The Role of P21-Activated Protein Kinase 1 in Metabolic Homeostasis

THE ROLE OF P21-ACTIVATED PROTEIN KINASE 1 IN METABOLIC HOMEOSTASIS

YU-TING CHIANG

A thesis submitted in conformity with the requirements for the degree of

Doctor of Philosophy

Graduate Department of Physiology University of Toronto

The Role of P21-Activated Protein Kinase 1 in Metabolic Homeostasis Yu-ting Chiang Doctor of Philosophy Department of Physiology University of Toronto 2014

Abstract

Our laboratory has demonstrated previously that the proglucagon gene (gcg), which encodes the incretin hormone GLP-1, is among the downstream targets of the Wnt signaling pathway; and that

Pak1 mediates the stimulatory effect of insulin on Wnt target gene expression in mouse gut nonendocrine cells. Here, I asked whether Pak1 controls gut gcg expression and GLP-1 production, and whether Pak1 deletion leads to impaired metabolic homeostasis in mice. I detected the expression of Pak1 and two other group I Paks in the gut endocrine L cell line GLUTag, and colocalized Pak1 and GLP-1 in the mouse gut. Insulin was shown to stimulate Pak1 Thr423 and β-cat

Ser675 phosphorylation. The stimulation of insulin on β-cat Ser675 phosphorylation, gcg promoter activity and gcg mRNA expression could be attenuated by the Pak inhibitor IPA3. Male Pak1-/- mice showed significant reduction in both gut and brain gcg expression levels, and attenuated elevation of plasma GLP-1 levels in response to oral glucose challenge. Notably, the Pak1-/- mice were intolerant to both intraperitoneal and oral glucose administration. Aged Pak1-/- mice showed a severe defect in response to intraperitoneal pyruvate challenge (IPPTT). In primary hepatocytes, however, IPA3 reduced basal glucose production, attenuated glucagon-stimulated glucose production, and inhibited the expression of Pck1 and G6pc. This implicates that the direct effect of group I Paks in hepatocytes is the stimulation of gluconeogenesis, and that the impairment in

IPPTT in aged Pak1-/- mice is due to the lack of Pak1 elsewhere. The defect in IPPTT in aged

Pak1-/- mice could be rescued by stimulating gcg expression with forskolin injection or by enhancing the incretin effect via sitagliptin administration. In summary, my study demonstrates that: 1) Pak1 positively regulates GLP-1 production, 2) Pak1/β-cat signaling plays a role in gut/liver axis or gut/pancreas/liver axis governing glucose homeostasis, and 3) Pak1-/- mice can be utilized as a novel model for metabolic research.

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Acknowledgements

I would like to express my deepest gratitude to my supervisor Dr. Tianru Jin, for his continuous support and guidance throughout my studies. He has exemplified the merging of a critical mind with a relentless pursuit of knowledge, which has been and always will be an inspiration to me, both in science and in life. I would like to extend my gratitude toward the members of my Supervisory Committee, my co-supervisor Dr. Michael Wheeler, as well as Dr.

Herbert Gaisano and Dr. Qinghua Wang, whose constant encouragement and advice have made the completion of this research project an enjoyable experience. Thanks to all the past and present members of the Jin lab and the TMDT 10th floor diabetes lab, you all made the past few years so much more colorful and memorable. Thanks to Joan and Wilfred for journeying with me, your assistance, advice, and friendship have been invaluable. I want to give special thanks to my parents, David and Lisa, who supported me unconditionally from afar and whose wisdom and love have always been just a Skype call away. Last but not least, I want to thank my sister Helen for the numerous dinner conversations revolving around science, research, Western blots, and mice. Your daily companionship as a sister, friend, and fellow scientist has been a true blessing.

This thesis is dedicated to the loving memory of my teacher and mentor Dr. Robert Carsten

(Jack) von Borstel (1925-2012) from the University of Alberta, whose immeasurable kindness and encouragements have been instrumental in the completion of all my post-graduate studies.

I can do all things through Christ who strengthens me. (Philippians 4:13) 

Table of Contents

ABSTRACT ...... II TABLE OF CONTENTS ...... V LIST OF FIGURES ...... IX LIST OF FIGURES ...... X LIST OF FIGURES ...... XI LIST OF TABLES ...... XII LIST OF APPENDICES ...... XIII LIST OF ABBREVIATIONS ...... XIV LIST OF PUBLICATIONS ...... XIX 1 INTRODUCTION ...... 1 1.1 DIABETES MELLITUS AND THE METABOLIC SYNDROME ...... 2 1.1.1 Diabetes mellitus and the metabolic syndrome ...... 2 1.1.2 Major metabolic hormones in glucose homeostasis ...... 4 1.1.2.1 Hormones ...... 4 1.1.2.2 The pancreas and islets of Langerhans...... 5 1.1.2.3 Insulin ...... 6 1.1.2.4 Glucagon ...... 11 1.1.2.5 Somatostatin ...... 13 1.1.2.6 Pancreatic polypeptide ...... 15 1.1.2.7 Leptin ...... 16 1.1.3 The liver as a central organ in glucose homeostasis ...... 18 1.1.3.1 Glycogenolysis and glycogenesis ...... 19 1.1.3.2 Glycolysis and gluconeogenesis ...... 22 1.2 THE INCRETIN HORMONE GLUCAGON-LIKE PEPTIDE 1 ...... 24 1.2.1 Proglucagon gene, GLP-1 production and degradation ...... 24 1.2.2 Mechanisms underlying proglucagon gene expression ...... 30 1.2.3 The functions of GLP-1 ...... 38 1.3 THE WNT SIGNALING PATHWAY AND PROGLUCAGON GENE EXPRESSION ...... 42 1.3.1 Overview of the Wnt signaling pathway ...... 42 1.3.2 Wnt signaling pathway and metabolic homeostasis ...... 44 1.3.3 Wnt signaling pathway effectors as mediators of proglucagon gene expression ...... 47 1.4 P21-ACTIVATED PROTEIN KINASE 1 AND ITS ROLE IN METABOLIC HOMEOSTASIS...... 48 1.4.1 Overview of the Pak family ...... 48 1.4.1.1 The discovery of Paks 1-3 ...... 48 1.4.1.2 The discovery of Paks 4-6 ...... 52 1.4.1.3 Structural features, activation mechanisms, and upstream regulators of Paks ...... 53

1.4.1.3.1 Structural features of Paks ...... 53 1.4.1.3.2 Activation mechanism of group I Paks ...... 55 1.4.1.3.3 Positive regulators of Pak1 ...... 58 1.4.1.4 Negative regulators of Pak1 ...... 59 1.4.1.5 Substrate specificity ...... 62 1.4.1.6 The role of Paks in tumorigenesis and cancer ...... 62 1.4.1.7 The functions of Paks ...... 64 1.4.1.7.1 Cell cycle progression ...... 64 1.4.1.7.2 Cell survival and apoptosis ...... 66 1.4.1.7.3 Cytoskeleton remodeling ...... 67 1.4.1.7.4 Host-pathogen response ...... 69 1.4.1.7.5 Gene transcription and mRNA splicing ...... 70 1.4.1.7.6 Endothelial and vascular biology ...... 72 1.4.1.7.7 Metabolic homeostasis...... 73 1.4.2 Pak1 and glucose transport in muscle ...... 73 1.4.3 Pak1 and insulin secretion in pancreas ...... 82 1.4.4 Pak1 as a mediator of the crosstalk between insulin and Wnt signaling pathways ...... 86 2 RATIONALE, HYPOTHESIS, AND RESEARCH AIMS ...... 88 2.1 RATIONALE ...... 89 2.2 HYPOTHESIS AND RESEARCH AIMS ...... 89

3 GENERAL MATERIALS AND METHODS ...... 91 3.1 CHEMICALS AND ANTIBODIES ...... 92 3.2 WESTERN BLOTTING ...... 92 3.3 RNA EXTRACTION AND REAL-TIME QUANTITATIVE REVERSE-TRANSCRIPTASE PCR ...... 93 3.4 EXPERIMENTAL ANIMALS, MAINTENANCE, AND GENOTYPING...... 94 3.5 MOUSE ORGAN WEIGHT MEASUREMENTS ...... 95 3.6 IMMUNOHISTOCHEMISTRY OF MOUSE INTESTINE AND PANCREAS ...... 95 3.7 STATISTICAL ANALYSES AND DENSITOMETRY ANALYSIS ...... 95

4 P21-ACTIVATED PROTEIN KINASE 1 MEDIATES THE CROSSTALK BETWEEN INSULIN AND ΒETA- CATENIN ON REGULATING PROGLUCAGON GENE EXPRESSION IN THE GUT ...... 96 4.1 ABSTRACT ...... 97 4.2 INTRODUCTION ...... 97 4.3 MATERIALS AND METHODS ...... 99 4.3.1 Cell lines and tissue culture ...... 99 4.3.2 Fetal rat intestinal cell isolation ...... 99 4.3.3 Plasmids, transfection, and luciferase reporter gene analysis ...... 100 4.3.4 Real-time quantitative reverse-transcriptase PCR ...... 101 vi

4.3.5 Northern blotting ...... 101 4.4 RESULTS ...... 102 4.4.1 Insulin stimulates Pak1 activation in gcg-expressing cells ...... 102 4.4.2 Insulin-stimulated gcg expression can be attenuated by Pak inhibition ...... 103 4.4.3 Pak inhibition attenuates insulin-stimulated β-cat Ser675 phosphorylation ...... 104 4.5 DISCUSSION ...... 111 4.6 ACKNOWLEDGEMENTS ...... 113

5 ABLATION OF P21-ACTIVATED PROTEIN KINASE 1 PERTURBS GLUCOSE HOMEOSTASIS ...... 114 5.1 ABSTRACT ...... 115 5.2 INTRODUCTION ...... 115 5.3 MATERIALS AND METHODS ...... 116 5.3.1 Real-time quantitative reverse-transcriptase PCR ...... 116 5.3.2 Mouse distal ileum GLP-1 extraction ...... 116 5.3.3 Mouse brain primary neuron isolation ...... 117 5.3.4 Intraperitoneal and oral tolerance tests ...... 118 5.3.5 Hormone measurements ...... 118 5.4 RESULTS ...... 119 5.4.1 Pak1−/− mice in mixed C57BL/6-129 background have normal phenotypes ...... 119 5.4.2 Pak1−/− mice in C57BL/6 background show impaired glucose disposal and reduced gut gcg expression level ...... 119 5.4.3 Pak1−/− mice have reduced brainstem gcg expression level ...... 121 5.4.4 Pak1−/− mouse brain neurons show abolished response to insulin on β-cat Ser675 phosphorylation ...... 121 5.4.5 Pak1-/- mice have reduced distal ileum weight ...... 121 5.4.6 Pak1-/- mice have comparable responses to intraperitoneal insulin tolerance test ...... 122 5.5 DISCUSSION ...... 131 5.6 ACKNOWLEDGEMENTS ...... 136

6 THE ROLE OF P21-ACTIVATED PROTEIN KINASE 1 IN HEPATIC GLUCOSE PRODUCTION ...... 137 6.1 ABSTRACT ...... 138 6.2 INTRODUCTION ...... 138 6.3 MATERIALS AND METHODS ...... 140 6.3.1 Mouse primary hepatocyte isolation ...... 140 6.3.2 Glucose production assay ...... 141 6.3.3 Real-time quantitative reverse-transcriptase PCR ...... 142 6.3.4 Intraperitoneal administration of forskolin and sitagliptin gavage in mice ...... 142 6.4 RESULTS ...... 142 6.4.1 Aged Pak1-/- mice exhibit more severe defects in IPPTT and GLP-1 secretion response ...... 142 vii

6.4.2 Inhibition of Group I Paks represses glucose production in primary hepatocytes ...... 143 6.4.3 Inhibition of group I Paks represses gluconeogenic gene expression in hepatocytes ...... 144 6.4.4 In vivo forskolin administration improves IPPTT and increases gut gcg mRNA levels in aged Pak1-/- mice ...... 145 6.4.5 Sitagliptin gavage reverses the IPPTT defect and stimulates plasma GLP-1 levels in aged Pak1-/- mice ...... 146 6.4.6 Aged Pak1-/- mice have reduced epididymal fat pad weight ...... 147 6.5 DISCUSSION ...... 158 6.6 ACKNOWLEDGEMENTS ...... 162

7 GENERAL DISCUSSIONS, CONCLUSION, AND FUTURE DIRECTIONS ...... 164 7.1 GENERAL DISCUSSIONS ...... 165 7.1.1 The crosstalk between insulin and Wnt signaling pathways and its effect on GLP-1 production ...... 165 7.1.2 The in vivo role of Pak1 deficiency...... 167 7.1.3 The gut/liver axis or gut/pancreas/liver axis ...... 172 7.1.4 Redundant functions of group I Paks ...... 175 7.1.5 Pak1-/- mice as a novel model for metabolic and aging studies ...... 177 7.2 OVERALL IMPORTANCE OF STUDY AND CONCLUSION ...... 179 7.3 FUTURE WORK ...... 182 7.3.1 Liver-specific Pak1 knockout mice ...... 182 7.3.2 IPA3 as a potential glucose-lowering drug ...... 182 7.3.3 The role of Pak1 and Wnt signaling in adipogenesis ...... 184 7.3.4 GLP-2 as an intestinotrophic factor and as a treatment for intestinal diseases ...... 188 8 REFERENCES ...... 190

9 APPENDICES ...... 240 9.1 AGED PAK1-/- MICE EXHIBIT REDUCED WHOLE BODY FAT ...... 241 9.2 AGED PAK1-/- MICE HAVE COMPARABLE HEPATIC FAT CONTENT ...... 241 9.3 AGED PAK1-/- MICE HAVE REDUCED CIRCULATING GLP-2 LEVELS ...... 241

viii List of figures

Chapter 1

Fig. 1.1 Insulin signaling pathway ...... 9 Fig. 1.2 Glycogenolysis and glycogenesis ...... 20 Fig. 1.3 Glycolysis and gluconeogenesis ...... 23 Fig. 1.4 Proglucagon and proglucagon derived peptides (PGDPs) ...... 26 Fig. 1.5 GLP-1 and its derivatives...... 28 Fig. 1.6 Cis- and trans-elements involved in the regulation of gcg promoter activity...... 31 Fig. 1.7 Transcriptional regulation of the proglucagon gene...... 33 Fig. 1.8 Schematic presentation of the function of GLP-1...... 39 Fig. 1.9 Overview of Wnt signaling pathway...... 43 Fig. 1.10 P21-activated protein kinases (PAKs) are effectors for selected small GTPases...... 50 Fig. 1.11 Structural features of Pak proteins...... 54 Fig. 1.12 Activation mechanism of Pak1...... 56 Fig. 1.13 Interaction domains and phosphorylation sites of Pak1...... 57 Fig. 1.14 Pak1 and its upstream positive and negative regulators...... 61 Fig. 1.15 Pak1 and its downstream effectors regulate a multitude of cellular functions...... 65 Fig. 1.16 Summary of the role of Pak1 in skeletal muscle and pancreas...... 74 Fig. 1.17 Overview of mechanisms underlying insulin-stimulated glucose uptake in skeletal muscle...... 77 Fig. 1.18 Overview of the mechanisms underlying glucose-stimulated insulin secretion in pancreas...... 83

Chapter 4

Fig. 4.1 Insulin activates Pak1 in gcg-expressing cell lines ...... 106 Fig. 4.2 Pak1 expression profiles in selected tissues of C57BL/6 and CD1 mice .... 107 Fig. 4.3 Insulin-activated gcg promoter and mRNA expression can be attenuated by IPA3 ...... 108 Fig. 4.4 Wnt ligand Wnt3A stimulates gcg promoter and mRNA expression in GLUTag cell line ...... 109 Fig. 4.5 Insulin stimulates β-cat Ser675 phosphorylation in GLUTag cells ...... 110

Chapter 5

Fig. 5.1 Pak1-/- mice exhibit impaired glucose disposal ...... 123 Fig. 5.2 Body weight monitoring of Pak1-/- mice in C57BL/6-129 mixed genetic background ...... 124 Fig. 5.3 Pak1-/- mice show abnormalities in plasma hormone levels and gut gcg expression ...... 125 Fig. 5.4 Pak1-/- mice exhibit comparable pancreatic islet architecture ...... 126 Fig. 5.5 Pak1-/- mice show reduced brainstem gcg mRNA level and Pak1 regulates gcg expression in brain neurons ...... 127 Fig. 5.6 Pak1-/- mice brain neurons show lack of response in insulin-stimulated β-cat Ser675 phosphorylation ...... 128 Fig. 5.7 Pak1-/- mice exhibited reduced weight of distal ileum ...... 129 Fig. 5.8 Pak1-/- mice and age-matched wild-type (WT) mice have comparable responses in intraperitoneal insulin tolerance test ...... 130 Fig. 5.9 Our current understanding of the role of Pak1 in glucose homeostasis...... 135

Chapter 6

Fig. 6.1 Aged Pak1-/- mice show severe defect in IPPTT ...... 148 Fig. 6.2 The group I Pak inhibitor IPA3 represses glucose production in primary hepatocytes...... 149 Fig. 6.3 IPA3 represses gluconeogenic gene expression in primary hepatocytes ... 150 Fig. 6.4 Forskolin injection improves IPPTT and increases gut gcg mRNA levels in aged Pak1-/- mice ...... 151 Fig. 6.5 Forskolin injection for one week generated no effect on body weight in aged Pak1-/- mice and wild-type control mice ...... 152 Fig. 6.6 Sitagliptin rescues the IPPTT and OGTT impairments in aged Pak1-/- mice ...... 153-155 Fig. 6.7 No changes in body weight during sitagliptin treatment in aged Pak1-/- mice ...... 154 Fig. 6.8 Aged Pak1-/- mice exhibit smaller epididymal fat pads ...... 155 Fig. 6.9 Summary of the role of Pak1 in metabolic homeostasis and the phenotypes of Pak1-/- mice ...... 163

Chapter 7

Fig. 7.1 Overall summary and significance of study ...... 181

xi List of tables

Chapter 1

Table 1 Examples from the two categories of GLP-1 based therapeutics ...... 29

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List of appendices

Appendix 1 Aged Pak1-/- mice exhibit reduced whole body fat ...... 242 Appendix 2 Aged Pak1-/- mice have comparable hepatic fat content ...... 243 Appendix 3 Aged Pak1-/- mice have reduced circulating GLP-2 levels ...... 244

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List of Abbreviations

AID Autoinhibitory domain APC Adenomatous polyposis coli AR Androgen receptor ARH Arcuate nucleus of the hypothalamus ATF3 Activated transcription factor 3 ATP Adenosine triphosphate BAD Bcl-2-associated death promoter Bak Bcl-2-homologous killer Bax Bcl-2-associated protein Bcl-2 B-cell lymphoma-2 BMMC Bone marrow derived mast cell Brn4 Brain-4 CAM Chorioallantoic membrane Camp Cyclic adenosine monophosphate CCK Cholecystokinin Cdc42 Cell division control protein 42 homolog Cdx-2 Caudal type homeobox-2 cGMP Cyclic guanosine monophosphate ChIP Chromatin immunoprecipitation CK1α Casein kinase 1 α CNS Central nervous system cpE carboxypeptidase E CRE cAMP response element CREB cAMP response element binding CRIPAK Cysteine-rich inhibitor of Pak1 CtBP1 C-terminal binding protein 1 of E1A DKO Double-knockout DLC1 Dynein light chain 1 DMEM Dulbecco’s Modified Eagles Medium DPP-IV Dipeptidyl peptidase IV Dvl Dishevelled ECL Enhanced chemiluminescence EGF Epidermal growth factor eIF4E Eukaryotic translatioin initiation factor 4E Epac Exchange protein activated by cAMP

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ER Endoplasmic reticulum Ex-4 Exendin-4

F-1,6-P2 Fructose-1,6-bisphosphate F6P Fructose-6-phosphate FBP Fructose-1,6-biphosphatase FBS Fetal bovine serum FFA Free fatty acid FGF Fibroblast growth factor FGFR Fibroblast growth factor receptor FoxO Forkhead box O FRIC Fetal rat intestinal cell Frz Frizzled G1P Glucose-1 phosphate G6P Glucose-6-phosphate G6P Glucose-6-phosphatase G6ph Glucose-6-phosphatase GAP GTPase activating protein GC Glucagon challenge Gcg Proglucagon gene GcgR Glucagon receptor GDI Guanine nucleotide dissociation inhibitor GDP Guanosine diphosphate GEF Guanine nucleotide exchange factor GI Gastrointestinal GIP Glucose-dependent insulinotropic peptide GK Glucokinase GLP-1 Glucagon-like peptide-1 GLP-1R Glucagon-like peptide 1 receptor GLUT1/2 Glucose transporter 1/2 GLUT4 Glucose transporter 4 Glyc-3-P Glyceraldehyde-3-phosphate

Glycogenn Glycogen chain GNG Gluconeogenesis GP Glycogen phosphorylase GPa Glycogen phosphorylase active form GPb Glycogen phosphorylase inactive form GPCR G-protein coupled receptor GPM Glucose production medium GRPP Glicentin-related pancreatic polypeptide GS Glycogen synthase

GSa Glycogen synthase active form

GSb Glycogen synthase inactive form GSIS Glucose stimulated insulin secretion GSK3 Glycogen synthase kinase-3 GST Glutathione-S-transferase GTP Guanosine triphosphate HD Homeobox domain HDL High density lipoprotein HGF Hepatocyte growth factor HGP Hepatic glucose production HIV Human immunodeficiency virus HMG High-mobility group hPIP1 Human Gβ-like WD-repeat protein 1 IBMX 3-isobutyl-1-methylxanthine IDDM Insulin-dependent diabetes mellitus IGF Insulin-like growth factor 1 IP1 Intervening peptide-1 IP2 Intervening peptide-2 IPGTT Intraperitoneal glucose tolerance test IPITT Intraperitoneal insulin tolerance test IPPTT Intraperitoneal pyruvate tolerance test IR Insulin receptor IRK Insulin receptor kinase IRS Insulin receptor substrate Isl-1 Insulin gene enhancer protein-1

KATP ATP-sensitive K+ channel LADA Latent autoimmune diabetes in adults LDL Low density lipoprotein LEPR Leptin receptor LIMK Lim kinase LKB1 Serine threonine kinase liver kinase B1 LPA Lysophosphatidic acid LRP5/6 Low-density lipoprotein receptor-related proteins 5 and 6 LTD Long-term depression LTP Long-term potential LUC Luciferase reporter gene MAPK Mitogen-activated protein kinase MAPKK6 Mitogen-activated protein kinase kinase 6 MBP Myelin basic protein MEF Mouse embryo fibroblast

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MHC Myosin heavy chain miRNA microRNA MLCK Myosin light chain kinase MODY Maturity onset diabetes of the young MPGF Major proglucagon fragment mTOR Mammalian target of rapamycin NADPH Nicotinamide adenine dinucleotide phosphate Nck Tyrosine kinase adaptor protein NIDDM Non-insulin-dependent diabetes mellitus NPY Neuropeptide Y OAA Oxaloacetate OGTT Oral glucose tolerance test Pak p21-activated protein kinase Pax Paired box PBD p21-GTPase-binding domain Pbx Pre-B cell leukemia transcription factor PC Pyruvate carboxylase PC1/3 Prohormone convertase 1/3 PC2 Prohormone convertase 2 PCA Passive cutaneous anaphylaxis PCBP1 PolyC-RNA-binding protein 1 PDE3B Phosphodiesterase 3B PDGF Platelet-derived growth factor PDK1 Pyruvate dehydrogenase lipoamide kinase isozyme 1 PEI Polyethylenimine PEP Phosphoenolpyruvate PEPCK Phosphoenolpyruvate carboxykinase PGDP Proglucagon derived peptide PI3K Phosphoinositide 3-kinase PK Phosphorylase kinase PyrK Pyruvate kinase PKA Protein kinase A PKa Phosphorylase kinase active form PKb Phosphorylase kinase inactive form PKB Protein kinase B POU3F4 POU class 3 homeobox 4 PP Pancreatic polypeptide PP1 Protein phosphatase-1 PTB Phosphotyrosine-binding

PtdIns(4,5)P2 Phosphatidylinositol (4,5) bisphosphate

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PYY Peptide YY qRT-PCR quantitative reverse transcriptase polymerase chain reaction RIA Radioimmunoassay RIPA Radioimmuno precipitation assay RLC Regulatory light chain ROCK Rho-associated coiled-coil-containing protein kinases SA SST analog SAD-A Synapses of amphids defective SNP Single nucleotide polymorphism SKIP Skeletal muscle and kidney enriched inositol phosphatase SSC Saline sodium citrate SST Somatostatin SSTR Somatostatin receptor T1D Type 1 diabetes T2D Type 2 diabetes TAG Triacylglycerol TxNIP Thioredoxin-interacting protein VSMC Vascular smooth muscle cell Y4R Y4 receptor β-cat β-catenin

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List of Publications

Original Research Articles:

1) Chiang Y, Ip W, Shao W, Song ZE, Chernoff J, Jin T. Sitagliptin normalizes impaired hepatic glucose production in aged p21-activated protein kinase 1 knockout mice. (Revised manuscript submitted, Endocrinology).

2) Chiang Y, Shao W, Xu XX, Chernoff J, Jin T. 2013. P21-activated protein kinase 1 (Pak1) mediates the cross talk between insulin and β-catenin on proglucagon gene expression and its ablation affects glucose homeostasis in male C57BL/6 mice. Endocrinology 154(1):77- 88.

3) Ip W, Shao W, Chiang Y, Jin T. 2013. GLP-1-derived nonapeptide GLP-1(28-36) represses hepatic gluconeogenic gene expression and improves pyruvate tolerance in high fat diet fed mice. Am J Physiol Endocrinol Metab 305(11):E1348-58.

4) Shao W, Wang Z, Ip W, Chiang Y, Xiong X, Chai T, Xu C, Wang Q, Jin T. 2013. GLP- 1(28-36) improves β-cell mass and glucose disposal in streptozotocin induced diabetes mice and activates PKA-β-catenin signaling in beta-cells in vitro. Am J Physiol Endocrinol Metab 304(12):E1263-72.

5) Shao W, Wang D, Chiang Y, Ip W, Xu F, Columbus J, Belsham DD, Irwin DM, Zhang H, Wen X, Wang Q, and Jin T. 2013. The Wnt signaling pathway effector TCF7L2 controls gut and brain proglucagon gene expression and glucose homeostasis. Diabetes 62(3):789- 800.

6) Ip W, Shao W, Chiang Y, Jin T. 2012. The Wnt signaling pathway effector TCF7L2 is upregulated by insulin and represses hepatic gluconeogenesis. Am J Physiol Endocrinol Metab 303(9):E1166-76.

7) Liu S, Liu R, Chiang Y, Song L, Li X, Jin T, Wang Q. 2012. Insulin detemir enhances proglucagon gene expression in the intestinal L cells via stimulating beta-catenin and CREB activities. Am J Physiol Endocrinol Metab 303(6):E740-51.

8) Shao W, Yu Z, Chiang Y, Yang Y, Chai T, Foltz W, Lu H, Fantus IG, Jin T. 2012. Curcumin prevents high fat Diet induced insulin resistance and obesity via attenuating lipogenesis in liver and inflammatory pathway in adipocytes. PLoS One 7(1):e28784.

9) Columbus J, Chiang Y, Shao W, Zhang N, Wang D, Gaisano HY, Wang Q, Irwin DM, Jin T. 2010. Insulin treatment and high-fat diet feeding reduces the expression of three TCF genes in rodent pancreas. J Endocrinol 207(1): 77-86.

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Review Articles:

1) Chiang Y, Jin T. P21-activated protein kinases and their emerging roles in metabolic homeostasis. (Revised article submitted, Am J Physiol Endo Metab)

2) Chiang Y, Ip W, Jin T. The role of the Wnt signaling pathway in incretin hormone production and function. 2012. Front Physiol 3:273.

3) Ip W, Chiang Y, Jin T. The involvement of the Wnt signaling pathway and TCF7L2 in diabetes mellitus: The current understanding, dispute, and perspective. 2012. Cell Biosci. 2(1):28.

Summary of publications:

Number of first-authored original research articles: 1 published, 1 revision submitted

Number of first-authored review articles: 1 published, 1 revision submitted

Number of co-authored publications: 8 published

1 Introduction

Figures 1.4 and 1.8 were modified from figures of a review article published by Chiang et al. The role of the Wnt signaling pathway in incretin hormone production and function. 2012. Front Physiol 3:273 [200].

Figure 1.7 was modified from a figure of a review article published by Jin T. Mechanisms underlying proglucagon gene expression. The Journal of endocrinology. 2008;198(1):17-28 [192].

Figures 1.10, 1.11, 1.12, and 1.13 were from a review article that has been submitted by Chiang et al. P21-activated protein kinases and their emerging roles in metabolic homeostasis (revised article submitted, Am J PhysiolEndoMetab).

1.1 Diabetes mellitus and the metabolic syndrome

1.1.1 Diabetes mellitus and the metabolic syndrome

Diabetes mellitus, commonly referred to as diabetes, is a medical condition in which blood glucose concentrations are elevated beyond the normal range. There are several forms of diabetes mellitus(1). The most common form is type 2 diabetes (T2D), also called maturity-onset diabetes or non-insulin-dependent diabetes mellitus (NIDDM). T2D is mainly characterized by:

1) insulin resistance, which is the inability of the target tissues of insulin to respond to insulin action and 2) pancreatic β cell failure resulting in defective insulin secretion. Another form is

Type 1 diabetes (T1D), also called juvenile-onset diabetes or insulin-dependent diabetes mellitus

(IDDM). T1D is characterized by the autoimmune destruction of the pancreatic β cells, leading to insulin deficiency. There are several key features that differ between these two forms of diabetes. The typical age of onset of T2D occurs at >40 years, while that of T1D occurs between

6 months and 25 years (1). The typical body physique of T2D patients is overweight or obese, while T1D patients are normally lean and even show weight loss at disease diagnosis (1).

Other distinct but more rare forms of diabetes have been identified (1). Gestational diabetes mellitus refers to diabetes occurring in pregnant women (2). It usually disappears post partum, but predisposes the mothers to increased risk of developing T2D later in life. Maturity onset diabetes of the young (MODY) refers to a group of conditions (MODY1 to MODY7), where each is inherited in a Mendelian fashion (3). The genetic basis of these forms is being uncovered; for example, MODY2 is caused by a mutation in glucokinase, which causes defects in pancreatic β cell insulin secretion in response to high glucose. Other types of MODY seem to result from mutations in transcription factors involved in β cell development or function. Lastly,

latent autoimmune diabetes in adults (LADA) refers to the autoimmune-mediated β cell destruction that occurs in adults, and hence is often misdiagnosed as T2D (4). An estimated 10% of those diagnosed with T2D have this rare form of diabetes, and LADA patients often progress to insulin treatment earlier than those with T2D.

The term ‘metabolic syndrome’ reflects the notion that insulin resistance, the core of

T2D, is associated with a wide array of other inter-related diseases. These dysfunctions include obesity and liver diseases; as well as vascular disorders, for example coronary heart disease, hypertension, and atherosclerosis (5-9). In addition, a number of diabetes-associated complications manifest in diabetic patients, such as diabetic nephropathy, retinopathy, and diabetic foot. The severity of insulin resistance in diabetic patients occurs in a continuum, and it is now recognized that the condition of insulin resistance is associated with numerous adverse metabolic changes in the body. Some common features associated with insulin resistance include: glucose intolerance, elevated plasma triglyceride levels, reduced plasma high density lipoprotein (HDL)-cholesterol concentrations, impaired endothelia functions, elevated blood pressure, increased blood coagulation, and higher blood uric acid concentration.

Although various modern medical interventions have been developed for the treatment of diabetes, it continues to spread at an alarming rate and is on the verge of becoming a global pandemic, affecting over 371 million people worldwide (10). Moreover, it is estimated that currently half of those affected remain undiagnosed, and diabetes is expected to impose enormous health and socioeconomic burden in almost every single country. Hence, the journey to find a cure for diabetes continues, and efforts are being made in multiple dimensions, including the development of approaches in regenerative medicine, new types of insulin delivery

machineries, novel glucose-lowering agents, and further improvements of current therapies such as the latest category of glucagon-like peptide-1 (GLP-1) based therapeutics.

1.1.2 Major metabolic hormones in glucose homeostasis

1.1.2.1 Hormones

The word “hormone” comes from the Greek word “horman” which means to “to set in motion or to excite” (11). A hormone is defined as a substance that is secreted into the bloodstream, and once it reaches its destination, it is able to regulate the activity of cells or organs. A distinction must be made, however, between hormones and metabolites. Hormones are regulators whose action affects changes in substances other than themselves, whereas a metabolite is also able to regulate cellular activities, but the change occurs within the substance itself. An example of a hormone is insulin; it is produced in the pancreas and travels through the bloodstream to the muscle tissue where it stimulates glucose uptake. Glucose, on the other hand, is a metabolite; it is produced in the liver and secreted into the bloodstream, and once it reaches the muscle tissue, it is taken up by the muscle cells.

Hormones are produced by glands, which are organs that are able to produce

“secretions”, be it a hormone that enters the bloodstream (e.g. insulin), a juice that enters an organ (e.g. gastric juice), or a substance that is released out of the body (e.g. sweat). There are two major categories of glands: exocrine (secretions to be delivered out of the body) and endocrine (secretions to be delivered to internal organs). The main endocrine organs in humans are the pancreas, the pituitary gland, the thyroid gland, and the adrenal glands. In addition to

these glands, tissues such as the adipose tissue, the heart, the kidney, the skeletal muscle, the brain, and the intestine are now being uncovered as “metabolic tissues”, referring to their ability to produce and secrete hormones.

1.1.2.2 The pancreas and islets of Langerhans

The pancreas is a central organ in controlling glucose homeostasis. It is a tadpole-shaped organ lying in between the stomach and the liver. As a polarized organ, the ‘head’ of the pancreas points towards the body’s midline, while its ‘tail’ end points towards the left side of the body. The pancreas is composed of both exocrine and endocrine tissues, where the exocrine function consists of the secretion of digestive juices into the small intestine, and the endocrine function consists of the secretion of an array of hormones. Exocrine cells make up almost 94% of the pancreas, and produce a digestive juice containing amylase, lipase, trypsin, chymotrypsin and other enzymes. This cocktail of digestive juices is secreted from the exocrine cells into small ducts, which then join together to form the pancreatic duct. The pancreatic duct exits the pancreas and is joined with the bile duct, with the other end entering the duodenum of the small intestine. Hence, the pancreatic digestive juices are released into the duodenum together with the bile salts, and together they aid in the neutralization of the acidic chyme and the breakdown of proteins and lipids. Endocrine cells were discovered by the German medical student Paul

Langerhans in 1869 and were described as small “islands of cells” scattered among the exocrine tissue; they are hence called “islets of Langerhans” (12), or are commonly referred as “islets”.

Although only representing a small percentage of the total pancreas mass, there are about one million islets in the adult pancreas.

The cytoarchitecture of an islet is somewhat defined, and it has been recognized that there are differences between species (13,14). In a rodent islet, the structural organization is clearly distinguished by the periphery portion consisting of α cells, which secrete glucagon, and the inner core, which is made up of β cells secreting insulin, δ cells secreting somatostatin, PP cells secreting pancreatic polypeptide (PP), and ε cells secreting ghrelin. In human islets, the organization is not as defined, with the α and the β cell types often intermingled with each other.

Each islet is surrounded by pancreatic arteries from which it receives blood supply, and by pancreatic veins, which join the hepatic vein and thus act as a transport system for pancreatic hormones to be delivered to the liver. Hence, the liver is the first organ to be exposed to the pancreatic hormones, and this has important implications on the metabolic functions of the liver

(for details see Section 1.1.3).

