approximately double its mass (Dimeric Bax). Blots are representative of at least three independent experiments. Immunoblotting with anti-β-actin, anti-β-tubulin and anti-COX IV antibodies were used as internal controls.

4.2.8 GIP Inhibits p38 MAPK and JNK via Akt-mediated Phosphorylation of ASK1 The regulatory link was then sought between Akt signalling and dual inhibition of p38 MAPK and JNK in STS-treated INS-1 cells. The Apoptosis Signal regulating Kinase-1 (ASK1) is an upstream MAPK kinase kinase that phosphorylates (activates) both MEK 3/6 and MEK 4/7, which then phosphorylate their downstream targets, p38 MAPK and JNK, respectively (Ichijo et al., 1997; Takeda et al., 2008). Akt has been shown to phosphorylate ASK1 at the Ser-83 residue (pS83) in several cell lines (Kim et al., 2001; Zhang et al., 2005), resulting in ASK1 inactivation, and this was associated with decreased phosphorylation at Thr-845 (pT845), a site that is important for its activation (Takeda et al., 2008; Zhang et al., 2005). It was therefore evaluated whether GIP inhibited ASK1 activity in INS-1 cells via elevating phosphorylated levels of ASK1 pS83 and preventing STS-induced increases in phosphorylated levels of ASK1 pT845. Since currently available antibodies directed against the phosphorylated ASK1 pS83 and pT845 epitope recognize the human protein but not that from rat, INS-1 cells were transfected with a pcDNA3 vector that expressed the wild type human ASK1 protein, and then treated without or with STS ± GIP and levels of phosphorylated ASK1 pS83 and pT845 determined via Western blotting analysis. In untransfected INS-1 cells, endogenous (rat) ASK1 protein was detected but, as expected, there were no detectable levels of phosphorylated ASK1 pS83 or pT845, and in transfected cells, the human ASK1 protein was efficiently expressed (Fig. 21A). In STS-treated INS-1 cells, the levels of phosphorylated human ASK1 pS83 were markedly reduced and phosphorylated human ASK1 pT845 levels were increased relative to untreated cells (control), whereas GIP treatment neutralized the effects of STS on the phosphorylation state of human ASK1. Since the phosphorylation state of endogenous ASK1 could not be detected in cell lysates from rodent cells, to evaluate whether GIP regulated endogenous ASK1 activity in STS-treated β-cells, untransfected cells were treated without or with STS ± GIP and then endogenous ASK1 protein was immunoprecipitated and ASK1 enzyme activity determined using an in vitro kinase assay. With this assay, treatment of INS-1 cells with STS caused a significant elevation in ASK1 enzyme activity, but this effect was greatly reduced in the presence of GIP stimulation (Fig. 21B&C), thus revealing that GIP regulated endogenous ASK1 signalling in INS-1 cells. To verify that the effects of STS on p38 MAPK and JNK were mediated by ASK1, INS-1 cells were transfected with a pcDNA3 vector expressing GFP (control) or kinase-dead ASK1 (dominant negative) and treated without or with STS. While there was no detectable effect in cells not treated with STS, the effects of STS on p38 MAPK, JNK, and caspase-3 were reduced

in INS-1 cells expressing kinase dead ASK1 relative to those expressing GFP (Fig. 22A). To then verify that the protective effects of GIP on STS-treated INS-1 cells was due to Akt- mediated phosphorylation of ASK1 pS83, INS-1 cells were transfected with a pcDNA3 vector expressing wild type ASK1 (ASK1WT) or ASK1 containing an alanine mutation at Ser-83

(ASK1S83A) and treated without (control) or with STS ± GIP. As validation of this mutant, Western blotting analysis with anti-ASK1 pS83 antibody showed detectable levels of phosphorylated ASK1 pS83 in INS-1 cells transfected with ASK1WT but not ASK1S83A (Fig. 22B). But more importantly, the ability of GIP to suppress the effects of STS on p38 MAPK,

JNK and caspase-3 was clearly reduced in INS-1 cells expressing ASK1S83A relative to those expressing ASK1WT. Lastly, to establish that phosphorylation of transfected human ASK1 pS83 and pT845 equated to effects of GIP on endogenous ASK1 in β-cells, responses to GIP were examined in human islets treated without or with STS ± GIP. Treatment of human islets with STS resulted in decreased levels of phosphorylated ASK1 pS83, increased levels of phosphorylated ASK1 pT845 and MEK 3/6, as well as increases in levels of cleaved caspase-3 (Fig. 22C). However, these effects were ablated in the presence of GIP, indicating that it had a similar role in regulating ASK1 activity in islets to that observed with INS-1 cells.

4.2.9 Inhibiting p38 MAPK and JNK is a Core Anti-apoptotic Mechanism of GIP Action It was next determined whether suppression of p38 MAPK and JNK with GIP could also promote survival responses to other pro-apoptotic stimuli. INS-1 cells were treated without or with 100 nM STS (mitochondrial stress), 500 nM thapsigargin (ER stress) or 5 µM etoposide (genotoxic stress) ± GIP for 4 h, followed by Western blotting analysis. In the presence of STS, thapsigargin or etoposide, levels of phosphorylated p38 MAPK and JNK were increased, levels of phosphorylated Bad at Ser-112 were decreased and levels of cleaved caspase-3 increased, and each of these effects was greatly reduced with GIP treatment (Fig. 23A). In parallel experiments extended to 6 h, levels of cell death were approximately 30% in cells treated with STS, thapsigargin or etoposide, and GIP significantly reduced these levels with all apoptosis- promoting agents (Fig. 23B). Treatment of INS-1 cells without or with STS, thapsigargin or etoposide ± SB 203580 or SP600125 demonstrated that inhibition of either p38 MAPK or JNK reduced cell death (Fig. 23C). Collectively these findings indicated that inhibition of p38 MAPK and JNK is a key component of GIP-mediated anti-apoptotic signalling mechanisms in stressed β-cells.

Figure 21. GIP Inhibits the Activity of Apoptosis Signal Regulating Kinase-1 (ASK1) in STS-treated INS-1 Cells. A) INS-1 cells transfected without or with GFP or human ASK1 were treated without or with STS (100 nM) ± 10 nM GIP for 4 h and Western blotting analysis performed on total cell lysates with anti-ASK1 pS83 (P-ASK1 83), anti-ASK1 pT845 (P-ASK1 845), anti-ASK1 or anti-β-actin antibodies. B) Untransfected INS-1 cells were treated without or with STS ± GIP for 4 h and ASK1 in vitro kinase assays performed on ASK1 protein that was immunoprecipitated with anti-ASK1 antibody. C) Mean ± SEM change in ASK1 in vitro kinase activity relative to control is shown (n=5). Immunoblotting with anti-β-actin and anti-GST (MEK 6) antibodies were used as internal controls. Note: anti-ASK1 antibody detects both human and rat ASK1 protein.

Figure 22. GIP Suppresses p38 MAPK and JNK Activity via Inhibition of ASK1 in INS-1 cells and Human Islets. A) INS-1 cells transfected with GFP or dominant negative ASK1 (ASK1kinase dead) were treated without or with STS (100 nM) for 4 h and Western blotting analysis performed on total cell lysates with indicated antibodies. B) INS-1 cells transfected with wild type ASK1 (ASK1WT) or ASK1 containing a S83A mutation (ASK1S83A) were treated without or with STS (100 nM) ± 10 nM GIP for 4 h and Western blotting analysis performed on total cell lysates with indicated antibodies. C) Human islets were treated without or with STS (100 nM) ± 10 nM GIP for 4 h and Western blotting analysis performed on total cell lysates with indicated antibodies. Shown in A) and B) are representative blots of 3 independent experiments. Immunoblotting with anti-β-actin antibodies were used as internal controls. Note: anti-ASK1 antibody detects both human and rat ASK1 protein. Blots are representative of at least three independent experiments.

Figure 23. GIP Mediated Suppression of p38 MAPK and JNK Promotes the Survival of INS-1 Cells Exposed to ER and Genotoxic Stress. A) INS-1 cells were treated without or with STS (100 nM), thapsigargin (500 nM), or etoposide (5 µM) ± 10 nM GIP for 4 h and Western blotting analysis performed on total cell lysates with indicated antibodies. Blots are representative of at least three independent experiments. Immunoblotting with anti-β-actin antibodies were used as internal controls. B) INS-1 cells were treated without or with STS, thapsigargin, or etoposide ± GIP for 6 h and cell death determined. Mean ± SEM of cell death (n=6); #, p < 0.05 as indicated. C) INS-1 cells were treated without or with STS, thapsigargin, or etoposide ± p38 MAPK inhibitor (5 µM SB 203580) or JNK inhibitor (5 µM SP600125) for 6 h and cell death determined. Mean ± SEM of cell death (n=6); #, p < 0.05 vs STS only; $, p < 0.05 vs thapsigargin only; %, p < 0.05 vs etoposide only.

4.3 Discussion In recent years incretins have been revealed to exert anti-apoptotic actions in β-cells. In support of these findings, the present studies showed that GIP promotes the survival of INS-1 cells and dispersed mouse and human islets exposed to glucolipotoxic stress (Fig. 9). In order to gain insight into its mode of action, the anti-apoptotic effects of GIP were examined on β-cells exposed to STS, an activator of the mitochondrial apoptosis pathway, with particular attention on the regulation of pro- and anti-apoptotic Bcl-2 family proteins. Previously, GIP was found to promote β-cell survival by regulating multiple signalling modules and gene transcription of Bcl-2 and Bax (Ehses et al., 2003; Kim et al., 2008b; Kim et al., 2005b; Trümper et al., 2002). Disruption of the signalling pathway involved in regulating Bcl-2 expression reduced the ability of GIP to suppress caspase-3 activation (Kim et al., 2008b). Therefore GIP appears to regulate the levels of mitochondria-associated Bcl-2 family members that are appropriate for maintaining cell integrity. As with other cells, β-cells are exposed to various stresses requiring responses to counter-act the redistribution of pro-apoptotic Bcl-2 family proteins from the cytoplasm to the mitochondria, and pore formation by Bax and Bak oligomers (Allen et al., 2005; Donath et al., 2008; Eizirik et al., 2008; Evans et al., 2002; Haataja et al., 2008; Hanke, 2001; Harwood et al., 2005; Kim et al., 2005c; Laybutt et al., 2002; Poitout and Robertson, 2008; Scheuner and Kaufman, 2008; Shi, 2002; Shimabukuro et al., 1998a). Studies were therefore performed on the regulatory effects of GIPR signalling on interactions of pro- and anti-apoptotic Bcl-2 family proteins with mitochondria and corresponding changes in the onset of apoptosis. Treating INS-1 β-cells with STS resulted in mitochondrial release of cytochrome C, cleavage of caspase-3 and apoptosis, whereas GIP suppressed these effects in a concentration- dependent manner (Fig. 10). Previous studies showed that GIP stimulates insulin secretion from β-cells via PKA and EPAC2 (see Section 1.7.1) and cAMP was implicated in GIP-mediated pro- survival effects (Ehses et al., 2003). The current results clearly demonstrated a dependence upon adenylate cyclase activation for the anti-apoptotic effects of GIP and contributions from autocrine insulin signalling appear unlikely (Fig. 11). Furthermore, it was evident that STS induced mitochondrial translocation of Bad and BimEL as well as activation of mitochondrial Bax (Fig. 12&13). The level of mitochondria-associated Bcl-2 remained constant during STS treatment, whereas that of Bcl-XL increased, indicating a role for Bcl-2 in regulating pro- apoptotic proteins bound under unstressed conditions, with Bcl-XL contributing during an apoptotic stimulus. GIP stimulation almost completely counter-acted the effects of STS on Bad