1.1.2.3 Insulin

Insulin production

Insulin is a peptide hormone made up of two peptide chains: the α chain consisting of 21 amino acids, and the β chain, consisting of 30 amino acids. The two chains are held together via two disulfide bonds, located at A-Cys7/B-Cys7 and A-Cys20/B-Cys19. The gene encoding insulin in human is the INS gene, located on the q-arm of chromosome 11 (15). The first precursor, preproinsulin, is translated from the mRNA transcript at the endoplasmic reticulum (ER) membrane. A signal peptidase situated at the ER membrane removes the 24 amino acid signal peptide, leading to the formation of proinsulin. Proinsulin then undergoes protein folding and disulfide bond formation in the ER lumen, and is exported from the ER to the Golgi apparatus,

where it is packed into secretory granules and cleaved by the endopeptidases prohormone convertase (PC) 2 and PC1/3. Cleavage by the last peptidase carboxypeptidase E (cpE) yields the final mature insulin hormone, and in equimolar ratio the 31 amino acid active C-peptide fragment. Several stressors, including oxidative stress, endoplasmic reticulum stress, and amyloid protein formation, have been recognized as contributing factors to accelerated  cell death, leading to  cell dysfunction and impaired insulin response in T2D (16-19).

Insulin secretion

Insulin secretion is tightly controlled by the metabolic or nutritional state of the body, and the pancreatic β cells function as “glucose sensors”. The consensus model is that, when exposed to high extracellular glucose concentrations, the β cell takes up glucose via glucose transporter

1/2 (GLUT1/2) transporters, and the glucose is converted into pyruvate through the glycolysis pathway in the cytosol. The end product of glycolysis, pyruvate, enters the mitochondria and undergoes the Citric Acid cycle, leading to the oxidation of glucose and the generation of energy in the form of adenosine triphosphate (ATP) (20). The increase in ATP/ADP ratio results in the closing of the ATP-sensitive KATP channel, cell membrane depolarization, and opening of the voltage-gated Ca2+ channel (20). The resulting exocytosis of insulin-containing granules is mediated by the intricate interplay between membrane-bound and membrane-associated signaling proteins, ion channels, and the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein machinery (21).

Multiple insulin secretory defects are present in T2D, including the loss of early-phase insulin secretion, lower basal and stimulated plasma insulin concentrations, and progressive loss

of insulin secretory response over time (20). The exquisite control of insulin granule exocytosis is mediated by a wide panel of transducer and adapter proteins, small GTPases and their effectors, and cytoskeletal proteins (21-24). For example, depletion of Munc proteins that induce the formation of SNARE complexes, or of Sec5, a downstream effector of Ral-GTPase, have been shown to abrogate insulin granule mobilization in  cells (25,26). It has also been reported that intracellular compartmentalization, mediated by the coupling of SNARE proteins to lipid rafts, and membrane lipid content, such as cholesterol levels, are involved in the regulation of insulin secretion (27,28). Glucose is not the only stimulus for insulin secretion; other stimuli include amino acids arginine (29) and leucine (30), acetylcholine (31), sulfonylurea (32), cholecystokinin (CCK) (33), leptin (34), and the well-known insulin secretion inducer GLP-1

(35,36).

Insulin action

As a hormone, insulin circulates freely in the bloodstream, and it exerts its effect in the target tissues through the activation of the insulin signaling pathway (Fig. 1.1). The insulin signaling pathway is important for the regulation of a multitude of cellular activities, including gene expression, protein synthesis, cell differentiation/growth/survival, fatty acid metabolism, glycogen synthesis, and glucose uptake among many others. The insulin receptor (IR) tetramer has two extracellular α subunits and two transmembrane β subunits. Upon the binding of insulin to the α subunits, the resulting conformational change leads to the autophosphorylation of Tyr residues of the β subunits. These phosphorylation events are sensed by the phosphotyrosine- binding (PTB) domains of the adapter protein insulin receptor substrate (IRS), in turn resulting in the phosphorylation of key Tyr residues of IRS proteins, and the activation of p110, the catalytic

8 Glucose Insulin

Glucose Plasma membrane GLUT4 uptake IR P P PTP1B P P Shc PIP2 PTEN PI3K IRS Grb SOS Ras

SHIP1/2 PP2A PDK1 PIP3 Raf Akt PDK1 TSC1 TSC2 Pak1

MEK GLUT4 GSK-3 vesicle Bad mTOR

AS160 Erk GS p70S6K 4EBP1

Apoptosis eIF4E

Glycogen FOXO Protein synthesis synthesis Cytoplasm

Nucleus

FOXO

Cell growth and proliferation Gene transcription Hepatic glucose production Adipogenesis GLP-1 production

Fig. 1.1 Insulin signaling pathway. An overview of major downstream pathways of insulin signaling. IR: insulin receptor; IRS: insulin receptor substrate; GLUT4: glucose transporter 4; Grb: growth factor receptor-bound protein; Shc: Src-homology containing protein; SOS: son-of-sevenless; PTP1B: protein tyrosine phosphatase-1B; PTEN: phosphatase and tensin homolog; SHIP: SH2 domain-containing inositol 5’-phosphatase

PIP2: phosphatidylinositol (4,5) biphosphate; PIP3: phosphatidylinositol (3,4,5) trisphosphate; PP2A: protein phosphatase 2A; PDK1: pyruvate dehydrogenase lipoamide kinase isozyme 1 AS160: Akt substrate of 160kDa; GS: glycogen synthase; Erk: extracellular signal-regulated kinase; FOXO: forkhead class of transcription factors; Bad: B-cell lymphoma/leukemia 2 (Bcl- 2)/Basal cell lymphoma-extra large (Bcl- xl)-associated death promoter ; PI3K: phoshoinositide 3- kinase; AKT/PKB: protein kinase B; mTOR: mammalian target of rapamycin; 4EBP1: eIF4E- binding protein 1; p70S6K: p70 ribosomal S6 kinase; Pak1: p21-activated protein kinase 1.

subunit of phosphoinositide 3-kinase (PI3K). p110 then phosphorylates phosphatidylinositol

(4,5) bisphosphate (PIP2), leading to the formation of PIP3 and the activation of its downstream effectors pyruvate dehydrogenase lipoamide kinase isozyme 1 (PDK1) and protein kinase B

(PKB) (also known as AKT). The insulin signaling cascade is both intricate and extensive and regulates a vast number of pathways, including the mammalian target of rapamycin (mTOR) pathway for protein synthesis, the glycogen synthase (GS) pathway for glycogen synthesis, the forkhead box O (FOXO) pathway for gene transcription, the AS160/Rab pathway for GLUT4 translocation, and the mitogen-activated protein kinase (MAPK) pathway for regulating cell growth and differentiation.

Insulin clearance

The IR-bound insulin is removed by receptor-mediated internalization. The mechanism is reported to involve the insulin receptor kinase (IRK), a tyrosine kinase that is activated by insulin binding (37). The insulin-IR-IRK complex is internalized into endosomes, followed by the proteolytic degradation of insulin. This endosome trafficking process has been suggested to serve two purposes: the clearance of insulin from circulation and the attenuation of insulin-mediated signal transduction responses (38). Although the detailed mechanism for insulin clearance is not completely understood, the liver is a major organ for insulin clearance. It is estimated that about

50-70% of the insulin reaching the liver is removed during its passage. Therefore, only a fraction of the insulin reaches circulation, resulting in the “dampening” of the insulin effect (39). This is another illustration of the importance of the liver in insulin signaling and glucose metabolism.

1.1.2.4 Glucagon

Glucagon production

Glucagon is generally viewed as the counter-regulatory hormone of insulin and is a single polypeptide of 29 amino acids long. Glucagon is encoded by the proglucagon gene (gcg), located on the q-arm of chromosome 2 (40). The mRNA transcript is translated into the 160 amino acid prohormone proglucagon, which then undergoes tissue-specific post-translational cleavage to give rise to different peptide hormones (for details see Section 1.2.1). Cleavage by PC2 gives rise to glucagon in the pancreatic α cells (41), and cleavage by PC1/3 produces GLP-1 in the intestinal L cells (42) and certain neurons in the hypothalamus and brainstem (43).

Regulation of glucagon production

Glucagon production, similar to that of insulin, is mainly controlled by circulating glucose concentration, and its major action is to stimulate hepatic gluconeogenesis during the fasting state (44). The release of glucagon is stimulated by hypoglycemia, epinephrine (45), amino acids arginine (46) and alanine (47), acetylcholine (48), leptin (34), and CCK (49).

Suppressors of glucagon secretion include insulin (50), somatostatin (51), and increased levels of free fatty acids (FFAs) (52). As in the case of insulin, a proportion of glucagon is removed as it passes through the liver; it is estimated that the reduction is about 20% (53).

Glucagon action

Although the main action of glucagon is the stimulation of hepatic glucose production

(HGP) during hypoglycemia, glucagon exerts a number of physiological effects in other organs.

One of these effects is in the central nervous system (CNS), where central infusion of glucagon lead to the activation of protein kinase A (PKA), which was associated with inhibition of hepatic gluconeogenesis (54). Other effects include the control of energy expenditure, lipid metabolism, and weight loss. Transient infusion of glucagon in humans has been demonstrated to increase resting energy expenditure; the infusion also caused a rise in plasma glucose levels, which was then abrogated by the infusion of GLP-1 due to its insulinotropic effect (55). The activation of the glucagon receptor (GcgR) using GcgR agonists resulted in hyperglycemia, reduced body fat, and lower plasma cholesterol (56). Injection of glucagon in humans also resulted in elevated circulating levels of fibroblast growth factor 21 (FGF21) (56). FGF21 is a protein secreted from liver, adipose tissue, and pancreas, and has been reported to exert beneficial effects in improving insulin sensitivity, and lipid and energy metabolism (57). Acute infusion of glucagon in humans was shown to lower hepatic lipoprotein particle production as well as inhibiting particle clearance, but did not have an effect on intestinal lipoprotein metabolism (58). Lastly, pharmaceutical dosages of glucagon administration in human subjects lead to the reduction of motility in the small (59) and large intestine (60).

Hyperglucagonemia

Hyperglucagonemia is widely observed in both type 1 and type 2 diabetic patients. Due to the inter-relational nature of glucagon and insulin, the challenge lies in dissecting their

individual effects. Animal studies have shown that β cell destruction leads to hyperglucagonemia, as a result of the attenuated intra-islet suppression of glucagon secretion by insulin (61). Long-term infusion of glucose in rats leads to the development of hyperglucagonemia, which was found to precede the reduction of plasma insulin (62). The rise in glucagon was accompanied by hyperglycemia and accelerated HGP, which are rescued by the infusion of anti-glucagon antibodies (62). The detailed role of glucagon in the pathogenesis of diabetes remains to be identified; however, it is apparent that overproduction of glucagon is a central defect in diabetes and glucagon suppression may serve as potential therapies for the treatment of diabetes (63).

1.1.2.5 Somatostatin

Somatostatin (SST) was discovered, isolated, and first characterized more than three decades ago (64-66). SST is synthesized in two bioactive forms: the predominant but less biologically active somatostatin-14 (SST-14), and the larger more potent somatostatin-28 (SST-

28) (67). The actions of SST are mediated through the binding to a family of somatostatin receptors belonging to the G-protein coupled receptor (GPCR) superfamily. Six somatostatin receptor (SSTR) subtypes have been identified and cloned, including sst1, sst2A and sst2B, sst3, sst4, and sst5 (68). In the human intestine, all six SSTR subtypes are expressed, and the implicated cellular signaling pathways modulated by SSTRs include cyclic adenosine monophosphate (cAMP), K+, Ca2+, Na+, protein lipase C, cyclic guanosine monophosphate

(cGMP), protein tyrosine phosphatases, and MAPK (69).

SST and SSTRs have been detected in almost every single tissue and organ, and reflecting this ubiquity, the functions of SST are extremely versatile. SST can function in the dimensions of a neurohormone, a neurotransmitter, or an autocrine/paracrine hormone (70). The main actions of SST can be classified into three major categories: in the central and peripheral nervous systems, in the endocrine and exocrine systems, and in the proliferation and differential of normal and tumor cells. Originally identified as a peptide produced in the hypothalamus that inhibits the secretion of growth hormone and thyroid-stimulating hormone, the central actions of

SST have been well characterized. Furthermore, the endocrine and exocrine functions of SST have been established, emphasizing its important role in nutrient and metabolism. SST inhibits the secretion of insulin, glucagon, and PP in the endocrine pancreas, and the secretion of bicarbonate and digestive enzymes in the exocrine pancreas. In the gastrointestinal (GI) tract,

SST inhibits the secretion of a multitude of peptide secretions including: gastrin, secretin, cholecystokinin, vasoactive intestinal peptide, gastric inhibitory polypeptide, motilin, enteroglucagon, and neurotensin (70). In addition to modulating pancreatic and GI secretions,

SST also regulates GI functions, such as inhibiting bowel motility, gastric emptying, GI transit, and intestinal nutrient absorption.

In the recent years, SST analogs (SAs) have been and are actively being developed as anti-proliferative agents for the treatment of various types of neuroendocrine tumors (71,72), including insulinoma, pancreatic fistula, thymomas, and hepatocellular carcinoma. Other uses of

SAs include treatment for pancreatitis and acromegaly.

1.1.2.6 Pancreatic polypeptide

Originally isolated in 1968 during the preparation of insulin, PP is the founding member of the 36 amino acid pancreatic polypeptide family (73,74); the other two members are peptide

YY (PYY) and neuropeptide Y (NPY). PP is released from pancreatic PP-cells and gut endocrine cells in response to food ingestion. The secretion of PP occurs in a biphasic manner, with the first phase resulting from vagal stimulation and the second prolonged phase resulting from hormonal stimulation (75). Elevated PP levels can remain up to 6 h postprandially (76).

PP is part of the gut-brain axis, referring to the bidirectional communication between the gut and the brain, and is importantly involved in appetite control and food intake. Transgenic overexpression of PP in murine pancreatic islets led to reduced food intake, which is abolished by the administration of anti-PP antiserum (77). Peripheral administration of PP in mice led to acute reduction of food intake and gastric emptying, and prolonged administration resulted in attenuated body weight gain and energy expenditure (78). PP-overexpressing mice exhibited the lean phenotype with lower food intake and gastric emptying rate (79). In the leptin-deficient

Lepob/ob (ob/ob) mouse model, repeated intraperitoneal PP injection attenuated body weight gain and ameliorates insulin resistance and hyperlipidemia (78). In humans, some reports have suggested that PP levels are lower in obese subjects (80,81), and peripheral PP administration reduced food intake and gastric emptying (82,83). Intravenous infusion of PP in healthy lean volunteers resulted in reduced appetite and decreased energy intake, where the inhibition of energy intake sustained for up to 12 h (76).

The action of PP is implicated to be mediated by the Y4 receptor (Y4R), a subtype of the

NPY receptor family, in the brainstem and hypothalamus. The involvement of the vagus nerve is demonstrated by the lack of PP anorectic effects following vagotomy in rodents (78). Y4R is

expressed in multiple neural centers in the brain, including AP, NTS, DVN, ARC, and PVN (84), and the major site of PP action is suggested to be the brainstem. Despite the clear appetite- suppressing role of exogenous PP administration in rodents and humans, central administration of PP stimulates food intake (85), suggesting that its physiological actions are dependent on the route of administration. This may occur due to the different distribution of receptor or activation sites, although the exact mechanisms remain undetermined. Although mechanistically PP could be a potential target for anti-obesity drugs, it is rapidly degraded in the circulation; however, the development of Y4 agonists may provide a potential avenue in treating obesity.

1.1.2.7 Leptin

Leptin is a 16 kDa hormone synthesized in adipose tissues, and it controls various metabolic processes and physiological behaviors, such as appetite regulation, body weight loss, neuroendocrine functions, and glycemia. Leptin is produced proportionally to the amount of adipose tissue in the body, and its effects are mediated through leptin receptors (LEP-Rs, also known as Ob-Rs) expressed in the central nervous system (86,87). The hypothalamus is a key site in the brain for the actions of insulin and leptin in regulating energy homeostasis, where their actions are mediated via the NPY and agouti-related peptide (AgRP) expressing neurons.

Neuronal insulin resistance was demonstrated to lead to impaired leptin-mediated regulation of neuronal signaling and gene expression and hence central lepin resistance (88).

A multitude of preclinical studies have reported the anti-obesity action of leptin (89).

Daily injection of recombinant leptin into healthy mice resulted in reduced caloric intake, increased energy expenditure, and almost complete elimination of adipose tissue (90). However,

these beneficial effects were not observed in the initial clinical trials, where leptin therapy was only effective in treating obese individuals who also suffered from congenital leptin deficiency

(91,92). Furthermore, the potential to develop leptin into an anti-obesity drug was halted by observations of leptin resistance, where obese patients have elevated leptin levels are resistant to exogenously administered leptin (93,94).

Nonetheless, leptin has multiple beneficial effects that have been confirmed in animal models and human subjects. In mouse models of T1D, leptin treatment ameliorated the deleterious effects of insulin deficiency (95,96), and leptin therapy along with insulin improved insulin sensitivity in T1D patients (97), revealing the anti-diabetic potential of leptin in T1D. In the T2D setting, leptin administration has been shown to improve insulin resistance as well as glucose and lipid imbalances in mouse models (98-101). On the other hand, leptin clinical trials indicated that leptin therapy is only marginally effective in improving diabetes and insulin resistance in obese individuals (102,103). However, this does not preclude the potential use of leptin in other types of T2D patients, for example lean T2D subjects.

Leptin administration was also shown to correct insulin resistance and hyperglycemia in the context of lipodystrophy in mice (104), and this was recapitulated in humans where leptin treatment improved insulin resistance, hyperglycemia, and hypertriglyceridemia in patients suffering from severe hypoleptinaemia and lipodystrophy (105,106). Due to the heterogeneous nature of lipodystrophy, not all patients have extremely low leptin levels. Furthermore, leptin therapy did not improve glycemia in lipodystrophic individuals with moderately low leptin levels

(107), thus posing a limitation on the potential of leptin therapy in the context of lipodystrophy.

The remarkable effectiveness of leptin in improving glucose and lipid profiles have been observed in select T1D and T2D animal models and certain T2D patients, as well as in

lipodystrophy patients, which are a small subgroup of T2D patients. Despite these promising findings, several potential pitfalls must be overcome before leptin therapy becomes an antidiabetic treatment option. For example, it has been demonstrated that leptin administration increases arterial pressure (108), thereby posing a risk of worsening the hypertension that is commonly observed in T2D patients. As leptin is known to stimulate PI3K, a major regulator of cell proliferation, its potential tumor-inducing effects must not be overlooked (109). Lastly, the pervasiveness of leptin resistance in T2D individuals, leading to hyperleptinemia, renders these subjects unsuitable for leptin therapy. Despite these challenges, leptin could still prove itself useful in the treatment of targeted populations of patients, for example once its beneficial effects can be ascertained in non-obese, leptin-sensitive T2D patients.

1.1.3 The liver as a central organ in glucose homeostasis

The liver lies beneath the diaphragm, and is supplied from below with blood from two major vessels: the hepatic artery (supplying about 20% of the blood) and the hepatic portal vein

(supplying about 80% of the blood). The hepatic portal vein carries blood that has passed through the intestinal tract, and is formed by further joining the veins from the stomach, the spleen, and the pancreas. The hepatic portal vein is rich in monosaccharides and amino acids absorbed from the intestine, and it also receives pancreatic hormones such as insulin and glucagon. Blood leaves the liver through the hepatic veins, which enter the inferior vena cava, the main blood vessel returning blood from the lower parts of the body towards the heart.

The majority of the liver (about 80% by mass) is composed of one cell type: hepatocytes.

In a liver cross-section, the hepatocytes form lobules, which appear as hexagonal units in a

stacked formation. At each corner of the hexagon there is a triad of three vessels: branches of the hepatic portal vein, the hepatic artery, and the bile duct. Blood flows from the hepatic portal vein and the hepatic artery into tiny passages called sinusoids, the equivalent of capillaries in other tissues. The sinusoids lead to the branch of the hepatic vein located at the center of the hexagon, and is redirected back to the heart. From the center, individual hepatocytes radiate out from the hepatic vein, where their specific arrangement reflects metabolic zonation. The hepatocytes on the outside of each lobule (periportal hepatocytes) are exposed to incoming blood from the hepatic portal vein and hepatic artery, and hence are well-oxygenated and rich in nutrients; therefore, the synthesis of glucose (gluconeogenesis) predominates. The cells near the center of each lobule (perivenous hepatocytes) are located near the hepatic vein and are mainly the site of glycolysis. Despite the zonation, each hepatocyte is able to perform either function, depending on varying physiological conditions. The biochemical processes of glucose metabolism under fasting and fed conditions that occur in the liver are outlined in the sections below.

1.1.3.1 Glycogenolysis and glycogenesis

Glycogenolysis

Glycogenolysis is the process by which liver metabolizes stored glycogen into glucose, to be released into the bloodstream under fasting condition. Figure 1.2 illustrates the biochemical reactions of glycogenolysis. Glycogenolysis is stimulated by glucagon, acting through the cAMP-PKA pathway. PKA phosphorylates phosphorylase kinase (PK), converting it from the inactive (PKb) to the active form (PKa). Active PKa then phosphorylates and activates glycogen phosphorylase (GP), converting it from the inactive form (GPb) to the active form (GPa). GPa

19 Blood Hepatocyte

+ Insulin GPb P PP1 PKa PKb P

GPa

PKA GLYCOGENOLYSIS + Glucagon

G1P + Glycogenn-1 Glycogenn

G6P GLYCOGENESIS + Insulin

GSa Glucose P PP1 GSK3 GSK3

+ Insulin P GSb GLUT2 Glucose P = Phosphorylation

Fig. 1.2 Glycogenolysis and glycogenesis. Glycogenolysis is stimulated by glucagon, acting through the cAMP-PKA pathway. PKA phosphorylates PK, converting it from the inactive (PKb) to the active form (PKa). Active PKa then phosphorylates and activates GP, converting it from the inactive form (GPb) to the active form (GPa). GPa subsequently catalyzes the release of one G1P unit from an existing glycogen chain (Glycogenn). G1P is converted to G6P and then to glucose, which is then released into the bloodstream via GLUT2. Glycogenolysis is inhibited by insulin, where insulin stimulates PP1, which converts the active GPa to inactive GPb via dephosphorylation. Glycogenesis is the formation of glycogen under fed conditions. Glucose is converted to G6P and subsequently to G1P, which is assembled onto an existing glycogen chain (Glycogenn-1) by active GSa. Glycogenesis is mainly controlled by the phosphorylation-mediated regulation of GS. GSK3 phosphorylates the active GSa and converts it to the inactive form GSb. Insulin signaling leads to the inactivation of GSK3 and activation of PP1, thereby promoting the formation of active GSa. PK, phosphorylase kinase; GP, glycogen phosphorylase; G1P, glucose-1-phosphate; G6P, glucose-6-phosphate; PP1, protein phosphatase-1; GS, glycogen synthase; GSK3, glycogen synthase kinase-3.

subsequently catalyzes the release of one glucose-1-phosphate (G1P) unit from an existing glycogen chain (Glycogenn). G1P is converted to glucose-6-phosphate (G6P) and then to glucose, which is then released into the bloodstream via GLUT2. Glycogenolysis is inhibited by insulin, where insulin stimulates protein phosphatase-1 (PP1), which converts the active GPa to inactive GPb via dephosphorylation.

Glycogenesis

Upon exposure to high glucose concentrations under fed conditions, the hepatocytes take up the glucose via the GLUT2 transporter and use them for glycogen synthesis, a process also called glycogenesis. Figure 1.2 illustrates the biochemical pathway of glycogenesis. Glucose is converted to G6P and subsequently to G1P, which is assembled onto an existing glycogen chain

(Glycogenn-1) by active glycogen synthase (GSa). Glycogenesis is mainly controlled by the phosphorylation-mediated regulation of GS. Glycogen synthase kinase-3 (GSK3) phosphorylates the active GSa and converts it to the inactive form GSb. Insulin signaling leads to the phosphorylation and hence inactivation of GSK3, and therefore promotes the formation of active

GSa. In addition, insulin activates PP1 and induces the conversion of inactive GSb into active

GSa via phosphorylation.

1.1.3.2 Glycolysis and gluconeogenesis

Glycolysis

The pathways of glycolysis and gluconeogenesis (GNG) catalyze opposite functions, and conditions that favor the one will suppress the other. Glycolysis is stimulated by starving conditions, whereas GNG is induced by fed conditions. Figure 1.3 illustrates the biochemical reactions of glycolysis, where the three enzymes that differ for glycolysis (GK, PFK, PK) listed at their corresponding steps. Glycolysis is the sequential conversion of glucose to pyruvate.

Glucose is converted to G6P by glucokinase (GK), which is then formed into fructose-6- phosphate (F6P). Phosphofructokinase (PFK) then converts F6P to fructose-1.6-bisphosphate (F-

1,6-P2), which through the intermediaries glyceraldehyde-3-phosphate (Glyc-3-P) and phosphoenolpyruvate (PEP), eventually forms pyruvate by the action of pyruvate kinase (PyrK).

Insulin positively regulates the gene expression of GK and PyrK, whereas glucagon suppresses gene expression of PyrK. Insulin also regulates via allosteric mechanisms, and stimulates PFK activation.

Gluconeogenesis

In addition to glycogenesis, another mechanism by which the body can produce glucose under fasting condition is GNG, which is the formation of glucose from non-carbohydrate substrates. In mammals, GNG is found predominantly in the liver, although to a lesser extent it also occurs in the kidney and intestine (110). The amino acids alanine, glycine, threonine, cysteine, serine, arginine, proline, histidine, glutamine, methionine, valine, asparagine, and aspartate are referred to as ‘glucogenic’ amino acids, based on their ability to enter GNG once

22 Glucose + Glucagon G + Insulin - Insulin L GK G6ph G6P Y C F6P O G L + Insulin L PFK FBP Y U S F-1,6-P2 C I O S Glyc-3-P DHAP N - Glucagon E + Insulin PyrK O Pyruvate PEP G E + Glucagon PEPCK - Insulin N OAA E S Pyruvate I OAA Lactate S PC LDH Pyruvate

Mitochondria

Fig. 1.3 Glycolysis and gluconeogenesis Glycolysis is the sequential conversion of glucose to pyruvate. Glucose is converted to G6P by GK, which is then formed into F6P. PFK then converts F6P to F-1,6-P2, which through the intermediaries Glyc-3-P and PEP, eventually forms pyruvate by the action of PK. Insulin positively regulates the gene expression of GK and PK, whereas glucagon suppresses gene expression of PK. Insulin also regulates via allosteric mechanisms, and stimulates PFK activation. GNG utilizes lactate as its main substrate. LDH converts lactate to pyruvate, which enters the mitochondrial matrix, which is then converted to OAA by PC. OAA is decarboxylated and phosphorylated to PEP by PEPCK. PEP is converted to Glyc-3-P, then to F-1,6-P2, and subsequently to F6P by FBP. F6P is converted to G6P and subsequently to glucose by G6ph. Long-term regulation of the gene encoding PEPCK is mediated by the suppressive effect of insulin and the inductive effect of glucagon. PEP is subsequently converted to F6P through multiple steps, and F6P is converted to G6P by glucose-6- phosphatase. Similarly, insulin suppresses, while glucagon stimulates, the gene expression of G6ph.

GK, glucokinase; F6P, fructose-6-phosphate; PFK, phosphofructokinase; F-1,6-P2, fructose-1,6- bisphosphate; Glyc-3-P; glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; PyrK, pyruvate kinase. LDH, lactate dehydrogenase; OAA, oxaloacetate; PC, pyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; F-1,6-P2, fructose-1,6-bisphophate; FBP, fructose-1,6- bisphosphatase; G6ph, glucose-6-phosphatase. 23

they are catabolized. The remaining two amino acids, leucine and lysine, cannot be utilized as

GNG substrates. In humans, the main precursors utilized for GNG are lactate, glycerol (from the breakdown of triacylglycerol, TAG), alanine, and glutamine (111).

The majority of the biochemical reactions of GNG are those of glycolysis reversed.

Figure 1.3 illustrates the biochemical pathway of GNG, where the four enzymes that differ from glycolysis (PC, PEPCK, FBP, G6ph) are listed at their corresponding steps. GNG begins in the mitochondrial matrix with the formation of oxaloacetate (OAA) from pyruvate by pyruvate carboxylase (PC). OAA is then decarboxylated and phosphorylated to phosphoenolpyruvate

(PEP) by phosphosenolpyruvate carboxykinase (PEPCK). PEP is converted to Glyc-3-P, F-1.6-

P2, and then to F6P by fructose-1,6-biphosphatase (FBP). F6P is converted to G6P and subsequently to glucose by glucose-6-phosphatase (G6Ph). Long-term control of the expression of Pck gene (the gene encoding PEPCK) is mediated by the suppressive effect of insulin and the inductive effect of glucagon. Similarly, insulin suppresses, while glucagon stimulates, the gene expression of G6pc (the gene encoding G6ph).

1.2 The incretin hormone glucagon-like peptide 1

1.2.1 Proglucagon gene, GLP-1 production and degradation

In 1902, two English physiologists, Sir William Maddock Bayliss and Ernest Henry Starling, speculated that after ingesting carbohydrates, the intestinal mucosa produces a hormone which travels to and stimulates endocrine secretions from the pancreas (112). They named these ‘gut factors’ with the term ‘secretin’ (112). In 1930, several scientists proposed the terms ‘incretin’

and ‘enterogastrone’, referring to a hormonal extract from the duodenum (113). Half a century later, interests were sparked with the discovery of the glucose-lowering effect of the incretin hormone glucose-dependent insulinotropic peptide (GIP) (114-116) and after the identification of the second incretin hormone, glucagon-like peptide 1 (GLP-1) (117,118). The incretin effect is defined as the amplification of pancreatic insulin secretion in response to orally ingested glucose, compared to intravenous glucose given at the same amount. Decades of research have yielded a substantial body of knowledge of the two incretin hormones, in particular of GLP-1.

GLP-1 is a peptide hormone produced in the L cells of the intestinal epithelium. GLP-1 is produced in all regions of the small intestine and colon, although the highest level of expression is localized to the distal ileum and colon (119). GLP-1 is encoded by the proglucagon gene (gcg), which among others also encodes glucagon produced in the pancreatic α-cells, as well as GLP-2, a gut growth factor (120). Fig. 1.4A illustrates the overall structure of the proglucagon peptide and the cleavage sites by the preprohormone convertases 2 and 1/3 (PC2 and PC1/3). The proglucagon undergoes post-translational splicing in tissue-specific manners, leading to unique expression profiles of proglucagon derived peptides (PGDPs) in different organs or cell types. In pancreatic α-cells, the main products are glucagon, glicentin-related pancreatic polypeptide

(GRPP), intervening peptide-1 (IP1) and major proglucagon fragment (MPGF) (Fig. 1.4B).

Although uncommon, under certain scenarios such as during embryonic development or when islets encounter stress, low levels of GLP-1 can be detected in pancreatic α-cells. In intestinal L cells and certain neuronal cells in the brain, GLP-1 is liberated by PC1/3-mediated cleavage of the precursor proglucagon (Fig. 1.4B). In addition to GLP-1, other products glicentin, GLP-2, intervening peptide-2 (IP2), GRPP and oxyntomodulin are also produced in the intestine and brain.

25 A Proglucagon peptide:

PC2 PC2 PC2 PC1/3 PC1/3 PC1/3 PC1/3

EDKRHS NTKRNR IAKRH ERHA RGRRDF LGRRHA TDRK

N GRPP Glucagon IP1 GLP-1 IP2 GLP-2 C

1 30 33 61 64 69 78 107 111 122 126 158 160

B Pancreas:

GRPP Glucagon IP1 MPGF

1 30 33 61 64 69 72 158

GLP-1

78 107

Intestine and brain:

Glicentin IP2 GLP-2

1 69 111 122 126 158 GLP-1

GRPP Oxyntomodulin 78 107

1 30 33 69

HAEGT FTSDV SSYLE GQAAK EPIAW LVKGR G

Fig. 1.4 Proglucagon and proglucagon derived peptides (PGDPs). A) Proglucagon, encoded by the gcg gene, is a pro-hormone with 160 amino acid residues. The peptide contains both PC2 and PC3 cleavage sites. The amino acid sequences of the cleavage sites are as indicated, bold letters represent amino acids adjacent to the cleavage sites. B) Schematic presentation of the cleavage products in pancreas (top) and intestine/brain (bottom). Although GLP-1 is not normally produced in the pancreas, during the embryonic stage or when islets are under stress, some pancreatic α-cell will produce GLP-1. In the intestine and brain, GLP-1 is a 31 amino acid peptide, with the amino acid sequence as indicated. PC2 and PC1/3, preprohormone convertases 2 and 1/3; GRPP, glycentin related polypeptide; IP1 and IP2, intervening peptide 1 and 2; MPGF, major proglucagon fragment. GLP-2, glucagon-like peptide 2.

Fig. 1.5 depicts the four currently identified and biologically active GLP-1 derivatives. GLP-1(7-

37) and GLP-1(7-36) amide are the two main biologically active forms of GLP-1 in circulation. A recently identified short form, GLP-1(9-36) amide, was initially presumed to be an inactive degradation product, but has recently been demonstrated to exert protective effects in the heart

(121-126). A nonapeptide, GLP-1(28-36) amide, has also been identified and demonstrated to exert metabolic functions as reported by a few studies (127,128).

The identification and study of the metabolic functions of the incretin hormones such as GIP and GLP-1 led to the coinage of the term “enteroinsular axis”, referring to the connection between the gut and pancreatic islets (129). Oral nutrient ingestion is a potent stimulus of GLP-1 secretion, and all the macronutrients (carbohydrates, fat, and protein) of a mixed meal contribute to GLP-1 secretion (130). Although both GIP and GLP-1 can elicit the incretin effect, the circulating levels of GLP-1 after a meal are much lower than those of GIP, and hence GLP-1 is a more potent stimulator of insulin secretion (131). A biphasic rise in postprandial plasma GLP-1 is observed in humans, peaking first at 15-20 min and then at 1-2 h (132,133). The half-life of

GLP-1 in circulation is only a few minutes, due to the rapid cleavage by the enzyme dipeptidyl peptidase IV (DPP-IV), thus limiting its direct use as clinical therapeutic agents. However, two new categories of T2D drugs have been developed based on established knowledge of the incretin hormone GLP-1. One category is the GLP-1 analogs, such as the injectable Exenatide (a synthetic form of Ex-4, marketed as Byetta®), and the second category is DPP-IV inhibitors, such as Sitagliptin (Januvia®) (134). Table 1 lists one example from each of the two categories of GLP-1 based therapeutics (135).

27 DPP-IV NEP24.11

HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR G GLP-17-37

HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR NH2 GLP-17-36 amide

EGT FTSDV SSYLE GQAAK EFIAW LVKGR NH2 GLP-19-36 amide

FIAW LVKGR NH2 GLP-128-36 amide

Fig. 1.5 GLP-1 and its derivatives. Amino acid sequences of the four currently identified GLP-1 derivatives. GLP-1 is a 31 amino acid peptide, numbered aa 78-107 of the proglucagon prohormone, but more commonly referred as aa 7-37 based on numbering of the MPFG fragment. The cleavage sites of DPP-IV and

NEP24.11 are as indicated, where DPP-IV generates the GLP-19-36 amide while NEP24.11 produces the nonapeptide GLP-128-36 amide. MPFG, major proglucagon fragment; DPP-IV, dipeptidyl peptidase-4; NEP 24.11, neutral endopeptidase 24.11.

Table 1 Examples from the two categories of GLP-1 based therapeutics.

Generic name Category Dosage Adverse reactions (Brand)

Nausea, vomiting, diarrhea, feeling jittery, dizziness, headache, dyspepsia, injection- Adults: site reactions, dysgeusia, GLP-1 5mcg SQ, 60 somnolence, generalized Exenatide (Byetta®) Analogs min before am pruritus and/or urticarial, & pm meals. macular or popular rash, angioedema, rare reports of anaphylactic reaction, abdominal pain, hypoglycemia.

(Monotherapy/Combination therapy): Upper respiratory tract infection, nasopharyngitis, DPP-IV Sitagliptin phosphate Adults: headache. inhibitor (Januvia®) 100mg qd.

(Combination therapy): Hypoglycemia.

1.2.2 Mechanisms underlying proglucagon gene expression

Overview of proglucagon gene transcriptional regulation

In the early 1980s, gcg was first identified based on cDNA sequences from rodents and humans (117,136-138). Gcg transcription is regulated by multiple signaling cascades and components, including homeobox domain proteins, Wnt signaling effectors, as well as downstream targets of cAMP and insulin signaling. Notably, these signaling pathways intertwine and crosstalk with each other, and together they provide sophisticated, tissue- or cell-type specific transcriptional regulation of gcg expression.

A schematic of the cis- and trans-elements involved in the regulation of gcg expression are presented in Fig. 1.6. The promoter region of gcg contains multiple cis-elements: G1, G2,

G3, G4, and G5 enhancer elements, a cAMP response element (CRE), and a GATA box. The homeobox domain (HD) proteins, including Isl1, Pax-2, Pax-6, Cdx-2, Brn-4, and Pbx have been shown to bind to G1and G3. Components of the cAMP signaling cascade, including cAMP and cAMP response element binding protein (CREB), as well as Ca2+ and Activated transcription factor 3 (ATF3), bind to the CREs located at the distal gcg promoter and within G2. In pancreatic α cells, insulin was shown to bind to G3 and suppress gcg expression. In the hypothalamic gcg-expressing cell line mHypoE-20/2, insulin and leptin were reported to stimulate gcg expression (139). Our laboratory further demonstrated that in intestinal L cells but not in pancreatic α cells, the Wnt effectors β-cat/TCF bind to G2 and are positive regulators of gcg expression.

30 ATF3 Pbx

Ca2+ Cdx-2 Brn-4 cAMP Insulin Isl-1 CREB Pax β-cat/TCF Pax GATA CRE G3 G2 G5 G4 G1 gcg

Fig. 1.6 Cis- and trans-elements involved in the regulation of gcg promoter activity. Gcg contains five enhancer elements (G1-G5). The homeodomain box proteins Pax bind to the G1 and G3. Cdx-2, Pbx, Isl-1, and Brn-4 bind to G1. The Wnt bipartite transcription factor β-cat/TCF, cAMP, calcium, and ATF3 bind to G2. Insulin was found to bind to G3 and suppress gcg expression in pancreatic α cells. Gcg contains two CREs, one at the distal promoter and one located within G2. The distal gcg promoter contains a GATA-binding motif.

In the gut, the regulation of gcg expression by G-protein coupled receptor signaling, insulin/IGF-1 signaling, and Wnt signaling, as well as their crosstalk with each other, is illustrated in Fig. 1.7. The cAMP-PKA cascade, activated in response to stimulation of GPCR or cAMP promoting agents such as forskolin, leads to CREB binding to the CRE at the distal promoter region. Insulin signaling activates PI3K, which crosstalks with both the cAMP-PKA as well as the Wnt signaling pathway to regulate gcg transcription. Insulin also exerts its effect through the Epac pathway. The crosstalk between insulin and Wnt signaling involves the participation of the key Wnt effector β-cat; however, the detailed mechanism is not known. The

β-cat/TCF bipartite transcription factor binds to the G2 enhancer element of proximal gcg promoter and induces gcg expression. Lastly, activation of Wnt signaling, either through Wnt ligand binding or treatment with lithium chloride (LiCl), leads to the stimulation of gcg expression.

Homeobox domain proteins

HD proteins are DNA-binding proteins that play important roles in the patterning of developmental processes. They were first discovered in Drosophila melanogaster to regulate homeotic genes, from which the name originates. To date, HD proteins are defined as possessing the HD motif, a conserved sequence of about 180 base pairs long, and are mostly classified as transcription factors. About a dozen HD proteins were found to be expressed in pancreatic α cells and/or intestinal L cells, including Insulin gene enhancer protein-1 (Isl-1), Paired box (Pax) proteins, Caudal type homeobox-2 (Cdx-2), Brain-4 (Brn4), Pre-B cell leukemia transcription factor (Pbx) proteins, and members of the Nkx family.

32 Peptide hormones Insulin/IGF-1 Wnt

GPCR RTK 7-TMR

PI3K GSK3β LiCl ?

Forskolin cAMP Epac β-cat ? PKA Transcription factors CREB TCF β-cat CRE G2 gcg

Fig. 1.7 Transcriptional regulation of the proglucagon gene. Cell signaling cascades crosstalk with each other in regulating gcg expression. The cAMP-PKA cascade, activated in response to stimulation of GPCR or cAMP promoting agents such as forskolin, leads to CREB binding to the CRE at the distal promoter region. Insulin signaling activates PI3K, which crosstalks with both the cAMP-PKA as well as the Wnt signaling pathway to regulate gcg transcription. Insulin also exerts its effect through the Epac pathway. The crosstalk between insulin and Wnt signaling occurs through the participation of the key Wnt effector β-cat. The β-cat/TCF bipartite transcription factor binds to the G2 enhancer element of proximal gcg promoter and induces gcg expression. Lastly, activation of Wnt signaling, either through Wnt ligand binding or treatment with lithium chloride (LiCl), leads to the stimulation of gcg expression.

Isl-1 is expressed in all pancreatic cell types that are hormone-producing, and it regulates the transcription of the insulin, gcg, and somatostatin genes (140-143). Isl-1 was found to mediate the activation of gcg transcription in the pancreatic α cell line InR1-G9, where it binds to

TAAT-rich motifs of the G1 enhancer element (142). Disruption of Isl-1 in mice led to arrested embryonic development, accompanied by the complete lack of pancreatic endocrine cells (144).

Pax-2 binds to G3 and G1 enhancer elements of the gcg promoter and activates gcg transcription in vitro (145), although this binding was not observed in rodent pancreas and intestinal tissues (146). The transgenic Pax-2 (1Neu) mice have normal islet α cells and intestinal

L cells, and no change in pancreatic and intestinal gcg expression (146), while another study indicates that these mice have enlarged pancreas volume, without affecting pancreatic insulin and glucagon content (145). Pax-6 plays an important role in islet cell development (147), and is expressed in the epithelium of pancreatic buds during development. Over-expression of Pax-6 stimulates gcg expression in intestinal endocrine L cells through its binding to G3 (Fig. 1.6A)

(148), and Pax-6 and Cdx-2 have additive effects in stimulating gcg promoter activity (149). The

Sey-/- mice, which carry a spontaneous mutation in the Pax6 gene, have abnormal islet organization, less α, β, δ and PP cells, accompanied by reduced insulin and glucagon production

(150).

Cdx-2 is a caudal-like HD protein, and is expressed in the pancreatic α cell line InR1-G9, and the intestinal GLUTag and STC-1 cell lines, and in mouse pancreas (151). Cdx-2 binds to two AT-rich motifs within the G1 enhancer element of the gcg promoter. Co-transfection of

Cdx-2 stimulates gcg promoter expression, while mutating the Cdx-2 binding sites abolishes this stimulation. Over-expression of Cdx-2 leads to increased gcg mRNA production in InR1-G9

(152). Two alternatively spliced isoforms of Cdx-2 exist in InR1-G9, with the longer form being the main stimulator of gcg expression (153). Cdx2-/- mice die as embryonic lethals, due to the

critical role of Cdx-2 in embryo implantation, and they exhibit multiple developmental abnormalities despite having normal pancreas development (154). Cdx-2 regulates its own transcription by binding to two AT-rich motifs within the proximal Cdx-2 promoter region, and based on this auto-regulatory mechanism it has been suggested that having one copy of the functional Cdx-2 allele is sufficient in maintaining normal development of pancreatic and intestinal gcg -expressing cells (155).

Brn-4, also referred to as POU class 3 homeobox 4 (POU3F4), is expressed in pancreatic

α cells but not β cells, and is an α cell specific transcription factor. Brn-4 stimulates gcg expression in pancreatic α cells (156), while induced expression in the pancreatic β cell line Ins-1 led to detectable glucagon levels (157). Furthermore, expression of Brn-4 using the β cell specific Pdx-1 promoter resulted in ectopic gcg expression in these insulin-expressing β cells, confirming the role of Brn-4 as a key controller of the pancreatic α cell lineage. Despite this,

Brn-4-/- mice display normal pancreatic bud formation, gcg -expressing cell numbers, and other physiological parameters (158). Brn-4 binds to motifs within the G1 enhancer element of the gcg promoter, and Brn-4 acts in a synergistic manner with Cdx-2 in stimulating gcg expression (159).

Pbx proteins were originally identified as Hox cofactors, as part of transcriptional complexes that regulate developmental programming. Pbx expression has been detected in embryonic pancreas, as well as in all four pancreatic hormone-producing cell types and acinar and ductal cells of adult mouse pancreas. Pbx-1 was shown to interact with Cdx-2 through a penta-peptide motif within Cdx-2, and co-transfection of Pbx-1 with Cdx-2 stimulated the activation of the gcg promoter by Cdx-2. When the penta-peptide motif is mutated, Cdx-2 stimulated gcg promoter expression was attenuated (160). Pbx-1-/- mice die prior to birth, with noticeable defects in the pancreatic endocrine and exocrine cells (161). These mice also

exhibited abnormalities in pancreas development, accompanied by islet malformation and hyperinsulinemia, suggesting its critical role in the growth of pancreatic cells and pancreas organ development (161).

The Nkx family of transcription factors consists of eleven members. The two members

Nkx2.2 and Nkx6.1 were originally identified as regulators of neural patterning and organ development, and subsequently were found to be involved in pancreas development, islet cell specification and differentiation, and maintaining proper β cell function. Nkx2.2 is expressed in early pancreatic progenitors as early as e9.5 (162), and is then limited to pancreatic β cells and a subset of α cells and PP cells (163). Nkx2.2-/- mice have defects in pancreatic α and β cell differentiation (162,163), and they die shortly after birth, and thus the role of Nkx2.2 in mature β cells remains unknown. Nkx2.2 is suggested to function as a transcriptional repressor during endocrine cell differentiation, as a dominant Nkx2.2 repressor derivative can rescue the specification of α and β cells during embryogenesis in the absence of endogenous Nkx2.2

(164,165). Nkx6.1 is implicated to regulate β cell proliferation and thereby maintaining β cell mass (166). Nkx6.1 has been reported to bind to the gcg promoter and repress gcg mRNA production (167). Adenovirus-mediated over-expression of Nkx6.1 increases β cell mass in isolated human and rat islets, whereas knockdown of Nkx6.1 using siRNA method led to the opposite effect (168). In Nkx6.1-/- mice, expression of Nkx6.1 transgenes in select progenitor cell populations rescued the formation and maturation of insulin-producing β cells (169). However, transgenic mice over-expressing β cells specific Nkx6.1, using an inducible Cre-recombinase- based system, failed to exhibit enhanced β cell proliferation, β cell mass, and glucose metabolism

(170). Nkx6.1-/-;Nkx2.2-/- double knockout mice exhibit similar phenotypes as the single Nkx2.2-/- mice, raising the possibility that Nkx6.1 functions downstream of Nkx2.2 (171).

The cAMP signaling cascade

The effectors of the cAMP signaling pathway are well-recognized activators of gcg expression in pancreatic α cells and intestinal L cells. The distal gcg promoter contains a CRE

(172,173), and the stimulatory effect of cAMP signaling has been established in multiple pancreatic α cell lines as well as in islet primary cell cultures (172,174-179). In intestinal L cell lines and primary fetal rat intestinal cells, membrane-permeable cAMP analogues and cAMP promoting agents increase gcg expression and GLP-1 production (173,180,181). However, deletion of the CRE motif, located between -291 and -298 bp of the rat gcg promoter, only partially attenuated the stimulatory effect of cAMP (182). The identification of a second CRE motif, located within the G2 enhancer element, led to the finding that this second CRE motif can mediate the effects of cAMP, calcium (183) as well as the ATF3 (179). The identification of the second CRE within G2, as well as two CRE-like elements in the distal gcg promoter, provided explanations for the only partial attenuation of cAMP-stimulated gcg expression following the mutation or deletion of the canonical CRE motif (183,184).

In the PKA-deficient InR1-G9 cell line, cAMP still exerts a stimulatory effect on gcg expression, albeit the effect being much smaller compared to that in intestinal L cell lines

GLUTag and STC-1 which do express PKA (173,176,185). The Exchange protein directly activated by cAMP (Epac) signaling cascade was then discovered as another signaling pathway for mediating the effect of cAMP (186,187), where Epac proteins exert their effects through the downstream Rap-Raf-MEK-ERK pathway (188,189). In intestinal L cell lines, PKA inhibition cannot fully block cAMP-stimulated gcg expression, and Epac pathway specific cAMP analogs stimulate gcg promoter and mRNA production (190).

The Wnt signaling pathway

Recent advances in our understanding of the regulation of gcg expression include the identification of the involvement of the developmental Wnt signaling pathway and its effectors, such as β-cat. For a detailed overview of the Wnt signaling pathway, please refer to Section

1.3.1. A general overview of the role of Wnt signaling in metabolic homeostasis is presented in

Section 1.3.2. The involvement of Wnt signaling in regulating gcg expression and GLP-1 production is described in detail in Section 1.3.3. In addition, one may refer to a number of recent review articles published by our lab and others, which outline the recent advances and perspectives on the role of Wnt signaling pathway in metabolic disorders (191-200).

1.2.3 The functions of GLP-1

Extensive investigations in the past two decades have revealed both pancreatic and extra- pancreatic functions of GLP-1. GLP-1 exerts its functions mainly through the glucagon-like peptide 1 receptor (GLP-1R), which belongs to the G-protein coupled receptor family and was originally isolated from a rat pancreatic islet cDNA library (201). Although early studies focused on the effects of GLP-1 in pancreatic β cells, numerous recent studies demonstrate the existence of a wide panel of extra-pancreatic functions of GLP-1, including both central and peripheral actions. Figure 1.8 illustrates the pancreatic and extra-pancreatic effects of GLP-1.

38 Gastric emptying ↓

Appetite ↓ Glucose uptake ↑

Insulin ↑ GLP-1 Glucagon ↓ Lipolysis ↑ Somatostatin ↑ β cell mass ↑

Gluconeogenesis ↓ Cardioprotection ↑

Fig. 1.8 Schematic presentation of the function of GLP-1. In the pancreas, stomach, heart and brain, the effects of GLP-1 are likely to be mediated by its specific receptor GLP-1R. As GLP-1(9-37) was also shown to exert protective effects in the heart and improve cardiac function, whether there is a yet to be identified receptor is under debate. It is not clear at this stage whether the effect of GLP-1 in liver, fat and muscle is mediated directly through GLP-1R.

Pancreatic functions of GLP-1

GLP-1 potentiates insulin secretion after a rise in blood glucose levels following nutrient

+ intake. Insulin secretion occurs via the closure of ATP-sensitive K channels (KATP), resulting in subsequent membrane depolarization, and a rise in intracellular Ca2+ level, which lead to the release of insulin-containing granules to the plasma membrane (202,203). Both PKA and Epac signaling pathways are involved in this process (204). GLP-1 has also been demonstrated to induce insulin secretion via inhibiting voltage-dependent K+ channels (202). In addition, GLP-1 enhances insulin biosynthesis and insulin gene transcription and mRNA stability (205). GLP-1 strongly inhibits glucagon secretion from α cells, and stimulates the secretion of somatostatin from δ cells (206,207). The combined effects of GLP-1 in stimulating insulin secretion while suppressing its counter-hormone glucagon lead to reduced hepatic glucose production, thereby contributing to the lowering of blood glucose levels. Additional pancreatic effects of GLP-1 are both tropic and protective, where GLP-1 increases β cell proliferation, induces β cell neogenesis from precursor cells, and inhibits β cell apoptosis (208-211). Lastly, our group and others have shown that in pancreatic β-cells, GLP-1 and Exendin-4 (Ex-4, a naturally occurring GLP-1R agonist) reduces the expression level of thioredoxin-interacting protein (TxNIP), a mediator of glucotoxicity (212,213). In summary, the beneficial effects of GLP-1 in pancreatic β cells are its incretin effect on stimulating insulin secretion, its proliferative effect on stimulating β cell growth and neogenesis, and its protective effect on reducing glucotoxicity (214).

Extra-pancreatic functions of GLP-1

GLP-1R has been detected in various tissues, including pancreatic islets, lung, stomach, heart, intestine, kidney, brainstem, hypothalamus and pituitary gland. Whether it is expressed in hepatocytes has been controversial (215-218).

GLP-1 functions as an enterogasterone, where it inhibits gastrointestinal motility and secretion, leading to reduced gastric emptying and improved postprandial glucose excursions

(219-221). Central GLP-1 signaling suppresses appetite, and chronic GLP-1 administration leads to weight loss associated with reduced appetite (222). GLP-1 produced in the nucleus of the solitary tract of the brain was shown to regulate food intake and blood glucose levels via activation of the GLP-1R in the hypothalamus and brainstem (223-227). GLP-1 also has beneficial effects on the cardiovascular system, where it enhances myocardial performance, reducing infarct size, and improves endothelial dysfunction (228,229). The clinical potential of

GLP-1 based therapeutics in alleviating cardiovascular burden, especially in T2D patients, are being actively investigated (230,231). Interestingly, the cardioprotective effects of GLP-1 were observed for both GLP-1(7-36) amide and GLP-1(9-36) amide (125,232,233). Ex-4 was found to stimulate glucose uptake in rat skeletal muscle (233), and GLP-1 was shown to bind to GLP-1R in hepatocytes and rat skeletal muscle in a cAMP-independent mechanisms (234). In adipocytes,

Ex-4 induces secretion of the lipolytic hormone adiponectin and stimulates lipolysis (235,236).

GLP-1 also regulates lipoprotein metabolism by inhibiting triglyceride-rich lipoprotein production (237). In the liver, GLP-1 has been reported to exert beneficial effects by reducing gluconeogenesis; however, the exact mechanism is unclear, as the expression of GLP-1R in hepatocytes has been controversial (215,218,238), and it has been suggested that the hepatic actions of GLP-1 may be through indirect mechanisms (239).

Numerous review articles have been published on the physiological actions of GLP-1, and one may refer to these selected articles for further details (228,239-254).

1.3 The Wnt signaling pathway and proglucagon gene expression

1.3.1 Overview of the Wnt signaling pathway

The Wnt signaling pathway was initially discovered in research on embryonic development in Drosophila, Xenopus, and other organisms, and subsequently intensively investigated in tumor biology. Wnt ligands, through their cell membrane bound receptors and co- receptors, exert many fundamental physiological and pathophysiological functions in different organs and cell lineages, including patterning and organogenesis, tumorigenesis and metastasis, as well as metabolic homeostasis.

Fig. 1.9 presents the key components of the Wnt signaling pathway. The major downstream effector of the canonical Wnt signaling pathway (referred to as Wnt signaling pathway hereafter) is the bipartite transcription factor β-cat/TCF, formed by β-cat and a member of the TCF family (TCF7, LEF-1, TCF7L1 and TCF7L2). The pool of free cytosolic β-cat is tightly controlled by proteasome-mediated degradation, carried out by a ‘destruction complex’ involving the tumor suppressor adenomatous polyposis coli (APC), axin, GSK3, and casein kinase 1α (CK1α). In the absence of Wnt activation, β-cat is phosphorylated by CK1α at Ser45, which primes β-cat for further phosphorylation events by GSK-3 at Ser33, Ser37, and Thr41.

These destabilizing phosphorylation events lead to the degradation of β-cat. Wnt signaling activation is initiated by the binding of Wnt ligand to its seven-transmembrane domain Frizzled

42 Inactive Wnt signaling Active Wnt signaling

Wnt

Frz LRP5/6 Frz LRP5/6 Cytoplasm Cytoplasm

Dvl axin Dvl CK1α CK1α axin APC GSK3 β-cat APC GSK3 Proteosomal degradation β-cat β-cat β-cat β-cat

Nucleus Nucleus CtBP β-cat TCF Groucho TCF

Wnt target gene Wnt target gene

Fig. 1.9 Overview of Wnt signaling pathway. In its active state, Wnt signaling effector β-cat is bound by the destruction complex consisting of CK1α, GSK3, APC, and axin, leading to its proteosomal degradation. At the same time, TCF is bound by corepressors Groucho and CtBP, thereby suppressing Wnt target gene expression. Upon Wnt ligand binding to the Frz receptor and LRP5/6 coreceptor, conformational changes lead to the association of Dvl with Frz, causing the disintegration of the destruction complex. Free β-cat accumulates in the cytosol and is translocated into the nucleus, where it binds with TCF and stimulates Wnt target gene expression.

(Frz) receptor and the Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) coreceptor.

Following Wnt ligand binding, the Frz receptor associates with Dishevelled (Dvl). This leads to the binding of Dvl with axin and thereby titrating axin away from the ‘destruction complex’. As a result, the inactivating phosphorylation of β-cat is inhibited and its degradation is prevented. Free cytosolic β-cat accumulates in the cytosol, and subsequently translocates into the nucleus to form the β-cat/TCF complex and to activate the transcription of Wnt target genes (Fig.

1.9). Unlike the GSK3- and CK1α-mediated phosphorylation events, other residues have been identified as ‘activating’ residues. PKA signaling is known to stimulate β-cat phosphorylation at

Ser675, an event that has positively associated with β-cat nuclear localization and activity

(255,256). Furthermore, both PKA and Akt were shown to phosphorylate β-cat at Ser552, which promotes β-cat nuclear localization and transcriptional activity (255,257,258). GSK3 is an important negative modulator of the Wnt signaling pathway, and lithium and other inhibitors of

GSK-3 have been established as pharmacological agents that mimic the function of Wnt ligands in stimulating the expression of Wnt downstream target genes.

1.3.2 Wnt signaling pathway and metabolic homeostasis

Several studies have demonstrated that TCF and/or β-cat serve(s) as effectors of a number of signaling cascades other than Wnt signaling, including several peptide hormones, such as insulin, IGF-1 and other growth factors that use cAMP as the second messenger, as well as the lipid metabolite lysophosphatidic acid (LPA) (259-261).

The Wnt effector TCF7L2

Although TCF7 (previously referred to as TCF-1) was originally isolated as a lymphoid transcription factor, members of this family are now well recognized to be transcriptional regulators of many physiological processes. Shortly after the identification of TCF7, isolated cDNAs for TCF7L1 and TCF7L2 were identified, and at that time these two members were named TCF-3 and TCF-4, respectively (262). Because the high-mobility group (HMG) boxes of the TCF7L1, TCF7L2, and TCF7 sequences show marked similarity, it was suggested that these members represent a subfamily of TCF7-like HMG box-containing transcription factors (262).

Subsequently, the genomic structure of the human TCF7L2 gene was identified and mapped to chromosome 10q25.3 (263).

At a time when little is known about the genetic basis of T2D, a genome-wide linkage study in an Icelandic population reported a suggestive link between T2D and chromosome 10q

(264). Subsequently, a genome-wide scan of Mexican American pedigrees revealed a susceptibility locus on 10q, which was linked to T2D and age of onset of T2D (265). In a landmark study in 2006, Grant et al. genotyped 228 microsatellite markers in Icelandic individuals with T2D and in healthy controls across a 10.5 Mb interval on chromosome 10q

(266). The microsatellite DG10S478, located within intron 4 of the TCF7L2 gene, was found to be associated with the risk of T2D (266). This observation was then replicated in a Danish cohort as well as a U.S. cohort (266). Two single nucleotide polymorphisms (SNPs), rs12255372 and rs7903146, were found to be in strong linkage disequilibrium with DG10S478 and also showed similar robust associations with T2D (266). Heterozygous carriers (38%) and homozygous carriers (7%) of the at-risk alleles have relative elevated T2D risks of 1.45 and 2.41, respectively

(266).

Following this pioneering report, the findings have been replicated in all main ethnic groups by numerous independent researchers (267-288). To date, meta-analysis of published studies show that TCF7L2 locus remains the most statistically significant genetic finding of T2D

(289). Despite the consistently observed genetic association between TCF7L2 and T2D, the mechanism through which intronic TCF7L2 SNPs confer T2D risk remains elusive. A number of studies point to the potential roles of these TCF7L2 SNPs in modulating incretin-stimulated insulin secretion in pancreatic β cells and in regulating hepatic gluconeogenesis

(282,283,287,288).

The Wnt effector β-cat

The transcriptional co-activator β-cat is recruited to chromatin DNA via its binding partner TCF, thereby driving the expression of Wnt target genes. It is critically involved in cell proliferation, and its overexpression is associated with many types of carcinomas. Mice overexpressing β cell specific active β-cat exhibited increased β cell proliferation and islet mass expansion (290). Treatment with GLP-1 and Ex-4 induced TCF/LEF-driven reporter gene expression in pancreatic β cells, and Ex-4 treatment stimulated β-cat activation and binding of β- cat/TCF to Wnt target gene promoter (291). PKA, a known effector of GLP-1 signaling, was found to induce β-cat activation through its Ser675 phosphorylation in pancreatic β cells (291).

Other Wnt signaling components

In addition to the Wnt effectors TCF7L2 and β-cat, several other Wnt signaling components were shown to participate in metabolic processes, in particular β cell proliferation and function. The Wnt ligand Wnt3a was found to stimulate pancreatic β cell proliferation and

Wnt target gene expression in β cell lines and islets, whereas mice over-expressing the negative

Wnt modulator Axin in a β cell specific manner exhibited β cell hypoplasia and defects in islet development (290). This was repeated in another study, where treatment with Wnt ligands induced insulin secretion in the presence of high glucose, and where addition of a soluble Frz antagonist abolished the effect (292). Glucokinase is a critical regulator of β cell glucose sensing, and gck gene expression was found to be activated by β-cat, where β-cat is a coactivator of PPARγ at the gck promoter (293). These in vitro findings depict the importance of Wnt signaling for β cell glucose sensing and islet development. The physiological role of the Wnt coreceptor LRP5/6 was established when LRP5/6-deficient mice exhibited marked glucose intolerance under chow diet, and showed increased plasma cholesterol levels in response to high fat diet (292). However, β-cat deficient mice show normal glucose tolerance after birth (294).

1.3.3 Wnt signaling pathway effectors as mediators of proglucagon gene expression

As illustrated in Fig. 1.7, our laboratory has made major contributions in the concept of

Wnt signaling-mediated regulation of incretin hormone production (295,296). Our laboratory demonstrated that gcg promoter expression can be stimulated by lithium, a general Wnt signaling activator, and by constitutively active S33Y mutant β-cat (295). Lithium also stimulated endogenous gcg mRNA expression and GLP-1 production in the mouse intestinal GLUTag and

STC-1 cell lines, as well as in fetal rat intestinal cell (FRIC) cultures (295). The stimulatory effect of lithium on gcg expression occurred in a tissue-specific manner, where it was only observed in intestinal endocrine L cells and not in pancreatic α cells (295). Activation of gcg promoter activity was found to be dependent on a TCF binding site within the G2 enhancer element of the gcg promoter (296).

As the G2 enhancer element has been shown to mediate the stimulatory effects of both cAMP and calcium on gcg promoter activity (175,183), our group investigated whether cAMP activates gcg expression via crosstalking with the Wnt signaling pathway in the gut. Using chromatin immunoprecipitation (ChIP), our group demonstrated a direct interaction between

TCF7L2 and the G2 enhancer element in vivo (296). Western blotting, RT-PCR, and immunostaining confirmed that TCF7L2 is abundantly expressed in both cultured intestinal

GLP-1 producing cell lines and intestinal epithelia of adult mice (296). Furthermore, treatment with dominant negative TCF7L2 attenuated both basal and lithium-stimulated gcg mRNA expression in the intestinal endocrine L cell line GLUTag (296).

1.4 P21-activated protein kinase 1 and its role in metabolic homeostasis

1.4.1 Overview of the Pak family

1.4.1.1 The discovery of Paks 1-3

Pak1 was initially discovered as a binding partner of the Rac and Cdc42 (Cell division control protein 42 homolog) GTPases in the rat brain (297). Fig. 1.10 shows the classification of

G proteins. Rac and Cdc42 are two subfamilies that belong to the Rho family of GTPases, along with the other subfamily RhoA. The Rho family is part of the Ras superfamily, which makes up the small (~21 kDa) monomeric G-proteins. Based on their small sizes, they are commonly referred to as p21 GTPases. In addition to the Rho family, there are seven other families: Ras,

Rab, Arf, Ran, Rap, Rheb, and Rit (Fig. 1.10). The p21 GTPases act as nucleotide exchange factors by binding to and catalyzing the hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP). They act as molecular switches, cycling between the active GTP-bound and inactive GDP-bound states, and are regulated by three categories of proteins. The guanine nucleotide exchange factors (GEFs) promote the dissociation of GDP from the GTPase, thereby freeing the GTPase for the binding of GTP at the plasma membrane, and hence act as activators of GTPases. GTPase activating proteins (GAPs) accelerate GTP hydrolysis and the subsequent production of GDP, thereby promoting the inactivation of GTPases. The guanine nucleotide dissociation inhibitors (GDIs) bind to and sequester the GTPase in the cytosol and thereby maintain the inactive state of the GTPases.

In addition to the small monomeric p21 GTPases, other GTPases exist as heterotrimeric

G proteins. These G proteins are made up of three subunits – the largest G subunit, and the two smaller G and G subunits – which together act as part of the GPCRs. Upon ligand binding to the GPCR, the receptor undergoes a conformational change that activates the GDP-bound G subunit, inducing the exchange of GTP in place of GDP. This is followed by the dissociation of the GTP-bound G subunit from the G-G dimer and the receptor, and the subsequent hydrolysis of GTP to GDP and the activation of adenylyl cyclase, which converts ATP to cAMP. Following

GTP hydrolysis, the inactive GDP-bound G subunit is recycled to the plasma membrane for binding to the receptor.

49 Superfamily Family Subfamily GTPase

Monomeric RhoA G proteins Ras Rho Ras Heterotrimeric Rac Rac1 G proteins Rab Cdc42 Rac2 Arf Rac3 Ran RhoG Rap Rheb Rit

PAKs

Fig. 1.10 P21-activated protein kinases (PAKs) are effectors for selected small GTPases. Pak proteins are downstream targets and functional effectors for Rac and Cdc42 small p21 GTPases, which are classified as the Rho family within the Ras superfamily of GTPases. The Rho subfamily contains five PAK-interacting members, known as Rac1, Rac2, Rac3, RhoG and Cdc42. The Ras superfamily contains over 90 members, known as monomeric G proteins, in contrast with heterotrimeric G proteins, which are downstream effector of G-protein coupled receptors.

In 1994, Manser et al. identified a group of novel serine/threonine kinases that bind to

Rac1 and Cdc42 in a gel overlay assay using [-32P]GTP-labeled glutathione-S- transferase(GST)-p21 fusion protein as substrate (297). Three kinases of 68kDa, 65kDa, and

62kDa were found to bind to Cdc42 and Rac1 but not RhoA, and were abundantly present in rat brain cytosol. Closer examination of the first kinase, termed p65 Pak, revealed that it preferentially binds to the GTP-loaded form of Cdc42/Rac, which leads to p65 Pak autophosphorylation and activation. As newly identified targets and effectors of the small p21

GTPases, these kinases were therefore coined p21-activated protein kinases (Paks), and the three molecular sizes were found to represent three isoforms of the same kinase family: -Pak (Pak1),

-Pak (Pak2), and -Pak (Pak3). Purification and sequence comparison of the first member,

Pak1, showed that it was related to the yeast protein kinase Ste20, which is involved in G-protein mediated pheromone-response pathways.

Following the initial discovery by Manser et al., several other studies further confirmed the role of Paks as targets and effectors of Cdc42 and Rac (298-300). The purification and cloning of the human Pak1 (termed hPAK65) from human exhibited >95% and ~63% sequence homology in its kinase domain with rat Pak1 and yeast Ste20, respectively (298). The hPAK65 exhibits identical specificity with endogenous brain Pak1, where it binds to Rac and Cdc42 in a

GTP-dependent manner, leading to the autophosphorylation of hPAK65 (298). Once activated,

Rac or Cdc42 are no longer required for hPAK65 activity (298). Pak1 is involved in inflammatory responses in phagocytic leukocytes, by acting as an effector of Rac in activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in a GTP-dependent manner (299). The cloning of the mouse homolog (termed m-PAK-3) from a mouse fibroblast cDNA library showed that m-PAK-3 activation was stimulated by binding to GTP-loaded forms of Cdc42 and Rac, but not RhoA (300). The m-PAK-3 contained potential SH3 domain binding

sites, bound to the SH3 domain of phospholipase C- in a highly specific fashion, and interacted with at least one SH3 domain of the adaptor protein non-catalytic region of tyrosine kinase adaptor protein (Nck) (300).

1.4.1.2 The discovery of Paks 4-6

Since the initial discovery of Paks 1-3, three more isoforms have been identified in mammals. Unlike Paks 1-3, which are collectively called group I Paks, Paks 4-6 differ significantly in their structural organization and regulation, and hence are referred to as group II

Paks. Pak4 was the first reported member from the group, and was identified from a PCR screen using degenerate primers based on Pak2. Pak4 had a fairly ubiquitous expression profile, with highest levels expressed in the prostate, testis, and colon (301,302). Pak6 was identified as an androgen receptor interacting protein in a yeast two-hybrid screen, and its expression was found to be highest in the testis and prostate (303) as well as in the brain, kidney, and placenta (304).

Pak5 is the last member of the family identified, and is primarily expressed in adult neuronal tissue, with higher levels detected in the cerebellum (305), cerebral cortex, and olfactory bulb

(305).

1.4.1.3 Structural features, activation mechanisms, and upstream regulators of Paks

1.4.1.3.1 Structural features of Paks

The structural features and domains of each Pak isoform are illustrated in Fig. 1.11. All

Paks have two major domains: an N-terminal p21-GTPase-binding domain (PBD) (aa 67-113 in

Pak1), and a highly conserved C-terminal catalytic kinase domain (KD) (aa 255-529 in Pak1). In the group I Paks, a prominent feature is an additional autoinhibitory domain (AID) (aa 83-149 in

Pak1), which overlaps but is not coincident with the PBD. The AID acts as an ‘inhibitory switch’ and is critical for the auto-inhibition of group I Paks. The six Pak isoforms have one or more proline-rich regions interspersed at the N-terminal and/or in the central region, where Pak1 has five, Pak2 has two, and Pak3 has four canonical PXXP SH3-binding motifs. For Pak1, the first, second canonical SH3-binding motifs interact with the adapter proteins Nck (306,307) and Grb2

(308), while Cloned out of library (Cool)/Pak-interacting exchange factor (PIX) protein binds to the non-canonical site (309). Pak1 also has a conserved binding site for the G subunit of the heterotrimeric G proteins near the end of the C terminus (310,311).