and BimEL translocation as well as Bax activation. Since CHX had no effect on Bad levels, caspase-3 activation or protection against cell death, transcriptional changes by GIP did not contribute significantly to its effects (Fig. 14). Reducing levels of Bax dimerization in STS- treated INS-1 cells by GIP stimulation was likely a key component of its survival effects, since it has been established that Bax oligomerization is critical for initiating the mitochondria-mediated apoptotic pathway (Harwood et al., 2005; Reed, 2006; Shi, 2002). Indeed, Bax channel inhibitors reduced levels of STS-induced cell death (Fig. 13B). How does GIP stimulation prevent the apoptotic effects of STS? Interestingly, the anti- apoptotic effects of GIP on INS-1 cells were blocked by the Akt inhibitor, Akt IV, whereas chemical inhibitors of PI3K/Akt, ERK 1/2 or PKA signalling had no effect (Fig. 15A). Akt IV also blocked the survival effects of forskolin, whereas inhibition of PKA was without effect (Fig. 15B). These results indicated that EPAC2-mediated activation of Akt, as found in studies described in Chapter 3, was involved in the survival effects of GIP, and indeed, the EPAC2 agonist, 8-cpt cAMP exhibited concentration-dependent anti-apoptotic actions in STS-treated INS-1 cells (Fig. 15C). By comparison, although IGF-I also promoted INS-1 cell survival, inhibition of Akt signalling with Akt VIII blocked this effect (Fig. 15D). Interestingly, STS + GIP treated INS-1 cells exhibited a large increase in Akt pT308 and pS473 levels in the presence of Akt IV, despite a complete ablation of Akt signalling (Fig. 16), whereas Ly294002 and Akt VIII ablated levels of Akt pT308 and pS473 yet had no inhibitory effect on Akt signalling, demonstrating that promotion of INS-1 cell survival involves the alternate mode of Akt activation identified in Chapter 3. Further evidence implicating a role for Akt was found by the potentiating survival effects of GIP in INS-1 cells transfected with HA-Akt1 (Fig. 15E&F). Treating INS-1 β-cells with STS elevated the phosphorylation (active forms) of p38 MAPK and JNK as well as MEK 3/6, the p38 MAPK kinase. These phosphorylations, plus cleaved caspase-3 levels were greatly reduced by GIP, in an Akt-IV sensitive manner (Fig. 17A). Overexpression of Akt-DN or knockdown of Akt 1&2 produced similar results to Akt-IV (Fig. 17B&C), demonstrating that Akt was central to the pro-survival actions of GIP in STS treated INS-1 cells. Inhibitor studies revealed that p38 MAPK and JNK mediated the effects of STS on dephosphorylation of Bad at Ser-112, mitochondrial translocation of Bad and BimEL and release of cytochrome C as well as activation of mitochondrial Bax and onset of cell death (Fig. 18&20). Since combined inhibition of p38 MAPK and JNK promoted a similar level of survival to that observed in INS-1 cells treated with GIP (Fig. 18C) and exerted similar actions on Bax activation

(Fig. 20B), this pathway was central to the pro-survival effects of GIP on STS-treated INS-1 cells. Supporting evidence was also found with MIN6 cells (Fig. 19). Similar results were obtained in INS-1 cells exposed to ER or genotoxic stress, indicating that suppression of p38 MAPK and JNK pathways is a common and powerful anti-apoptotic mechanism by which GIP acts (Fig. 23). Since Bad and BimEL have well established pro-apoptotic actions (Chipuk et al., 2010), and Bad was previously shown to play important roles in β-cells (Danial et al., 2003; Danial et al., 2008), the effects of GIP on their cellular distribution likely played an important role in reducing STS-induced INS-1 cell death. However, the precise mechanisms whereby p38 MAPK and JNK promoted activation of Bax could not be determined. Both kinases can phosphorylate Bax at Thr-167 (Kim et al., 2006a) and, in ceramide treated A549 cells, protein phosphatase 2A was shown to dephosphorylate Bax at Ser-184 (Xin and Deng, 2006), an Akt target site that, when phosphorylated, inactivates the pro-apoptotic actions of Bax (Xin and Deng, 2005). These events could therefore result in Bax activation and mitochondrial translocation, and their involvement could be an interesting avenue for future β-cell research. The link between GIP signalling and the suppression of p38 MAPK and JNK activity in INS-1 β-cells and human islets involved Akt-mediated suppression of ASK1. Although human and rodent β-cells have been shown to exhibit differences in proliferative responses (Parnaud et al., 2008), the current studies indicate that the core incretin-mediated anti-apoptotic pathways are conserved, although further studies are needed to establish whether ASK1 plays a central role in β-cell death associated with human diabetes. Stimulation with GIP elevated levels of phosphorylated ASK1 at Akt target site Ser-83 (Fig. 21&22), inhibiting ASK1 activation (Kim et al., 2001) as well as the downstream activation of p38 MAPK and JNK (Ichijo et al., 1997; Takeda et al., 2008). Similarly, GIP prevented phosphorylation of ASK1 pT845 (Fig. 21&22), which is critical for ASK1 activation (Takeda et al., 2008; Zhang et al., 2005). Consistent with this, GIP prevented STS-induced elevations in ASK1 in vitro kinase activity (Fig. 21 B&C) and phosphorylation of the downstream ASK1 target, MEK 3/6 (Fig. 17A&22C). Furthermore, dominant-negative ASK1 suppressed STS-induced elevations in levels of phosphorylated p38 MAPK and JNK as well as cleaved caspase-3 (Fig. 22A), and INS-1 cells expressing ASK1 protein that lacked the Ser-83 Akt phosphorylation site were resistant to the effects of GIP on p38 MAPK, JNK and caspase-3 (Fig. 22B). These findings thus revealed that the effects of STS and GIP on INS-1 cell death/survival were relayed via signals sent to ASK1.

Chapter 5 Effects of GIPR Activation on β-cells in Diabetic Rodents 5.1 Introduction As mentioned in Section 1.6.5, both GLP-1 receptor agonists and DPP-IV inhibitors improve β-cell function and glycaemic control in patients with T2D (Amori et al., 2007), whereas controversy exists regarding the anti-diabetic potential for GIPR agonists (McIntosh et al., 2009) mainly because T2D patients exhibit greatly reduced insulin responses to GIP and elimination of GIP signalling promotes resistance to obesity in rodents (Irwin and Flatt, 2009; Meier and Nauck, 2004; Miyawaki et al., 2002; Nauck et al., 1993), indicating that GIPR agonists would be ineffective in restoring β-cell function and may increase obesity in T2D patients. Conversely however, pharmacological doses of DPP-IV resistant GIP analogues are insulinotropic in rodents that are unresponsive to physiological levels of GIP (Hinke et al., 2002; Irwin et al., 2005), normalizing glycaemia improves β-cell sensitivity to GIP in diabetic rats (Piteau et al., 2007) and T2D patients (Hojberg et al., 2009; Meneilly et al., 1993), and alternative GIPR signalling modules such as those with anti-apoptotic actions may remain functional in diabetes (Section 1.8.1). Since GIPR signalling was shown to promote the survival of cultured β-cells, as discussed in Chapter 4 and in previous studies (Ehses et al., 2003; Kim et al., 2005b; Trümper et al., 2002), the current studies examine the in vivo effects of a DPP-IV resistant GIP analogue in rodent models of diabetes, with a particular focus on β-cell function and survival as well as glycaemic control.

5.2 Results 2 5.2.1 D-Ala GIP1-30 (D-GIP1-30) is a DPP-IV Resistant GIP Analogue with Equivalent

Islet Actions to Native GIP

Since the in vivo effects of native GIP (GIP1-42) are transient due to rapid N-terminal cleavage by DPP-IV and a C-terminally truncated form of GIP containing amino acids 1-30

(GIP1-30) was found to exhibit similar biological activity to GIP1-42 in studies on cell lines (Hinke et al., 2001; McIntosh et al., 2009), the in vivo effects of GIPR activation on β-cells in the current studies were examined using a truncated GIP analogue containing a D-alanine substitution at position 2 (D-GIP1-30), a modification that has been shown to render GIP1-42 DPP- IV resistant (Kuhn-Wache et al., 2000) while retaining biological activity on β-cells (Hinke et al., 2002).

Initially, the acute insulinotropic effects of D-GIP1-30 were evaluated in Vancouver Diabetic Fatty (VDF) rats, an obese and mildly diabetic sub-strain of the Zucker Fatty rat (Lynn et al., 2001). Compared to lean rats, obese VDF rats exhibited mild fasting hyperglycaemia, and marked hyperglycaemia during an oral glucose tolerance test (OGTT; Fig. 24A), with elevated fasting insulin levels and blunted insulin responses (Fig. 24B). In intraperitoneal (i.p.) glucose tolerance tests (IPGTT), subcutaneous (s.c.) injections of D-GIP1-30 (8 nmol/kg BW) resulted in a moderately improved glucose profile in obese VDF rats, with profound reductions in glucose excursions in lean rats (Fig. 24C) and increased insulin responses in both obese VDF and lean rats, compared to rats injected with PBS (Fig. 24D). To investigate potential off-target actions, the effects of D-GIP1-30 on glucose tolerance were assessed by administering s.c. injections of -/- PBS or D-GIP1-30 (8 nmol/kg BW) to WT and GIPR knockout (GIPR ) mice immediately prior to an IPGTT (2 g glucose/kg BW). D-GIP1-30 improved glucose tolerance in WT but not in GIPR-/- mice (Fig. 25A), thus demonstrating specificity of action. Similarly, treatment of static -/- mouse islet cultures with D-GIP1-30 potentiated insulin release from WT islets but not GIPR islets, and this occurred at 11 mM, but not 3 mM, glucose (Fig. 25B), consistent with the known glucose threshold required for GIP-stimulated insulin secretion (McIntosh et al., 2009). Thus these findings reveal that D-GIP1-30 exhibits insulinotropic actions via GIPR activation.

To assess its potency on β-cells, the insulinotropic effect of D-GIP1-30 was compared to

GIP1-42 on pancreas isolated from obese VDF and lean rats and then perfused with linear gradients of peptide (0 to 1 nM). In the presence of 16.7 mM glucose, the peptides demonstrated equivalent insulinotropic potencies (Fig. 26A) whereas, compared to lean rats, responsiveness of pancreata from obese VDF rats to either peptide was greatly attenuated, which is consistent with earlier findings (Lynn et al., 2001). Furthermore, the in vitro effects of D-GIP1-30 and GIP1-42 on β-cell survival were also compared by monitoring the onset of cell death in STS-treated INS-1 cells co-treated with D-GIP1-30 or GIP1-42 (0-100 nM). Both suppressed INS-1 cell death with similar maximal effects and although D-GIP1-30 demonstrated slightly reduced mean efficacy

(EC50 values: D-GIP1-30 978±134 pM vs GIP1-42 509±114 pM), the difference was not statistically significant (Fig. 26B). Together these findings reveal that D-GIP1-30 and GIP1-42 demonstrate almost identical potency and biological activity on β-cells.

Figure 24. A DPP-IV Resistant GIP Analogue (D-GIP1-30) Exhibits Potent Insulinotropic Actions in Lean and Obese Vancouver Diabetic Fatty (VDF) Zucker Rats. A) OGTTs were performed on fasted lean (n=3) and obese VDF (n=6) rats and blood glucose levels measured. Mean ± SEM; ***, p < 0.001 vs lean rats. B) Insulin levels were determined from blood samples collected in A. Mean ± SEM with significance as indicated. C) Intraperitoneal (i.p.) glucose tolerance tests (IPGTT) were performed on fasted lean (n=3) and obese VDF (n=4) rats that received the glucose immediately following subcutaneous (s.c.) injections with PBS or D-GIP1-30 (8 nmol/kg bodyweight (BW)) and blood glucose levels measured. Mean ± SEM; **, p < 0.01, ***, p < 0.001 vs lean controls; #, p < 0.05 vs VDF controls. D) Insulin levels were determined from blood samples collected in C. Mean ± SEM; ***, p < 0.001 vs lean controls; #, p < 0.05, ###, p < 0.001 vs VDF controls.

Figure 25. The GIP Receptor (GIPR) is Required for the Insulinotropic Effects of D-GIP1- +/+ -/- 30. A) IPGTTs were performed on fasted wild type (GIPR ) mice and GIPR knockout (GIPR ) mice that received the glucose immediately following s.c. injections with PBS or D-GIP1-30 (8 nmol/kg BW), and blood glucose levels measured. Mean ± SEM (n=3); *, p < 0.05 vs GIPR+/+ mice treated with PBS. B) Islets from GIPR+/+ and GIPR-/- mice were incubated for 2 h in 3 or 11 mM glucose ± 10 nM D-GIP1-30 and secreted insulin levels determined. Mean ± SEM (n=3); statistical significance is indicated.

Figure 26. D-GIP1-30 Demonstrates Equivalent Islet Actions to GIP1-42. A) Pancreas perfusions with 4.4 mM glucose, then 16.7 mM glucose + D-GIP1-30 or GIP1-42 (0-1 nM) were performed on lean and obese VDF rats and insulin levels determined in perfusate. Mean ± SEM (n=3). Pancreas perfusions were performed by Gary Yang, as described in Materials and Methods. B) INS-1 cells were treated without or with 100 nM staurosporine + increasing concentrations of D-GIP1-30 or GIP1-42 (0-100 nM) for 6 h and cell death determined. Mean ± SEM (n=4); $$$, p < 0.001 vs control (no staurosporine); ###, p < 0.001 vs staurosporine alone. In the upper right is the calculated EC50 value for GIP1-42 and D-GIP1-30.

5.2.2 Effects of D-GIP1-30 in Streptozotocin Treated Rats The capacity for chronic GIPR activation to promote β-cell survival was then examined by determining the effects of D-GIP1-30 on lean Zucker rats that had undergone exposure to the β- cell toxin, streptozotocin (STZ). Rats were treated twice daily with PBS or D-GIP1-30 (8 nmol/kg BW) from day -2 to day 1, as outlined in Fig. 27A. On day 0, animals received a single i.p. injection of STZ (35 mg/kg BW) and blood glucose levels were monitored from day -2 to day 4; controls did not receive any treatment. OGTTs were performed on day 5 and pancreas samples collected for histological analysis on day 6. As expected, rats receiving STZ had elevated morning blood glucose levels and reduced glucose tolerance and insulin responses during OGTTs compared to untreated rats (Fig. 27A-C). However all parameters were significantly improved in STZ treated rats receiving D-GIP1-30 injections, when compared to rats receiving

PBS, indicating that D-GIP1-30 partially protected β-cells from STZ exposure. Histological analyses of pancreas samples revealed that islets in STZ-treated rats had obvious structural derangements as well as apparent alpha cell expansion and increased localization to the islet core

(Fig. 27D). However, these derangements were much less severe in rats treated with D-GIP1-30.

Consistent with a pro-survival effect, β-cell areas in STZ treated rats receiving D-GIP1-30 injections were significantly greater than those receiving PBS injections (Fig. 27E&F).

5.2.3 Effects of D-GIP1-30 in Vancouver Diabetic Fatty (VDF) Rats

To examine effects of D-GIP1-30 on β-cell function, OGTTs were performed on obese

VDF rats before and after 10 days of twice daily treatment with s.c. injections of D-GIP1-30 (8 nmol/kg BW) or vehicle control (PBS). Although GIP has often been considered a „pro-obesity hormone‟, there were no significant differences in body weights at the end of treatment (Fig.

28E); in fact, weight gain was significantly reduced in D-GIP1-30 treated (2.2±0.3 g/day) versus PBS treated (3.6±0.3 g/day) obese VDF rats (Fig. 28F). Noteworthy, since it was critical to examine sustained effects of D-GIP1-30, the treated rats did not receive OGTTs until ~48 h following final injections, thus allowing complete renal clearance of circulating peptide. It was found that D-GIP1-30 treatment significantly improved glucose tolerance and acute insulin responses (0 to 30 min), whereas PBS treatment had no effect (Fig. 28A-D). Following the OGTTs (~24 h), islets were isolated and protein samples collected from

PBS and D-GIP1-30 treated obese VDF rats along with age matched lean rats. In comparison to lean rats, Western blotting analysis revealed that islets from PBS treated obese VDF rats

expressed significantly higher levels of pro-apoptotic proteins (p53, Bax, Bad, Bim, CHOP and cleaved caspase-3) and the anti-apoptotic protein Bcl-2 (Fig. 28G&H). However, the increased pro-apoptotic protein levels were reduced by treatment with D-GIP1-30, resulting in a decrease in the Bax/Bcl-2 ratio, an important contributing factor to regulating the apoptosis program.

5.2.4 Effects of D-GIP1-30 in Akita Mice, a Mouse Model of β-cell ER Stress As mentioned in Section 1.6.3, ER stress plays an important role in β-cell dysfunction and apoptosis in T2D (Eizirik et al., 2008; Scheuner and Kaufman, 2008). Furthermore, GIP promoted survival of INS-1 cells exposed to the ER stress-inducing agent, thapsigargin (Chapter

4; Fig. 23) and D-GIP1-30 reduced protein levels of the ER-associated C/EBP homologous protein (CHOP) in the VDF rat (Fig. 28 G&H). The Akita mouse has a Cys96Tyr mutation in the insulin2 gene that causes production of misfolded insulin and has been widely used as a model of chronic β-cell ER stress and diabetes (Scheuner and Kaufman, 2008; Wang et al., 1999). Of note, Akita mice exhibit progressively elevated levels of β-cell apoptosis and diabetes as early as 4 weeks of age, and deleting the chop gene greatly preserves β-cell function and survival as well as glycaemic control (Oyadomari et al., 2002). Moreover, deleting chop has been shown to protect β-cells in multiple mouse models of diabetes (Song et al., 2008). It was therefore proposed that GIPR signalling may promote β-cell survival by decreasing the pro-apoptotic effects of β-cell ER stress via actions such as reducing CHOP protein levels. Homozygous Akita mice die early post-natally due to severe complications of diabetes, and studies were therefore performed on heterozygote males, which remain relatively healthy while still exhibiting hyperglycaemia. The effects of D-GIP1-30 on β-cells under ER stress were examined by treating WT and Akita mice with s.c. injections of D-GIP1-30 (15 nmol/kg BW) or vehicle control (PBS) twice daily for 18 days, starting from 4 weeks of age (shown in Fig. 29A as day 0). In WT mice, treatment with PBS or D-GIP1-30 exhibited no significant effect. Akita mice exhibited a progressive rise in morning glucose levels throughout the study. In comparison to PBS treated Akita mice, treatment with D-GIP1-30 significantly reduced glucose levels from day 12 to day 18 (Fig. 29A) without significant changes in body weight (Fig. 29B).

The effects of D-GIP1-30 on morning glucose levels in Akita mice were likely a result of improvements in β-cell function. This possibility was examined by performing OGTTs on WT and Akita mice ~48 h following final injections, allowing complete peptide clearance. As with morning glucose, WT mice treated with PBS or D-GIP1-30 had similar glucose profiles and non-

significantly different insulin profiles, though mean fasting insulin was higher in PBS treated

WT mice. Importantly, Akita mice treated with D-GIP1-30 demonstrated superior glucose tolerance when compared to those treated with PBS (Fig. 29C&D). Furthermore, D-GIP1-30 treated Akita mice exhibited an enhancement in first-phase insulin secretion and an improvement in β-cell function as indicated by the integrated rise in early insulin responses relative to glucose excursions (Fig. 29E&F). Thus stimulation of the GIPR with D-GIP1-30 caused sustained β-cell improvements in Akita mice.

To determine whether D-GIP1-30 improved β-cell survival in Akita mice, histological analyses were performed on pancreas samples collected ~24 h following OGTTs. Unexpectedly, islets from Akita mice were similar in size to those in WT mice, although islets were frequently observed that exhibited a more elongated morphology, coupled with α-cells residing in the islet core (Fig. 29G). Furthermore, whereas islets from WT mice treated with PBS or D-GIP1-30 had similar levels of β-cell proliferation and apoptosis, islets from Akita mice treated with D-GIP1-30 exhibited markedly reduced levels of β-cell apoptosis (Fig. 29I) but similar levels of β-cell proliferation (Fig. 29H), when compared to those treated with PBS. The improvements in β-cell function and glycaemic control in Akita mice treated with D-GIP1-30 may therefore reflect the improved survival of β-cells under ER stress. The CHOP protein plays a critical role in the loss of β-cells in response to ER stress, and GIPR signalling may modulate CHOP actions by reducing CHOP protein levels. To investigate this possibility, studies were performed on INS-1 and Akita mouse-derived β-cells (β-cellsAkita) exposed to the ER stressors, tunicamycin or thapsigargin, ± D-GIP1-30. In time-course studies, treating INS-1 cells with tunicamycin or thapsigargin resulted in the rapid phosphorylation, and consequent inactivation, of the general protein translation regulator eukaryotic initiation factor 2α (eIF2α) by 2 h, accompanied by a robust increase in CHOP protein levels by 4 h, along with a modest rise in levels of cleaved caspase-3 in tunicamycin-treated cells and a robust rise in thapsigargin-treated cells by 10 h (Fig. 30A&B). Surprisingly, D-GIP1-30 had no effect on levels of phosphorylated eIF2α and actually enhanced CHOP protein levels, despite greatly reducing levels of cleaved caspase-3 in tunicamycin and thapsigargin treated INS-1 cells. Comparison of responses to treatment with tunicamycin or thapsigargin ± D-GIP1-30 for 8 h demonstrated milder Akita effects of D-GIP1-30 on cleaved caspase-3 levels in β-cell than in INS-1 cells (Fig. 30C&D).

These findings can be used to argue against the proposition that D-GIP1-30 improves the function and survival of β-cells under ER stress by reducing CHOP protein levels.

Figure 27. D-GIP1-30 Protects β-cells in Streptozotocin (STZ) Treated Rats. A) Glucose levels were monitored 2 days prior to (day -2) and 4 days (day 4) following an i.p. injection of STZ (35 mg/kg BW; on day 0) to lean rats treated twice daily with PBS or D-GIP1-30 (8 nmol/kg BW) from day -2 to day 1, as well as in untreated lean rats. B) On day 5, OGTTs were performed on rats described in A and blood glucose levels measured. C) Insulin levels were determined from blood samples collected in B. For A-C, Mean ± SEM (n=4); *, p < 0.05, **, p < 0.01, ***, p < 0.001 vs rats treated with STZ and PBS.

Figure 27 continued. D) Representative images of pancreases collected on day 6 and stained with hematoxylin & eosin or with insulin (green), glucagon (red) and DAPI (blue); scale bar = 100 µm. E) Representative sections of pancreases described in D that were stained for insulin positive (beta-cell) area via peroxidase catalyzed reaction with 3,3'-Diaminobenzidine. The width of these sections represents 2 centimeters. F) Mean ± SEM of β-cell (insulin positive) area relative to pancreas area (n=4; 4 sections per animal); statistical significance is shown.