53 Auto-Inhibitory Domain Kinase Domain

N PBD AID KD C

P21-Binding Domain

Pak1 N C Group I Pak2 N C Pak3 N C

Pak4 N C Group II Pak5 N C Pak6 N C

PXXP SH3-binding motifs PBD AID ED-rich regions PXP SH3-binding motifs KD

Fig. 1.11 Structural features of Pak proteins. Common features of Pak members include the p21-binding domain (PBD, orange), the catalytic kinase domain (KD, green), and multiple PXXP canonical SH3-binding motifs (brown) near the N- terminus and/or in the central region. The group I PAKs have a common auto-inhibitory domain (AID, black), which interacts with the KD in the PAK-PAK homodimer formation and thereby leading to the inactivation of PAK kinase activity. Members of group I Paks also contain acid-rich domains known as ED-rich regions (light blue) as well as a non-canonical PXP Pix/Cool SH3- binding motif (dark blue) in the central region.

1.4.1.3.2 Activation mechanism of group I Paks

As the most extensively studied and representative member of the Pak family, the crystal structure of Pak1 in an auto-inhibited conformation was determined by Lei et al. in 2002 (312).

A simplified depiction of the Pak1 activation mechanism is illustrated in Fig. 1.12. Inactive Pak1 exists as a homodimer in a trans-inhibited conformation, where the N-terminal AID of one Pak1 molecule binds to and inhibits the KD of the other Pak1 molecule. The current understanding of the activation mechanism consists of a model where the binding of the p21 GTPases Cdc42 or

Rac to the PBD domain disrupts Pak1 dimerization, leading to a series of conformational changes that destabilize the folded structure of the inhibitory switch, thereby inducing the dissociation of the two Pak1 molecules from each other. Once freed from each other, the Pak1 molecules undergo sequential phosphorylation steps leading to its activation.

Pak1 contains seven auto-phosphorylation sites, which are Ser21, Ser57, Ser144, Ser149,

Ser199, Ser204, and Thr423, as illustrated in Fig. 1.13 (313,314). The key event of Pak1 activation is its phosphorylation at the Thr423 residue, located within the KD, which is required for maintaining relief from the auto-inhibition and the full activation of the catalytic kinase activity towards exogenous substrates (315-317). An acidic substitution at this site (T423E) renders Pak1 constitutively active. Following the Thr 423 phosphorylation event, Pak1 autophosphorylates at Ser 141, an event that is associated with the initiation and/or maintenance of its activation, and which is required for GTPase- but not sphingosine-dependent activation

(318).

Inactive Pak1 homodimer

p21

P Thr 423

Active Pak1 molecules

Thr 423

Fig. 1.12 Activation mechanism of Pak1. In its active state, Pak1 forms a homo dimer in a trans-inhibited formation where the catalytic domain (green) is bound by the AID (black) of the other Pak1 molecule. The binding of p21 GTPases to the PBD domain (orange) leads to the destabilization of the AID, resulting the dissociation of the two Pak1 molecules from each other. Pak1 then undergoes a series of phosphorylation and autophosphorylation events, the key activating residue being Thr423 (yellow circle), which renders Pak1 as fully active in its catalytic activity.

56 Rac Cdc42 CHP Pak1 TC10 LKB1 hPIP1 Wrch-1 Merlin Nck Grb2 Pix/Cool SKIP Thr109 CRIPAK Nischarin P N C P P P P P P P P P Ser21 Ser57 Tyr131 Ser144 Ser199 Thr212 Thr423 Ser204 Ser223 Pdk-1 Akt Src p35/Cdk5 CK2 Cdc2 SAD-A Erk1/2

Fig. 1.13 Interaction domains and phosphorylation sites of Pak1. Illustration of the PAK1 molecule. The small p21 GTPases, including Rac, Cdc42, CHP, TC10, and Wrch-1, bind to the PBD (orange) and stimulates its activation. Ser21, Ser57, Ser144, Ser149, Ser199, Ser204, and Thr423 are seven autophosphorylation sites. The adapter proteins Nck and Grb2 bind to two PXXP motifs (brown) near the N terminus. The non-canonical PXP motif (dark blue) interacts with the adapter protein Pix/Cool. The endogenous inhibitors of PAK1 include hPIP1, Merlin, LKB1, CRIPAK, and Nischarin, with their sites of interaction as indicated (red line).

1.4.1.3.3 Positive regulators of Pak1

Pak1 is activated by a wide panel of upstream regulators, including GTPases such as

Rac1, Rac2, Rac3 (297,319,320), Cdc42 (297), but not by Rho A-G or by other Ras superfamily members. When bound to Pak, the intrinsic- and GAP-stimulated GTP hydrolysis of the GTPase is inhibited, hence making the Pak PBD a useful affinity reagent for detecting Rac and Cdc42 activation (321). Small GTPases including Rac and Cdc42, as well as atypical small GTPases such as CHP (322), TC10 (323), and Wrch-1 (324) bind to the PBD and stimulate Pak1 activation (Fig. 1.13). The CHP/Pak1/Pix signaling has been shown to regulate cell adhesion during zebrafish embryonic development (325). TC10 was found to activate Pak1 when examining insulin-stimulated prolactin gene expression in the rat pituitary cells (326). Wrch-1 is a Wnt-1 stimulated small GTPase, which stimulates Pak1 Thr423 phosphorylation in the COS-7 cell line (324).

The adapter proteins Nck and Grb2 interact with the N-terminal canonical proline-rich motifs, while Pix/Cool binds to the non-canonical motif in the centre region (Fig. 1.13).

Upstream activators of Pak1, such as Akt, Src, p35/Cdk5, Cdc2, Erk1/2, Pdk1, and SAD-A induce the phosphorylation of Ser and Thr residues, some of which overlap with the autophosphorylation sites of Pak1 (Fig. 1.13).

Hormones and growth factors that regulate Pak1

An overview of the positive and negative factors that regulate Pak1 activity is presented in Fig. 1.14. In addition to the small GTPases, Pak1 is activated by tumorigenic factors, growth factors, and hormones. Thrombin-stimulated Pak1 activation was observed in vascular smooth

muscle cells, and expression of the kinase-dead Pak1 mutant was shown to attenuate stress fiber formation and cell migration (327) (Fig. 1.14). The epidermal growth factor (EGF) stimulates

Pak1 activation in breast cancer cells, and expression of a dominant-negative Pak1 was shown to attenuate EGF-induced cell migration (328) (Fig. 1.14). Pak1 is a point of convergence for platelet-derived growth factor (PDGF) and lysophosphatidic acid in regulating collagen matrix contraction in human fibroblasts (329). Over-expression of PDGF is known to stimulate Erk activity in medulloblastoma cells, and this was found to be mediated through the PAK1-MEK-

Erk signaling cascade (330). Pak1 was also shown to mediate the function of estrogen in stimulating FKHR phosphorylation, leading to FKHR nuclear exclusion and the inhibition of cell apoptosis (331) (Fig. 1.14). Noteworthy, the growth factor insulin-like growth factor-1 (IGF-1), as well as the metabolic hormone insulin, were found to activate Pak1 as well (Fig. 1.14). IGF-1 activates RUNX2, a transcription factor that promotes endothelial cell migration, invasion, and proliferation. Pak1 inhibition attenuates RUNX2 DNA-binding (332). Importantly, we and others have also demonstrated that Pak1 mediates the function of the metabolic hormone insulin in muscle and intestine (333,334).

1.4.1.4 Negative regulators of Pak1

Considerable efforts have been made to develop highly selective and potent Pak inhibitors, which can be used as part of Pak-based therapeutics in targeting cancer. One method is to identify endogenous Pak1 inhibitors. The domains of Pak1 that interact with its negative regulators are illustrated in Fig. 1.13, while the list of these negative regulators is presented in

Fig. 1.14. Interaction of Pak1 N-terminal regulatory domain with the human Gβ-like WD-repeat protein called human Pak1 interacting protein 1 (hPIP1), which shares sequence homology with

the Pak regulator Skb15 in fission yeast, abolishes Cdc42/Rac-stimulated Pak1 kinase activity

(335) (Fig. 1.14). Merlin, the product of the Nf2 tumor-suppressor gene, inhibits the activation of

Pak1 by binding to its PBD (336) (Fig. 1.14). The serine threonine kinase liver kinase B1

(LKB1, also known as serine/threonine kinase 11 or STK11), the product of another tumor- suppressor gene, suppresses Pak1 activity by phosphorylating it at Thr109 in the PBD (337) (Fig.

1.14). The cysteine-rich inhibitor of Pak1 (CRIPAK) was identified in a yeast two-hybrid screen, which suppresses Pak1-mediated ER transactivation in breast cancer cells (338) (Fig. 1.14). The skeletal muscle and kidney enriched inositol phosphatase (SKIP) binds to the KD of Pak1 and inhibits its scaffolding activity (detailed in Section 1.4.2) (Fig. 1.14). The integrin binding partner Nischarin selectively inhibits Pak1 kinase activity and Pak1-mediated cell migration through its direct interaction with Pak1 C-terminal domain (339) (Fig. 1.14). Two serine/threonine phosphatases, POPX1 and POPX2, inactivate Pak1 via dephosphorylation, and represent yet another component of the signaling pathways that regulate Pak activity (340).

The microRNAs (miRNAs) are non-coding RNAs that inhibit the expression of their targets in a sequence-specific manner, and miRNA-7 (miR-7) was found to suppress Pak1 expression via interacting with its 3’-untranslated region of Pak1 mRNA in human cancer cells

(341). In a cellular model for breast cancer, levels of Pak1 protein were upregulated whereas those of miR-7 and its upstream activator HoxD10 were found to be downregulated (341). In another study, two homologs of the miRNA miR-126, miR-126a and miR-126b, were identified in the zebrafish genome and were found to downregulate Pak1 mRNA expression.

Overexpression of miR-126a/b overexpression lead to decreased Pak1 transcript levels, whereas knockdown of miR-126a/b causes increased Pak1 transcript levels in endothelial cells (342).

60 Positive regulators Negative regulators

p21 Tumorigenic Growth Endogenous Hormones GTPases factors factors inhibitors

• SKIP • Rac1 • Merlin • Cdc42 • EGF • Estrogen • LKB1 • CHP • Thrombin • PDGF • Insulin • CRIPAK • TC10 • IGF-1 • Nischarin • Wrch-1 • hPIP1

Pak1

Fig. 1.14 Pak1 and its upstream positive and negative regulators. Small p21 GTPases, including Cdc42 and Rac1, as well as atypical ones CHP, TC10, and Wrch-1, can stimulate Pak1. Pak1 can also be activated by other upstream factors, including tumorigenic factors, growth factors, and hormones such as insulin. Several endogenous inhibitors of PAK1 have been identified, including the kinase LKB1, the phosphatase SKIP, and other factors such as Merlin, CRIPAK, Nischarin, and hPIP1.

1.4.1.5 Substrate specificity

Many downstream substrates for the group I Paks, Pak1 in particular, have been identified. A portion of these substrates are also targeted by the group II Paks. Thus, it is not clear as to what degree these two groups of Paks recognize similar substrates and the extent of their redundancy. Based on their sequence divergence, however, one would predict that the two groups of Paks exhibit at least some degree of substrate specificity. Using a degenerate peptide library method, characterization of the consensus phosphorylation motifs of Pak1, Pak2, and

Pak4 showed that Pak1 and Pak2 exhibit virtually identical substrate specificity, which is distinct from that of Pak4 (343). Comparison of crystal structures of Pak4, Pak5, and Pak6 to those of group I Paks revealed significant differences between the two groups in the rearrangements of the C helix, which would suggest conformational differences and thereby contributing to substrate specificity (344).

1.4.1.6 The role of Paks in tumorigenesis and cancer

Pak proteins have been found to be upregulated and have been extensively researched in a variety of cancers. The mechanisms underlying tumorigenesis and cancer metastasis include increased copy number of Pak1 gene, hyperactivation of GTPases or upstream activators, as well as downregulation or inhibition of endogenous negative regulators of Pak (345-347). Pak1 deregulation is well-documented in breast cancer, where more than 50% of human breast cancers display overexpression and/or hyperactivation of Pak1 (348). Other cancers where altered expression of Pak kinases occurs include the following cancer types: brain (349), esophagus

(350), liver (351), kidney (352), pancreas (353), colon (350,354,355), bladder (356), ovarian

(357-359), prostate (360), and T-cell lymphoma (361). A detailed review of Pak kinases and their role in cancers has been summarized by Dummler et al (362).

Pak small-molecule inhibitors

Significant progress has been made in developing Pak1 small molecule inhibitors, with the initial studies focusing on ATP-competitive inhibitors. Oncogenic Ras mutants such as v-Ha-

Ras are upstream activators of Pak1, and the synthetic derivative of the ATP antagonist K252a, called CEP-1347, was found to directly inhibit Pak1 activity and able to block Ras-induced transformation in vitro and in vivo in nude mice (363). The cyclooxygenase inhibitor derivative

OSU-03121 was found to reduce the levels of phosphorylated Pak1, whereas the octahedral ruthenium complex FL-172 targets the Pak1 ATP-binding site and shows an ability to inhibit

Pak1 in mammalian cells (364).

In addition to ATP-competitive inhibitors, allosteric inhibitors that can interact with Pak1 outside of the ATP-binding sites are also under development. The most recent discovery is the group I Pak inhibitor IPA3 (2,2’-dihydroxy-1,1’-dinaphtyldisulfide), which bound covalently to the Pak1 regulatory domain and prevented Pak1 from binding to its upstream activator Cdc42

(365,366). IPA3 selectively inhibited the group I Paks and not the group II Paks, and had no effect on pre-activated group I Pak kinases (365,366).

1.4.1.7 The functions of Paks

An overview of the functions of Paks, focusing on the group I Paks, are presented in Fig. 1.14, where each column represents a category of related functions, and each category is described in further detail in the sections below.

1.4.1.7.1 Cell cycle progression

Pak proteins play critical roles in cell cycle progression (Fig. 1.15, column 1). Pak1 localizes to specific structures during mitosis, such as chromosomes, centrosomes, mitotic spindles, and the contraction ring during cytokinesis (367). During mitosis, two Pak-binding proteins, GIT1 and Cool/PIX, bind to and activate Pak1 independently of Rac or Cdc42.

Activated Pak1 then in turn phosphorylates and activates two protein kinases, Aurora-A at

Thr288 and Ser342 (368), and Polo-like 1 (Plk1) at Ser49 (369), both of which are important regulators of mitotic events. During the early phase of mitosis, Pak1 co-localizes with Histone

H3 on condensing chromosomes and phosphorylates Histone H3 on Ser10, leading to the initiation of chromosome condensation (370). Another downstream target of Pak1 is cyclin D1, and constitutively active Pak1 has been shown to induce transcription of cyclin D1 via activation of the transcription factor NFκB (348,371). Paks have also been shown to regulate the MAPK pathway, where Pak1 phosphorylates two mediators of the MAPK pathway, MEK1 at Ser298 and Raf1 at Ser338 (372-376), and could potentially regulate cell cycle progression through this pathway as well. Phosphorylation of MEK1 and Raf1 by Pak is not sufficient to activate these two MAPK pathway effectors, but enhances their activation by the respective upstream activators Ras and Raf1.

64 Pak1

(1) (2) (3) (4) (5)

Cell cycle Cell survival Cytoskeleton Host-pathogen Transcription progression and apoptosis remodeling response and splicing

Aurora-A Raf1 LIMK Erk MAPK

PIK1 NFB Myosin NFAT histone H3

histone H3 FKHR Myo3p CtBP1 AR

NFκB BimL RLC PCBP1

MEK1 DLC1 MLC FKHR

Raf1 MHC CtBP

MLCK SHARP

Snail

β-cat

Fig. 1.15 Pak1 and its downstream effectors regulate a multitude of cellular functions. Pak1 and its downstream targets regulate cell cycle progression (1), cell survival and apoptosis (2), cytoskeleton remodeling (3), host-pathogen response (4), and gene transcription and mRNA splicing (5).

1.4.1.7.2 Cell survival and apoptosis

Initiation of apoptosis is carried out by Bcl-2-associated protein (Bax) and Bcl-2 homologous killer (Bak), two families of pro-apoptotic proteins that localize to the mitochondria and induce pore formation in the mitochondria outer membrane, thereby allowing cytochrome C to escape into the cytoplasm and leading to the activation of the pro-apoptotic caspase cascade.

This series of events can be blocked by the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2), which binds to and forms heterodimers with Bax or Bak, thus preventing apoptosis. The regulation of apoptosis is further enhanced by Bcl-2-associated death promoter (BAD) proteins, where dephosphorylated BAD forms heterodimers with and inactivates Bcl-2, subsequently allowing Bax/Bak-mediated apoptosis to proceed. Akt, on the other hand, can phosphorylate

BAD, leading to the formation of BAD/14-3-3 homodimers, which leaves Bcl-2 free to inhibit

Bax/Bad-triggered apoptosis.

The pro-survival and anti-apoptotic functions of Pak1 are presented in Fig. 1.15 column

2. Pak1 is anti-apoptotic and protects cells from intrinsic apoptotic signals via its downstream target Raf1. Pak1, as well as Pak5, phosphorylates Raf1 at Ser338 and stimulates its translocation to the mitochondria (377,378). In the mitochondria, Raf1 binds to Bcl-2 and forms the protective Raf1-Bcl-2 complex. Like Akt, Raf1 can phosphorylate the pro-apoptotic protein

BAD at Ser112, a site that renders BAD unable to bind to Bcl-2, and therefore maintaining Bcl-2 activity and its inhibition of apoptosis. FKHR is involved in the induction of apoptotic genes, and Pak1 directly phosphorylates FKHR and sequesters it in the cytosol, thereby inhibiting its ability to induce transcription of FKHR target genes (331).

Paks mediate their pro-survival effects through the transcription factor NFκB. NFκB has been shown to regulate genes involved in cell survival, proliferation, and angiogenesis, and is often active in tumor cell types. Pak1 has been reported to activate NFκB, but the exact mechanism is still unclear (348,371,379,380).

Other downstream targets that are inactivated by Pak1 are BimL and dynein light chain 1

(DLC1) (381). In a similar manner to BAD, BimL is a pro-apoptotic protein that binds to and inactivates Bcl-2. Upon initiation of apoptosis, DLC1-BimL dimers are released from the dynein motor complex, where BimL then interacts with and inactivates Bcl-2 at the mitochondria. This process is inhibited by Pak1, where Pak1 binds to and phosophorylates BimL, preventing it from binding to Bcl-2 and resulting in an anti-apoptotic action.

1.4.1.7.3 Cytoskeleton remodeling

The regulation of the cytoskeleton by Pak kinases is well-established (Fig. 1.15 column

3), and the process may be dependent or independent on the Pak kinase activity (382,383). It is known that the formation of lamellipodia and filopodia is regulated through the effects of Rac and Cdc42 on the cytoskeleton, respectively. The role of Pak1 in the induction of lamellipodia, filopodia, formation of membrane ruffles, and cell motility was identified through its interaction with its downstream target LIM domain kinase (LIMK) (384). Pak1 phosphorylates LIMK at

Thr508, leading to LIMK activation (385,386). Active LIMK catalyzes the phosphorylation of cofilin, which suppresses its activity in acting as an actin capping and severing protein and subsequently results in increased amount of cellular filamentous actin (387).

In addition, several of the Pak-interacting proteins are main components of thin, thick, and intermediate filaments. Myosin activity is crucial for the intracellular movement machinery underlying cytoskeletal rearrangements, and myosins have been identified as targets of Pak proteins. Pak1 was initially identified as the mammalian homolog of the yeast protein Ste20, which is a kinase that modulates cell morphology and polarity in Saccharomyces cerevisiae

(388). This budding yeast has two myosin-I isoforms, encoded by Myo3 and Myo5 genes. Myo3p was found to be a phosphorylation target (at Ser357) for Ste20p and Cla4p, another member of the yeast Pak family, both in vitro and in vivo (389). Phosphorylation of myosin-I was required for yeast budding and was found to regulate the reorganization of actin cytoskeleton (390).

In Drosophila melanogaster, Pak1 phosphorylates the regulatory light chain (RLC) of nonmuscle myosin II on Ser21 and Thr20, sites that are homologous to Ser19 and Thr18 in the mammalian smooth muscle myosin RLC (391). In Xenopus laevis, activated Pak1 phosphorylates the regulatory light chain (MLC) of myosin II on Thr18 and Ser19 (392).

Mammalian Pak1 phosphorylates MLC on Ser19 (393-395), and MLC phosphorylation by Pak1 and Pak3 in neuronal cells promotes dendritic spine morphogenesis by local stabilization of the actin network (396). Pak1 also phosphorylates the myosin heavy chain (MHC) of myosin VI, which plays a central role in membrane trafficking and cell migration (397).

Pak1 can also indirectly regulate the phosphorylation of myosin by myosin light chain kinase (MLCK), where Pak1 phosphorylates MLCK and thereby decreasing its activity (398).

Cdc42-activated Pak2 can phosphorylate MLCK on Ser439 and Ser991, which inhibits MLCK activity and thereby hindering the development the isometric tension in smooth muscle and nonmuscle cells (399).

1.4.1.7.4 Host-pathogen response

The group I Paks are highly expressed in most leukocytes, and Pak proteins are being recognized to play important roles in immune function and responses (Fig. 1.15 column 4). Pak1 acts downstream of the Nck/Vav/SLP-76 signaling complex and is required for T-cell mediated activation of Erk and NFAT (400). This is consistent with the observation that macrophages from Pak1-/- mice display reduced Erk activation in response to growth factors and defective lamellipodia stability, since the effect of lamellipodial dynamics can be mimicked by Erk inhibitors (401). Pak1 activation is required for chemotaxis of leukocytes in response to the chemokine CXCR2 (402), and expansion of the active Pak1 pool via its interaction with G,

Cool/PIX, and Cdc42, is needed for chemotaxic signaling in myeloid cells (403). More recent studies have shown that Pak1 plays a key role in mast cells, which are important regulators of allergic diseases, where activation of high affinity IgE receptors lead to the release of proinflammatory mediators (404). Mast cell-deficient mice which received locally reconstituted

Pak1-/- mice bone marrow derived mast cells (BMMCs) showed decreased allergen-induced vascular permeability and reduced IgE receptor degranulation, indicating that Pak1 is a target for modulating acute mast cell responses in allergic diseases (404).

Mammalian Pak kinases have also been implicated in several types of host/pathogen responses, in particular in Human Immunodeficiency Virus (HIV) pathogenesis, where one or more Pak isoforms were identified to interact with the HIV-encoded protein Nef. Structural studies show that Pak2 associates with a hydrophobic binding surface on Nef (405), which leads to Pak2 activation (406). Nef, but not Nef mutants that are incapable of Pak binding, are able to block T-cell induced apoptosis during HIV replication, suggesting that Pak-Nef is required for its anti-apoptotic effects during HIV pathogenesis (407).

Pak proteins are also involved in the pathogenesis of other virus types. Alphaherpesvirus encode a protein kinase, US3, which induces actin cytoskeleton reorganization leading to virus spread. US3 is known to directly bind to and activate Pak, and in mouse embryo fibroblasts

(MEFs) from Pak2-/- mice, US3-mediated stress fiber disassembly is impaired, whereas Pak1-/-

MEFs show impaired US3-mediated cell projections, which suggest that each Pak isoform plays a distinct role in the cell/virus interactions and that Pak proteins are required for efficient herpesvirus spread (408).

Group I Paks are implicated in other stages of the virus life cycle as well. The participation of Pak1 is essential for vaccinia virus entry, involving macropinocytosis and membrane blebbing (409). This process may also involve Pak-mediated activation of C-terminal binding protein 1 of E1A (CtBP1, also known as brefeldin A-ribosylated substrate BARS), an event necessary for the fission of the macropinocytic cup (410). For the human adenovirus serotype Ad3, activation of the viral Pak1 and its phorphorylation target CtBP1 are required for

Ad3 infectious entry; this is consistent with the observation that a phosphorylation-defective

S147A-CtBP1 blocks Ad3 infection, and demonstrates the role of Pak1-CtBP1 in adenovirus entry (409).

1.4.1.7.5 Gene transcription and mRNA splicing

Three nuclear localization signals have been identified in Pak1 N-terminus (411). In addition to its well-characterized kinase activity, it is increasingly recognized that Paks also participate in nuclear events, such as transcription and mRNA splicing (Fig. 1.15 column 5).

One of the main targets of Pak1 is presumably through the activation of MAPK (300,412,413).

Endogenous Pak1 was found to localize to the nucleus of interphase cells and bind to histone H3

(367).

Pak6 was found to interact with androgen receptor (AR) and translocate into the nucleus upon androgen stimulation (303). Pak2 has been identified to undergo caspase-mediated cleavage, and the resulting Pak2 fragment containing the nuclear localization signal relocated into the nucleus and stimulated programmed cell death (414). Yeast two hybrid screening of a mammary gland cDNA library using Pak1 as bait identified polyC-RNA-binding protein 1

(PCBP1), which controls mRNA translation, as an interacting partner of Pak1 (415). Pak1 activation led to enhanced nuclear retention of PCBP1, its recruitment to the eukaryotic translation initiation factor 4E (eIF4E) promoter, and the stimulation of eIF4E expression, which subsequently resulted in alternative splicing of eIF4E target genes (415).

Estrogen was found to rapidly activate Pak1 in mammary cancer cells in a PI3K- dependent manner (331). Pak1 in turn directly phosphorylates and activates FKHR (also known as FOXO1), resulting in the nuclear exclusion of FKHR and the repression of FKHR target gene expression (331). CtBP is a ubiquitous transcriptional corepressor during development and oncogenesis. Pak1 was shown to bind to and phosphorylate CtBP at Ser151, inducing CtBP cellular redistribution and thereby blocking its corepressor function (416). A single amino acid substitution (S158A) of CtBP, as well as the use of siRNA-mediated Pak1 knockdown, inhibited

CtBP phosphorylation by Pak1 (416).

The Notch signaling pathway is important for the determination of cell fate and differentiation in many organs, and dysregulation of Notch and Pak1 have been observed in many types of cancers. SHARP, a component of Notch signaling, was identified as a Pak1- interacting protein in a yeast two hybrid screen. Pak1 phosphorylated SHARP at Ser3486 and

Thr3568, leading to the stimulation of SHARP repressor activity on Notch target gene expression

(417). Snail is a zinc finger phosphprotein that regulates cell epithelial-mesenchymal transition, a process that underscores the transition of early stage tumors to invasive malignancies. Pak1 regulates Snail repressor activity by phosphorylating it at Ser246, inducing its nuclear localization and its repressor function on target gene expression (418).

1.4.1.7.6 Endothelial and vascular biology

Rac1 has been established as a well-known regulator of vascular development, where endothelial-cell-specific Rac1 deletion results in embryonic lethality. The Rac1-/- embryos exhibit defective vessel development, and vascular development was completely absent in the yolk sacs of Rac1 knockout mice (419). The involvement of Pak1 in vascular development is less understood. Early studies have suggested a role for Pak1 in cultured endothelial cells (420).

Subsequently, a chicken chorioallantoic membrane (CAM) model was developed to study the role of Pak1 in angiogenesis, which uses βFGF-soaked filters placed on the chorioallantoic membrane of developing chicken embryos, and a peptide mimicking the effects of dominant- negative Pak1 was able to impair βFGF-mediated angiogenesis (421). In zebrafish, mutation in

Pak2α resulted in embryonic brain haemorrhage with intact gross development of the vasculature and normal hemostatic function (422). More recently, Pak4 null embryos show abnormalities in both yolk sacs and placentas, with lack of vasculature throughout the extra-embryonic tissue and abnormal formation of the labyrinthine layer of the placenta (423).

1.4.1.7.7 Metabolic homeostasis

In the last decade, Pak kinases are beginning to emerge as important regulators of metabolic homeostasis. Specifically, Pak1 has been demonstrated to participate in the regulation of glucose homeostasis in two key processes: 1) insulin-stimulated glucose uptake in skeletal muscle (detailed in Section 1.4.2), and 2) glucose-stimulated insulin secretion in β cells (detailed in Section 1.4.3) (Fig. 1.16).

1.4.2 Pak1 and glucose transport in muscle

Discovery of the kinase PK65

In 1996, Tsakiridis et al. used an in-gel kinase assay to examine the presence of insulin- responsive renaturable kinases in lysates of L6 myotubules (333). Using histone VI-S as substrate, they observed that insulin treatment induced the phosphorylation of a kinase of 65kDa, and they referred to this kinase as ‘PK65’ (Protein Kinase 65 kDa) (333). PK65 was found to be activated within minutes upon stimulation with insulin (333). When myelin basic protein (MBP) was used as substrate, a modest induction of PK65 was detected, suggesting that histone VI-S is the preferred substrate compared to MBP (333). Tsakiridis et al. then further investigated the dependence of PK65 on insulin activation, and found that treatment with erbstatin abolished the activation of PK65 by insulin, implying that stimulation of PK65 requires tyrosine kinase activity

(333). To identify whether PI3K, a critical node of insulin signaling, was involved, they pretreated myotubes with wortmannin prior to insulin activation, and found that wortmannin blocked the insulin stimulation of PK65, while not affecting insulin-induced tyrosine

73 FEEDING

Elevated insulin Elevated glucose

Muscle Pancreas

Cdc42 Cdc42 Rac1 Pak1 Rac1 GLUT4 translocation insulin Pak1 secretion

Glucose uptake Insulin response

Fig. 1.16 Summary of the role of Pak1 in skeletal muscle and pancreas. Upon feeding, the rise in blood glucose level leads to elevated circulating insulin levels. In the muscle, insulin stimulates GLUT4 translocation via the PI3K-Cdc42-Rac1-Pak1 signaling axis and the PI3K- Akt-AS160 axis, in promoting muscle glucose uptake (left). In the pancreas, increased blood glucose level elicits the postprandial release of insulin form the β-cells, involving the Cdc42-Pak1-Rac1 signaling cascade (right).

phosphorylation of IRS-1 or the two tyrosine phosphorylation and activation of Erk 1 and 2

(333). In contrast, the PKC inhibitor BIM did not affect insulin-stimulated PK65 activation, and neither did the inhibitor of p70S6k rapamycin (333).

Identification and renaming of PK65 as Pak1

In their in-gel kinase assay, PK65 from myotubes co-migrated with a 65 kDa fMLP- stimulated renaturable kinase from neutrophils, which at that time was suggested to be the neutrophil PAK65 (now known as Pak1 and referred to as Pak1 hereafter) (333). In order to identify whether PK65 was indeed Pak1 in myotubes, Tsakiridis et al. used anti-Pak1 antibodies and examined by immunoblotting and immunoprecipitation experiments using control and insulin-stimulated myotubes, and confirmed that PK65 present in the myotubes was indeed Pak1

(333). They further confirmed the identity of PK65/Pak1 using GST-Cdc42Hs coupled to agarose beads preincubated with GDP or GTPS followed by incubation with cell lysates (333).

PK65/Pak1 activity was significantly enhanced in the presence of GTPS compared to GDP

(333).

Pak1 activates MAPK pathway

Since transfection of dominant negative PK65/Pak1 was found to inhibit Rac and Cdc42 activation of p38 MAPK in Cos-7 cells (412), and tyrosine phosphorylation is an essential step in the activation of MAPK, Tsakiridis et al. further examined the effect of insulin on the MAPK pathway (333). Lysates from control and insulin-treated myotubes pretreated with or without

wortmannin were probed with anti-MAPK antibodies, and insulin was found to induce tyrosine phosphorylation of p38 MAPK within 5 min (333). Unlike the activation of Pak1 by insulin, activation of MAPK by insulin was not blocked by wortmannin, suggesting that Pak1 and

MAPK are activated by insulin through different pathways in myotubes (333).

The PI3K/Akt2 and PI3K/Rac/Pak1 axes in muscle glucose uptake

An overview of the mechanisms underlying skeletal muscle glucose uptake and the role of Pak1 is illustrated in Fig. 1.17.

Insulin is known to stimulate GLUT4 vesicle translocation to the cell surface, leading to glucose uptake in skeletal muscle tissue (424). This process requires the tyrosine phosphorylation of IRS-1, which recruits and activates PI3K. Insulin signaling bifurcates at

PI3K, with one arm leading to Akt2 activation, and another arm leading to Rac1 activation

(424,425). Akt2 phosphorylates and inactivates its target AS160, which is a GAP and in the absence of insulin stimulation leads to the inactivation of the GTPases. Hence, insulin stimulation leads to the activation of Akt2 and the GPTases Rab8A and Rab13, ultimately resulting in GLUT4 vesicle translocation and glucose uptake in muscle (426).

Both the Rac1 arm and the Akt2 arm promote glucose uptake in the skeletal muscle, and it has been proposed that each arm act independently of each other in promoting GLUT4 vesicle translocation (427,428). The role of PI3K/Rac signaling in the regulation of glucose uptake in muscle cells have been further elucidated, and F-actin remodeling is one of the major downstream mechanisms for Rac-mediated GLUT4 translocation. Actin cytoskeleton reorganization is involved in insulin-stimulated GLUT4 vesicle translocation, as

76 Glucose Muscle contraction Insulin Exercise

GLUT IR 4

PI3K IRS

Akt2 Rac1

AS160 RalA ROCK Pak1 SKIP

LIMK Rab8A Rab13 cofilin ?

? F-actin remodeling

GLUT4 vesicle

Fig. 1.17 Overview of mechanisms underlying insulin-stimulated glucose uptake in skeletal muscle. Insulin binding to the IR leads to the activation of PI3K, which bifurcates into the Akt2 arm and the Rac1 arm. The Akt2-mediated inactivation of AS160 enables the activation of the GTPases Rab8A and Rab13, which promote GLUT4 translocation. The Rac1 arm stimulates GLUT4 translocation through various mechanisms, including Pak1 which potentially involves LIMK/cofilin signaling, ROCK, and RalA, all of which lead to F-actin remodeling. Muscle contraction and exercise activate Rac1/Pak1 signaling and stimulates GLUT4 vesicle translocation. The phosphatase SKIP is an endogenous negative regulator of Pak1 in the muscle and inhibits its scaffolding activity. IR: Insulin receptor; PI3K: Phosphoinositide 3- kinase; LIMK: LIM domain kinase; ROCK: Rho-associated coiled-coil-containing protein kinases ; SKIP: Skeletal muscle and kidney enriched inositol phosphatase .

pharmacological agents that disrupt actin dynamics, such as latrunculin B, inhibited GLUT4 translocation to the plasma membrane and lead to reduced glucose uptake in the muscle cells

(425,429-431). Insulin is known to regulate actin dynamics in cultured muscle cells and in the mouse L6 myotubes (432,433). Insulin-induced actin cytoskeleton reorganization in muscle cells requires PI3K activity (431,434,435), and insulin-stimulated GLUT4 translocation and actin remodeling can be blocked either by depleting Rac1 or by inducing conditions that mimic insulin resistance in cultured muscle cells (424,436). Expression of a constitutively-active Rac1 elicited

GLUT4 vesicle translocation even in the absence of insulin, suggesting that the presence of active Rac1 suffices in stimulating GLUT4 translocation, at least in the in vitro setting (436).

Treating cultured muscle cells with glucose oxidase mimics insulin resistance in vitro, and this leads to the inhibition of Rac1 activation, accompanied by the loss of Rac1-mediated Pak1

Thr423 phosphorylation (424). These observations highlighted the requirement of Rac1 signaling in insulin-stimulated muscle glucose uptake, involving the process of cytoskeleton remodeling.

Consistent with the observations made in the above in vitro investigations, insulin was shown to stimulate Rac1 activation in mouse skeletal muscle, and mice Rac1 ablation in the muscle only showed reduced insulin-induced GLUT4 vesicle translocation in the muscle tissue

(437). The aforementioned observations collectively indicated the essential role of Rac1 in mediating the stimulatory effect of insulin on GLUT4 membrane translocation, and that Pak1 is among the downstream targets of insulin and Rac1 in the muscle.