Figure 28. D-GIP1-30 Improves Islet Function and Diminishes Islet Pro-apoptotic Protein Levels in Obese VDF Rats, While Not Promoting Weight Gain. A) OGTTs were performed on obese VDF rats ~24 h prior to and ~48 h following 10 days of twice daily treatment with PBS or D-GIP1-30 (8 nmol/kg BW) and blood glucose levels measured. Mean ± SEM (n=6); **, p < 0.01 vs same VDF rats prior to treatment. B) Integrated glucose profile for OGTTs described in A. Mean ± SEM (n=6); significant differences as shown. C) Insulin levels were determined from blood samples collected in A. Mean ± SEM (n=6); *, p < 0.05 vs same obese VDF rats prior to treatment. D) Integrated acute insulin response (from 0 to 30 min) for insulin profiles described in C. Mean ± SEM (n=6); significant differences as shown. E&F) Shown are absolute body weights (E) before and after (day 0 and 10) study and the relative increases in body weight (F) from day 0. Mean ± SEM (n=6); *, p < 0.05 vs obese VDF rats treated with PBS.

Figure 28 continued. G) Islets were isolated from obese VDF rats and age matched lean rats ~24 h following OGTTs and Western blotting analysis performed on cell lysates with indicated antibodies. H) For quantification, protein levels were normalized to β-actin and expressed relative to lean controls. Mean ± SEM (n=3); $, p < 0.05 vs lean; #, p < 0.05 vs obese VDF rats treated with PBS. Immunoblotting with anti-β-actin antibodies were internal controls.

Figure 29. D-GIP1-30 Improves β-cell Function and Survival and Glucose Tolerance in Akita Mice. A&B) WT and Akita mice were treated twice daily with PBS or D-GIP1-30 (15 nmol/kg BW) from day 0 to day 18 and morning glucose levels (A) and body weights (B) determined. Mean ± SEM (n=5); **, p < 0.01, ***, p < 0.001 vs Akita mice treated with PBS. C) OGTTs were performed on fasted WT and Akita mice ~48 h following the last day of treatment and blood glucose levels were measured. Mean ± SEM (n=5); *, p < 0.05, **, p < 0.01, ***, p < 0.001 vs Akita mice treated with PBS. D) Integrated glucose profile for OGTTs described in C. Mean ± SEM (n=5); significant difference as shown. E) Insulin levels were determined from blood samples collected in C. Mean ± SEM (n=5); **, p < 0.01 vs Akita mice treated with PBS. F) Integrated acute insulin response (from 0 to 15 min) for profiles described in E. Mean ± SEM (n=5); statistically significant differences are shown.

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Figure 29 continued. G) Representative images of pancreases collected ~24 h following the OGTTs. Pancreases were stained with insulin (green), glucagon (red) and DAPI (blue); scale bar = 50 µm. H) Mean percent ± SEM of β-cells undergoing proliferation as determined via PCNA positive nuclei (n=3). I) Mean percent ± SEM of β-cells undergoing apoptosis as determined via TUNEL positive nuclei (n=3); statistical significance is shown in panel.

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Figure 30. The Mechanism by Which D-GIP1-30 Promotes the Survival of Cultured β-cells Under ER Stress Does Not Involve Suppression of CHOP Protein Levels. A) INS-1 cells were treated without or with tunicamycin (2.5 µg/ml) or thapsigargin (200 nM) ± 10 nM D-GIP1- 30 for 0-10 h and Western blotting analysis performed with the indicated antibodies. B) Mean ± SEM (n=4); *, p < 0.05, ***, p < 0.001 vs PBS controls. C) INS-1 cells were treated in parallel with DMSO, tunicamycin (2.5 µg/ml) or thapsigargin (200 nM) ± 10 nM D-GIP1-30 for 8 h and Western blotting analysis performed with the indicated antibodies. D) Akita derived β-cells were treated in parallel with DMSO, tunicamycin (2.5 µg/ml) or thapsigargin (200 nM) ± 10 nM D- GIP1-30 for 8 h and Western blotting analysis performed with the indicated antibodies. Immunoblotting with anti-β-actin antibodies were internal controls.

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5.2.5 Effects of D-GIP1-30 in Zucker Diabetic Fatty (ZDF) Rats

Since GIPR activation with D-GIP1-30 improved β-cell responses to glucose and β-cell survival in STZ-treated and VDF rats as well as Akita mice, the effects of D-GIP1-30 treatment on glycaemic control and β-cell area were examined in male obese Zucker diabetic fatty (ZDF) rats. This model was chosen because male obese ZDF rats incur an aggressive onset of β-cell apoptosis and are one of the most commonly used and well characterized models of T2D (Shimabukuro et al., 1998b; Topp et al., 2007). Male lean and obese ZDF rats (starting at 6 weeks of age) were monitored from day -6 to day 18 (see Fig. 31A), and treatment with PBS or

D-GIP1-30 (8 nmol/kg BW) began at day 0. Lean rats treated with PBS or D-GIP1-30 showed no significant changes in any parameter throughout the study. No differences between the groups of obese ZDF rats were observed between day -6 to day 0 but, following onset of treatment, glycaemia was lower in obese ZDF rats treated with D-GIP1-30, reaching significance by day 9

(Fig. 31B). The difference in glycaemia between D-GIP1-30 and PBS treated groups increased over the subsequent 9 days (day 18 glucose values: D-GIP1-30 11.1±0.3 mM vs PBS 17.7±0.9 mM). Body weights did not differ between groups of obese ZDF rats (Fig. 31C), but food intake was significantly reduced in D-GIP1-30 treated obese ZDF rats by day 15 (Fig. 31D). More striking was the markedly reduced water intake in the D-GIP1-30 treated obese ZDF rats as early as day 12 (Fig. 31E). The elevated water intake in PBS treated obese ZDF rats correlated with glucose levels, indicating that D-GIP1-30 prevented the onset of diabetes-induced polydipsia in obese ZDF rats; polyuria was also evident in rats demonstrating polydipsia. On the final treatment day (day 18), glucose levels were monitored every 3 h over a 24 h period. Obese ZDF rats treated with D-GIP1-30 had significantly lower glucose levels than PBS treated obese ZDF rats at all time points (Fig. 31F). Collectively these findings indicated that D-GIP1-30 exerted potent anti-diabetic effects on obese ZDF rats.

The anti-diabetic effects of D-GIP1-30 were likely a result of improved β-cell function and mass. This was examined by performing OGTTs on lean and obese ZDF rats ~48 h following final injections, to allow complete peptide clearance. Lean rats treated with PBS or D-GIP1-30 had similar glucose and insulin profiles. However, obese ZDF rats treated with D-GIP1-30 had reduced fasting glycaemia and greatly improved glucose tolerance compared to PBS treated obese ZDF rats (Fig. 32A). Importantly, D-GIP1-30 was so effective in obese ZDF rats that the fasting and 2 h post-prandial glucose levels were similar to lean rats. Insulin measurements revealed that obese ZDF rats had markedly elevated insulin levels compared to lean rats,

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consistent with an insulin resistant phenotype (Fig. 32B). However, obese ZDF rats treated with

D-GIP1-30 had significantly greater insulin responses following a glucose challenge, and plotting this response with respect to HOMA SI calculations revealed that β-cell compensation was much greater in obese ZDF rats treated with D-GIP1-30 (Fig. 32C). Histological analyses were performed on pancreas samples collected ~24 h following OGTTs. As expected (Topp et al., 2007), many islets from PBS treated obese ZDF rats were greatly enlarged compared to lean rats, but with a discontinuous appearance and commonly having some α-cell infiltration into the islet core (Fig. 32D). In contrast, although most islets from D-GIP1-30 treated obese ZDF rats exhibited even greater enlargement (many exceeding a millimeter in diameter), they maintained structural integrity with α-cells residing in the islet periphery. Furthermore, β-cell area in obese ZDF rats treated with D-GIP1-30 was significantly greater than in those treated with PBS (Fig. 32G&H). The areas in PBS and D-GIP1-30 treated lean rats were similar. Staining for apoptotic (Fig. 32E) and proliferating (Fig. 32F) β-cells revealed that enhanced β-cell area in D-GIP1-30 treated obese ZDF rats was mainly due to a significant reduction in β-cell apoptosis, although there was a modest increase in β-cell proliferation. Collectively these findings indicated that D-GIP1-30 exerted potent anti-diabetic effects in obese ZDF rats via improving β-cell function and survival.

5.2.6 Cultured Adipocytes Differentially Respond to D-GIP1-30 and GIP1-42 As previously mentioned, GIP has been considered a pro-obesity hormone (Irwin and Flatt, 2009) as a result of its ability to promote lipogenesis (McIntosh et al., 2009). However the lack of weight gain in lean and obese ZDF rats (Fig. 32C) and reduced weight gain in obese VDF rats (Fig. 28F) indicated that D-GIP1-30 might exhibit reduced lipogenic effects, compared to

GIP1-42. It has previously been established that GIP1-42 increases lipoprotein lipase (LPL) activity in cultured 3T3-L1 adipocytes (Kim et al., 2007b). In collaborative studies, performed by Dr.

Su-Jin Kim, cultured 3T3-L1 adipocytes were treated with D-GIP1-30, GIP1-30, GIP1-42, or D-GIP1-

42 (0-1000 nM) and LPL activity determined 24 h later. Although GIP1-42 and D-GIP1-42 promoted equivalent increases in LPL activity, D-GIP1-30 and GIP1-30 had markedly reduced effects; indeed, concentrations as high as 1 µM D-GIP1-30 or GIP1-30 were incapable of producing maximal responses, revealing that the C-terminus of native GIP is important for stimulatory actions in adipocytes, but not in β-cells. [Since these experiments were not performed by the author of this thesis, the results have been included as Appendix B].

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Figure 31. D-GIP1-30 Improves Glycaemic Control in Obese ZDF Rats. A) Schematic depicting the treatment protocol in which lean or obese ZDF rats (starting at 6 weeks of age) were monitored every 3 days from day -6 to day 18 and treated twice daily with PBS or D-GIP1- 30 (8 nmol/kg BW) from day 0 to day 18. B-E) Routine monitoring involved measurements of blood glucose (B), body weight (C), food intake (D), and water intake (E). Mean ± SEM (n=6); *, p < 0.05, **, p < 0.01, ***, p < 0.001 vs ZDF rats treated with PBS. F) On day 18, blood glucose levels were determined every 3 h over a 24 h period. Mean ± SEM (n=6); **, p < 0.01, ***, p < 0.001 vs obese ZDF rats treated with PBS.

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Figure 32. D-GIP1-30 Improves β-cell Function and Survival and Glucose Tolerance in Obese ZDF Rats. A) OGTTs were performed on fasted lean and obese ZDF rats (described in figure 8) ~48 h following the last day of treatment and blood glucose levels were measured. Mean ± SEM (n=6); **, p < 0.01, ***, p < 0.001 vs obese ZDF rats treated with PBS. B) Insulin levels were determined from blood samples collected in A. Mean ± SEM (n=6); **, p < 0.01 vs obese ZDF rats treated with PBS. C) Integrated acute insulin responses (from 0 to 30 min) for profiles described in B were plotted with respect to HOMA SI. Mean ± SEM (n=6). Note that, in C), the clear square symbol representing PBS-treated lean rats is hidden behind the clear circle representing D-GIP1-30-treated lean rats.

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Figure 32 continued. D) Representative images of pancreases collected ~24 h following the OGTTs. Pancreases were stained with hematoxylin & eosin or with insulin (green), glucagon (red) and DAPI (blue); scale bar = 100 µm. E) Mean percent ± SEM of β-cells undergoing apoptosis as determined via TUNEL positive nuclei (n=6); statistical significance is shown.

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Figure 32 continued. G) Representative sections of pancreases described in D that were stained for insulin positive (beta-cell) area via peroxidase catalyzed reaction with 3,3'- Diaminobenzidine. The width of these sections represents 1.5 centimeters. H) The mean ± SEM of β-cell (insulin positive) area relative to pancreas area (n=3; 4 sections per animal); statistical significance is shown.

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5.3 Discussion The main target for anti-diabetic therapies has been to develop a sustained reduction in glycaemia, in order to lower the incidence of morbidities such as retinopathy, renal dysfunction and peripheral neuropathy (Brownlee, 2005). Prospective studies show that many insulin resistant individuals are capable of maintaining euglycaemia via compensatory responses (Kahn, 2003), but that β-cell dysfunction and reduced β-cell mass are characteristics of those that develop type 2 diabetes (Defronzo, 2009; Kahn et al., 2009). Therapies that improve the functional capacity and mass of β-cells should therefore offer important benefits to patients. There is increasing evidence supporting an important role for GIPR signalling in the promotion of β-cell function and survival. Profound insulinotropic effects are achieved with physiological concentrations of GIP in normal animals (McIntosh et al., 2009) and with pharmacological doses of DPP-IV resistant GIP analogues in diabetic rodents (Green and Flatt, 2007; Hinke et al., 2002; Irwin et al., 2005), and, as shown in Chapter 4 and a number of earlier studies (Ehses et al., 2003; Kim et al., 2008b; Kim et al., 2005b; Trümper et al., 2002; Trümper et al., 2001), GIPR activation promotes pro-survival responses in cultured β-cells via multiple signalling modules and reduces expression and activity of pro-apoptotic Bcl-2 family proteins. However, although there have been extensive studies on the β-cell secretory actions of long- acting forms of GIP (Irwin and Flatt, 2009), a paucity of information exists on their β-cell protective effects. The truncated analogue D-GIP1-30 demonstrated similar β-cell effects to the intact peptide, potentiating acute insulin responses and improving glucose tolerance in both obese VDF and lean rats (Fig. 24C&D) as well as stimulating insulin secretion from the isolated perfused pancreas preparation (Fig. 26A), and studies using GIPR-/- mice revealed that this required the functional GIPR (Fig. 25). D-GIP1-30 also exhibited similar effects to GIP1-42 on the survival of β-cells in STS-treated INS-1 cells (Fig. 26B).

It is important to note that the beneficial effects of D-GIP1-30 on glucose homeostasis were observed in glucose tolerance tests performed at least 48 h following the last treatment, when peptide would be cleared from the circulation. These sustained responses therefore resulted from protective effects on islet survival, and they were observed in all four of the animal models examined. In STZ-treated rats, D-GIP1-30 afforded partial protection of β-cells, resulting in greater glycaemic control and insulin responses (Fig. 27) whereas, in a previous study, protective effects of the GLP-1R agonist exendin-4, but not D-GIP1-42, were reported in studies on STZ- induced diabetes in mice (Maida et al., 2009). Although the reasons are not clear, a more

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aggressive STZ-treatment regimen was utilized compared to the current study, resulting in much greater β-cell destruction, and dose and pharmacokinetic profiles were not rigorously evaluated to ensure appropriate comparisons. Additionally, higher peptide dosing in their study may have also resulted in GIPR down-regulation (McIntosh et al., 2009) and species differences could also play a role. Nevertheless, in the current studies on male obese VDF rats (Fig. 28), Akita mice (Fig. 29), and obese ZDF rats (Fig. 31&32), significantly improved glycaemic control and compensatory insulin responses resulted from D-GIP1-30 treatment. The impact of D-GIP1-30 treatment on diabetes progression in obese ZDF rats was also evident in the delayed onset and reduced severity of polydipsia (Fig. 31E), attributed to the improvements in glycaemia. Similarly, since GIP has not been shown to exert any major effects on food intake in rodents (Kerr et al., 2009a; Kerr et al., 2009b; McIntosh et al., 2009), the small decrease observed (Fig. 31D) was likely secondary to the improved glycaemia, perhaps resulting from altered hypothalamic sensing of peripherally-derived signals.

Increased β-cell area was a major factor underlying D-GIP1-30 induced improvements in glycaemia in obese ZDF rats (Fig. 32G&H), enabling stronger compensatory insulin responses (Fig 32C). This was mainly a result of reduced levels of β-cell apoptosis (Fig. 32E) since there were no significant effects on β-cell proliferation (Fig. 32F) and a similar effect was observed in Akita mice (Fig 29H&I). However, previous in vitro studies on cultured β-cell lines (Ehses et al., 2002; Trümper et al., 2002; Trümper et al., 2001) and primary islets (Friedrichsen et al., 2006) have shown that activation of GIPR signalling is capable of stimulating proliferation. The lack of effect of D-GIP1-30 in the obese ZDF rats and Akita mice may be due to the already existing elevated levels of β-cell proliferation in both models compared to lean or WT controls, respectively (Fig. 29H&32F). There is, however, suggestive evidence in the literature for an effect on proliferation in vivo. A porcine model with β-cell specific transgenic expression of a dominant negative GIPR was found to exhibit deficient β-cell mass as a result of reduced levels in β-cell proliferation (Renner et al., 2010) and long-acting GIP analogues were found to increase islet area and number in ob/ob mice (Irwin et al., 2006), although the relative contributions of proliferative and anti-apoptotic effects were not established. In agreement with the studies presented in Chapter 4 and previous in vitro studies on

GIP1-42 (Ehses et al., 2003; Kim et al., 2008b; Kim et al., 2005b; Trümper et al., 2002; Trümper et al., 2001), treatment with D-GIP1-30 greatly decreased islet pro-apoptotic protein levels in obese VDF rats (Fig 28G), an important factor in reducing β-cell loss. Levels of Bcl-2 were also

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elevated in the PBS treated obese VDF rats. However, since it was the only protein examined which was not decreased by D-GIP1-30, there was an overall reduction in the Bax/Bcl-2 (pro- apoptotic/anti-apoptotic) ratio. In obese VDF rats of this age, increases in β-cell Bcl-2 levels may reflect natural stress responses in attempts to promote survival, and so at any one time of tissue sampling, increases in both pro- and anti-apoptotic proteins may be detected, as compensatory responses occur. Similar observations have been previously reported, for example with apoptotic β-cells in cultured and developing rat islets (Hanke, 2000, 2001) and following serum deprivation in MIN6 β-cells (Mizuno et al., 1998). Intriguingly, it is possible that the milder diabetes that develops in obese VDF rats, when compared to obese ZDF rats, is due to a more robust anti-apoptotic response, since the latter exhibit reduced β-cell Bcl-2 levels (Shimabukuro et al., 1998b), although no direct evidence has been obtained to support this suggestion.

With Akita mice, although D-GIP1-30 produced relatively small reductions in basal glucose levels, there were marked improvements in glucose tolerance and β-cell survival, providing the first in vivo evidence that GIPR signalling counter-acts the pro-apoptotic actions of β-cell ER stress. This was initially thought to occur by reducing β-cell CHOP levels. However, since D-GIP1-30 appeared to cause slight potentiation of the elevated CHOP protein levels in cultured INS-1 cells and β-cellsAkita exposed to tunicamycin- or thapsigargin-induced ER stress, this appears to be unlikely. In cells exhibiting healthy recovery from ER stress, CHOP plays an important role in dephosphorylating eIF2α and re-initiating protein translation (Scheuner and Kaufman, 2008). In a study investigating GLP-1R signalling in β-cells under ER stress (Yusta et al., 2006), there were similar increases in CHOP protein levels to those found in the current study and it was suggested that incretins accelerated this recovery phase. More recently, GLP-1 stimulation was found to enhance production of ER chaperone proteins, which would enable a stronger recovery from ER stress (Cunha et al., 2009). In developing GIP-based therapies for T2D a major caveat has been the possibility of GIP promoting obesity (Irwin and Flatt, 2009; Meier and Nauck, 2004). Such an effect would be consistent with the lipogenic actions of GIP (McIntosh et al., 2009) and with studies demonstrating that mice lacking functional GIP responses show resistance to the development of obesity (Althage et al., 2008; Irwin and Flatt, 2009; Miyawaki et al., 2002). However, neither mice administered long acting analogues of GIP1-42 (Kerr et al., 2009a; Kerr et al., 2009b) nor GIP-overexpressing transgenic mice (Ding et al., 2008) exhibit increases in body weight, food intake, adiposity or insulin resistance, questioning whether GIPR agonists would promote

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obesity in patients with type 2 diabetes. Additionally, there are only weak data linking over- nutrition, GIP hypersecretion and obesity in humans (McIntosh et al., 2009). Nevertheless, although the findings clearly need to be substantiated by studies on primary adipocytes and in vivo, the unexpected difference observed in stimulatory effects of D-GIP1-30 (or GIP1-30) and

GIP1-42 (or D-GIP1-42) on LPL activity in 3T3-L1 adipocytes is intriguing (Appendix B). The high affinity binding region of GIP resides in amino acids 6-30, but the N-terminus has proven critical for actions on the pancreatic islet (McIntosh et al., 2009) and, as shown in the current as well as a previous study (Hinke et al., 2001), GIP1-30 and GIP1-42 exert very similar β-cell effects. However, the C-terminal 12 amino acids have been previously shown to be important for actions on some tissues, as GIP1-30 exhibited much lower potency than GIP1-42 for inhibiting gastric acid secretion from the perfused rat stomach (Rossowski et al., 1992). Since, in addition to islets and adipose tissue, GIP also appears to act as a physiological regulator in bone, the gastrointestinal tract, cardiovascular system and brain (McIntosh et al., 2009), there may still be tissue specific differences in responses to GIP1-42 and GIP1-30 that need to be identified. Additionally, the recent demonstration of production of a C-terminally truncated version of GIP in pancreatic α-cells (Fujita et al., 2010b) and in sub-populations of GIP-expressing cells in the gut (Fujita et al.,

2010a) indicate that GIP1-30 may play a physiological role as either an autocrine or paracrine regulator of islet cell function and, possibly, as an endocrine hormone. Since K-cell derived

GIP1-42 is secreted mainly during a meal, whereas α-cell secretion is elevated during the inter- digestive phase, there may be differences in the temporal activity of the two peptides; whether administration of GIP1-30 and GIP1-42 analogues during fasting and feeding results in selective tissue target effects is currently unknown. Additionally, the basis for the differential cellular activity is unclear. It is possible that the two peptides induce alternative conformational changes in the GIPR residing in different tissues, possibly due to variations in the membrane environment. However, it is more likely that cell-specific splice variants of the GIPR account for the different responses (Harada et al., 2008). This possibility could impact on the development of clinically relevant GIP analogues.