The generation of Pak1-/- mice enabled the direct assessment of the contribution of Pak1 in muscle glucose uptake. Following insulin injection, muscle samples collected from Pak1-/- mice showed significantly reduced GLUT4 vesicle translocation to the plasma membrane when compared with muscle samples from control mice (438). Cofilin has been reported to act downstream of the PAK1 effector LIMK, and is critically involved in the regulation of actin

filaments in a marsupial kidney epithelial cell line (439). In line with the role of

Pak1/LIMK/cofilin signaling in cytoskeletal reorganization in epithelial cells, the skeletal muscle from Pak1-/- mice was shown to lack the response to insulin treatment in stimulating cofilin phosphorylation (438). Hence, the PI3K/Pak1/LIMK/cofilin axis may be one of the mechanisms underlying insulin-stimulated GLUT4 translocation.

A very recent study by Sylow and colleagues provided extended evidence that Rac1-Pak1 signaling centrally regulates muscle glucose uptake in intact muscle tissue, and that this signaling axis was rendered defective under the insulin-resistant state (429). First, they demonstrated that insulin indeed stimulates Rac1 and Pak1 activation in both mouse and human skeletal muscle

(429). Pharmacological inhibition of Rac1 resulted in the loss of insulin-stimulated glucose uptake, independent of Akt status, in isolated mouse skeletal muscle (429). Muscle-specific deletion of Rac1 in mice resulted in reduced insulin-stimulated muscle glucose uptake, associated with attenuated insulin-stimulated Pak1 Thr423 phosphorylation in the muscle (429).

Importantly, skeletal muscle isolated from high fat diet fed mice showed reduced Rac1 expression, associated with blunted Pak1 Thr423 phosphorylation in response to insulin treatment (429). These observations in the experimental animals were at least partially recapitulated in humans, where skeletal muscle insulin resistance induced via intralipid infusion resulted in a loss of insulin-stimulated Pak1 Thr423 phosphorylation during a hyperinsulinemic- euglycemic clamp study (429).

The Rho-associated coiled-coil-containing protein kinases (ROCKs) are serine/threonine kinases that act as downstream effectors of certain small GTPases, and ROCKs have been shown to participate in insulin signaling and energy homeostasis (440). Inhibition of ROCK1 impaired

GLUT4 translocation, while over-expression of ROCK1 resulted in enhanced insulin-sensitizing effect on glucose transport in adipocytes (441). In myoblasts, ROCK1 depletion lead to

attenuated insulin-stimulated glucose transport, associated with defects in actin cytoskeleton remodeling (441). Hence, ROCKS which are similar to Paks and act as downstream effectors of

GTPases, are potentially involved in GLUT4 translocation and glucose transport in the muscle as well (424,440,441).

Furthermore, the small GTPase RalA has been reported to function as the effector of

Rac1 in mediating its stimulatory effect on muscle glucose uptake (442). Depletion of RalA using the siRNA-mediated knockdown approach was shown to reduce Rac1-stimulated GLUT4 vesicle plasma membrane translocation in the mouse L6 myotubes (442), and therefore represent yet another potential mechanism for Rac-mediated GLUT4 vesicle translocation.

The joint requirement of both the PI3K-Akt2 arm and the PI3K-Rac arm in insulin- stimulated GLUT4 translocation has been proposed. Pharmacological inhibition of both Akt2 and Rac1 signaling cascades was shown to be required to completely block insulin-stimulated glucose uptake in mouse muscle cells (428). In addition, both Akt2 and Rac1 signaling pathways were found to be dysfunctional in the muscle tissue of the insulin-resistant ob/ob mouse model

(428). Interestingly, in examining the involvement of Pak1 as the effector of Rac1 signaling, muscle tissue isolated from Akt2-/- mice appear to have blunted insulin-stimulated Pak1 Thr423 phosphorylation, while pharmacological inhibition of Akt2 does not alter Pak1 activation (428).

To explain this obvious discrepancy, it has been proposed that although the Rac1 and Akt2 arms do not crosstalk with each other in regulating glucose uptake, the presence of Akt2 is still required for the activation of the Rac1 cascade (428). In summary, the critical role of Rac1 in regulating actin dynamics underlying muscle glucose uptake has been demonstrated in numerous investigations (424,425,427,436). Pak1 has been shown to regulate actin filament dynamics in a number of other cell types, including neuronal cells and pancreatic -cells (detailed in the

Section 1.4.3) (443-446). However, the direct function of Pak1 on actin remodeling and glucose uptake in muscle, for example whether it involves LIMK/cofilin or other yet-to-be-identified mechanisms, remains to be investigated.

Even though the majority of studies to date have focused on assessing Pak1 in the muscle as a protein kinase, Ijuin et al has recently identified a potential novel mechanism for Pak1 in regulating muscle glucose uptake (447). The phosphatase SKIP has been shown to inhibit insulin/PI3K/Akt signaling, and repress insulin-stimulated GLUT4 translocation as well as glucose uptake in the mouse L6 myoblasts (448-450). It has been postulated that the underlying mechanism involves the binding of SKIP to Pak1, which blocks Pak1 scaffolding function without affecting its kinase activity in mouse myoblasts (447) (Fig. 1.15). Hence, the potential dual function (kinase versus scaffold function) of Pak1 in insulin-stimulated muscle glucose uptake remains to be identified.

In addition to insulin activation, muscle contraction represents another mechanism by which muscle glucose uptake can be stimulated (451). The activity of Rac1 was observed to be elevated during contraction-stimulated glucose uptake in skeletal muscle, and this was concomitant with enhanced Pak1 Thr423 phosphorylation (452). Pak1 activation and muscle glucose uptake following contraction were attenuated by either Rac1 inhibition or the use of F- actin depolymerizing agents in vitro, and a similar defect was observed in muscle-specific Rac1-/- mice (429,452). Furthermore, exercise enhanced Pak1 Thr423 phosphorylation in mouse and human skeletal muscle, suggesting that Rac/Pak1 signaling plays a role in muscle glucose disposal during exercise (429,452).

1.4.3 Pak1 and insulin secretion in pancreas

Insulin is released from pancreatic β cells in two distinct phases following glucose stimulation. The first phase of insulin release is triggered by an elevation of intracellular calcium levels, leading to the fusion of predocked insulin-containing granules to the plasma membrane.

The second phase of insulin release involves the mobilization of stored granule pools to the cell surface, and represents the sustained insulin release for an hour or longer following glucose stimulation. F-actin remodeling is known to be involved in the process of granule mobilization to the SNARE sites at the cell surface, suggesting that actin remodeling is key to insulin granule release.

An overview of the mechanisms underlying GSIS and the role of Pak1 is presented in

Fig. 1.18.

Cdc42/Pak1/Rac axis regulates actin cytoskeleton remodeling

In 2007, Wang et al. proposed that Cdc42-induced signaling is involved in actin reorganization in the islet β cell (453). Using siRNA-based approaches, Wang et al. first demonstrate that siRNA-mediated Cdc42 knockdown attenuated glucose-induced insulin secretion in the mouse pancreatic β cell line MIN-6 (453). Isolated mouse islets were used for insulin release kinetics, and insulin secretion following glucose treatment in the control islets showed biphasic responsiveness (453). On the other hand, islets transduced with siCdc42 showed

~50% reduction in the second phase of insulin release, without any changes to the first phase insulin release and the basal insulin level (453).

82 Insulin secretion Glucose K+ GLP-1

GLUT K VGCC GLP-1R 4 ATP

Ca2+ Glucose AC Glycolysis

Pyruvate cAMP ↑

Kreb’s cycle

PKA Epac ATP Cdc42 ↑

Pak1 SAD-A

Rac1 MEK

Insulin granule- Erk containing vesicle

F-actin remodeling

Fig. 1.18 Overview of the mechanisms underlying glucose-stimulated insulin secretion in pancreas. Glucose is taken up into the cell via the GLUT4 transporter, and is converted to pyruvate through the glycolytic pathway. The pyruvate serves as a substrate for the Kreb’s cycle, and results in the rise of intracellular ATP concentration. This leads to the closing of the ATP-sensitive K channel (KATP) and membrane depolarization, resulting to the opening of the voltage-gated Ca2+ channel (VGCC). The influx of Ca2+ stimulates the mobilization of insulin granule-containing vesicles to the plasma membrane, leading to insulin release. The small GTPase Cdc42 stimulates insulin secretion via its effector Pak1, which acts through Rac1 and MEK/Erk signaling, leading to F-actin remodeling and insulin vesicle release. The SAD-A kinase has been shown to stimulate Pak1 in stimulating insulin secretion, and it has been proposed that this kinase may mediate the stimulation of insulin secretion by GLP-1. SAD-A: synapses of amphids defective .

Having established the importance of Cdc42 in the second phase of insulin release, Wang et al. sought to determine the role of Rac (453). Using both pancreatic β cell lines and isolated islets, they observed that Cdc42 activation precedes Rac activation during glucose-stimulated insulin release (453). Following glucose treatment, Rac activation was found to be dependent on

Pak1 activation, while Pak1 activation was dependent on Cdc42 activation (453). Pak1 depletion abolished Cdc42-mediated Rac activation, placing Pak1 as a linker in the Cdc42/Pak1/Rac axis in glucose-stimulated insulin release in pancreatic β cells (453).

The critical role of F-actin remodeling in GSIS has been recognized, where F-actin filaments were shown to act as barriers at the plasma membrane to prevent the docking and fusion of insulin granules under un-stimulated conditions (454-456). Rac1 and a number of other small GTPases have been implicated in various steps of GSIS in pancreatic β cells (457). In a murine β-cell line, Rac1 activation was shown to be stimulated by high glucose treatment (458).

The expression of a dominant-negative Rac1 in β-cells abolished its cytosol-to-membrane translocation upon high glucose stimulation, which was associated with reduced GSIS (458).

Very recently, Asahara and colleagues demonstrated the role of Rac1 in regulating GSIS via modulating F-actin using both in vitro and in vivo approaches (459). In the pancreatic  cell line MIN-6, Rac1 activation was observed following high glucose treatment (459). Rac1 knockdown leads to the persistence of intact F-actin in the presence of high glucose, associated with reduced insulin secretion following glucose stimulation (459). The study utilizing islets isolated from  cell specific Rac1-/- mice also recapitulated the observations of defective insulin secretion following high glucose treatment, without significant alterations in islet morphology

(459).

Although Pak1 is evidently involved in the second phase of GSIS, the exact mechanism by which Pak1 evokes F-actin remodeling in response to glucose is still not completely understood. An early study identified the role of the Cdc42-Pak1-Rac1 axis in regulating F-actin rearrangement and insulin secretion in  cells (453). It was further demonstrated that glucose- induced cortical F-actin remodeling also involves the Cdc42-PAK1-MEK-Erk pathway, and that glucose-induced insulin granule exocytosis can be blocked by the PAK inhibitor IPA3 (460,461).

The downstream effects of the PAK1-MEK-Erk signaling cascade may include MLCK, an actin- associated kinase which has been implicated in  cell insulin secretion (461,462).

In a recent study by Kalwat and colleagues, they utilized live cell imaging techniques to further elucidated the mechanistic requirement of Pak1 in glucose stimulated insulin granule exocytosis (460). Pretreatment of mouse pancreatic  cells with IPA3 inhibited glucose-induced

F-actin remodeling and ablated glucose-stimulated insulin granule localization to the plasma membrane (460). In mouse  cells, glucose treatment stimulated Raf1 phosphorylation and activation, while IPA3 treatment or Cdc42 inhibition abolished this activation (460). Similarly,

IPA3 treatment blunted MEK to Erk signaling, while the presence of a MEK-Erk inhibitor disrupted F-actin remodeling and resulted in defective insulin granule release (460). Altogether, these in vitro findings implicate the functional requirement of Pak1 in transmitting the signal of

Cdc42 to the downstream Raf1-MEK-Erk cascade, which leads to the potentiation of insulin granule mobilization through F-actin remodeling (460).

Despite a number of the studies supporting the participation of Pak1 in insulin granule mobilization, how Pak1 activation is stimulated by high glucose treatment in pancreatic  cells remains unknown. The synapses of amphids defective (SAD-A) kinase is a serine/threonine kinase closely related to the AMP-activated kinases (463). SAD-A is exclusively expressed in

the brain and pancreas, and has been implicated in synaptic function and neuronal development

(464). In examining the role of SAD-A in insulin secretion, Nie et al first showed that treatment with high glucose induces SAD-A activation, and that SAD-A in turn can stimulate Pak1 Thr423 phosphorylation and Pak1 kinase activity in a murine pancreatic  cell line (465). SAD-A was also shown to interact with recombinant GST-PBD of Pak, which can be completely abolished by a point mutation that inactivates the SAD-A kinase activity (465). Over-expression of SAD-A resulted in enhanced cortical F-actin formation, while expression of a kinase-dead SAD-A K48M mutant induced cortical actin filament disintegration (465). Conversely, shRNA-mediated SAD-

A knockdown attenuated GSIS in the mouse  cell line MIN-6, and adenoviral expression of either K48M SAD-A or the dominant-negative Pak1 mutant K299R was able to significantly reduce GSIS in mouse islets (465). Taken together, SAD-A is at least one of the direct upstream activators of Pak1, in regulating insulin granule exocytosis in  cells. SAD-A has been postulated as the downstream effect of PKA and calmodulin-dependent protein kinase kinase

(CaMMK) (465), while the relationship between SAD-A and Rac1 remains to be investigated.

The potential role of SAD-A in mediating the stimulation of insulin secretion by GLP-1 signaling remains to be identified.

1.4.4 Pak1 as a mediator of the crosstalk between insulin and Wnt

signaling pathways

Hyperinsulinemia and type 2 diabetes patients have higher correlated risk of developing colorectal cancer, while the Wnt signaling pathway is centrally involved in the development and progression of various tumors, including colorectal tumors (355,466). Activation of the Wnt

signaling pathway occurs upon the binding of Wnt ligands to the Frizzled receptor and LRP5/6 co-receptor, resulting in the nuclear translocation of β-cat. Nuclear β-cat will subsequently interact with a member of TCF transcription factor family, forming the bipartite transcription factor β-cat/TCF. A number of pro-proliferation genes, such as c-Myc and cyclin D1, are known downstream targets of β-cat/TCF.

Both insulin and IGF-1 were shown to stimulate Pak1 Thr432 phosphorylation in muscle and intestinal cells (333,334,467,468), while our laboratory has demonstrated that intraperitoneal injection of insulin in mice resulted in Pak1 Thr432 phosphorylation in the liver, fat, heart, small and large intestines (334). Expression of dominant-negative K299R Pak1 attenuated insulin- induced c-Myc and cyclin D1 protein expression, and reduced insulin-stimulated binding of β- cat/TCF to the human c-Myc gene promoter (334). Furthermore, Pak1 knockdown resulted in a drastic reduction of c-Myc and cyclin D1 expression, accompanied by attenuated Erk phosphorylation in the presence and absence of insulin stimulation (334). Insulin stimulates β-cat phosphorylation at Ser675, an event that is positively correlated with β-cat nuclear translocation and activity (255,256,334). Pak1 knockdown attenuated insulin-stimulated β-cat Ser675 phosphorylation, the binding of β-cat to c-Myc promoter, and c-Myc gene expression (334).

Collectively, these findings revealed the potential role of Pak1 as a linker underlying the crosstalk between the insulin/IGF-1 and the Wnt signaling pathways.

2 Rationale, hypothesis, and research aims

2.1 Rationale

Previous studies have identified Pak1 as a mediator of insulin-stimulated glucose uptake in skeletal muscle and glucose-stimulated insulin secretion in pancreatic  cells (333,453). In examining the crosstalk between insulin and Wnt signaling pathways, our lab has identified the role of Pak1 in mediating the proliferative effect of insulin in intestinal cancer cell lines. Insulin was also shown to concurrently activate Pak1 and the Wnt effector -cat, leading to the stimulation of Wnt target genes c-Myc and cyclin D1 (334). These studies by our group and others collectively emphasize the function of Pak1 as an effector of insulin signaling, and as a linker in the crosstalk between insulin and Wnt signaling pathways. Hence, I aimed to examine the role of Pak1 in mediating the effect of insulin in vitro and in vivo, in the context of intestinal gcg expression and GLP-1 production, and the deleterious effects of Pak1 ablation on glucose homeostasis.

2.2 Hypothesis and research aims

Central hypothesis

Pak1 is a linker between insulin and Wnt signaling pathways. It is centrally involved in regulating gcg expression and GLP-1 production, and hence is an important modulator of glucose metabolism and homeostasis.

Aim I

To assess whether Pak1 mediates the crosstalk between insulin and β-cat in regulating proglucagon gene expression in the in vitro setting (presented in Chapter 4).

Aim II

To assess whether Pak1 ablation perturbs glucose homeostasis in mice (presented in Chapter 5).

Aim III

To assess whether Pak1 regulates hepatic glucose production in vitro and in vivo (presented in

Chapter 6).

3 General materials and methods

3.1 Chemicals and antibodies

The group I Pak inhibitor 2,2’-dihydroxy-1,1’-dinaphthyldisulfide (IPA3), PKA inhibitor

H89, adenylyl cyclase activator forskolin, and inhibitor of cAMP phosphodiesterase 3-isobutyl-

1-methylxanthine (IBMX) were obtained from Sigma Aldrich (St. Louis, MO, USA). Sitagliptin

(Januvia®) was the product of Merck Canada Inc. (Montreal, Canada). The GLP-1 receptor

(GLP-1R) agonist exendin-4 (Ex-4) was the product of Genscript (Piscataway, NJ, USA).

Phospho-β-cat (Ser675), phospho-Pak1 (Thr423)/Pak2 (Thr402), Pak1, Pak2, Pak3, phospho-GSK3α (Ser21)/β (Ser9), GSK3β, Akt1, and phospho-CREB1 (Ser133) antibodies were purchased from Cell Signaling (Beverly, MA, USA). Total β-cat antibody was purchased from

Santa Cruz Biotechnology (Santa Cruz, CA, USA). β-Actin antibody was the product of Sigma

Aldrich. Phospho-Akt1(Ser473) antibody was from Signalway Antibodies (College Park, MD,

USA).

3.2 Western blotting

Cells were harvested using radioimmuno precipitation assay (RIPA) buffer (200 μL for a single well from 6-well plates), centrifuged at 16,110 × g for 5 min at 4°C, and supernatant is collected and placed on ice. The protein content was determined using Bradford protein assay.

In a cuvette, 1 μL of the supernatant was added to 1 mL of Bradford reagent (Sigma Aldrich) and gently mixed by vortexing. The protein concentration was determined by spectrophotometer reading at 595 nm and quantified using a bovine serum albumin standard curve.

The Western blotting procedure was conducted as follows. The protein samples (20-40

µg) were resolved by SDS-PAGE with 10% polyacrylamide gels before transfer to a nitrocellulose (Pierce, Rockford, Ill) membrane. Blots were incubated on shaker at room temperature in 5% non-fat milk in 1 × Tris-buffered saline Tween-20 (TBST, 137mM NaCl,

20mM Tris, 0.1% Tween-20, pH 7.6) for 1 h, followed by overnight incubation on shaker in primary antibody (1/1000 dilution) in 5% BSA in 1 × TBST at 4°C. The next day, blots undergo

1 h incubation on shaker at room temperature with horseradish peroxidase-conjugated secondary antibody (1/3000-1/5000 dilution) (Santa Cruz Biotechnology Inc, Dallas, TX, USA). Blots were then developed using enhanced chemiluminescence (ECL) (Pierce, Rockford, IL, USA) and exposed to Maximum Sensitivity double emulsion imaging film (Kodak).

3.3 RNA extraction and real-time quantitative reverse- transcriptase PCR

Cellular RNA was extracted using TRI reagent (Invitrogen Life Technologies, Carlsbad,

CA) based on a modification of the manufacturer’s protocol. Cells were harvested using 1 mL of

TRI reagent and stored at −80°C. On the day of extraction, samples were thawed on ice, and 200

μL of chloroform was added followed by vigorous shaking and incubation at room temperature for 5 min. The sample was centrifuged at 4°C for 10 minutes at 16,110 × g, and supernatant was collected into new microfuge tube. To this tube, 500 μL of isopropanol was added followed by inversion mixing and incubation at −20°C for 20 min for RNA precipitation. The sample was centrifuged at 16,110 × g at 4°C for 10 minutes, and the RNA pellet was washed with 1 mL 75% ethanol. The resulting RNA pellet was air-dried and reconstituted in the appropriate amount of

UltraPure DNase/RNase-free distilled water (Invitrogen Life Technologies, Carlsbad, CA). The

RNA solution is incubated at 60°C for 10 min, and subsequently cooled on ice and stored at

−80°C. For real-time quantitative reverse transcriptase polymerase chain reaction (real-time qRT-PCR), the RNA was reverse-transcribed into cDNA using high-capacity cDNA reverse transcription kit (Invitrogen Life Technologies, Carlsbad, CA, USA) as per manufacturer’s protocol.

3.4 Experimental animals, maintenance, and genotyping

The generation of the Pak1-/- mouse line has been described previously (404), and were provided by Dr. Jonathan Chernoff (Fox Chase Cancer Center, Philadelphia, PA, USA). We obtained the Pak1-/- mouse line in the mixed C57BL/6-129 background, and performed the backcross to the C57BL/6 genetic background for 7 generations. These mice were then intercrossed to produce Pak1−/− and wild-type offspring. Adult male mice were used in all studies. The mice were maintained on a standard chow diet and water ad libitum with 12h-12h light-dark cycles.

Extraction of genomic mouse tail DNA and genotyping was conducted using the KAPA mouse genotyping kit (KAPA Biosystems Inc, Woburn, MA, USA), as per manufacturer’s instructions. The KAPA Express Extract solution is added to the mouse tail biopsy, and the sample was incubated at 50-60°C for 20 min. After the completion of the enzymatic digestion, the resulting sample containing mouse tail DNA was mixed with the KAPA2G Fast Genotyping

Mix solution and the genotyping primers for PCR amplification.

The forward primer (5’-GCC CTT CAC AGG AGC TTA ATG A-3’) together with the

Pak1-specific reverse primer (5’-GAA AGG ACT GAA TCT AAT AGC A-3’) resulted in the amplification of a 240 bp product from the WT allele. The use of the same forward primer together with a specific reverse primer for the neomycin cassette (5’-CAT TTG TCA CGT CCT

GCA CGA-3’) amplified a 360 bp product from the targeted allele.

3.5 Mouse organ weight measurements

During animal euthanasia, organs such as epididymal fat pad, liver, and 5 cm of distal ileum were dissected and weighed using a digital bench-top balance from Mettler Toledo.

3.6 Immunohistochemistry of mouse intestine and pancreas

The co-immunostaining studies were conducted by the pathology laboratory at the

Toronto Centre for Phenogenomics (Toronto, Ontario, Canada), as per their established standard protocols.

3.7 Statistical analyses and densitometry analysis

Results are presented as average ± SEM, and they were analyzed using a two-tailed unpaired Student t test or one-way ANOVA followed by post hoc analysis as appropriate, with a p < 0.05 being considered statistically significant. Densitometry analyses for Western blots were performed using ImageJ.

4 P21-activated protein kinase 1 mediates the crosstalk between insulin and β-catenin on regulating proglucagon gene expression in the gut

Data presented in this chapter, as well as those in Chapter 5, have been published by Chiang et al. 2013. P21-activated protein kinase 1 (Pak1) mediates the cross talk between insulin and β- catenin on proglucagon gene expression and its ablation affects glucose homeostasis in male C57BL/6 mice. Endocrinology 154(1):77-88 (468).

All experiments were performed and figures contributed by Yu-ting Chiang. The mouse hypothalamic gcg-expressing cell line mHypoE-20/2 was a gift of Dr. Denise Belsham (469).

The K299R kinase-dead Pak1 (dominant negative Pak1) plasmid was provided by Jeffrey Field (470).

4.1 Abstract

In gut endocrine L cells, the Wnt signaling pathway effector β-cat/TCF7L2 mediates the stimulatory effect of insulin on proglucagon (gcg) expression and glucagon-like peptide-1 (GLP-

1) production. In several other cell lineages, insulin was shown to to stimulate Pak1. Here, we determined the role of Pak1 in gcg expression in two gut gcg-expressing cell lines and in a brain gcg-expressing cell line. Insulin stimulated Pak1 activation through its Thr423 phosphorylation in gut gcg-expressing cell lines, associated with increased gcg mRNA levels. This stimulation was attenuated by the Pak inhibitor 2,2′-dihydroxy-1,1′-dinaphthyldisulfide (IPA3) or expression of dominant-negative Pak1. Both insulin and cAMP-promoting agents activated β-cat Ser675 phosphorylation, which was attenuated by IPA3 or protein kinase A inhibition, respectively. We hence suggest that Pak1 mediates the cross talk between insulin and Wnt signaling pathways on gut and brain gcg expression.

4.2 Introduction

The proglucagon gene (gcg) encodes three major peptide hormones, including glucagon, glucagon-like peptide-1 (GLP-1) and GLP-2, which are importantly involved in maintaining glucose homeostasis and the growth of small intestines (120,471). Glucagon produced by pancreatic α-cells is a major counter-regulatory hormone of insulin, whereas GLP-1 produced by gut endocrine L cells is among the two important incretin hormones (472). GLP-1 is also expressed in certain neuronal cells in the brainstem, which controls satiety and peripheral glucose homeostasis by yet-to-be-further-defined mechanisms (246).

Gcg expression is controlled by a number of transcription factors and cell signaling molecules or pathways in cell-type specific manners, in response to nutritional and hormonal regulation (151,153,192,473,474). We have demonstrated previously that the G2 enhancer element within the proximal gcg promoter region is responsible for the stimulation of gcg transcription by the Wnt signaling pathway effectors transcription factor 7-like 2 (TCF7L2)

(previously known as TCF-4) (266) and β-catenin (β-cat) (295,296). β-cat and a member of the

TCF family form the bipartite transcription factor β-cat/TCF, which not only mediates the effect of Wnt ligand stimulation but also the effects of other cellular signaling molecules, including a battery of peptide hormones that use cAMP as the second messenger; insulin, IGF-I and other growth factors; and the lipid metabolite lysophosphatidic acid (475). Although insulin represses pancreatic gcg expression, glucagon production and secretion (476-478), it evidently stimulates gut gcg expression and GLP-1 production in vitro and in vivo in the MKR hyperinsulinemic and insulin-resistant mouse model (479-481). Interestingly, insulin was shown to use the same G2 enhancer element that mediates Wnt activation in stimulating gcg promoter expression in the gut endocrine L cells, and this stimulation is likely to be mediated by a PI3K-dependent but Akt- independent mechanism (479).

Paks are a highly conserved group of serine/threonine kinases, which are implicated in a number of cellular processes including cell proliferation, cell polarity, and actin cytoskeleton reorganization (482). They were initially recognized as key mediators of the Rho family

GTPases, Cdc42 and Rac, which are importantly involved in many biological functions, including insulin secretion (483). Functional studies have implicated Pak1 in tumorigenesis and tumor metastasis (466). Pak1 expression and activation are elevated in cancer cells of numerous types. Pak1 Thr423 phosphorylation, indicating Pak1 activation, can be stimulated by insulin or

IGF-I (334). In mouse muscle cells, this activation is PI3K dependent (333). In the gut endocrine

L cells, Cdc42 was shown to regulate actin remodeling, ERK1/2 activation, Pak1 activation, as well as GLP-1 secretion in response to insulin treatment (480). We have reported previously that in intestinal nonendocrine cells, insulin or IGF-I stimulates the Pak1 Thr423 phosphorylation in an Akt-independent manner, associated with increased β-cat Ser675 phosphorylation and Wnt target gene expression (334).

4.3 Materials and methods

4.3.1 Cell lines and tissue culture

The mouse intestinal gcg-expressing GLUTag and STC-1 cell lines were available in our laboratory. The mouse hypothalamic gcg-expressing cell line mHypoE-20/2 was a gift of Dr.

Denise Belsham (469). Tissue culture media, serum, and antibiotics were purchased from Sigma

Aldrich (St. Louis, MO, USA). GLUTag and STC-1 were maintained in Dulbecco’s Modified

Eagles Medium (DMEM) containing 10% fetal bovine serum (FBS) and penicillin/streptomycin

(P/S, 100 U/mL and 100 μg/mL, respectively). InR1G9 was grown in RPMI 1640 medium with

10% FBS and P/S. Cell cultures were maintained in incubators at 37°C and supplied with 5%

CO2.

4.3.2 Fetal rat intestinal cell isolation

Fetal rat intestinal cells (FRIC) were isolated for tissue culture via a method developed by

Dr. Patricia Brubaker (484). Briefly, on day 1 the intestines from a litter of 19 to 21 day gestation

fetal Wistar rats were collected and pooled. The intestines were minced using sterilized surgical scissors, and intestinal tissue pieces underwent two sequential 30 min incubations with an enzyme cocktail solution (10 mL Hanks buffer solution containing 40 mg collagenase, 50 hyaluronidase, and 5 mg deoxyribonuclease I). After enzymatic digestion, the dispersed cells were washed and seeded into monolayer cultures with DMEM supplemented with 10% FBS for

24 h and grown at 37°C and 5% CO2 for further treatment.

4.3.3 Plasmids, transfection, and luciferase reporter gene analysis

The 2.4kb-gcg-LUC, G2S-TK-LUC, and−302bp-gcg-LUC plasmid constructs have been described previously (151,173). The K299R kinase-deficient Pak1 (dominant negative Pak1) plasmid was provided by Dr. Jeffrey Field (470).

Polyethylenimine (PEI) was obtained from Sigma Aldrich, St. Louis, MO, USA) and was used as the transfection reagent, with a protocol modified from the manufacturer’s instructions.

Cells were seeded on day 1 at 60-70% confluency or 0.7-1.0 × 105 cells. For each single well from a 6-well plate, the DNA-PEI complex is prepared by mixing DNA and PEI at a ratio of 1:3 w/w in Opti-MEM transfection medium (Invitrogen Life Technologies, Burlington, Ontario,

Canada). The microfuge tube containing the DNA-PEI complex was incubated at room temperature for 15 min, and mixed gently by inverting the tube. After 15 m incubation, the

DNA:PEI mixture was added to the cells and transfection is performed for 24 h. On day 2, cells were serum starved overnight prior to subsequent drug treatments and luciferase reporter gene analysis on day 3.

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The luciferase reporter gene (LUC) analysis was carried out by a method described previously with minor modifications (296). Briefly, cells were washed with PBS and scraped off the plate with rubber scrapers after incubating with 0.3 mL of LUC harvesting buffer (50 mM

Tris/MES, 1mM dithiothreitol and 0.1% Triton X-100). The collected samples were vortexed for

40 seconds and centrifuged at 16,110 × g at 4°C for 10 minutes. Supernatant was collected and placed on ice. From that, 100 μL of the supernatant is dispensed into a clean tube, followed by the addition of 15 μL ATP cocktail solution (50mM Tris/MES, 0.18M MgOAc and 40mM ATP) and 100 μL of LUC harvesting buffer. LUC activity is immediately measured using a luminometer (Lumat LB 9507 from Fisher Scientific).

4.3.4 Real-time quantitative reverse-transcriptase PCR

Gcg mRNA levels were normalized to 18S ribosomal RNA levels. The primer pairs used are as follows: gcg forward, 5′-TGG ACT CCC GCC GTG CCC AA-3′, gcg reverse, 5′-CGA

CTT CTT CTG GGA AGT CTC GCC T-3′; 18S forward, 5′-CGG ACA TCT AAG GGC ATC

A-3′, 18S reverse, 5′-AAG ACG GAC CAG AGC GAA A-3′.

4.3.5 Northern blotting

The method for Northern blotting was modified from an existing protocol (151). In brief, following RNA extraction, the RNA sample was resolved by gel electrophoresis. The gels contained 12% agarose and 12.5% v/v 37% formaldehyde, prepared in 1 × MOPS buffer (40mM

MOPS, 10mM sodium acetate, 1mM EDTA, pH 8.3). The resolved RNA on the gel was then

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transferred to a nylon membrane by passive upward transfer overnight, using 10 × Saline sodium citrate (SSC) solution (300 nM sodium citrate, 1 M sodium chloride, pH 7.0). The next day, the

RNA on the membrane is UV-crosslinked for 5 min. The radioactive labeled nucleotide probe was prepared using the commercial Amersham Nick Translation dCTP kit (GE Healthcare Life

Sciences, Baie d’Urfe, Quebec, Canada) as per manufacturer’s instructions. The membranes were hybridized in rollers containing the P32-labelled nucleotide probe in prehybridization buffer

(5% dextran sulphate, 40% formamide, 4 × SSC, 7mM Tris, 1 × Denhardt, 100 µg/mL salmon sperm DNA) at 42°C overnight. Following hybridization, the membranes were washed twice in 2

× SSC rolling at 55°C for 15 min, and placed into cassettes with Maximum Sensitivity double emulsion imaging film (Kodak) for exposure at −80°C for 1-3 days.

4.4 Results

4.4.1 Insulin stimulates Pak1 activation in gcg-expressing cells

To investigate the role of Pak1 in gcg expression and glucose homeostasis, we first of all examined Pak1 protein expression in the gut and brain gcg-expressing cell lines and in various mouse tissues. As shown in Fig. 4.1A, Pak1 is expressed in the GLUTag cell line along with the other two group I Paks (Pak2 and Pak3). In mouse tissues, Pak1 shows a ubiquitous expression pattern with abundant levels in a number of organs that are potentially important in glucose homeostasis, including brain, distal ileum, pancreas, liver, muscle, and fat (Fig. 4.2). Pak1 is also expressed in the heart, colon, lung, and kidney (Fig. 4.2). Furthermore, Pak1 expression was colocalized with GLP-1 in the mouse gut endocrine L cells by coimmunostaining (Fig. 4.1B).

Similar to what we have observed in the gut nonendocrine cells (334), in the GLUTag cell line,

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insulin also stimulated Pak1 Thr423 phosphorylation (Fig. 4.1C). Furthermore, insulin treatment led to the stimulation of Pak1 Thr423 phosphorylation in STC-1, another mouse intestinal gcg- expressing cell line (Fig. 4.1D) and in the mouse gcg-expressing neuronal cell line mHypoE-

20/2 (Fig. 4.1E).

4.4.2 Insulin-stimulated gcg expression can be attenuated by Pak inhibition

We then assessed the effect of Pak inhibition on insulin- and cAMP-stimulated gcg promoter activity. Figure 4.3A shows that insulin-stimulated Pak1 activation can be partially attenuated by pretreating the GLUTag cells with IPA3, a highly selective, non-ATP-competitive inhibitor targeting the autoregulatory domain of group I Paks (365). Upon transfection of the

2.4kb-gcg-LUC construct (151) into the GLUTag cell line, both insulin and forskolin/3-isobutyl-

1-methylxanthine (F/I) treatment stimulated LUC reporter activity as we have anticipated (Fig.

4.3B). The stimulation by insulin was blocked by IPA3 pretreatment (Fig. 4.3B), whereas the stimulation by F/I was moderately but significantly attenuated by IPA3 pretreatment (Fig. 4.3C).

Next, we tested the effect of Pak inhibition on insulin- or cAMP-stimulated G2S-TK-

LUC expression. The TCF-binding motif containing G2 element is known to mediate the stimulation by cAMP, calcium, and insulin (183,296,479). Similarly, the stimulatory effect of cAMP and insulin on G2S-TK-LUC expression was attenuated by IPA3 pretreatment (Fig.