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Chapter 6 Further Thoughts and Conclusions 6.1 On the Regulation of Akt in β-cells by GIP In Chapter 3, it was reasoned that understanding how GIP regulates Akt activity would provide important insights into its ability to regulate β-cell mass. Akt is a protein- serine/threonine kinase involved in multiple incretin-mediated responses (Kim et al., 2005b; Lingohr et al., 2002a; Liu and Habener, 2008; Trümper et al., 2002; Wang et al., 2004), as well as playing a central role in glucose homeostasis (Whiteman et al., 2002) and the promotion of β- cell survival (Dickson and Rhodes, 2004; Elghazi et al., 2006). Transgenic mice with β-cell specific expression of a constitutively active Akt develop enhanced islet mass, improved glucose tolerance and resistance to STZ-induced diabetes (Bernal-Mizrachi et al., 2001; Tuttle et al., 2001), and transplanted human islets that express constitutively active Akt are more effective in improving glucose tolerance in diabetic mice (Rao et al., 2005). Conversely, transgenic mice with β-cell specific expression of a kinase-dead Akt develop diabetes due to defects in insulin secretion (Bernal-Mizrachi et al., 2004). However, it is worth noting that the authors of this study observed no significant effect of kinase-dead Akt on β-cell mass and argued that this finding indicated that Akt does not contribute to β-cell growth. However, the level of reduction in β-cell Akt signalling in these mice was not well characterized and independent studies showed that Akt2-/- mice (Garofalo et al., 2003) and Akt2-/-/Akt1+/- mice (Chen et al., 2009) exhibit major reductions in β-cell function and mass as well as severe diabetes. Moreover, in studies using transgenic and knockout mouse models (Balcazar et al., 2009; Fatrai et al., 2006; Rachdi et al., 2008), Bernal-Mizrachi and colleagues revealed that Akt plays a critical role in the proliferation of β-cells via regulating protein levels of cyclin-dependent protein kinase-4 as well as cyclin D1, cyclin D2 and p21. This involved Akt-mediated inhibition of tuberous sclerosis complex (TSC) 1/2, resulting in enhanced activity of mTORC1, a critical nutrient-sensing and growth-promoting protein complex (Wullschleger et al., 2006). Thus, there is significant evidence demonstrating that Akt plays an important role in regulating the growth and function of pancreatic β-cells. Correspondingly, it has been argued by Rhodes (Rhodes, 2005) that diminished signalling via the IRS-2 / PI3K / Akt module plays a major role in the susceptibility of β-cells to apoptosis in T2D, and enhancing β-cell Akt signalling has been proposed as an alternative therapy for T2D, with a view to maintaining or increasing β-cell function and mass (Dickson and Rhodes, 2004; Elghazi et al., 2006). Therefore, the discovery that GIP promotes Akt activation in β-cells in a non-canonical manner has important implications. 113

Unfortunately, despite extensive experimental evidence that GIP enhances Akt signalling in β-cells, the link between GIPR signalling and Akt activation remains unclear. Indeed, Akt activation in the absence of phosphorylation at Thr-308 has generally been considered impossible (Yang et al., 2002a). However, in studies using cultured COS-7, NIH 3T3 or 293T cells exposed to transient heat shock, Kikkawa and colleagues identified a similar phenomenon in which Akt is activated without requiring phosphorylation at Thr-308 or Ser-473 (Konishi et al., 1999; Konishi et al., 1997; Matsuzaki et al., 2004). In stressed cells, they and others (Wu et al., 2007; Zheng et al., 2006) have identified Akt associated with a high molecular mass protein complex containing heat shock proteins that they propose act in a chaperone-like manner and manipulate the conformation of Akt into an activated state (Mearow et al., 2002; Wu et al., 2007; Zheng et al., 2006). A similar mechanism could explain the effects observed in the studies described in Chapter 3, whereby GIP stimulates EPAC2, which then activates Akt via modulation of such a protein complex. Conversely, in the original study that identified the Thr- 308 and Ser-473 residues, Ser-124 and Thr-450 were also found to be phosphorylated, though a role in regulating Akt function was not found (Alessi et al., 1996a). Additionally, the tyrosine kinase, Src, was found to phosphorylate Akt at Tyr-315 and Tyr-326, events that appeared to be important for priming Akt activity (Chen et al., 2001; Jiang and Qiu, 2003). Moreover, it was shown that treatment of mouse β-cells with glucosamine diminishes Ser-473 phosphorylation by replacement with O-linked N-acetylglucosamine (Kang et al., 2008a). While the relevance of this finding needs to be clarified, it provides a precedent and supports the possibility that GIP could activate Akt by promoting an unidentified post-translational modification. Of additional complexity, GIP stimulation acutely enhances phosphorylation of Akt at Ser-473, without requiring it for activity, but in longer stimulation periods it also enhances phosphorylation of Akt at Thr-308. As shown in Fig. 1, a modest rise in Akt pT308 was observed by 30 min and levels continued to rise for at least a 4 h period (data not shown). Moreover, in studies by others (Trümper et al., 2001), INS-1 cells stimulated with GIP for 1 h exhibited elevated PI3K activity, and this was potentiated with high glucose stimulation and associated with a 2-fold elevation in IRS-2 protein levels and 4-fold elevation in phosphorylation of IRS-2 at tyrosine residues. Since IRS-2 is critical for coupling insulin and IGF-I receptor signalling to PI3K (Saltiel and Kahn, 2001; Taniguchi et al., 2006), prolonged GIP stimulation appears, therefore, to also promote canonical Akt activation via the IRS-2 / PI3K / Akt pathway in β-cells, with corresponding phosphorylation of Thr-308 and Ser-473. The mechanism by which GIPR

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signalling increases IRS-2 protein levels is likely due to its ability to elevate CREB pS133 levels (Ehses, 2003), as this is precisely the mechanism for GLP-1R signalling (Jhala et al., 2003; Park et al., 2006). A potential explanation for the staggered kinetics in Thr-308 and Ser-473 phosphorylation in response to GIP stimulation may derive from the fact that PDK1 is the Thr- 308 kinase (Stephens et al., 1998), whereas the mTORC2 protein complex is the major Ser-473 kinase (Sarbassov et al., 2005). Most studies on mTOR have focused on the mTORC1 protein complex that plays a critical role in balancing the rate of ribosome biogenesis, protein synthesis, cell growth and cell metabolism in proportion to cellular nutrient status (Proud, 2007, 2009; Wullschleger et al., 2006). While in mammals both are comprised of mTOR and mLST8, key differences in these protein complexes are that mTORC1 is inhibited by rapamycin and additionally contains the raptor protein, whereas the mTORC2 complex is relatively insensitive to rapamycin and includes rictor and SIN1 (Huang and Manning, 2009; Wullschleger et al., 2006). It is intriguing that mTOR lies both upstream and downstream of Akt, with mTORC2 as the Akt Ser-473 kinase and Akt enhancing mTORC1 activity by inhibiting its negative regulator, TSC1/2 (Huang and Manning, 2009). However, the mechanisms of mTORC2 regulation are not well understood, especially in regard to how PI3K promotes mTORC2 activity (Laplante and Sabatini, 2009). Interestingly, in contrast to its inhibitory role on mTORC1, TSC1/2 appears to promote mTORC2 activity (Huang et al., 2008). Moreover, Dedhar and colleagues demonstrated that interaction between integrin-linked kinase and mTORC2 plays an important role in promoting phosphorylation of Akt at Ser-473 (McDonald et al., 2008a; McDonald et al., 2008b). Other Ser-473 kinases have also been reported (Bozulic et al., 2008; Surucu et al., 2008). Thus, while GIP may stimulate mTORC2 activity in β-cells leading to its effect on Akt phosphorylation at Ser-473, this proposal is merely speculative since its mode of action is unclear. It should be noted, however, that Ser-473 phosphorylation is ablated in the presence of PI3K inhibitors (Fig. 2), so it seems that, at a minimum, basal PI3K activity is involved in GIP stimulation of Ser-473 phosphorylation. The overall complexity of these alternate modes of Akt activation stimulates curiosity into their underlying functions, and clues may lie in the fact that Akt exerts numerous intracellular actions and moves throughout the cell to multiple localities, including the cytoplasm, nucleus, golgi, ER and mitochondria (Parcellier et al., 2008). Thus, the multiple mechanisms of Akt activation may be linked to its differing roles and answers to this question await future studies.

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Based on current evidence and extrapolating from findings with regard to GLP-1R signalling, a proposed model for the mechanism by which GIP activates Akt is depicted in Fig. 33. Stimulation of β-cells with GIP rapidly elevates Akt activity in a manner involving EPAC2. In parallel is the phosphorylation of Akt at Ser-473, and this is proposed to occur by enhancing mTORC2 activity. With prolonged stimulation, GIP may enhance CREB pS133 levels and consequently increase IRS-2 protein levels, as occurs with GLP-1 (Jhala et al., 2003). Similarly, GIPR signalling in β-cells, like GLP-1R signalling (Cornu et al., 2010; Cornu et al., 2009), may enhance IGF-IR expression and the secretion of IGF-II, thus forming an autocrine circuit that enhances IGF-IR signalling in β-cells. Insulin may also form a similar circuit but, based on the study by Thorens and colleagues (Cornu et al., 2009), insulin appears not to have a strong effect on Akt in β-cells. As described (Section 1.6.2), the IGF-IR undergoes autophosphorylation at tyrosine residues, resulting in the binding of IRS-2 through its PTB domain (Saltiel and Kahn, 2001; Taniguchi et al., 2006). Receptor-induced phosphorylation of tyrosine residues in the mid- region of IRS-2 forms a docking site for PI3K through its SH2 domain and ultimately couples receptor signalling to PI3K activation. PI3K then generates PIP3 at the plasma membrane, which is bound by Akt and PDK-1 through their PH domains (Alessi et al., 1996a; Hanada et al., 2004). PDK-1 phosphorylates Akt at Thr-308 (Alessi et al., 1996a; Hanada et al., 2004; Manning and Cantley, 2007; Stephens et al., 1998) and mTORC2 phosphorylates Akt at Ser-473 (Sarbassov et al., 2005), resulting in Akt activation. The overall effects of GIPR signalling through acute stimulation of Akt signalling via EPAC2 combined with increasing IRS-2 levels and IGF-IR production and IGF-II secretion greatly enhances Akt signalling and promotes its potent actions on β-cell growth, survival and function (Section 1.7.3).

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Figure 33. Proposed Mechanism by Which GIP Activates Akt in β-cells. Stimulation of the GIPR promotes rapid activation of Akt via EPAC as well as Akt phosphorylation at Ser-473 via mTORC2 (shown in black arrows). Longer stimulation periods may promote Akt activation and phosphorylation at Thr-308 and Ser-473 via enhancing IRS-2 expression and IGF-II / IGF-I receptor signalling (red arrows).

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6.2 On the Anti-apoptotic Actions of GIPR Signalling in β-cells In Chapter 4, it was demonstrated that GIPR signalling promotes β-cell survival by interacting with pro- and anti-apoptotic members of the Bcl-2 family of proteins. A series of studies by Korsmeyer and colleagues were essential for developing an understanding of the regulatory roles and actions of the Bcl-2 protein family (Cheng et al., 2003; Cheng et al., 2001; Danial et al., 2003; Danial and Korsmeyer, 2004; Hockenbery et al., 1990; Korsmeyer, 1999; Korsmeyer et al., 1993; Ruiz-Vela et al., 2005; Scorrano et al., 2003; Wang et al., 1996a; Wei et al., 2001; Yang et al., 1995; Yin et al., 1995). However, while it is clear that Bax and Bak are the „gate-keepers‟ for initiating cytochrome C release, the biochemical mechanisms involved in initiating Bax and Bak activation remain controversial (Chipuk et al., 2010), and currently, resolution of this question is considered the “holy grail of apoptosis research” (Youle and Strasser, 2008). In a recent study, Cheng and colleagues (Kim et al., 2006b) provided evidence indicating that BH3-only domain containing proteins such as Bim and Bid initiate apoptosis via direct interactions with Bax and Bak, whereas Bcl-2 and Bcl-XL antagonize this initiation by sequestering Bim and Bid without directly interacting with Bax or Bak. Other „sensitizer‟ proteins, such as Bad, were proposed to promote apoptosis by inhibiting Bcl-2 and Bcl-XL, preventing them from binding to Bim and Bid. In a subsequent study (Kim et al., 2009), they described the underlying mechanism by which Bim and Bid binding promotes Bax and Bak conformational changes that enables oligomerization, mitochondrial pore formation and release of cytochrome C, and further support for this model has been provided by others (Gavathiotis et al., 2008). In striking contrast however, studies from Huang and colleagues (Fletcher and Huang, 2008; Fletcher et al., 2008; Willis et al., 2007) and others (Dewson et al., 2008) have provided evidence for a quite different model in which anti-apoptotic proteins such as Bcl-2 and Bcl-XL prevent apoptosis via direct interactions with Bax and Bak, thus inhibiting their activation, whereas Bim and Bid interact with Bcl-2 and Bcl-XL, preventing their inhibitory actions on Bax and Bak. Reconciliation of these differing models and development of an understanding of the relationships between Bax/Bak pore formation and the permeability transition pore should eventually reveal critical aspects of the underlying apoptotic mechanisms. In recent years, Bcl-2 family proteins have been shown to insert into ER membranes and modulate the unfolded protein response as well as regulate functions unrelated to apoptosis such as mitochondrial fission and fusion dynamics, regulation of ER Ca2+ stores and autophagy (Chipuk et al., 2010; Danial, 2008; Galluzzi et al., 2008; Hetz et al., 2006; Scorrano et al.,