4.3D). To further verify the inhibitory effect of IPA3 on cAMP signaling, we tested its effect in

GLUTag cells using the −302bp-gcg-LUC fusion gene, which contains the cAMP response element (485). Evidently the stimulatory effect of cAMP elevation on LUC expression can be moderately but significantly attenuated by IPA3 pretreatment (Fig. 4.3E). These, along with the observed stimulatory effect of Wnt ligand Wnt3A on gcg promoter and gcg mRNA expression in

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the GLUTag cell line (Fig. 4.4), indicated the involvement of Pak in mediating the crosstalk between cAMP and Wnt signaling pathways as well as between insulin and Wnt signaling pathways. We have also verified by qRT-PCR (Fig. 4.3F) and Northern blotting (Fig. 4.3G) that

IPA3 pretreatment partially blocked the stimulation by insulin on gcg mRNA expression in

GLUTag cells. Finally, we directly tested the effect of functional knockdown of Pak1, using the dominant-negative Pak1 K299R plasmid construct. Cotransfection of Pak1 K299R significantly attenuated the stimulation by insulin on the expression of the 2.4 kb-gcg-LUC fusion gene (Fig.

4.3H). Insulin has been shown to repress gcg expression in pancreatic islets (476,477), in contrast to its stimulatory effect on gcg expression in the gut (479).

4.4.3 Pak inhibition attenuates insulin-stimulated β-cat Ser675 phosphorylation

PKA-mediated stimulation of β-cat Ser675 phosphorylation has been recognized as an event leading to enhanced β-cat transcriptional activity and Wnt downstream target gene expression (255,256). Insulin has also been shown to stimulate β-cat Ser675 phosphorylation in intestinal nonendocrine cells (334). We demonstrate here that in the GLUTag cell line, F/I or insulin treatment led to increased β-cat Ser675 phosphorylation (Fig. 4.5A and B). cAMP- stimulated β-cat Ser675 phosphorylation can be blocked by PKA inhibition (H89) but not by

IPA3 (Fig. 4.5C). Insulin-stimulated β-cat Ser675 phosphorylation, however, can be partially attenuated by IPA3 pretreatment (Fig. 4.5D). Thus, in the gut endocrine L cell line GLUTag, both PKA and Pak activation lead to β-cat Ser675 phosphorylation. Based on this new observation and our previous findings (296,479), we suggest that insulin is able to activate gut

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gcg transcription via increasing β-cat Ser675 phosphorylation and concurrently involving the activation of Pak1.

105 A B a b c Pak1 Pak2 Pak3 70 kDa

β-actin

C Fold change 1 1.2 1.3 1.6 2.8 2.9 p-Pak1/2 (Thr423/Thr402) Pak1 p-Akt1 (Ser473) Akt1 Time (min) 0 5 10 20 30 60 Insulin

D Fold change 1 2.9 3.2 3.3 3.1 2.7 p-Pak1 (Thr423) Pak1 p-GSK3α/β (Ser21/Ser9) GSK3β β-actin Time (min) 0 5 10 20 30 60 Insulin

E Fold change 1 5.4 7.2 3.2 3.5 1.7 p-Pak1 (Thr423) Pak1 p-Akt1(Ser473) Akt1 p-GSK3α/β (Ser21/Ser9) GSK3β β-actin Time (min) 0 5 10 20 30 60 Insulin

Fig. 4.1 Insulin activates Pak1 in gcg-expressing cell lines. A, Detection of the expression of group I Paks (Pak1, 68 kDa; Pak2, 61 kDa; Pak3, 65 kDa) in the GLUTag cell line. Approximately 40 g whole-cell lysate proteins were loaded into each lane for Western blotting, with indicated antibody. B, Colocalization of Pak1 and GLP-1 in mouse distal ileum cryptic L cells. A 5-cm region of mouse distal ileum was isolated and prepared for coimmunostaining of Pak1 (brown) and GLP-1 (pink). Arrow points to the same crypt region under various magnification views. Bar indicates 200 μm (a); 50 μm (b); 20 μm (c). C–E, Insulin stimulated Pak1 Thr423 phosphorylation in GLUTag (C), STC-1 (D), and mHypoE-20/2 (E) cell lines. All images are representative blots (n ≥ 3). Fold change indicates the change in p-Pak1 Thr423 levels compared to the basal level. 106 Pak1

C57BL/6 γ-tubulin

Pak1 CD1 γ-tubulin

Fig. 4.2 Pak1 expression profiles in selected tissues of C57BL/6 and CD1 mice. A representative blot is shown. Protein lysates were prepared from indicated tissues of 8-weeks-old male C57BL/6 or CD1 mouse. Equal amount of proteins (40 µg) were loaded for Western blotting with Pak1 and γ-tubulin antibodies.

107 A B 2.4kb-gcg-LUC p-Pak1/2 (Thr423/Thr402) Basal Insulin 2.0 Pak1 *** * 1.5 p-GSK3α/β (Ser21/Ser9) 1.0 GSK3β 0.5 DMSO + + - - IPA3 - - + + 0.0 Insulin - + - + Luciferase change)(fold DMSO IPA3

C D E 2.4kb-gcg-LUC G2S-TK-LUC -302bp-gcg-LUC

Basal F/I Basal Insulin F/I Basal F/I 16 4 * 7 * *** * 14 *** *** 6 12 3 *** 5 10 4 2 * 8 3 6 2 4 1 2 1 Luciferase change)(fold Luciferase change)(fold Luciferase change)(fold 0 0 0 DMSO IPA3 DMSO IPA3 DMSO IPA3

F G H Basal Insulin 2.0 Basal Insulin 4 * * gcg ** * 1.5 3 18S 1.0

(fold change) (fold 2 IPA3 - + - + 1 Basal Insulin 0.5 mRNA mRNA

0 Luciferase change)(fold 0.0 gcg DMSO IPA3 Vector Pak1 (K299R)

Fig. 4.3 Insulin-activated gcg promoter and mRNA expression can be attenuated by the Pak inhibitor IPA3. A, Insulin-stimulated Pak1 activation can be attenuated by IPA3 in GLUTag cells. GLUTag cells were serum starved for 16 h, and pretreated with IPA3 (10 μM) or dimethylsulfoxide (DMSO) (vehicle) for 1 h, followed by insulin treatment for 10 min. Line indicates noncontiguous lanes on the same blot. B–E, The stimulatory effect of insulin and cAMP on gcg promoter was attenuated by IPA3 pretreatment. GLUTag cells were transfected with 1 μg of 2.4kb-gcg-LUC (B and C), G2S-TK-LUC (D), or −302bp-gcg-LUC (E) for 18 h, followed by serum starvation for 16 h. The cells were then pretreated with IPA3 (10 μM) or DMSO for 1 h, followed by insulin (100 nM) or F/I (10 μM each) treatment for 4 h. LUC activity is presented as fold change against the control DMSO treatment (n ≥ 3 with triplicates in each experiment). F and G, Insulin-stimulated gcg mRNA expression was attenuated by IPA3. GLUTag cells were serum starved for 16 h, followed by IPA3 (10 μM) or DMSO pretreatment for 1 h. Total RNA was extracted for qRT-PCR (results normalized against 18S) (F) or Northern blotting (representative blot in which the line indicates the noncontiguous lanes on the same blot, n ≥ 3) (G). H, Dominant-negative Pak1 (K299R) cotransfection attenuated insulin-stimulated gcg promoter activity. GLUTag cells were cotransfected with Pak1(K299R) and 2.4 kb-gcg-LUC constructs (n ≥ 3). *, P ≥ 0.05; **, P ≥ 0.01; ***, P ≥ 0.001.

108 A B Basal Wnt3A Wnt11

* N.S.

3 * 2 * N.S. 1.5 *** 2 (fold change)(fold 1 1 mRNA mRNA 0.5 gcg

Luciferase change)(fold 0 0 Control Wnt3A Wnt3A DMSO IPA3 (0.25 nM) (2.5 nM)

Fig. 4.4 Wnt ligand Wnt3A stimulates gcg promoter and mRNA expression in GLUTag cell line. A, GLUTag cells were transfected with 1 µg of 2.4kb-gcg-LUC overnight, serum-starved for 16 h, and treated with indicated concentration of Wnt3A for 2 h. Cells are subsequently harvested for LUC analysis. B, GLUTag cells were treated with 2.5 nM Wnt3A or Wnt11 (a non-canonical Wnt ligand as control) for 2h, followed by RNA extraction and qRT-PCR.

109 A Fold change 1 3.8 2.9 4.5 3.2 2.4 p-β-cat (Ser675) β-cat p-CREB1 (Ser133)

β-actin Time (min) 0 5 10 30 60 120

F/I

B Fold change 1 3.2 3.7 3.3 3.9 2.5 p-β-cat (Ser675) β-cat p-Akt1 (Ser473) Akt1 β-actin Time (min) 0 5 10 30 60 120 Insulin

C Fold change 1 2.6 1 1.3 1 0.9 p-β-cat (Ser675)

β-cat p-CREB1 (Ser133) β-actin

F/I - + - + - + DMSO IPA3 H89

D Fold change 1 2.8 1 1.2 p-β-cat (Ser675)

β-cat Insulin - + - +

DMSO IPA3

Fig. 4.5 Insulin stimulates β-cat Ser675 phosphorylation in GLUTag cells. A and B: Both F/I and insulin stimulated β-cat Ser675 phosphorylation in GLUTag cells. GLUTag cells were serum-starved for 18 h, followed by F/I (10 µM each) (A) or insulin (100 nM) (B) treatment. C and D: F/I-stimulated β-cat S675 phosphorylation was attenuated by pretreatment with the PKA inhibitor H89 but not with IPA3 (C), while insulin-stimulated β-cat Ser675 phosphorylation was attenuated by IPA3 pre-treatment (D). GLUTag cells were serum- starved for 16 h, followed by 1 h IPA3 (10 µM) or H89 (10 µM) pre-treatment and 30 min F/I (10 µM each) or insulin (100 nM) treatment. All images are representative blots (n=3). Fold change indicates the change in β-cat Ser675 levels compared to the basal level.

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4.5 Discussion

Gcg encodes several important peptide hormones that are critically involved in glucose homeostasis and satiety (192,472). Because gut GLP-1 and pancreatic glucagon exert opposite effects on glucose homeostasis, significant effort has been made to identify molecular mechanisms that control gcg expression in cell-type specific manners (192). Evidently cAMP elevation stimulates gcg expression in both the gut endocrine L cells and pancreatic α-cells, although the levels of stimulation could vary (173,175). Insulin is known to repress gcg expression and glucagon secretion in pancreatic α-cells (476-478). We found previously, however, that both the Wnt pathway effector β-cat/TCF7L2 and insulin specifically stimulate gcg expression in gut endocrine L cells (296,479) and that this stimulation led to increased gut

GLP-1 production (296). We suggest that Wnt signaling as well as the crosstalk between Wnt and insulin signaling pathway are among the cell-type specific mechanisms that control gut gcg expression. Recently, García-Martínez et al. (486) have demonstrated that the Wnt/β-cat signaling pathway also stimulates the production of another incretin hormone, glucose-dependent insulinotropic peptide, indicating that Wnt signaling is a common regulator of incretin hormone production.

Here we present evidence showing that, similar as in the skeletal muscle as demonstrated by other groups (333,438), Pak1 is among the downstream targets of the insulin signaling pathway in gut and brain gcg-expressing cells. In addition, insulin stimulates both Pak1 Thr432 phosphorylation/activation and β-cat Ser675 phosphorylation. Insulin-stimulated gcg transcription in gut endocrine L cells can be attenuated by either the chemical inhibitor IPA3 or the expression of dominant-negative Pak1 (K299R). These in vitro results collectively suggest

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that: 1) Pak1 is important for gut and brain gcg expression, and 2) Pak1 mediates the stimulatory effect of insulin on gcg transcription, using the Wnt signaling pathway bipartite transcription factor β-cat/TCF as the effector. This is further supported by a very recent study, showing that in another cell lineage Rac1/Pak1 cascade directly controls β-cat Ser675 phosphorylation and its full activation (487).

Paks were initially discovered as effectors of the small GTPases, Cdc42 and Rac1.

Although the implication of Paks in cell motility and tumorigenesis has been well recognized

(466), relatively less is known about their involvement in metabolic homeostasis. Insulin signaling is important in stimulating GLUT4 activity in muscle and adipocytes (488). Tsakiridis et al. (333) demonstrated that insulin rapidly stimulates Pak1 in differentiated L6 muscle cells, and the activation can be blocked by the PI3K inhibition. It has been implicated that this stimulation mediates the effect of insulin on GLUT4 activation (425). In pancreatic β-cells, there is accruing evidence that small G protein signaling is critically involved in insulin secretion

(489). Wang et al. (453) showed that small interfering RNA-mediated depletion of Pak1 abolished Rac1 but not Cdc42 activation and blocked the second phase of glucose-stimulated insulin release. Subsequently Kowluru et al. demonstrated that Raf, Rac, and ERK are importantly involved in glucose-stimulated insulin secretion, thus further supporting the pivotal role of the small G protein signaling in pancreatic β-cells (490).

The role of Wnt ligands, Wnt signaling pathway, and its effectors β-cat and TCF7L2 in glucose homeostasis has received widespread attention in the past few years (271,293,479), especially after the discovery that certain single nucleotide polymorphisms within the intronic region of TCF7L2 are strongly associated with the risk of type 2 diabetes (266,271,293,491,492).

A number of studies have shown that in pancreatic β-cells, TCF7L2 plays an important role in

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facilitating insulin secretion, preventing β-cell apoptosis as well as in stimulating the expression of receptors for the two incretin hormones (287,288). We show here that β-cat is a downstream target of the insulin/Pak1 signaling cascade in regulating gcg expression in intestinal L cells. It will be interesting to determine whether some of the observed beneficial effects of TCF7L2 in pancreatic β-cells revealed to date are mediated by the Wnt signaling effector β-cat/TCF7L2, with the participation of the insulin/Pak1/β-cat signaling cascade.

Group 1 Paks may exert certain redundant functions in neurons or other cell types. We conclude that Pak1 mediates the crosstalk between cAMP and Wnt signaling pathways, as well as the cross talk between insulin and the Wnt signaling pathways in gcg-expressing cell lines.

Functionally, these cross talks are important for gcg expression and gut GLP-1 production.

Questions that remain to be answered include: 1) whether the two other group I Paks (Pak2 and

Pak3) are also involved in gut and brain gcg expression and 2) whether brain insulin-Pak1 signaling cascade could provide therapeutic targets for improving peripheral glucose homeostasis. Indeed, brain Pak1 is also controlled by the FOXO signaling pathway, another downstream target of the insulin signaling (493).

4.6 Acknowledgements

We thank Dr. Jeffery Field for providing the K299R Pak1 plasmid and Dr. Denise Belsham for providing the mHypoE-20/2 cell line.

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5 Ablation of p21-activated protein kinase 1 perturbs glucose homeostasis

Data presented in this chapter (except Fig. 5.8 and 5.9), and those presented in Chapter 4, have been published by Chiang et al. 2013. P21-activated protein kinase 1 (Pak1) mediates the cross talk between insulin and β-catenin on proglucagon gene expression and its ablation affects glucose homeostasis in male C57BL/6 mice. Endocrinology 154(1):77-88 (468).

All experiments were performed and figures contributed by Yu-ting Chiang. The Pak1-/- mouse line was provided by Dr. Jonathan Chernoff (Fox Chase Cancer Center).

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5.1 Abstract

We have previously identified the role of Pak1 as an effector of insulin-stimulated the proglucagon gene (gcg) expression, and as a linker for the crosstalk between insulin and Wnt signaling pathways in the intestinal and brain gcg-expressing cell lines. In order to assess the in vivo physiological role of Pak1 in metabolic homeostasis, we have characterized the Pak1−/− mice. Gut gcg levels were reduced in male Pak1−/− mice, associated with impaired glucose tolerance after an ip or oral glucose challenge. These mice had lower circulating active GLP-1 levels after a glucose challenge, as well as reduced distal ileum GLP-1 content after insulin treatment. Finally, the Pak1−/− mice exhibited reduced brainstem gcg mRNA levels and abolished β-cat Ser675 phosphorylation in brain neurons after insulin treatment. These observations suggest that Pak1 mediates the crosstalk between insulin and Wnt signaling pathways on gut and brain gcg expression, and its ablation reduced circulating postprandial GLP-

1 levels, which contributed to the defects in glucose homeostasis in Pak1−/− mice.

5.2 Introduction

Pak1 deficient mice were previously shown to exhibit defects in the second phase of pancreatic insulin secretion, peripheral insulin resistance, and ip glucose tolerance test (IPGTT)

(438). Notably, islets obtained from T2D patients have ~80% reduction in Pak1 protein levels, and IPA treatment resulted in impaired glucose stimulated insulin secretion (GSIS) in human islets (438). These observations implicate the importance of Pak1 in pancreatic β cell insulin secretion and the manifestation of glucose intolerance.

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I have previously identified Pak1 as an effector of insulin-stimulated gcg expression in both intestinal and brain gcg-expressing cells (468). Insulin stimulated Pak1 Thr423 phosphorylation and activation, associated with insulin-mediated β-cat phosphorylation at

Ser675 (468). Gcg promoter activity and mRNA production following insulin treatment was attenuated in the presence of the group I Pak inhibitor IPA3, and expression of dnPak1 blocked insulin-stimulated gcg promoter expression (468). As the incretin effect is estimated to contribute 50-70% of postprandial insulin secretion, and is importantly involved in the regulation of glucose homeostasis, in this study I aimed to assess the in vivo roles of Pak1 in gcg expression and GLP-1 production, and the effect of Pak1 ablation on glucose homeostasis (438).

5.3 Materials and methods

5.3.1 Real-time quantitative reverse-transcriptase PCR

Gcg mRNA levels were normalized to 18S ribosomal RNA levels. The primer pairs used are as follows: gcg forward, 5′-TGG ACT CCC GCC GTG CCC AA-3′, gcg reverse, 5′-CGA

CTT CTT CTG GGA AGT CTC GCC T-3′, 18S forward, 5′-CGG ACA TCT AAG GGC ATC

A-3′, and 18S reverse, 5′-AAG ACG GAC CAG AGC GAA A-3′.

5.3.2 Mouse distal ileum GLP-1 extraction

The extraction of GLP-1 from mouse distal ileum sections were performed using a protocol developed by Dr. Patricia Brubaker (494). The procedure involved the extraction of

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peptides from distal ileum tissue using the Classic C18 Sep-Pak cartridges from Waters (Milford,

MA, USA). The distal ileum section was dissected from mice, and homogenized using a polytron in extraction medium (1% trifluoroacetic acid, TFA, pH-adjusted to 2.5 using diethylamine). The cartridges were pre-moistened using 10 mL Reagent A (80% 2-propanol with 0.1% TFA), and washed with Reagent B (0.1% TFA solution in water). The tissue homogenate was added slowly to the cartridge using a syringe, and the flow-through was discarded. After washing with 10 mL of Reagent B, the bound peptides were eluted using the appropriate volume of Reagent A. The purified peptides were collected into a clean tube and stored at −80°C for further GLP-1 RIA measurements.

5.3.3 Mouse brain primary neuron isolation

The protocol for isolating mouse brain primary neurons was modified from an existing protocol (495). On day 1, the cerebellums were dissected from neonatal (P0 to P7) mice, rinsed with glucose-PBS solution (2 mg/mL glucose in 1 × PBS at pH 7.4), placed into a 15mL tube, and minced using sterilized surgical dissecting scissors. Immediately, the trypsin/DNase solution

(10 mg/mL trypsin, 1 mg/mL DNase, 5 mg/mL MgSO4, 2 mg/mL glucose, prepared in 1 × PBS at pH 7.4) was added at a volume to cover the tissue pieces, and the tube is incubated at 37°C for

3 minutes. Using a 20 G needle, the tissue solution was drawn up and then expelled, with the entire process repeated 15-20 times to mechanically shear the tissue pieces. After the process has been completed, the tissue homogenate was loaded onto a Percoll gradient with 30% and 60%

Percoll, and centrifuged at 150 × g at 4°C for 2 minutes. The neuron-enriched fraction resides at the 30%-60% interface, and was collected into a new tube. The neuron-enriched fraction is resuspended in culturing medium (DMEM with 10% FBS and 2% glucose), and plated at 2.5-5 ×

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4 10 cells/mL on polylysine-coated dishes. The primary neurons are incubated in 37°C at 5% CO2 for overnight. On day 2, if needed, the neurons were serum starved, for subsequent treatments on day 3.

5.3.4 Intraperitoneal and oral tolerance tests

For ip glucose tolerance test (IPGTT), ip pyruvate tolerance test (IPPTT), ip insulin tolerance test (IPITT), mice were fasted for 16 h prior to the test, and for glucagon challenge

(GC) mice were fasted for 6 h prior to the test. An ip bolus of glucose (2 g/kg), pyruvate (2 g/kg), insulin (0.75 U/kg) or glucagon (15 μg/kg) was administered, respectively, and tail vein blood samples were collected at indicated time points followed by AccuChek glucometer

(Roche, Basel, Switzerland) measurements.

For oral glucose tolerance test (OGTT), mice were fasted for 16 h prior to the test. An oral glucose load (2 g/kg) was given via gavage, followed by the same blood collection and glucose measurement method as described above.

5.3.5 Hormone measurements

Mice blood was collected from tail vein followed by the isolation of the serum or plasma fraction. Hormone analyses were conducted using the following commercial kits: Insulin, mouse insulin ELISA kit from Crystal Chem Inc. (Downer’s Grove, IL, USA); total GLP-1, RIA kit from EMD Millipore (Billerica, MA, USA) and ELISA kit from Meso Scale Discovery

(Rockville, MD, usa); active GLP-1, ELISA kit from Meso Scale Discovery with an antibody

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recognizing GLP-17-36 amide; glucagon, RIA assay from EMD Millipore; GLP-2, Yanaihara

(Shizuoka, Japan). All protocols were conducted as per manufacturers’ instructions.

5.4 Results

5.4.1 Pak1−/− mice in mixed C57BL/6-129 background have normal phenotypes

A recent study by Wang et al. (438) demonstrated that Pak1−/− mice in the C57BL/6 genetic background exhibit defects in glucose disposal, associated with peripheral insulin resistance and impaired GLUT-4 translocation in skeletal muscle after insulin stimulation. Here, we focused on determining the effect of Pak1 ablation on gut and brain gcg expression, GLP-1 content, and glucose homeostasis.

5.4.2 Pak1−/− mice in C57BL/6 background show impaired glucose disposal and reduced gut gcg expression level

The absence of Pak1 protein expression in various tissues was confirmed by Western blotting (Fig. 5.1A). In the C57BL/6 background, the adult male Pak1−/− mice showed a trend of increased body weight starting at the age of 19 wk (Fig. 5.1B). This trend, however, was less obvious when the mice were in the mixed C57BL/6-129 genetic background (Fig. 5.2).

Importantly, the Pak1−/− mice showed impairments in IPGTT (Fig. 5.1C), which is in agreement with the findings by Wang et al. (438), as well as in OGTT (Fig. 5.1D). Furthermore, the Pak1−/−

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mice exhibited increased blood glucose levels in IPPTT (Fig. 5.1E), indicating these mice may have elevated hepatic glucose production.

To determine whether Pak1 ablation affects circulating hormone levels, mice were challenged with glucose gavage. Wild-type mice showed a significant increase in serum insulin levels at 30 min after glucose gavage. The increase in the Pak1−/− mice, however, was not detectable (Fig. 5.3A). Furthermore, serum glucagon levels were significantly lower in the

Pak1−/− mice, both before and 30 min after the glucose gavage (Fig. 5.3B). We, however, did not detect any notable defects in the architecture of Pak1−/− mice in their pancreatic islets, as shown by insulin and glucagon co-immunostaining (Fig. 5.4).

We then examined whether Pak1 ablation affects gut gcg expression in vivo. The Pak1−/− mice showed significantly reduced gut gcg mRNA levels, assessed by qRT-PCR (Fig. 5.3C).

This reduction, however, was not appreciably associated with a change of circulating total GLP-

1 level, either during fasting or 5 min after oral glucose gavage (Fig. 5.3D). We then measured active GLP-1 levels (GLP-17-36 amide) and observed that wild-type mice had increased serum active GLP-1 levels after glucose gavages; however, this increase was absent in the Pak1−/− mice

(Fig. 5.3E). We then assessed distal ileum GLP-1 content by dissecting a 5-cm region of the distal ileum followed by treatment with or without insulin for 2 h. The distal ileum GLP-1 content was induced by insulin treatment in the wild-type mice but not in the Pak1−/− mice (Fig.

5.3F).

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5.4.3 Pak1−/− mice have reduced brainstem gcg expression level

Gcg is expressed in the brainstem and central infusion of GLP-1 or GLP-1 receptor agonist inhibits food intake. As Pak1 is also abundantly expressed in the mouse brain, I then aimed to assess whether Pak1 ablation would affect brain gcg expression. As shown in Fig.

5.5A, Pak1−/− mice had dramatically reduced brain gcg mRNA level as measured by qRT-PCR.

Furthermore, I observed that in the brain gcg-expressing mHypoE-20/2 cell line, insulin or F/I treatment stimulated gcg promoter expression. The stimulation can be significantly attenuated by

IPA3 pretreatment in both cases (Fig. 5.5B).

5.4.4 Pak1−/− mouse brain neurons show abolished response to insulin on β-cat Ser675 phosphorylation

Brain neuron cells from Pak1−/− mice and wild-type controls were prepared for insulin or

F/I treatment. Whereas cAMP-stimulated β-cat Ser675 phosphorylation was partially attenuated in the Pak1−/− mice (Fig. 5.6A), insulin-stimulated β-cat Ser675 phosphorylation was abolished in the Pak1−/− mice, although insulin was still able to stimulate Akt Ser473 phosphorylation (Fig.

5.6B).

5.4.5 Pak1-/- mice have reduced distal ileum weight

The distal ileum section was dissected from the mice the age of 16 wks and 8 wks. A 5 cm longitudinal segment was isolated from each mouse, and organ weight measurement was

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performed (Fig. 5.7). At both age groups, the Pak1−/− mice have about 30% reduction in distal ileum weight.

5.4.6 Pak1-/- mice have comparable responses to intraperitoneal insulin tolerance test

At the age of 7wks, ip insulin tolerance test was performed in Pak1−/− and age-matched wild-type control mice (Fig. 5.8). Both groups of mice exhibited comparable responses to ip insulin, where blood glucose levels fell within the first 60 min post ip insulin followed by subsequent normoglycemia after the first 60 min.

122 A

Pak1 β-actin Pak1-/- mouse Wild-type mouse

Wild-type Pak1-//-- B C IPGTT 3000

30 50 **

* 2000 25

40 AUC * ** 1000 30 20 0 20 15 Wild-type Wild-type 10 -/- 10

Body weightBody(g) Pak1-/- Pak1 Pak1--/--

Glucose (mmol/L) Glucose Pak1 0 5 4 6 8 10 12 19 21 24 0 20 40 60 80 100 120 Age (weeks) Time (min)

D E Wild-type Pak1-//-- Wild-type Pak1-//-- OGTT Pak1 IPPTT

2000 20 * 20 1500 **

AUC 1000 * AUC 1000 15 15 500 * * 0 0 10 10 *** Wild-type Wild-type Glucose (mmol/L) Glucose Glucose (mmol/L) Glucose Pak1-//- - Pak1--/-- 5 5 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Time (min) Time (min)

Fig. 5.1 Pak1-/- mice exhibit impaired glucose disposal. A, Lack of Pak1 protein expression in Pak1-/- mouse tissues. Wild-type mouse pancreas serves as positive control. B, Pak1-/- mice showed a trend of increased body weight starting at wk 19 (n ≥ 6). C, Representative result of IPGTT (30 wk old, n ≥ 10). Inset shows area under the curve (AUC). D, Representative result of OGTT (31 wk old, n ≥ 12). Inset shows AUC. E) Representative result of IPPTT (12 wk old, n ≥ 6 for both groups). Inset shows AUC. *, P ≥ 0.05; **, P ≥ 0.01; ***, P ≥ 0.001.

123 50 Wild-type Pak1--/-/ -

20 Body weightBody(g) 10

0 4 6 8 10 12 19 21 24 Age (weeks)

Fig. 5.2 Body weight monitoring of Pak1-/- mice in C57BL/6-129 mixed genetic background. Measurements performed with n=5 mice per group.

124 A B □ Wild-type ■ Pak1-/- □ Wild-type ■ Pak1-/-

80 ** 2.0 ** ** *

60 1.5 )

1.0 40 (pg/mL

insulin insulin (ng/mL) 0.5 20 Serum Serum glucagon

0.0 0 Serum Serum Fasting 30 min Fasting 30 min 60 min C D □ Wild-type ■ Pak1-/- 40 1

1.5 *** -

1.0 20 change) mRNA

totalGLP

0.5 (pg/mL) 10 gcg (fold

0.0 Serum 0 Wild-type Pak1Pak1-/--/- Fasting 5 min

E F

□ Wild-type ■ Pak1-/- □ Wild-type ■ Pak1-/-

8 100 1 - * *** *** 80 ***

6 content 1

- 60 activeGLP

pg/mL) 4 ( 40 (pg/mL) 2 Serum Serum 20

0 ileum Distal GLP 0 Fasting 5 min 15 min Basal Insulin

Fig. 5.3 Pak1-/- mice show abnormalities in plasma hormone levels and gut gcg expression. A, Pak1-/- mice show attenuated elevation of insulin 30 min after glucose challenge (12 wk old, n ≥ 4). B, Pak1-/- mice exhibit reduced glucagon levels during fasting and postglucose gavage. After mice (12 wk old and n ≥ 4 per group) were fasted for 16 h, blood samples were taken before and after glucose gavage (2 g/kg, in PBS) at indicated times. Serum glucagon levels were measured by RIA (Millipore). C, Distal ileum gcg mRNA level was significantly reduced in Pak1-/- mice. gcg mRNA levels were determined by qRT-PCR (16 wk old, n ≥ 6 for both groups). D and E, Assessment of serum total (D) and active (E) GLP-1 levels in the Pak1-/- mice. The Pak1-/- mice showed no significant difference in serum total GLP-1 levels under fasting condition and 5 min postglucose gavage (n ≥ 4 for both groups, aged 16 wk). For assessment of serum-active GLP-1 levels, mice were faster for 16 h followed by three glucose gavages spaced at 5-min intervals. The Pak1-/- mice exhibited attenuated rise in serum active GLP-1 at 5 min after the third glucose gavage (24 wk old, n ≥ 9). F, Insulin- stimulated GLP-1 content in the distal ileum is attenuated in the Pak1-/- mice. The distal ileum 5-cm segments are dissected out from 16 h-fasted mice (28 wk, n ≥ 8 –9) and treated with or without insulin (100 nM, 2 h). Tissue GLP-1 content was normalized to tissue protein content. *, P ≥ 0.05; **, P ≥ 0.01; ***, P ≥ 0.001. 125 a b

Wild-type

c d

Pak1-/-

Fig. 5.4 Pak1-/- mice exhibit comparable pancreatic islet architecture. Pancreas sections of a Pak1-/- mice and an age-matched wild-type controls (age 9 or 10wks) were prepared for co-immunostaining of insulin (brown) and glucagon (red). Arrow points to the islet shown in the insets, bar indicates a and c: 1 mm, b and d: 100 µm.

126 Basal Insulin F/I A B

1.6 5

1.4 *** ** 4 ** 1.2 * (fold change)(fold

1.0 3

(fold change)(fold 0.8 2 0.6 *

0.4 Luciferase 1

gcg mRNA mRNA gcg 0.2 0.0 0 Wild-type Pak1Pak1--//-- DMSO IPA3

Fig. 5.5 Pak1-/- mice show reduced brainstem gcg mRNA level and IPA3 attenuates insulin- and forskolin-stimulated gcg promoter activity in brain neurons. A, Pak1-/- mice show reduced brainstem gcg expression levels by qRT-PCR (16 wk old, n ≥ 6 for both groups). B, IPA3 attenuates insulin- and F/I- stimulated 2.4 kb-gcg-LUC promoter activity in the mHypoE-20/2 cell line. The cells were transfected with 2.4 kb-gcg-LUC for 24 h, pretreated with IPA3 or dimethylsulfoxide (DMSO) and stimulated with insulin or F/I for 4 h, followed by LUC analysis (n ≥ 3, triplicates each). *, P ≥ 0.05; **, P ≥ 0.01; ***, P ≥ 0.001.

127 A

Fold 1 2.1 1 1.3

p-β-cat (Ser675) β-cat p-CREB1 (Ser133) β-actin

F/I - + - + Wild-type Pak1-/-

Fold 1 1.2 1.5 1 0.6 0.6

p-β-cat (Ser675) β-cat p-Akt1 (Ser473) Akt1 Insulin 0 60 120 0 60 120 (min) Wild-type Pak1-/-

Fig. 5.6 Pak1-/- mice brain neurons show lack of response in insulin-stimulated β-cat Ser675 phosphorylation. A, Neuronal cells from Pak1-/- mice show attenuated F/I-mediated β-cat Ser675 phosphorylation. Representative blot (n ≥ 3) is shown with brain neuronal cells from Pak1-/- mice and wild-type controls. Fold change of p-β-cat Ser675 is shown, with each basal state defined as 1-fold. B, Neuronal cells from Pak1-/- mice lack the β-cat Ser675 phosphorylation in response to insulin stimulation (representative blot, n ≥ 3 for both groups). Fold change of p-β-cat Ser675 is shown, with each basal state defined as 1-fold.

128 A B

0.12 ** 0.12 ***

0.10 (g) 0.10

0.08 0.08 weight(g) 0.06 0.06

0.04 0.04

0.02 0.02 Distal ileum ileum Distal weight Distal ileum ileum Distal 0.00 0.00 Wild-type Pak1Pak1--//-- Wild-type Pak1Pak1-/-/--

Fig. 5.7 Pak1-/- mice exhibited reduced weight of distal ileum. Male mice were sacrificed for organ extraction and organ weight measurement at the age of 16 wks (A) (n≥6) or 8 wks (B) (n≥6). Pak1-/- mice showed significant reduction in distal ileum weight (measurement performed using 5 cm segments).

129 12.0

10.0

8.0

6.0 +/+ WildPak1WT -type -/- 4.0 Pak1 -/- Blood glucose level glucose Blood 2.0

0.0 0 10 30 60 90 120 Time (min)

Fig. 5.8 Pak1-/- mice and age-matched wild-type (WT) mice have comparable responses in intraperitoneal insulin tolerance test. Male mice were subjected to intraperitoneal insulin tolerance test at the age of 7 wks (n≥6).

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5.5 Discussion

In an ex vivo study by Wang et al., Pak1−/− islets exhibited profound defects in the second phase of insulin secretion (438). They subsequently conducted their in vivo study, revealing that

Pak1−/− mice showed impaired IPGTT and IPITT (438). I aimed to expand the characterization of the Pak1−/− mice, from the angle of incretin hormone production and glucose homeostasis. In addition to confirming the defects in IPGTT, I demonstrated the defect in OGTT in the Pak1−/− mice in the C57BL/6 background.

In my study, reduced brain gcg mRNA level was observed in the Pak1−/− mice. Because brain GLP-1 signaling is known to affect insulin action in peripheral tissues including the liver

(225,496); hence, it will be interesting to determine whether deleting Pak1 specifically in the brain would affect glucose disposal.

The Pak1−/− mice exhibited a significant reduction of distal ileum weight both at 8 wks and at 4 months (Fig. 5.7), which points to a potential role of Pak1 in the production of GLP-2, another gcg-encoded hormone involved in the growth of small intestines (120). Preliminary results support this notion, and show that the Pak1−/− mice have reduced circulating GLP-2 levels

(details see Section 7.3.4 and Appendix 3).

In my study, the Pak1−/− mice did not exhibit a substantial defect in IPITT (Fig. 5.8) as previously observed by Wang et al. (438). In my study, the Pak1−/− mice showed reduced serum glucagon levels, both during fasting and at 30 min after feeding (Fig. 5.3B). This implicates that

Pak1 is potentially involved in pancreatic glucagon secretion, although reduced glucagon secretion was not observed by Wang et al. in their ex vivo Pak1−/− islet study (438).

Alternatively, this may be due to a compensatory mechanism to maintain a proper circulating

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insulin and glucagon ratio, as the Pak1−/− mice exhibited reduced serum insulin levels as well.

This yet-to-be-identified compensatory mechanism may undermine the relatively modest metabolic defects observed in these Pak1−/− mice in the absence of a challenge. The reduction of both serum insulin and glucagon levels at basal states would allow the Pak1−/− mice to maintain euglycemia in the absence of oral glucose challenge.

Although gut gcg mRNA levels are reduced significantly in the Pak1−/− mice, we did not see reduced levels of circulating total GLP-1, neither during fasting nor 5 min after an oral glucose challenge. A potential explanation is the existence of compensatory mechanisms to maintain blood GLP-1 at a desirable level. We, however, cannot eliminate the possibility that current technology for GLP-1 detection is not sensitive enough for revealing the subtle difference in the Pak1−/− mice vs. the wild-type controls. Indeed, plasma GLP-1 levels are within the nanomolar range, which are much lower compared with that of insulin or glucagon. To date, the measurement of plasma GLP-1 levels has not been developed as a diagnostic tool for diabetes, although a few studies have reported that certain type 2 diabetes patients have reduced plasma GLP-1 levels (497,498). In the insulin-resistant and hyperinsulinemic MKR mouse model, elevated serum GLP-1 levels were observed during fasting, although feeding-stimulated

GLP-1 secretion was attenuated (480). In my study, the Pak1−/− mice did exhibit a reduced active serum GLP-1 level after glucose gavage. Whether Pak1 signaling is involved in protecting active

GLP-1 from DPP-IV-mediated degradation is worth to be investigated.

Group 1 Paks may exert certain redundant functions in neurons or other cell types.

Knockout mice lacking either Pak1 or Pak3 were shown to bear no abnormalities in overall neuronal morphology and brain development (499). On the contrary, mice lacking both Pak1 and

Pak3 exhibited severely impaired postnatal brain growth, resulting in a dramatic reduction in

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brain volume (499). We present here that Pak1 depletion significantly reduced brain gcg expression. This, along with the findings that both cAMP elevation and insulin treatment increased β-cat Ser675 phosphorylation in brain neurons and that Pak1−/− neurons show attenuated or blocked responses to cAMP and insulin stimulation, suggests that Pak1 mediates the crosstalk between cAMP and Wnt signaling pathways as well as the crosstalk between insulin and the Wnt signaling pathways in the brain. Functionally, this crosstalk is important for brain gcg expression and GLP-1 production. Questions that remain to be answered include: 1) whether the two other group I Paks (Pak2 and Pak3) are also involved in brain gcg expression and 2) whether the brain insulin-Pak1 signaling cascade could provide therapeutic targets for improving peripheral glucose homeostasis. Indeed, brain Pak1 is also controlled by the forkhead box O (FOXO) signaling pathway, another downstream target of the insulin signaling (493).

Figure 5.9 summarizes our current understanding of the key roles of Pak1 in glucose homeostasis. In muscle, Pak1 mediates the effect of insulin in stimulating GLUT4 translocation

(333,438). In the pancreas, Pak1 is required for the second phase of glucose-induced insulin secretion, possibly via glucose-stimulated Erk1/2 activation (438). Since we show that Pak1-/- mice exhibited reduced serum glucagon level, whether Pak1 mediates pancreatic glucagon secretion needs to be further investigated. In this study, we have also shown that in the gut and brain, Pak1 controls gcg expression and GLP-1 production. This is at least partially mediated by the stimulation of Ser675 phosphorylation of β-cat, the major effector of the Wnt signaling cascade. Impaired IPPTT in Pak1-/- mice observed in the current study could be an indirect effect on liver gluconeogenesis, due to attenuated insulin signaling and reduced GLP-1 production.

Whether more severe abnormalities occur in the Pak1-/- mice after a high-fat diet challenge is worth investigating. It will also be interesting and worthy to further assess the contribution of

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Pak1 in metabolic homeostasis in mouse models with tissue-specific knockout approaches and to determine the roles of the other two group I Paks in metabolic homeostasis.

134 Cdc42/Rac1 Insulin

Pak1

β-cat

Glucose transport: Hormone gene expression: Hormone secretion: • Muscle (GLUT4) • Gut (gcg) • Gut (GLP-1) • Brain (gcg) • Pancreas (insulin)

Fig. 5.9 Our current understanding of the role of Pak1 in glucose homeostasis. In the muscle, Pak1 is important for insulin-stimulated GLUT4 translocation together with Cdc42/Rac1 small GTPases. The current study shows the role of Pak1 in mediating the stimulatory effect of insulin on gut and brain gcg expression, involving the Wnt effector β-catenin. Furthermore, Pak1 plays a role in the secretion of metabolic hormones, including insulin and GLP-1.

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5.6 Acknowledgements

We thank Ms. Christina Li for technical assistance for the mouse blood collection, and Dr.

Jonathan Chernoff for providing the Pak1-/- mouse line.

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6 The role of p21-activated protein kinase 1 in hepatic glucose production

Data presented in this chapter (except Fig. 6.9) have been included in a first-author manuscript by Yu-ting Chiang et al (revised manuscript submitted, Endocrinology).

All experiments were performed and figures contributed by Yu-ting Chiang. The Pak1-/- mouse line was provided by Dr. Jonathan Chernoff (Fox Chase Cancer Center).

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6.1 Abstract

Pak1 plays a role in insulin secretion and GLP-1 production. Pak1-/- mice were found to carry a defect in IPPTT, leading us to speculate whether Pak1 represses hepatic gluconeogenesis.

We show here that the defect in IPPTT became more severe in aged Pak1-/- mice. In primary hepatocytes, IPA3, a potent inhibitor of group I Paks, reduced basal glucose production, attenuated forskolin- or glucagon-stimulated glucose production, and attenuated the stimulation of forskolin on the expression of Pck1 and G6pc. These in vitro observations imply that the direct effect of Paks in hepatocytes is the stimulation of gluconeogenesis, and that the impairment in IPPTT in Pak1-/- mice is due to the lack of Pak1 elsewhere. Consecutive i.p. injection of forskolin for two weeks increased gut proglucagon gene (gcg) expression, associated with improved IPPTT in aged Pak1-/- mice and wild-type controls. In addition, administration of the DPP-IV inhibitor sitagliptin for 1 wk reversed the defect in IPPTT in aged Pak1-/- mice, associated with increased plasma GLP-1 levels. Our observations indicate a potential role of

Pak1 in the gut/liver axis or gut-pancreas-liver axis in controlling glucose disposal, and affirmed the therapeutic application of GLP-1 and DPP-IV inhibitors in attenuating hepatic gluconeogenesis.

6.2 Introduction

Group I Paks function as critical effectors that link Rho GTPases (Rac1 and Cdc42) to cytoskeleton reorganization, and hence are involved in regulating various important cellular functions including cell survival, differentiation, proliferation, polarity and hormone secretion

(383,384,489,500,501). An early investigation in muscle cells revealed that insulin stimulates

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Pak1 via a PI3K-dependent mechanism (333). This activation is likely responsible for muscle cell GLUT4 translocation (425). The utilization of small interference RNA knockdown, dominant negative Pak1 cDNA transfection and other approaches has also implicated the role of

Pak1 in mediating the second phase of glucose-stimulated insulin secretion (453) and the secretion of the incretin hormone GLP-1 from intestinal endocrine L cells (480). Utilizing the

Pak1-/- mice, Wang and colleagues conducted an ex vivo study and reported that Pak1-/- islets exhibited a substantial defect in second phase of insulin secretion (499).

As a serine and threonine protein kinase, Pak1 phosphorylates a battery of downstream target proteins, including β-catenin (β-cat) (502,503), which forms a bipartite transcription factor with a member of the TCF family in controlling Wnt pathway downstream target gene expression. Our laboratory demonstrated that Pak1 acts as a downstream effector of insulin signaling in the crosstalk with the Wnt signaling pathway in colon cancer cell lines (334). In the gut endocrine L cells, however, this crosstalk leads to increased expression of the proglucagon gene (gcg) and the synthesis of GLP-1 (468). Pak1-/- mice show reduced gut gcg mRNA expression and reduced plasma GLP-1 levels upon glucose challenge (468). Pak1-/- mice were also shown to carry a moderate impairment in response to pyruvate (468), a major substrate for gluconeogenesis, leading us to wonder whether Pak1 exerts a repressive effect on hepatic glucose production.

The liver is the major organ for glucose production during fasting as well as for the conversion of glucose into glycogen after feeding. During fasting, elevation of glucagon levels leads to the activation of key transactivators of gluconeogenic genes, while after feeding, elevation of insulin leads to the inactivation of certain key transactivators such as FOXO proteins. Glucagon and insulin also mediate the effect of stress and other signaling cascades in

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regulating glucose homeostasis (499,503-506). Although the detailed underlying mechanism is still unclear, GLP-1 was shown to repress hepatic gluconeogenesis in both insulin-dependent and

-independent manners (507,508). In the current study, we observed that aged Pak1-/- mice show more profound intolerance in response to pyruvate challenge. Surprisingly, in vitro analysis with primary hepatocytes indicated that group I Paks are likely positive regulators of gluconeogenesis, suggesting that the defect in aged Pak1-/- mice in response to pyruvate challenge is due to the lack of Pak1 in extra-hepatic organs. Activation of GLP-1 signaling through either i.p. forskolin injection or the administration of the DPP-IV inhibitor sitagliptin increased gut gcg expression or the plasma GLP-1 levels, along with the restoration of tolerance to i.p. pyruvate challenge. Our observations indicate a potential role of Pak1 in controlling hepatic glucose production through the gut-GLP-1-liver axis. These observations affirm the therapeutic application of GLP-1 and

DPP-IV inhibitors in attenuating hepatic gluconeogenesis.

6.3 Materials and methods

6.3.1 Mouse primary hepatocyte isolation

The protocol for mouse primary hepatocyte isolation was adapted from a published protocol (509). The 6-10 weeks old C57BL/6 male mice were anesthetized using isoflurane, followed by cannulation of the hepatic portal vein using a 23 G needle and the cutting of the inferior vena cava (IVC). Perfusion of the liver was initiated with supplemented Hank’s

Balanced Salt Solution (HBSS, 5mM glucose, 0.5mM EGTA, 25mM HEPES at pH7.4) for 5-10 min, and then switched to digestion medium (DMEM with 5 mM glucose, 100 U/mL penicillin,

0.1 mg/mL streptomycin, 15 mM HEPES, 100 U/mL type IV collagenase) for an additional 5-

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10min digestion. After digestion was complete, the liver was excised and transferred to a sterile dish containing the same digestion medium. Primary hepatocytes obtained from the digested liver were passed through a 74 μm cell strainer, followed by three cycles of washing and final resuspension in isolation medium (DMEM with 25mM glucose, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 15mM HEPES, 100 nM dexamethasone, 10 % FBS). The viability and yield of the cells were determined using trypan blue staining, and preparations achieving ≥ 90% viability were used for experimental procedures.

6.3.2 Glucose production assay

Following mouse primary hepatocyte isolation, the hepatocytes were plated on collagen- coated (5 μg/cm2 type I collagen) 12-well plates at 60-70% density and cells were incubated for

16-24h for attachment. The evening prior to glucose production assays, cells were changed to serum-free and phenol red-free glucose production medium (GPM) (DMEM with 5 mM glucose,

44 mM NaHCO3, 2 mM L-glutamine, penicillin/streptomycin, 5 mM HEPES, 10 nM dexamethasone). For glucose production, cells were washed and incubated in fresh GPM plus 10 nM of pyruvate/lactate substrate, followed by pretreatment of IPA3 or DMSO vehicle control for

1h. After 1h, treatment was initiated with various agents such as forskolin, glucagon, insulin, or exendin-4. Medium was collected after 4 h and amount of glucose was measured using a glucose assay kit from Sigma Aldrich (St. Louis, MO, USA). Cells were harvested in protein lysis buffer to determine protein content, which was used to normalize glucose production measurements.

141

6.3.3 Real-time quantitative reverse-transcriptase PCR

DNA sequence information for oligonucleotide primer pairs utilized in this study is as follows: Pck1 (5'-CAT AAC GGT CTG GAC TTC TCT GC-3', 5'-GAA TGG GAT GAC ATA

CAT GGT GCG-3'); G6pc (5'-CTC TGG GTG GCA GTG GTC GG-3', 5'-AGG ACC CAC

CAA TAC GGG CGT-3'); Gcg (5’-TGG ACT CCC GCC GTG CCC AA- 3’, 5’-CGA CTT CTT

CTG GGA AGT CTC GCC T- 3’); 18s (5’-CGG ACA TCT AAG GGC ATC A-3’, 5’-AAG

ACG GAC CAG AGC GAA A-3’).

6.3.4 Intraperitoneal administration of forskolin and sitagliptin gavage in

mice

Forskolin was administered as daily ip injections (5 mg/kg per day) for the duration of two weeks. Sitagliptin (Januvia®) was administered daily as oral gavage (dose 300 mg/kg per day) for a total duration of 5 weeks. Prior to each gavage, a fresh sitagliptin working stock was prepared by dissolving the tablets in the appropriate volume of saline buffer.

6.4 Results

6.4.1 Aged Pak1-/- mice exhibit more severe defects in IPPTT and GLP- 1 secretion response

We observed previously that compared to wild type littermates, Pak1-/- mice had moderately elevated blood glucose levels in response to pyruvate injection (468). Hepatic

142

glucose production is controlled by multiple metabolic hormones in response to nutritional and environmental changes. The three major hormones are glucagon, insulin and GLP-1, involving a battery of transcription factors including FOXOs, the effectors of stress and ageing signaling pathway. To assess whether the severity of the defect in IPPTT correlates with ageing, we conducted pyruvate tolerance test in a set of Pak1-/- mice and age-matched control mice at the age of 12 months. Although there was no appreciable difference between the knockout mice and the control mice in fasting blood glucose levels, the aged Pak1-/- mice exhibited profound defect in IPPTT, with a ~30% increase in blood glucose AUC0-120 (Fig. 6.1A). To assess the hormonal responses of the aged Pak1-/- mice to glucose challenge, we measured plasma insulin, GLP-1, and glucagon levels. The aged Pak1-/- mice had attenuated insulin levels at 15 min and 30 min after i.p. glucose challenge (Fig. 6.1B), along with a ~50% reduction in circulating total GLP-1 levels at 5 min post oral glucose gavage (Fig. 6.1C). The plasma glucagon levels of the aged

Pak1-/- mice after an i.p. glucose challenge were comparable to the wild-type controls, with a trend of lower basal glucagon level (Fig. 6.1D). The glucagon tolerance of the aged Pak1-/- mice was similar to that of the wild-type controls (Fig. 6.1E). Together, a profound defect in IPPTT was observed in the aged Pak1-/- mice, associated with a severe impairment in GLP-1 response, along with impairment in insulin secretion, but no appreciable defect in glucagon secretion or glucagon tolerance.

6.4.2 Inhibition of Group I Paks represses glucose production in primary hepatocytes

We examined the direct effect of Pak kinases on glucose production in mouse primary hepatocytes utilizing IPA3, a highly specific non-ATP-competitive chemical inhibitor of group I

143

Paks (365). Pretreating hepatocytes with IPA3 attenuated basal as well as glucagon- or forskolin- stimulated glucose production (Fig. 6.2A). Ex-4 treatment generated no appreciable effect on glucose production in this in vitro setting. However, IPA3 pretreatment prior to the addition of

Ex-4 significantly lowered glucose production (Fig. 6.2A). Fig. 6.2B shows that the repressive effect of IPA3 on glucose production is dosage dependent. Since IPA3 inhibits all three members of the group I Pak family (Pak1, Pak2, and Pak3) (365), we wondered whether ablation of Pak1 alone would attenuate the stimulation by forskolin or glucagon. Hence, we isolated hepatocytes from Pak1-/- mice. As shown in Fig. 6.2C, Primary hepatocytes isolated from wild-type and

Pak1-/- mice were stimulated with glucagon or forskolin, in presence or absence of IPA3, followed by glucose production measurements. In wild-type hepatocytes, IPA3 attenuated the glucagon- and forskolin-stimulated glucose production. Pak1-/- hepatocytes have reduced basal glucose production, and IPA3 blunted the stimulation of glucagon and forskolin on glucose production, suggesting that IPA3 treatment had an additive effect with Pak1 ablation.

6.4.3 Inhibition of group I Paks represses gluconeogenic gene expression in hepatocytes

To further determine the role of group I Paks in hepatic glucose production, we assessed gluconeogenic gene expression in Pak1-/- mice and in mouse primary hepatocytes treated with

IPA3. Compared to livers of wild-type controls, liver tissues of aged Pak1-/- mice had increased expression of Pck1 and G6pc, which encode PEPCK and G6P, respectively, two rate-limiting enzymes for gluconeogenesis (Fig. 6.3A). Unlike in the aged mice, the livers of young Pak1-/- mice had similar Pck1 and G6pc mRNA levels compared to wild-type controls (Fig. 6.3B). IPA3 on its own was observed to moderately increase the expression of Pck1 (Fig. 6.3C) and G6p

144

mRNA (Fig. 6.3D). However, IPA3 pre-treatment significantly attenuated the stimulatory effect of forskolin on Pck1 mRNA expression in the presence or absence of insulin treatment (Fig.

6.3C). Similarly, a robust stimulation of G6pc expression by forskolin was observed in primary hepatocytes, and this stimulation was significantly attenuated by IPA3 pre-treatment, in the presence and absence of insulin treatment (Fig. 6.3D). These observations further support our suggestion that in hepatocytes per se, group I Paks positively regulate gluconeogenic genes, and that the elevated hepatic gluconeogenesis in aged Pak1-/- mice is likely due to the absence of

Pak1 elsewhere.

6.4.4 In vivo forskolin administration improves IPPTT and increases gut gcg mRNA levels in aged Pak1-/- mice

GLP-1 is known to repress hepatic gluconeogenesis in vivo, and this effect was initially attributed entirely to its role as an incretin (507). A few recent studies have suggested that GLP-1 can repress hepatic glucose production in an insulin-independent manner (508,510). Gcg expression can be stimulated by PKA activation (173,195,485) and we have observed recently that in vivo administration of forskolin, an activator of adenylyl cyclase, increased gut gcg mRNA levels (511). To investigate whether the hepatic defect in the aged Pak1-/- mice is at least partially due to reduced gut gcg expression, we tested the rescue ability of forskolin administration (5 mg/kg/day). As shown in Fig. 6.4A, aged Pak1-/- mice and age-matched (14 months old) control mice were subjected to daily i.p. forskolin injection for 14 days. IPPTT was performed before and after the forskolin administration. Nine mice utilized in the above IPPTT as well as 10 additional age-matched mice (5 wild type mice and 5 Pak1-/- mice) which received

PBS injection were euthanized at the end of the treatment period for the collection of distal ileum

145

followed by the measurement of gut Gcg mRNA levels. During the two-week period, forskolin injection generated no significant change in body weight (Fig. 6.5). As shown in Fig. 6.4B, forskolin injection significantly improved IPPTT in both aged Pak1-/- mice and age-matched wild-type control mice, although the aged Pak1-/- mice maintained a relatively worse phenotype.

Furthermore, forskolin injection lowered fasting plasma glucose levels in these aged mice (Fig.

6.4B). Forskolin injection also increased gut gcg mRNA levels in both aged wild-type control mice and Pak1-/- mice. Notably, gut gcg levels in the aged Pak1-/- mice receiving forskolin injection were even higher than that of wild-type controls receiving PBS injection (Fig. 6.4C).

Thus, although the in vitro effect of forskolin in isolated hepatocytes is the stimulation of gluconeogenesis, it functions as the gcg transcriptional stimulatory agent when administrated in vivo. The improvement of IPPTT in the aged Pak1-/- mice by forskolin administration suggests that reduced gut gcg expression is among the underlying causes of the IPPTT defect in aged

Pak1-/- mice.

6.4.5 Sitagliptin gavage reverses the IPPTT defect and stimulates plasma GLP-1 levels in aged Pak1-/- mice

We then tested whether the administration of the DPP-IV inhibitor sitagliptin would improve IPPTT and OGTT. In this set of experiments, mice at the age of 46 wks were utilized.

As shown in Fig. 6.6A, aged Pak1-/- mice and wild-type controls were subjected to IPPTT after receiving PBS or sitagliptin via daily oral gavage for one week (300 mg/kg per day). Following the IPPTT, PBS or sitagliptin was continually administrated via daily gavage for 4 additional wks. Within this period, other procedures were performed including blood collection for plasma

GLP-1 assessment, OGTT, IPITT, and GC. During the five wk period, the effect of PBS or

146

sitagliptin gavage did not generate a significant difference in mouse body weight change (Fig.

6.7). The overall effect of sitagliptin administration on mouse glucose disposal is summarized as follows. Firstly, both aged wild-type and Pak1-/- mice that received sitagliptin daily gavage for one week showed marked improvement in IPPTT compared to the respective PBS control groups, and sitagliptin was able to almost completely normalize the defect in the aged Pak1-/- mice (Fig. 6.6B). Secondly, sitagliptin administration for 2 weeks significantly improved OGTT

-/- -/- in aged wild-type as well as aged Pak1 mice (Fig. 6.6C). AUC0-120 in aged Pak1 mice after sitagliptin gavage was comparable with that in age-matched control mice receiving PBS gavage

(Fig. 6.6C). Thirdly, in the aged Pak1-/- mice, sitagliptin treatment enhanced GLP-1 secretion, as indicated by elevated circulating GLP-1 levels at basal and 5 min post oral glucose gavage (Fig.

6.6D). Finally, sitagliptin administration did not result in a noticeable change in insulin tolerance

(Fig. 6.6E) or glucagon tolerance (Fig. 6.6F) in the aged wild-type and Pak1-/- mice. Together, these results suggest that sitagliptin administration significantly improved both IPPTT and

OGTT, associated with enhanced GLP-1 levels, while having no substantial effect on insulin or glucagon sensitivity.

6.4.6 Aged Pak1-/- mice have reduced epididymal fat pad weight

At the time when the aged Pak1-/- mice were euthanized, organs including liver, distal ileum 5 cm segment, and epididymal fat pad were dissected and weighed. The aged Pak1-/- mice show modest yet significant reduction in body weight (Fig. 6.8A). The aged Pak1-/- mice demonstrated no substantial abnormalities in liver and distal ileum weight (Fig. 6.8B and 6.8C), but exhibited reduced epididymal fat pad weight (Fig. 6.8D). The weight measurements were performed using both absolute weight as well as were normalized to body weight.

147 A IPPTT Wild-type Pak1-KO 20 * ** 2000

* 15 ** * /L) 1500

10 mmol ( 1000 AUC 5 500 Wild-type Pak1-KO 0 0 0 20 40 60 80 100 120 Blood glucose Blood Time (min)

B Wild-type Pak1-KO C Wild-type Pak1-KO ns * 1.6 * * 120 * ** * ns

100

1.2 80 0.8 60 1 (pg/mL) - 40

Insulin Insulin (ng/mL) 0.4 GLP 20 0 0.0 Basal 15 E 30 Basal 5 min 15 min min min

D E Wild-type Pak1-KO Wild-type Pak1-KO GC 120 20 2000

* 100 15 1500 80

60 10 1000 40 AUC 5 Wildtype 500

Glucagon (pg/mL) Glucagon 20 Pak1-KO

0 glucose Blood(mmol/L) 0 0 Basal 15 min 30 min 0 30 60 90 120 Time (min)

Fig. 6.1 Aged Pak1-/- mice show severe defect in IPPTT. Aged Pak1-/- (KO) mice exhibited a profound defect in response to i.p. pyruvate injection (IPPTT), associated with impaired insulin and GLP-1 secretion. (A) IPPTT. (B) Plasma insulin levels before and after glucose i.p. injection. (C) Plasma GLP-1 levels before and after glucose gavage. (D) Plasma glucagon levels before and after glucose i.p. injection. (E) Glucagon challenge (GC) test. Wild-type mice, n=5; Pak1-/- mice, n=5. Age-matched 12 months old mice were used for each set of experiment. * p < 0.05, ** p < 0.01, *** p < 0.001.

148 A 20 DMSO IPA3

* 15 * * *

* protein)

g 10 * production per µ 5 g/mL g/mL Glucose Glucose µ ( 0 Basal Glucagon Forskolin EX4

B DMSO IPA3 (1 μM) IPA3 (20 μM)

18 * * ** ** 16 * *

14 ** 12 * protein)

g 10

production 8 per µ 6

Glucose Glucose 4 (µg/mL (µg/mL 2 0 Basal Forskolin (1 μM) Forskolin (10 μM) C

20 * ** # ##

protein) 15 Wildtype, DMSO ug *** * Wildtype, IPA3 * 10 Pak1-KO, DMSO # # Pak1-KO, IPA3

Glucose productionGlucose 5 (μg/mL (μg/mL per

0 Basal Glucagon Forskolin

Fig. 6.2 The group I Pak inhibitor IPA3 represses glucose production in primary hepatocytes. (A) IPA3 attenuated basal, as well as glucagon- or forskolin-stimulated glucose production in primary hepatocytes. (B) IPA3 dose-dependently attenuates basal and forskolin-stimulated glucose production in primary hepatocytes. (C) Primary hepatocytes isolated from wild-type and Pak1-/- mice were stimulated with glucagon or forskolin, in presence or absence of IPA3, and followed by glucose production assay. In wild-type hepatocytes, IPA3 attenuate the glucagon- and forskolin-stimulated glucose production. Pak1-/- hepatocytes have reduced basal glucose production, and IPA3 blunted the stimulation of glucagon and forskolin on glucose production. Each experiment was performed using triplicate wells, n = 3 experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; # p < 0.05, # # p < 0.01, # # # p < 0.001 which indicates the comparison to the basal level of each respective treatment in (C). 149 A Wild-type Pak1-KO B Wild-type Pak1-KO *** 4 4

3 3 ** 2 2

Fold change Fold 1

Fold changeFold 1

0 0 Pck1 G6pc Pck1 G6pc

C D Basal Forskolin Forskolin + Insulin Basal Forskolin Forskolin + Insulin 4 * 40 ** *

* * 3 * 30 ** 20 ***

fold change) 2 fold change) ( (

* Pck1 1 G6pc 10

0 0 DMSODMSO IPA3 IPA3 DMSODMSO IPA3 IPA3

Fig. 6.3 IPA3 represses gluconeogenic gene expression in primary hepatocytes (A) Aged mice liver pck1 and g6pc mRNA levels (wild-type mice, n=4, Pak1-/- mice, n=5) (B) Young mice liver pck1 and g6pc mRNA levels (wild-type mice, n=6, Pak1-/- mice, n=6) (C) IPA3 attenuates forskolin-stimulated pck1 mRNA levels in mouse primary hepatocytes (D) IPA3 attenuate forskolin-stimulated g6pc mRNA levels in mouse primary hepatocytes. The aged mice were 14 months old and the young mice were10 weeks old. Age-matched wild-type and Pak1-/- mice were used within each set of experiment. For mouse primary hepatocyte studies, each experiment was performed using triplicate wells, n = 3 experiments. All target gene expression levels were normalized to ribosomal 18S mRNA levels. * p < 0.05, ** p < 0.01, *** p < 0.001.

150 A

IPPTT IPPTT

Number of weeks (forskolin injection) 0 1 2

B IPPTT 18

16 Wild-type Pak1-KO 14 ** 12 2000 ### ** ** 10 # # 1500 *** && ###

& ## 8 ## * & & 1000

& AUC 6 * ** *** 500

*glucose Blood(mmol/L) 4 Wild-type pre-treatment Pak1-KO pre-treatment 0 2 Wild-type post-treatment Pak1-KO post-treatment 0 0 20 40 60 80 100 120 Time (min)

Wild-type Pak1-KO C 2.5 ** ns * 2.0

1.5 * 1.0 (fold)

cg 0.5 G

0.0 PBS Forskolin

Fig. 6.4 Forskolin injection improves IPPTT and increases gut gcg mRNA levels in aged Pak1-/- mice. (A) Schematic of forskolin injection regimen consisting of two weeks of daily i.p. forskolin injection (5mg/kg per day) (B) IPPTT of aged wild-type (n=5) and Pak1-/- mice (n=4) before and after forskolin injection (C) Mice distal ileum gcg expression levels. Nine mice utilized in above IPPTT as well as 10 additional age- matched mice (5 wild type mice and 5 Pak1-/- mice) receiving PBS injection were utilized in this assay. Gcg mRNA expression levels were normalized to ribosomal 18S mRNA levels. In the IPPTT, statistical significance is indicated as follows: * Wild-type mice before versus after treatment; & Pak1-/- mice before versus after forskolin treatment; # wild-type mice after treatment versus Pak1-/- mice after treatment. *,&,# p < 0.05, **,&&,## p < 0.01, ***,&&&,### p < 0.001.

151 A B

40 WT PAK1-KO 0

30 -1 * ** -2 n.s. 20 -3 Body weightBody(g) 10 Wild-type Pak1-KO Changein body weight(g) 0 0 1 2 Weeks (post forskolin injection)

Fig. 6.5 Forskolin injection for one week generated no effect on body weight in aged Pak1-/- mice and wild-type control mice. (A) Body weight measurement in 14 months old Pak1-/- (KO) and aged-matched wild-type (WT) control mice during the two week duration of forskolin injection. (B) Change in body weight in the aged Pak1-/- and wild-type control mice. n.s. indicates not statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

152 A

Blood Euthanasia

IPPTT OGTT IPITT GC

Number of weeks (sitagliptin gavage) 0 1 2 3 4 5

B IPPTT 18

12 Wild-type Pak1-KO

10 2000 * * 8 & 1500

** && 6 && ** AUC *** 1000 Blood glucose Blood(mmol/L) 4 Wild-type, PBS & Wild-type, sitagliptin 500 2 Pak1-KO, PBS Pak1-KO, sitagliptin 0 0 0 20 40 60 80 100 120 PBS Sitagliptin Time (min)

Fig. 6.6 Sitagliptin rescues the IPPTT and OGTT impairments in aged Pak1-/- mice. (A) Schematic of sitagliptin oral gavage regimen consisting of daily gavages (300 mg/kg per day) for a total duration of 5 weeks. During the 5-week treatment period, aged wild-type and Pak1-/- mice receiving either PBS or sitagliptin underwent the tests described in B-F. (B) IPPTT. In the IPPTT, statistical significance is indicated as follows: * Wild-type mice receiving PBS vehicle versus sitagliptin; & Pak1-/- mice receiving PBS vehicle versus sitagliptin. Wild-type mice PBS group n=3, sitagliptin group n=3; Pak1-/- mice PBS group n=4, sitagliptin group n=5). *,&,#,$ p < 0.05, **,&&,##,$$ p < 0.01, ***,&&&,###,$$$ p < 0.001.

153 C OGTT 18 #

# 16

14 $

&& 12 Wild-type Pak1-KO 2000 ** 10 * *** 8 && 1500 &&& && & 6 ** * Blood glucose Blood(mmol/L) 1000 AUC 4 Wild-type, PBS Wild-type, sitagliptin 500 2 Pak1-KO, PBS Pak1-KO, sitagliptin 0 0 0 20 40 60 80 100 120 PBS Sitagliptin Time (min)

D Basal 5min

250 ** *** 200

150

100 * 1 (pg/mL) - 50 GLP 0 PBS Sitagliptin

Fig. 6.6 Sitagliptin rescues the IPPTT and OGTT impairments in aged Pak1-/- mice (continued) (C) OGTT, (D) plasma GLP-1 levels at basal and 5 min post oral glucose challenge. In the IPPTT, statistical significance is indicated as follows: * Wild-type mice receiving PBS vehicle versus sitagliptin; & Pak1-/- mice receiving PBS vehicle versus sitagliptin; # wild-type mice receiving PBS versus Pak1-/- mice receiving PBS; $ wild-type mice with sitagliptin versus Pak1-/- mice with sitagliptin. Wild-type mice PBS group n=3, sitagliptin group n=3; Pak1-/- mice PBS group n=4, sitagliptin group n=5). *,&,#,$ p < 0.05, **,&&,##,$$ p < 0.01, ***,&&&,###,$$$ p < 0.001.

154 E 12 IPITT Wild-type Pak1-KO

1500 10

8 1000

6 AUC Blodd glucoseBlodd (mmol/L) 4 Wild-type, PBS 500 Wild-type, sitagliptin 2 Pak1-KO, PBS Pak1-KO, sitaglliptin 0 0 0 50 100 PBS Sitagliptin Time (min) F

GC 16 Wild-type Pak1-KO

14 1500

10 1000 8

6 Blood glucose Blood(mmol/L) Wild-type, PBS AUC 500 4 Wild-type, sitagliptin 2 Pak1-KO, PBS Pak1-KO, sitagliptin 0 0 0 20 40 60 80 100 120 PBS Sitagliptin Time (min)

Fig. 6.6 Sitagliptin rescues the IPPTT and OGTT impairments in aged Pak1-/- mice (continued) (E) IPITT, (F) Glucagon challenge (GC). Wild-type mice PBS group n=3, sitagliptin group n=3; Pak1-/- mice PBS group n=4, sitagliptin group n=5.

155 40

Body weightBody(g) Wild-type, PBS 10 Wild-type, sitagliptin 5 Pak1-KO, PBS Pak1-KO, sitagliptin 0 0 1 2 3 4 5 Weeks (post sitagliptin gavage)

Fig. 6.7 No changes in body weight during sitagliptin treatment in aged Pak1-/- mice. Sitagliptin was administered consecutively with daily gavages (300 mg/kg per day) for a total duration of 5 weeks. During this time period, body weight was recorded for all the treatment versus control mice groups. Wild-type mice PBS group n=3, sitagliptin group n=3; Pak1-/- mice PBS group n=4, sitagliptin group n=5.

156 50 A * 40 30 20 Bodyweight(g) 10 0 WT KO B Liver (normalized) Liver 0.05

2 * (g) 0.04 1.5 0.03

1 0.02

0.5

Weight(g) 0.01 Weight/bodyweight 0 0 WT KO WT KO C 0.15 Distal ileum 0.004 Distal ileum (normalized)

* (g) 0.003 0.1

0.002 0.05

Weight(g) 0.001

0 Weight/bodyweight 0 WT KO WT KO D Epididymal fat Epididymal fat (normalized) 2 0.05 ** * (g) 1.5 0.04

0.03 1 0.02 Weight(g) 0.5 0.01

0 Weight/bodyweight 0 WT KO WT KO

Fig. 6.8 Aged Pak1-/- mice exhibit smaller epididymal fat pads. (A) Body weight measurement. (B) Absolute and normalized liver weight (C) Absolute and normalized distal ileum weight (5 cm section) (D) Absolute and normalized epididymal fat pad weight. Male aged Pak1-/- knockout (KO) mice were used along with age-matched wild-type (WT) controls. Normalized weights are calculated by adjusting the absolute organ weights with body weight of the animal. WT, n=4. KO, n=5.

157

6.5 Discussion

Although Pak1 is known to mediate insulin-stimulated glucose transport in muscle cells

(333), and its ablation affects the second phase of glucose-stimulated insulin secretion in vitro and ex vivo (453,499), the metabolic defects in young Pak1-/- mice are relatively moderate, especially in the absence of a challenge (468,499). We show here that aged Pak1-/- mice carry a profound defect in IPPTT, which can be drastically improved by GLP-1 activation, either through increasing its production or reducing its inactivation. These, along with the direct repressive effect of IPA3 on glucose production in primary hepatocytes, revealed the important physiological significance of Pak1 in regulating GLP-1 production, especially in aged mice. This study also further supports the therapeutic application of GLP-1R agonists and DPP-IV inhibitors in attenuating hepatic gluconeogenesis. Furthermore, we suggest that aged Pak1-/- mice may serve as a novel model in studying the gut-liver axis or the gut-pancreas-liver axis in metabolic homeostasis.