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2003). Studies defining the functional attributes of Bcl-2 family proteins with respect to β-cell function and survival may therefore be critical for understanding the development of β-cell dysfunction in T2D. In an elegant study from Danial and Korsmeyer (Danial et al., 2003), Bad was found to associate with glucokinase in hepatic mitochondria and play an important role in integrating glycolysis and mitochondrial respiration. The importance of this interaction was demonstrated in Bad-/- mice that developed glucose intolerance due to impairments in hepatic glucose metabolism. Subsequently, Danial and colleagues demonstrated that Bad exerted similar actions in β-cells and played an important role in β-cell glucose metabolism and insulin secretion (Danial et al., 2008). It appears that the actions of Bad on β-cell function, as opposed to apoptosis, were due to differing modifications in which phosphorylation of Ser-155 was critical for transporting glucokinase to the mitochondria, whereas dephosphorylation of Bad at Ser-112 and/or Ser-136 promoted Bad release from protein scaffolds and mitochondrial translocation, consequently contributing to initiation of apoptosis (Danial, 2008). In addition to these findings, others have shown that pro-inflammatory cytokines activate the mitochondrial apoptotic pathway in human and rat islets as well as INS-1 β-cells (Grunnet et al., 2009), and Thomas and colleagues have attempted to define the contributions of pro-apoptotic proteins to β-cell apoptosis by exposing islets isolated from knock-out mouse models to pro-inflammatory or glucotoxic stress and examining both cytochrome C release and the onset of apoptosis (McKenzie et al., 2008; McKenzie et al., 2010). As expected, over-expression of Bcl-2 increased β-cell survival but, interestingly, β-cells from Bid knock-out mice were protected from cytokine- induced but not glucose-induced apoptosis, whereas β-cells from Bim or Puma knock-out mice were protected from glucose-induced apoptosis. Furthermore, β-cells from Bak knock-out mice were protected from cytokine-induced but not glucose-induced apoptosis, whereas β-cells from Bax knock-out mice were significantly protected from apoptosis under all conditions, thus indicating differing roles for these proteins in regulating the onset of apoptosis, with Bax performing a critical role. In mice with β-cell specific deletion of Bcl-XL, β-cells developed normally but were more susceptible to the onset of apoptosis in response to pro-inflammatory and ER stress (Carrington et al., 2009), indicating that Bcl-XL is not fundamental to β-cell survival but contributes under conditions of β-cell stress. Furthermore, in studies on diabetic mice receiving islet transplants, over-expression of a protein called the X-linked inhibitor of apoptosis (XIAP), which acts by preventing activation of caspase-9 and caspase-3, in human (Emamaullee et al., 2005) or mouse islets (Plesner et al., 2005) resulted in improved islet graft

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survival and markedly greater ability to normalize glycaemia compared to control islets. Clearly therefore, members of the Bcl-2 family of proteins play critical roles in β-cell survival and function, and modulation of their actions may have significant therapeutic implications. In β-cells exposed to stress, GIP stimulation prevented mitochondrial translocation of Bad and BimEL, and activation of mitochondrial Bax, thus preventing the release of cytochrome C, activation of caspase-3 and onset of apoptosis (Chapter 4). The underlying mechanism of action involved Akt-mediated inhibition of ASK1, resulting in the dual suppression of p38 MAPK and JNK. Interestingly, this appears to be the first study identifying an important role for ASK1 in the initiation of β-cell apoptosis. In other cell types such as lymphocytes, cardiac tissue, neurons and endothelial cells, ASK1 has been shown to operate as a redox sensor that, upon exposure to excessive levels of reactive oxygen species (ROS), is capable of initiating the mitochondria-mediated apoptotic pathway via activation of p38 MAPK and JNK (Matsuzawa and Ichijo, 2008; Takeda et al., 2008). It should be emphasized that, whereas sustained stress causes pro-apoptotic ASK1 signalling, low or short-term stress generates only transient episodes of ASK1 signalling that, in contrast, plays a role in promoting cell survival and/or differentiation (Matsuzawa and Ichijo, 2008). In agreement with its pro-apoptotic role, sustained activation of p38 MAPK and JNK and the onset of apoptosis were significantly diminished in mouse embryonic fibroblasts from ASK1-/- mice exposed to tumour necrosis factor-α (TNFα) or hydrogen peroxide (Tobiume et al., 2001). Stressors that activate ASK1 include oxidative stress, ER stress, Ca2+ overload and receptor mediated inflammatory signals. The underlying mechanism coordinating ASK1 activation involves a protein complex described as the “ASK1 signallosome” that exists as a 1500-2000 kDa complex under „healthy conditions‟, but, following exposure to oxidative stress, ER stress or pro-inflammatory cytokines such as TNFα, forms a protein complex >3000 kDa in molecular mass, bound to which are proteins such as the TNF- receptor-associated factor (TRAF) family proteins and inositol-requiring enzyme (IRE)-1 (Matsuzawa and Ichijo, 2008; Noguchi et al., 2005; Takeda et al., 2008). Bound to the inactive ASK1 complex is the anti-oxidative protein, thioredoxin (Trx), the reduced form of which binds the N-terminal region of ASK1, preventing ASK1 signalling, whereas when intracellular levels of ROS exceed the anti-oxidative capacity of the cell, Trx is oxidized and dissociates from ASK1 (Matsuzawa and Ichijo, 2008; Takeda et al., 2008). ASK1 then commences to activate MEK 3/6 and MEK 4/7, resulting in the activation of p38 MAPK and JNK, respectively. Furthermore, even if Trx is released from ASK1, an additional regulatory mechanism exists for ASK1 in

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which Akt phosphorylates Ser-83 and prevents ASK1 activation (Kim et al., 2001), as found with GIP (Chapter 4). This constitutes an intriguing scenario in which, even in the presence of elevated intracellular stress, extracellular signals can relay instructions to the cell, such as via Akt, resulting in the interception of ongoing pro-apoptotic signals, and highlighting the complex balance between life and death signalling. Studies from Ichijo and colleagues were the first to discover the ASK-1 signallosome and describe its role in integrating or „sensing‟ cellular stress (Matsuzawa and Ichijo, 2008; Takeda et al., 2008). They described how elevated mitochondrial metabolism, ER stress or prolonged activation of the TNF-α receptor (TNF-R) causes production of ROS that oxidizes Trx, resulting in derepression of ASK1, whereas activation of IRE-1 via ER stress or TNF-R signalling via elevations in circulating TNF-α, results in binding with TRAF2, a major component of the pro- apoptotic ASK1 signallosome. This revealed an elegantly integrated protein network that enables pro-apoptotic responses to excessive nutrient or prolonged pro-inflammatory cytokine exposure. Interestingly, it appears that ASK1 also contributes to insulin resistance (Bian et al., 2010; Bluher et al., 2009; Nishikawa et al., 2007), and this involves JNK-induced serine phosphorylation of IRS-1 (Imoto et al., 2006). Thus in the context of obesity, ASK1 may contribute to diabetes pathogenesis by promoting insulin resistance and β-cell apoptosis. Intriguingly, Hotamisligil and colleagues recently proposed that a putative protein complex they termed the metabolic inflammasome (metaflammasome) was involved in integrating metabolic and inflammatory stress during obesity and played a critical role in the development of insulin resistance and metabolic disease (Hotamisligil, 2010; Nakamura et al., 2010). The prediction of this complex was based on evidence linking activation of inflammatory and stress signalling pathways in response to nutrient excess, ER stress and inflammation to the development of metabolic disease as well as evidence revealing that ER stress and inflammation are intricately linked to each other. Core effectors of the metaflammasome include JNK, IKK and PKR, which are coordinately regulated and mediate a multitude of actions such as promoting insulin resistance via serine phosphorylation of IRS proteins and promoting apoptosis in β-cells. Conversely, activation of IRE-1 and TNF-R were proposed to play critical roles in modulating metaflammasome activity. While some of its effectors were defined, the integrating „sensor‟ of the metaflammasome remains unclear, and an intriguing possibility is that it may be one and the same as the ASK1 signallosome. However, although these complexes exhibit functional similarities, such as activating JNK and responding to IRE-1 and TNF-R, the actual existence of

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a metaflammasome and its structural components must first be established before any structural similarity to the ASK1 signallosome can be determined. Such studies will be of great interest and could greatly advance our understanding of the origins of diabetes and metabolic disease. In addition to inhibiting ASK1, the effects of GIPR signalling on β-cell survival also involve changes in transcriptional activity. As discussed (Section 1.7.3 & 6.1), GIP increases CREB-mediated expression of IRS-2 and Bcl-2 in a manner involving PKA and Akt signalling (Kim et al., 2008b; Trümper et al., 2001), as well as inhibiting Foxo-1 mediated expression of Bax via Akt-dependent phosphorylation of Ser-256 (Kim et al., 2005b). Furthermore, chronic GIPR signalling in VDF rats reduced islet protein levels of p53, Bax, Bad, Bim, CHOP and cleaved caspase-3 (Fig. 28G). Though the mechanism by which CHOP levels were reduced is uncertain, CHOP enhances Bim expression in response to ER stress (Puthalakath et al., 2007), and thus GIPR signalling likely reduced Bim levels via reducing CHOP. Moreover, since the p53 transcription factor enhances expression of pro-apoptotic genes such as Bax, Puma, Noxa, Bid and Bad (Chipuk and Green, 2006; Haupt et al., 2003; Jiang et al., 2006; Leu et al., 2004; Mihara et al., 2003; Mihara and Moll, 2003; Vousden and Lane, 2007), GIPR signalling probably reduced Bax and Bad protein levels by attenuating p53 protein levels via Akt-mediated activation of MDM2 (Fig. 1) through phosphorylation at Ser-166, an event that is known to promote p53 delivery to the proteosome and consequent degradation (Harris and Levine, 2005). A role for reducing p53 activity in contributing to the anti-apoptotic effects of GIPR signalling is supported by studies showing that p53 promoted apoptosis in β-cells exposed to hyperglycaemia (Ortega-Camarillo et al., 2006), hyperlipidaemia (Lovis et al., 2008) or cytokines (Kim et al., 2005c), and Akt-mediated survival of INS-1 β-cells exposed to lipotoxic stress involved down- regulation of p53 activity (Wrede et al., 2002). However, GIP-induced reduction in Foxo-1 activity was probably also important for β-cell survival since Foxo-1 negatively regulates PDX- 1, a crucial factor in β-cell function and survival (Buteau and Accili, 2007; Kitamura and Ido Kitamura, 2007; Kitamura et al., 2002). Considering the studies described in the thesis and those in the literature, a model describing the anti-apoptotic pathways of GIPR signalling in β-cells is proposed, as follows (Fig. 34). In β-cells exhibiting oxidative, ER and/or pro-inflammatory stress due to chronic exposure to glucose, lipids and/or pro-inflammatory cytokines, activation of IRE-1 and TNF-R signalling promotes TRAF2 coupling with the ASK1 signallosome. Note that TRAF2 is not shown, but considered part of the activated ASK1 signallosome protein module, and though not described in

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detail, other pro-inflammatory receptors in β-cells such as the interleukin 1-β receptor (Donath et al., 2008), could exhibit similar actions, and the receptor in Fig. 34 is therefore simply shown as a „death receptor‟. Elevations in ROS levels are capable of oxidizing Trx, resulting in its dissociation from ASK1 and enabling activation of ASK1 signalling. ASK1 activates MEK 3/6 and MEK 4/7, which then activate p38 MAPK and JNK, respectively. In parallel, elevations in Foxo-1 and p53 transcriptional activity occur, resulting in elevated expression of pro-apoptotic proteins such as Bax, Bad and Puma. The elevation in p38 MAPK and JNK signalling results in Bad and BimEL mitochondrial translocation, Bax oligomerization, mitochondrial pore formation, cytochrome C release and the onset of apoptosis. However, GIPR stimulation promotes Akt-mediated phosphorylation of ASK1 at Ser-83, resulting in ASK1 inhibition and consequently suppression of p38 MAPK and JNK signalling. Furthermore, Akt inhibits Foxo-1 activity via phosphorylation at Ser-256, and also inhibits p53 activity by promoting its degradation through activation of MDM2 via phosphorylation at Ser-166, ultimately resulting in a reduction in Bax, Bad and Puma expression. GIPR signalling also promotes Akt and PKA mediated activation of CREB, resulting in elevated expression of the anti-apoptotic protein Bcl-2 as well as the potent growth promoting signalling molecule, IRS-2. Thus, GIPR signalling antagonizes pro-apoptotic signalling at both transcriptional and post-translational levels, and future studies addressing the roles of these components should reveal important insights into the regulation of β-cell apoptosis.

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Figure 34. Proposed Anti-apoptotic Actions of GIPR Signalling in β-cells. GIPR stimulation in β-cells exposed to oxidative, ER or pro-inflammatory stress prevents the pro-apoptotic actions of ASK1, Foxo-1 and p53 via combined responses involving Akt as well as PKA. White labels indicate pro-apoptotic proteins and black labels indicate anti-apoptotic proteins. Shown with black arrows are the effects of GIPR signalling. Shown with red arrows are effects prevented by GIPR signalling. Note that the ASK1 signallosome is suggested to also be the recently described metaflammasome, and that Trx is not shown in order to signify its dissociation from ASK1. The term „death receptor‟ refers to TNF-R as well as other receptors such as the interleukin 1β receptor.

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6.3 On the Proposed Hypothesis and Future Directions In Section 1.8.2, it was hypothesized that activation of the GIPR in β-cells enhances critical anti-apoptotic signalling networks and promotes β-cell survival, which in rodent models of T2D results in an elevation in β-cell mass and improvement in glycaemic control. The Bcl-2 family of proteins, Foxo-1 and p53 play key roles in the promotion of apoptosis and both JNK and p38 MAPK mediate pro-apoptotic actions in response to nutrient, ER and pro-inflammatory stress, whereas Akt is an established anti-apoptotic protein in β-cells. Studies presented in this thesis showed that GIPR signalling exerts major effects on each of these components of apoptotic signalling in β-cells (Sections 3.3, 4.3, 6.1 and 6.2). Further support for an important pro-survival role was demonstrated in Chapter 5, in which chronic GIPR activation with D-GIP1-

30 promoted β-cell survival and enhanced glycaemic control in rodent models of diabetes. Thus, data from these studies are consistent with the hypothesis proposed. Despite supportive findings, the anti-apoptotic effects of GIPR signalling observed with in vitro studies have not been definitively linked to the anti-apoptotic effects of GIPR signalling observed with in vivo studies. Moreover, according to the proposed model (Fig. 34), GIPR signalling should promote the survival of β-cells exposed to pro-inflammatory cytokines, but this has not yet been tested with in vivo or in vitro studies. Indeed, it remains possible that some anti- apoptotic effects of GIPR signalling were secondary, due to improvements in β-cell insulin responses and lowering of glycaemia with consequent reductions in β-cell stress. Though discriminating between direct and indirect pathways is difficult in vivo, studies using genetic mouse models may provide more clarity. For example, investigating the effects of GIPR signalling in mice with β-cell specific expression of a Ser83Ala mutant ASK1 would likely test the proposed link between Akt and the mitochondrial apoptotic pathway, as would studies on mice with β-cell specific expression of constitutively active or dominant-negative Akt, Foxo-1 or p53. First however, it remains to be established in vivo that ASK1 actually contributes to the reductions in β-cell mass in models of T2D. The STZ rat model served as an adequate „proof-of-principle‟ study for demonstrating that GIPR signalling promotes anti-apoptotic actions in vivo. However, the leptin-receptor deficient Zucker rats reflected the human situation much more closely since they are hyperphagic and hyperlipidaemic and have obesity-associated insulin resistance, with VDF rats exhibiting mild hyperglycaemia and ZDF rats exhibiting marked hyperglycaemia and β-cell apoptosis. Since β-cell ER stress has been strongly implicated in the pathogenesis of T2D (Eizirik et al.,

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2008; Marchetti et al., 2007; Scheuner and Kaufman, 2008), studies investigating the in vivo effects of GIPR signalling on the Akita mice that model β-cell ER stress-induced diabetes was a useful adjunct. Interestingly, similar types of mutations to that causing ER stress in Akita mice have been found to cause permanent neonatal diabetes mellitus in humans (Colombo et al., 2008; Edghill et al., 2008; Stoy et al., 2007). Therefore the anti-apoptotic effects of GIPR signalling in β-cells of Akita mice indicates that incretin therapies may have important glucose lowering actions in this patient population. The effects of GIPR signalling on β-cells in Akita mice appeared not to involve regulatory actions on CHOP, and so further studies on other factors involved in ER stress such as Wolfram syndrome 1 (Fonseca et al., 2010), CCAAT/enhancer- binding protein-β (Matsuda et al., 2010) or the ER chaperone BiP (Cunha et al., 2009) may reveal important mechanistic insights. Of further interest, islets in T2D patients consistently exhibit fibril formation of human islet amyloid polypeptide (Kahn et al., 1999), and this has been considered a critical contributing component to the loss of β-cell mass and function (Hull et al., 2004), though the mechanisms of action remain controversial (Haataja et al., 2008; Zraika et al., 2010). Studies investigating the effects of GIPR signalling in mice or rats with β-cell specific transgenic expression of human islet amyloid polypeptide would be informative. Overall, the effects of GIPR signalling were examined in multiple rodent models of diabetes and provided significant support to the hypothesis, but further studies are required to establish clear links. Furthermore, studies on alternative animal models as well as cultured human islets should provide important information for relating effects to T2D in humans. It is worthy of note that the in vivo studies involved twice-daily injections, as opposed to a chronic infusion. Thus, robust elevations in GIPR signalling would have occurred in a biphasic fashion. This may have been important for the anti-apoptotic effects of D-GIP1-30, since the GIPR is known to undergo homologous desensitization (see Section 1.4.1) and it is possible that β-cells respond more effectively to intermittent, rather than constant, GIPR signalling. Interestingly, in an elegant study investigating the Wnt signalling pathway in cell lines as well as Xenopus embryos (Goentoro and Kirschner, 2009), it was revealed that fold-changes and not absolute levels of β-catenin dictated biological outcomes, and it was argued that the underlying nature of this system buffered stochastic, genetic, and environmental variations and enabled a much more reliable response to signalling events. It is possible that β-cells evolved a similar response mechanism to GIP, which naturally undergoes rapid elevations following meal ingestion and then undergoes rapid inactivation via DPP-IV degradation.

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As discussed (Section 1.7), incretins potentiate GSIS, increase insulin biosynthesis and promote elevations in β-cell mass. It is quite likely therefore that GIP/GLP-1 combination therapy will have strong glucose-lowering actions in T2D patients. In a recent presentation by Richard DiMarchi and Matthias Tschöp on “Novel Glucagon-Like Chimera Peptides: Virtues of Combinatorial Pharmacology” at the Keystone Symposia on Islet Biology and Diabetes (Proceedings held at the Whistler Conference Centre; April 15, 2010), a GIP/GLP-1 co-agonist was shown to exhibit greater anti-diabetic effects than a 10-fold higher dose of the currently marketed GLP-1R agonist, Liraglutide, and it was argued that this form of therapy would be unlikely to demonstrate the negative side-effects of Liraglutide and Byetta, such as nausea (Section 1.6.5). Furthermore, T2D patients treated with DPP-IV inhibitors are, effectively, receiving GIP/GLP-1 combination therapy. In view of the pleiotropic actions of GIP and GLP-1 (Fig. 35), it is essential to develop a better understanding of their actions in order to attempt prediction of long-term outcomes. With respect to GIP, a deeper understanding of its lipogenic role is needed and, based on findings in Chapter 5, it is important to establish whether GIP analogues with tissue-selective GIPR activation can be developed. Similarly, the effects of GIP on bone, brain and the cardiovascular system require further study. Ultimately, numerous questions still remain regarding the effects of incretins and their potential as therapeutics. However, studies provided in this thesis have clearly established a generally unappreciated potential role for GIP-based therapies in promoting β-cell survival and preserving β-cell mass in T2D. Of additional interest is whether similar findings would also occur in the situation of type 1 diabetes mellitus (T1D), which is caused by a near complete loss in β-cell mass due to auto- immune induction of β-cell apoptosis (Donath et al., 2008; Eizirik et al., 2008). Moreover, islet transplantation markedly improves glycaemic control in patients with T1D, but this is greatly limited by a marked rise in β-cell apoptosis in islets following transplant (Warnock et al. 2007). Therefore, GIP-based therapeutics may be capable of exhibiting broader-ranging benefits by promoting survival of pancreatic β-cells in patients with T2D, T1D and certain forms of neonatal diabetes as well as β-cells in islet transplants. Such exciting possibilities should incite more intense research efforts into the actions of GIPR signalling, since only then will we realize the potential therapeutic applications of this remarkable hormone system.

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Figure 35. The Pleiotropic Actions of Incretin Hormones. For detailed discussions on the actions of GIP as well as GLP-1 in the tissues shown, see Section 1.5, 1.7, 3.3, 4.3 and 5.3.

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Appendices

Appendix A. An Illustration of the Canonical Mechanism of Akt Activation

This illustration ascribes to the mechanism described in Section 3.1. The IGF-I receptor is shown here as a representative of the receptor tyrosine kinase family, which have been the proto-typical receptor family utilized for determining the Akt activation mechanism. However, it has generally been thought that the incretin receptors, which are members of the class B G-protein coupled receptor family, stimulate the same mechanism of Akt activation in β-cells. Note that the phosphorylation of the Thr-308 residue of Akt is widely considered essential for kinase activation. Though the Thr-308 and Ser-473 sites are discussed in this thesis, in actual fact, these sites correspond only to Akt1 in mammals, which contain two additional Akt genes, Akt2 and Akt 3. Analogous sites in Akt2 are Thr-309 and Ser-474, whereas in Akt3 the sites are Thr-305 and Ser-472. However, in this thesis, Thr-308 and Ser-473 are meant to represent the analogous sites for all Akt isoforms in assays that do not discriminate between isoforms.

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Appendix B. Cultured 3T3-L1 Adipocytes Differentially Respond to D-GIP1-30 and GIP1-42

3T3-L1 cells were cultured onto 96-well culture plates and induced to differentiate into adipocytes as previously described (Kim et al., 2007b). 3T3-L1 adipocytes were serum starved in 3 mM glucose DMEM containing 0.1% BSA overnight and then treated for 24 h with increasing concentrations (0-1000 nM) of GIP1-42, D-GIP1-42, GIP1-30 or D-GIP1-30 in the presence of 1 nM insulin and then LPL activity determined. Mean ± SEM (n=7); statistical significance is shown. LPL enzyme activity assays were performed using the manufacturers protocol (Roar Biomedical Inc.) and presented as relative activity normalized to protein concentration.

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Appendix C. Certificates of Approval

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