We found previously that gut gcg transcription and GLP-1 production can be positively regulated by the Wnt ligand Wnt3a (468), as well as the Wnt pathway effector β-cat/TCF7L2

(295,296,479). Pak1 mediates the crosstalk between insulin and Wnt signaling pathways (334).

In the gut, insulin treatment activates Pak1 through stimulating its Thr423 phosphorylation (334).

This activation is associated with increased β-cat Ser675 phosphorylation (334). The interaction between Pak1 and β-cat was also demonstrated to be required for the stimulation of β-cat signaling by gastrins in a gastric mucosa cell line (503,512). Recently, Zhu et al reported that

Rac/Pak1 cascade controls β-cat activation in colon cancer cells, and that Pak1 directly phosphorylates β-cat at the Ser675 residue (487). Furthermore, in mammary epithelial cells, the loss of Pak1 leads to diminished β-cat and its target gene expression (513). We have

158

demonstrated previously that in young Pak1-/- mice, gut Gcg mRNA level was significantly lower than in their control littermates; this was associated with reduced plasma GLP-1 and insulin levels in response to oral glucose challenge (468). We suggest that the defect in gcg expression and GLP-1 production is at least one of the underlying causes responsible for the increased hepatic glucose production in aged Pak1-/- mice, as impaired IPPTT can be efficiently reversed by either forskolin or sitagliptin treatments in vivo. Unlike the direct use of GLP-1 analogues, treatment with sitagliptin enabled us to measure plasma GLP-1 levels as the endpoint and thereby to attribute any changes in GLP-1 levels to the drug treatment. Further studies examining the effect of sitagliptin on hepatic gluconeogenic gene expression will be useful in clarifying the role of DPP-IV inhibitors in suppressing hepatic glucose production. It is worth to point out that forskolin or sitagliptin also significantly improved IPPTT in aged wild type mice, supporting the notion that GLP-1 analogues and DPP-IV inhibitors can be utilized in reducing hepatic gluconeogenesis (507,508).

Extensive studies during the past two decades have shown that the physiological significance of GLP-1 reaches far beyond its role as an incretin hormone (246). Extra-pancreatic functions of GLP-1 include the induction of satiety (227), reduction of gastric emptying (219), cardiac protection (233), hepatic glycogen storage (225), and repression of hepatic glucose production (510). However, the measurement of plasma GLP-1 level has not been recognized as a diagnosis tool for any metabolic disorders. We suggest that this is at least partially due to the fact that current GLP-1 detection methods are not sensitive enough to reveal subtle differences among the subjects. Indeed, GLP-1 levels in plasma samples are within the pg range (5-20 pg/mL), much lower compared with that of glucagon and insulin. While conducting blood GLP-

1 measurements in the transgenic mice utilized in this study, data obtained from different batches of experiments showed significant inter-assay variation.

159

We did not see appreciable defects in the Pak1-/- mice that are related to the extra- pancreatic functions of GLP-1. Surprisingly, aged Pak1-/- mice (over 14 months old) show significantly reduced body weight and epididymal fat when compared with age-matched wild type control animals (Fig. 6.8A and 6.8D). As Pak1 can be activated by different signaling cascades and has a large profile of downstream targets, it is possible that Pak1 exerts its multiple metabolic regulatory effects in tissue specific manners. Some of these effects may be deleterious while others may be beneficial, making the “net outcome” comparable, especially in the absence of a nutritional challenge. To further dissect the metabolic regulatory function of Pak1, cell-type specific gain-of-function as well as loss-of-function approaches will be needed. Furthermore, the three group I Pak members may exert redundant functions. We found that IPA3 inhibited both basal and glucagon- or forskolin-stimulated glucose production in primary hepatocytes of wild type animals. Pak1-/- hepatocytes, however, showed normal response to forskolin and glucagon activation on glucose production, indicating that the functional inactivation of all three Pak members is required to block the stimulatory effect of forskolin or glucagon on hepatic gluconeogenic gene expression. The involvement of Pak2 and Pak3 in gut gcg expression and

GLP-1 production, as well as in regulating glucose production in response to glucagon stimulation in hepatocytes per se, are worth further investigation.

Glucagon and insulin are two major metabolic hormones that control hepatic gluconeogenesis in response to fasting and feeding, respectively. Glucagon also mediates stress signaling in up-regulating hepatic glucose production (503,506). The stimulatory effect of glucagon is mainly mediated by cAMP/PKA activation, followed by increased phosphorylation of CREB and hepatic gluconeogenic gene expression. The repressive effect of insulin on hepatic glucose production is at least partially achieved via inactivating FOXO, which is also among the transactivators of gluconeogenic genes. As both insulin signaling and FOXO signaling may alter

160

during ageing, we aimed to examine whether aged Pak1-/- mice would show more deleterious metabolic defects compared to young animals. Based on the tolerance tests we have conducted, we have principally eliminated the possibility that defect of elevated hepatic gluconeogenesis is due to increased levels of glucagon or sensitivity of hepatocytes to glucagon signaling. We have also ruled out the possibility that elevated hepatic gluconeogenesis is due to reduced sensitivity of hepatocytes to insulin signaling. Thus, we suggest that the defect lies, at least partially, in the gut/liver axis or the gut/pancreas/liver axis. Whether the defect is due to impaired insulin- dependent effect of GLP-1, or impaired insulin-independent effect of GLP-1, or both, needs to be further investigated.

Significant effort has been made to verify whether GLP-1 possesses an insulin- independent effect on hepatic glucose production. An early report revealed that infusion of GLP-

1 resulted in lower rates of hepatic glucose production in human subjects (507). This effect was, at that time, attributed to the incretin effect. A few studies had claimed the difficulty in determining the existence of insulin-independent effects of GLP-1 on peripheral glucose turnover (514-516). However, a very recent pancreatic clamp study in human subjects demonstrated that infusion of physiological post-prandial levels of GLP-1 led to reduced hepatic glucose production by 27%, while no appreciable effect on whole-body glucose disposal was observed (508). We tested here the effect of sitagliptin, which has been shown to attenuate diet- induced adipose tissue inflammation and liver steatosis in a mouse study (517). The beneficial effect of sitagliptin on the liver in glucose homeostasis is worthy of further investigation in human subjects.

As the link between Rho GTPases (Rac1 and Cdc42) and cytoskeleton reorganization,

Fig. 6.9 presents a summary of the metabolic functions of Pak1, as well as our interpretation of

161

the phenotypes observed in the aged Pak1-/- mice. In pancreas, Cdc42/Pak1/Rac is involved in glucose-stimulated insulin secretion. In muscle cells, Pak1 mediates the stimulatory effect of insulin on GLUT4 membrane translocation and glucose uptake. In intestinal L cells, Pak1 is required for insulin-stiumulated gcg expression and GLP-1 production. The direct effect of Pak1, and potentially group I Paks in general, is to positively regulate gluconeogenesis. Both insulin and GLP-1 are known to suppress hepatic glucose production. In aged Pak1-/- mice, reduced gut gcg expression and lower circulating GLP-1 levels contribute to the elevation of hepatic glucose production, potentially by overriding the direct effects of Pak.

Aged Pak1 knockout mice may serve as a novel tool for dissecting the gut/pancreas/liver axis in hepatic glucose production, as well as for the testing of GLP-1 based therapeutics.

6.6 Acknowledgements

Technical assistance for using the plate reader for mouse plasma GLP-1 measurements was provided by Dr. Manuel Gil-Lozano and Dr. Patricia Brubaker, and equipment for detection was supported by the 3D (Diet, Digestive Tract and Disease) Centre funded by the Canadian

Foundation for Innovation and Ontario Research Fund (project number 19442). Technical assistance and hepatocyte isolation pump was kindly provided by Dr. Khosrow Adeli. Technical assistance for mouse primary hepatocyte isolation was provided by Wilfred Ip.

162 p21 GTPases Insulin signaling PKA signaling

Pak1

Insulin secretion  Glucose uptake  GLP-1 production  Glucose production 

Fig. 6.9 Summary of the role of Pak1 in metabolic homeostasis and the phenotypes of Pak1-/- mice. In pancreas, Cdc42/Pak1/Rac is involved in glucose-stimulated insulin secretion. In muscle cells, Pak1 mediates the stimulatory effect of insulin on GLUT4 membrane translocation and glucose uptake. In intestinal L cells, Pak1 is required for insulin- and forskolin-stiumulated gcg expression and GLP-1 production. The direct effect of Pak1, and potentially group I Paks in general, is to positively regulate gluconeogenesis. Both insulin and GLP-1 are known to suppress hepatic glucose production. In aged Pak1-/- mice, reduced gut gcg expression and lower circulating GLP-1 levels contribute at least partially to the elevation of hepatic glucose production, potentially through indirect mechanisms.

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7 General discussions, conclusion, and future directions

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7.1 General discussions

7.1.1 The crosstalk between insulin and Wnt signaling pathways and its effect on GLP-1 production

Gcg encodes several peptide hormones that are involved in glucose homeostasis, among many other functions. Tissue-specific cleavage of the pre-hormone proglucagon by PC1/3 leads to the production of GLP-1 in the intestinal endocrine L cells and in certain neuronal cells in the brain. The transcriptional regulation of gcg involves multiple signaling pathways and their components. Our laboratory has showed that insulin stimulated gcg expression and GLP-1 production in the intestinal L cells (479). Similarly, treatment with the Wnt activator LiCl induced gcg expression and GLP-1 synthesis, both of which were attenuated by the expression of a dominant negative TCF7L2 (295,296,479). Insulin treatment also enhanced the binding of β- cat/TCF7L2 to the G2 enhancer element of the gcg promoter, and overexpression of β-cat stimulated gcg promoter activity (479). These observations collectively indicate that the crosstalk between insulin and Wnt pathways is involved in regulating gut gcg expression and GLP-1 production.

The concept that Pak1 can serve as an effector of insulin/IGF-1 signaling has been exemplified in multiple cell types. In nematodes, Pak1 was shown to act downstream of insulin/IGF-1-PI3K signaling in regulating neuronal migration, a process that also involved

FOXO transcription factors (518). In myeloma cells, HGF and IGF-1 induced cell migration was positively correlated with Pak1/2 activation; IGF-1 stimulated Pak1 Thr423 phosphorylation; and

Pak1/2 depletion resulted in reduced migration (467). Previous research from our group showed that insulin stimulated Pak1 activation in intestinal cancer cells and mouse tissues including the

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intestine (334). In the current study, I found that insulin stimulated Pak1 Thr423 phosphorylation in intestinal and brain gcg-expressing cells. Treatment with the Wnt ligand Wnt3a activated gcg promoter activity and endogenous gcg mRNA expression in the intestinal L cells, and this activation was abolished in the presence of the Pak inhibitor IPA3.

The cAMP-PKA signaling was known to stimulate β-cat Ser675 phosphorylation in several cell lineages (255,256). Our group further identified that insulin/IGF-1signaling is able to stimulate β-cat activation in intestinal cell lineages (334,519). The phenomenon of insulin/IGF-

1-mediated β-cat activation has been reported in multiple other cell types. Insulin and IGF-1 activated β-cat signaling and promoted carcinogenesis in hepatocytes (261). IGF-1 was found to elevate β-cat levels and augment androgen-mediated gene transcription in prostate cancer cells

(520). In pancreatic β cells, insulin stimulated β-cat mRNA production, total cellular β-cat protein levels, and its nuclear translocation (521). In the current study, I found that insulin- stimulated β-cat activation is at least partially mediated through the linker Pak1, where treatment with IPA3 attenuated insulin-mediated β-cat Ser675 phosphorylation. Two very recent studies demonstrated that β-cat can be directly phosphorylated by Pak1 at Ser675 residue in the in vitro setting (487,513), thereby further supporting my hypothesis of the presence of the insulin/Pak1/β-cat axis in the crosstalk.

In the intestinal endocrine L cell line GLUTag, Pak1 has been verified to be the target of

Cdc42 (522). Both Cdc42 and Pak1 were required for insulin-induced actin remodeling, the mechanism underlying GLP-1 secretion (522). Chemical reagents disrupting filamentous actin potentiated insulin-induced GLP-1 secretion, whereas depletion of either Cdc42 or Pak1 is sufficient to attenuate actin remodeling and GLP-1 secretion following insulin treatment (522).

Consistent with my study, insulin was found to induce Pak1 Thr423 phosphorylation (522).

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Cdc42/Pak1 mediated the insulin stimulation through a MEK/Erk-dependent mechanism in potentiating GLP-1 secretion in intestinal L cells (522), in line with the reported role of

MEK/Erk as Pak effectors. These findings suggest that Cdc42/Pak1 is involved in insulin- stimulated GLP-1 secretion in intestinal L cell lines; however, the physiological significance of the Cdc42/Pak1 signaling cascade in mediating GLP-1 secretion in vivo remains to be addressed.

7.1.2 The in vivo role of Pak1 deficiency

The Pak1-/- mice were generated using target disruption of the Pak1 allele in embryonic stem cells, resulting in an allele that contains a neomycin cassette and that lacks a 2kb genomic

DNA encoding the p21-binding domain (404). These mice are viable and fertile, have normal life span, and reproduce at the predicted Mendelian frequency. Hematopoietic analysis showed that

Pak1-/- mice have normal peripheral blood indices and cell counts (404).

In this study, I hypothesized that Pak1 participates in the crosstalk between insulin and

Wnt signaling pathways in regulating gcg transcription. I first examined this hypothesis using gcg-expressing cell lines: two intestinal cell lines (GLUTag and STC-1) and a hypothalamic neuronal cell line (mHypoE-20/2). I then investigated the in vivo role of Pak1 in regulating gcg expression and GLP-1 production using the Pak1-/- mouse model. The Pak1-/- mice exhibited drastically reduced intestinal gcg mRNA levels, decreased distal ileum GLP-1 content, and lower circulating levels of active GLP-1. Although the difference in basal plasma total GLP-1 levels was not substantial, this may be partially due to the technical challenges in GLP-1 detection. In my study, I observed that the quantification of plasma GLP-1 levels is challenging, due to: 1) the relatively low levels of GLP-1 in the circulation as a result of the rapid DPP-IV mediated

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degradation, and 2) the range of sensitivity of the RIA assay is often not sufficient to detect minute changes in GLP-1 concentrations. Clinical studies have conducted simultaneous measurements of GLP-1 and GLP-2, and the changes in plasma GLP-1 and GLP-2 levels were shown to correlate with each other (523,524). Hence, GLP-2 assay may be a useful supplementary method to confirm changes in plasma GLP-1 concentrations; in my study, the reduced circulating GLP-2 levels indirectly support the presence of GLP-1 defect. Furthermore,

GLP-1 measurements in the latter parts of my study (in Chapter 5) were conducted using the more sensitive ELISA method.

GLP-1 and GLP-2 are co-secreted in a 1:1 ratio, and both are targets of DPP-IV-mediated degradation. I was able to show the concomitant reduction of plasma GLP-2 levels in the Pak1-/- mice, which indirectly supports the notion of reduced GLP-1 production and secretion in these mice (details described in Section 7.3.4).

Gcg is known to be expressed in certain neuronal cells within the brainstem and hypothalamus. I showed that Pak1-/- mice have reduced brainstem gcg mRNA levels, and brain neurons isolated from Pak1-/- mice lack the response of insulin-stimulated β-cat Ser675 phosphorylation. My findings collectively suggest that the insulin/Pak1/β-cat crosstalk mechanism regulates gcg expression in both the gut and the brain. The localized impairments in gcg expression and GLP-1 production in the Pak1-/- mice were associated with intolerance to both ip and oral glucose administration, as well as impaired tolerance to pyruvate challenge, demonstrating that Pak1 deficiency leads to global defects in glucose homeostasis.

In addition to metabolic homeostasis, a number of recent reports demonstrate the in vivo role of Pak1 in mast cell immune responses, muscle glucose uptake, pancreatic insulin secretion, synaptic plasticity, and cardiac functions (404,438,525-527).

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Following the generation of the Pak1-/- mice, closer examinations have revealed that genetic deletion of Pak1 resulted in subtle immune defects (404,528). Wild-type mice bone marrow-derived mast cells (BMMCs), which are a type of granulated cells in the bone marrow, showed normal granule release in response to antigen stimulation. BMMCs from Pak1-/- mice, however, exhibited lack of granule response after IgE challenge, accompanied by an inability to disassemble F-actin (404). These defects in allergen-stimulated granule release and in cytoskeleton reorganization were recapitulated in vivo. Passive cutaneous anaphylaxis (PCA) is a localized allergic reaction where mice are injected with intradermal IgE followed by allergen sensitization and dye administration. Dye extravasation can be quantified and represents allergen-induced IgE crosslinking. The Pak1-/- mice show reduced dye quantity and intensity, confirming the in vivo role of Pak1 in mast cell-mediated PCA reaction (404).

The in vitro requirement of Pak1 in GLUT4 vesicle translocation was shown in isolated mouse myotubes almost two decades ago (333). Insulin stimulated Pak1 Thr423 phosphorylation in myocytes, and this stimulation was mediated through Cdc42/Rac and involving the MAPK pathway (333). The in vivo role of Pak1 in glucose uptake was demonstrated in a recent study by

Wang et al. (438). Wild-type and Pak1-/- mice were injected with insulin or control vehicle solution, followed by isolation of skeletal muscle tissue (438). Insulin was confirmed to stimulate Pak1 Thr423 phosphorylation and activation in mouse skeletal muscle (438). Muscle tissue homogenates from Pak1-/- mice showed reduced GLUT4 vesicles in the plasma fraction, indicating a defect in insulin-stimulated GLUT4 vesicle translocation (438). The reduction of

GLUT4 was correlated with attenuated insulin-stimulated Erk activation, but not Akt activation, suggesting that Pak1 ablation selectively impairs the downstream pathways of insulin signaling after its bifurcation. A previous study by our group showed similar selectivity, where insulin- mediated Pak1 activation in the intestine was PI3K- and Erk-dependent, but Akt-independent

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(334). Related to my own study, whether the same selective activation of downstream insulin signaling pathways occurs in insulin-stimulated Pak1 activation in intestinal L cells can be examined further. The use of specific inhibitors against PI3K, MEK, Erk, and Akt will be useful in conducting this line of investigation.

The importance of Cdc42/Pak1/Rac signaling in the second phase of insulin release in pancreatic β cell lines and mouse islets has been demonstrated (453). The GEF protein Cool/PIX, a regulator of Pak1, has also been identified to be expressed in pancreatic β cells and to regulate insulin secretion (529,530). The in vivo role of Cdc42/Pak1 in insulin secretion and glucose homeostasis was recently identified by Wang et al (438). In their study, siRNA-mediated Cdc42 depletion inhibited the second phase of GSIS and Pak1 activation in human islets, and IPA3 attenuated the second phase of GSIS in human islets and MIN-6 cell line (438). Assessment of the Pak1-/- mice showed impaired glucose tolerance and insulin tolerance, and islets isolated from Pak1-/- had reduced second phase insulin secretion in response to glucose stimulation (438).

In my study, the Pak1-/- mice exhibited impaired ip glucose tolerance, and I further revealed the defect in oral glucose tolerance (468). However, I did not observe an appreciable defect in insulin tolerance as reported by Wang et al. A plausible explanation is the substantial age difference of the mice utilized in the two studies. Insulin intolerance is known to worsen with age progression, and since the mice utilized in my study were much younger (7 wks versus 4-6 mo by Wang et al.) this may have been a contributing factor to the discrepancy. Wang et al. showed that Pak1-/- mice islets bear no abnormalities in islet architecture, including islet density, size, and β cell mass (438). Similarly, I showed that Pak1-/- islets had comparable islet density, size, α cell and β cell mass (468). In their study, Wang et al. showed that Pak1-/- mice islets have comparable pancreatic insulin content, glucagon content, and glucagon release (438). My

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functional assessment of the Pak1-/- mice demonstrated that glucose challenge failed to elicit an insulin response, and basal plasma glucagon level was reduced (468).

Rac/Pak1 signaling has been implicated in neuronal and brain development. The Pak1-/- mice have normal gross brain structure, and at the cellular level, neurons from Pak1-/- mice show normal synaptic and spine structures (526). However, synaptic plasticity, as assessed by long- term potential (LTP) and long-term depression (LTD), is notably altered in Pak1-/- neurons (526).

The Pak1-/- neurons showed reduced polymerized F-actin in dendritic spines and misregulated cofilin phosphorylation, suggesting that Pak1 may play a role in synaptic function (526). In my study, Pak1-/- brain neurons lack the response of β-cat activation in response to insulin stimulation, suggesting that Pak1 ablation results in molecular defects in brain neurons.

GLP-1 is known to act centrally in appetite suppression and regulation of energy homeostasis, and it has been suggested that neuronal GLP-1 signaling in the brainstem and hypothalamus mediate these effects (43,226,227,531). In parallel, a substantial body of evidence suggest that insulin as well as GLP-1 exert neuroprotective effects, and that GLP-1 based therapeutics are potential candidates for the treatment of neurological diseases (253,532,533). In my study, the Pak1-/- brain neurons exhibited impaired insulin signaling; however, the Pak1-/- mice did not have abnormalities in body weight gain. Expanding the current study to assess potential functional impairments of the Pak1-/- mice, such as reduced brain GLP-1 production, changes in appetite regulation and energy expenditure, and neurological defects, is worth pursuing.

The involvement of Pak1 in cardiac function has been assessed in two different types of

Pak1-/- mice. Whole body ablation of Pak1 promoted chronic stress-induced cardiac hypertrophy, which was associated with increased Erk activation and reduced PP2A activation (534). A

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different group used a conditional gene deletion system to specifically knockout Pak1 in cardiomyocytes (Pak1cko) (527). The Pak1cko mice had enlarged cardiomyocyte cross-section and increased interstitial fibrosis (527). Transverse aortic constriction is a methodology to examine stress-induced cardiac failure. The Pak1cko mice exhibited several stress-induced heart defects including increased heart weight to tibia ratio, decreased contraction, and cardiohypertrophy, and cardiomyocytes from Pak1cko mice have impaired JNK signaling (527).

My investigations, along with those made by others, have demonstrated the essential requirement for Pak1 in multiple cell types and organs. Pak1 deficiency leads to molecular, cellular, functional, and physiological defects in vivo, and is linked to pathophysiological manifestations in mouse models.

7.1.3 The gut/liver axis or gut/pancreas/liver axis

To date, studies evaluating the existence of direct effect of GLP-1 on hepatocytes have been conflicting, and the question of whether GLP-1R is expressed in hepatocytes has been controversial. An early study showed that GLP-1 at physiological concentrations can stimulate the formation of glycogen in isolated rat hepatocytes (535). The binding of GLP-1(7-36)amide to the plasma membrane has been reported in rat liver and hepatocytes (536). GLP-1R expressed on nerve terminals in the hepatic portal vein were shown to mediate the hepatic effects of GLP-

1(537), and peripheral GLP-1 infusion into the hepatic portal region suppressed appetite (538).

The expression of GLP-1R mRNA and protein has been reported in rat liver tissues (539).

However, other studies do not support a direct role for GLP-1 in hepatocytes. Earlier examination of the tissue distribution of rat GLP-1R mRNA showed that it was not within the

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detection level in hepatocytes (215). In one study, GLP-1 failed to repress glucose output in isolated and perfused rat liver (540). In another study, GLP-1 did not block the gluconeogenic action of glucagon in isolated rat hepatocytes (541). In assessing the glycogenic effects of GLP-

1(7-36)amide, insulin but not GLP-1(7-36)amide was shown to stimulate the incorporation of glucose into glycogen in isolated rat hepatocytes (542).

In my study, Ex-4 treatment alone did not reduce glucose output in isolated mouse hepatocytes. However, it is worth pointing out that recent studies from another group and from our team have demonstrated the function of the nonapeptide GLP-1(28-36) amide in vitro and in vivo. Ip et al. from our laboratory has demonstrated the direct repressive effect of GLP-1(28-36) amide on glucose output in isolated mouse hepatocytes (543). Ip et al. then further demonstrated that GLP-1(28-36) amide repressed hepatic glucose production in mice (543). Another group showed that GLP-1(28-36) amide inhibited weight gain and reduced hepatic TG stores in high fat diet fed mice (128). Thus, the GLP-1 metabolite GLP-1(28-36) amide, previously assumed to be inactive, exerts physiological functions and may do so via an alternate GLP-1R. Comparison of the effects of Ex-4 versus various GLP-1 cleavage products, including the recently identified

GLP-1(28-36) amide, on hepatic glucose production can be further pursued.

Clinical studies in assessing the pancreatic and extra-pancreatic effect of GLP-1 resulted in conflicting conclusions. GLP-1 administration enhanced insulin-independent glucose disposal in healthy subjects (544) and in elderly T2D patients (545). The insulinomimetic effect of GLP-1 was demonstrated through GLP-1 infusion in obese T2D patients, where GLP-1 augmented insulin-mediated glucose clearance (546). On the contrary, a number of other studies contradicted the direct beneficial effect of GLP-1 in men. Infusion of GLP-1 at physiological levels in healthy subjects did not acutely improve insulin sensitivity (547), and it did not

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augment insulin-mediated glucose uptake in healthy subjects (548,549). Alternatively, reported beneficial effects of GLP-1 on glucose elimination were proposed to be dependent on its action on pancreatic glucoregulatory hormones (514). Specifically, the suppressive effect of GLP-1 on hepatic glucose production was attributed to the indirect regulation via insulin and glucagon

(515).

A handful of recent studies generated evidence supporting the direct role of GLP-1 in hepatocytes and the suppression of hepatic glucose production in humans. GLP-1R was found to be expressed in human hepatocytes (550), and was shown to internalize upon GLP-1 stimulation in human hepatocyte cell lines as well as human primary hepatocytes (218). In men, infusion of

GLP-1 during pancreatic clamp reduced endogenous glucose production, confirming the presence of an extra-pancreatic effect in vivo (510). Infusion of GLP-1(9-36)amide during euglycemic clamp potently inhibited hepatic glucose production in healthy human subjects (551).

In my study, the aged Pak1-/- mice exhibited impaired tolerance to ip pyruvate challenge

(IPPTT), suggesting that Pak1 is involved in hepatic glucose metabolism. Treatment with IPA3 led to a reduction in gluconeogenic gene expression and glucose production in isolated mouse hepatocytes, implicating group I Paks as positive regulators of glucose production. Consecutive injection of forskolin, a gcg-stimulating agent, ameliorated the defect in pyruvate intolerance.

Similarly, treatment with the DPP-IV inhibitor sitagliptin completely reversed the defect in

IPPTT. Both these findings support the notion that the impaired IPPTT in aged Pak1-/- mice is at least partially caused by defective gut gcg expression and GLP-1 production. I showed that the aged Pak1-/- mice also exhibited reduced insulin response following glucose challenge. Hence, it is difficult to distinguish between the pancreatic versus extra-pancreatic effects of GLP-1 in my study. However, considering the controversies revolving GLP-1 on hepatic glucose production,

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my study provided a foundational assessment of the role of Pak1 in GLP-1 production and hepatic glucose metabolism. The generation of tissue-specific Pak1-/- mice can be utilized as mouse models to study direct versus indirect Pak1 and GLP-1 effects in future studies.

Furthermore, specific deletion of Pak1 in gut endocrine L cells, or the expression of a dominant negative Pak1 construct in the gut endocrine L cells, will be useful in vivo models in assessing tissue-specific function of Pak1.

7.1.4 Redundant functions of group I Paks

The group I Pak inhibitor IPA3 is an allosteric non-ATP-competitive inhibitor against

Paks 1-3, while having no effect on the more distantly-related Paks 4-6 (365). In my investigation, IPA3 treatment attenuated insulin-stimulated gcg expression in intestinal L cells.

However, one cannot rule out the possibility that Paks 1-3 may act in a redundant manner in vitro, and more importantly, in vivo as well. Considering this functional redundancy, the physiological significance of the insulin/Pak1/β-cat axis in gut GLP-1 production may have been undermined in the Pak1-/- mice. To examine overall group I Pak function, knockout mouse models lacking multiple Pak isoforms can be generated.

Notably, I have shown that the intestinal endocrine L cell line GLUTag expresses all three members of the group I Paks. In addition to pharmacological approaches (i.e. the use of

IPA3), I also utilized dominant negative Pak1, which leads to the functional knockdown Pak1 only (without affecting Pak2 and Pak3). In order to further identify the actions of each Pak member in the in vitro setting, more sophisticated knockdown approaches are required. There is strong interest in the development of Pak inhibitors as anti-cancer drugs. However, currently

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there are no reported effective Pak inhibitors for clinical use; and for research purposes, no compound has been identified that targets a single Pak isoform (552). Hence, one may turn to genetic approaches, such as small interference RNA (siRNA) or small hairpin RNA (shRNA) based methods. In expanding the current study, one can knockdown the expression of each of the three Paks in vitro, and determine whether the knockdown affects basal as well as insulin- or

PKA-stimulated gcg expression and GLP-1 production.

The functional redundancies of group I Pak isoforms have been reported in vivo. The

Pak1-/- mice do not exhibit phenotypic abnormalities, but closer examination reveals defects in synaptic transmission (526). The Pak3-/- mice are healthy, fertile, and possess normal locomotor functions (415). However, Pak3-/- mice have significantly reduced late phase LTP, and display deficient memory retention in taste aversion tests, both of which are events associated with mental retardation (415). Notably, a lesion in PAK3 gene is associated with X-linked nonsyndromic mental retardation in humans (383). Unlike the relatively subtle deficits observed in Pak1-/- mice and Pak3-/- mice, the Pak1-/-;Pak3-/- double-knockout (DKO) mice have drastic loss of brain volume (499). The DKO mice also exhibited functional synaptic defects, including altered cofilin activation and actin filament properties, and behavioral defects, such as memory retention deficits and hyperactivity (499).

Using isolated mouse hepatocytes, I showed that IPA3 pretreatment attenuated the stimulatory effect of glucagon or forskolin on gluconeogenic gene expression. Combined with the observation that the Pak1-/- mice have worsened tolerance to ip pyruvate, one may conclude that the defect in hepatic glucose homeostasis is not due to the lack of Pak1 in the liver. My study demonstrates that the direct effect of group I Paks on hepatocytes is to function as positive regulators for gluconeogenesis. As hepatocytes isolated from Pak1-/- mice showed normal

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response to glucagon or forskolin treatment, I speculate that either Pak2, or Pak3, or both Pak2 and Pak3, are positive regulators of hepatic gluconeogenesis and can compensate for the lack of

Pak1. To verify this speculation, the siRNA or shRNA knockdown approaches can be utilized.

7.1.5 Pak1-/- mice as a novel model for metabolic and aging studies

As outlined in the previous sections, functional metabolic defects were observed in multiple organs in the Pak1-/- mice. Ablation of Pak1 led to attenuated insulin-stimulated glucose uptake in skeletal muscle and impaired second phase of GSIS in the pancreas. Expanding the investigation into the gut and liver, my studies demonstrated that Pak1-/- exhibited reduced levels of circulating active GLP-1, glucose intolerance, and aberrant hepatic glucose production.

Altogether, these observations corroboratively present Pak1-/- mice as a suitable model for examining metabolic homeostasis.

In my study, the defective GLP-1 production underscoring oral glucose intolerance and dysregulated hepatic glucose production was observed in significantly aged Pak1-/- mice. Aging is associated with multiple dimensions of the metabolic syndrome, including T2D, hypertension, and macrovascular diseases (553). Aging is a known risk factor for deteriorating glycemic control, and is associated with a progressive increase in T2D prevalence (554,555). This correlation was originally simply attributed to impaired β cell function and insulin response

(556,557). As the incretin effect is centrally involved in regulating glucose homeostasis, it stands to reason that GLP-1 production and/or function may be involved in age-related T2D pathophysiology.

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In humans, aging was suggested to be associated with a modest slowing of gastric emptying (558,559), which is a major determinant in postprandial timing and intensity of GLP-1 release. The GLP-1 response following a mixed meal was shown to be reduced in T2D patients

(560), and a longitudinal study demonstrated the correlation between reduced plasma GLP-1 levels and impaired glucose tolerance in elderly T2D men (497).

My study showed that aged Pak1-/- mice exhibited blunted insulin response following glucose challenge, and this may be due to reduced β cell mass. Supporting this line of thinking, age-dependent loss of β cell regeneration has been documented in mice (561,562) and in human

(563). I also showed that the aged Pak1-/- mice have lower basal glucagon levels, with no further reduction following glucose challenge. This is consistent with a previous study, where pancreases from young versus aged Zucker rats were perfused to examine age-related changes in pancreatic glucagon secretion (564). High glucose treatment suppressed glucagon secretion in pancreas from young rats, whereas basal glucagon secretion was lower and glucose did not elicit any response in pancreas from aged rats (564). Together, these observations imply that there may be age-dependent overall deterioration in pancreatic hormone production.

Wistar rats develop glucose intolerance and diminished insulin response as they progress with age (565,566). Notably, GLP-1 delivery via osmotic pumps or direct infusion in aged

Wistar rats resulted in improved glucose tolerance and enhanced insulin response to glucose challenge, suggesting that exogenous GLP-1 administration is able to reverse age-related glucose intolerance (567,568). The same phenomenon was observed in mice, where administration of a long-acting GLP-1R agonist led to improved glycemic response, elevated GSIS, and enhanced insulin sensitivity (569).

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In line with these observations, my study provided extended evidence for the beneficial role of GLP-1 in regulating glucose homeostasis in aged animals. I showed that administration of forskolin in aged Pak1-/- mice lead to increased gcg expression associated with improved pyruvate tolerance. More importantly, administration of sitagliptin resulted in the concurrent improvement of pyruvate tolerance and oral glucose tolerance in the aged Pak1-/- mice. The effectiveness of sitagliptin utilized in my study was demonstrated by the significantly elevated circulating GLP-1 levels in the aged Pak1-/- mice receiving this treatment.

The majority of aging-related clinical studies focus on the pancreatic effects of GLP-1, and it has been demonstrated that the β cell response to GLP-1 is impaired in healthy elderly subjects (570,571). However, it has also been suggested that age-dependent loss of glycemic control may be in part due to alterations in hepatic glucose production (553). As noted previously, the potential beneficial effects of GLP-1 on hepatic glucose homeostasis in humans is being actively investigated. In my study, I examined the role of GLP-1 in hepatic glucose homeostasis using the aged Pak1-/- mice as a novel mouse model. My study reaffirms the clinical importance of GLP-1 based therapeutics as a glucose-lowering agent, particularly in elderly T2D patients.

7.2 Overall importance of study and conclusion

The overall summary of this study is illustrated in Fig. 7.1. In examining the proliferative role of insulin, our group has previously reported the role of Pak1 mediating the crosstalk between insulin and Wnt signaling pathways in the context of colorectal cancer. In this study, we aimed to investigate the function of Pak1 in incretin hormone production and glucose

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homeostasis. We demonstrated the novel role of Pak1 as a central linker in the crosstalk between insulin and Wnt signaling pathways; and as a regulator of gcg expression in gut and brain gcg- expressing cells.

Ablation of Pak1 in mice led to reduced intestinal gcg expression and lower circulating postprandial GLP-1 and insulin levels, and culminated in glucose intolerance even in the absence of challenge. In aged Pak1-/- mice, a defect in elevated hepatic glucose production was observed by more severe intolerance to intraperitoneal pyruvate injection, which was accompanied by reduced plasma GLP-1 levels. However, treatment with IPA led to reduced gluconeogenic gene expression and glucose production in mouse primary hepatocytes, suggesting that the direct effect of Pak is to stimulate glucose production. It is plausible that the impairment in pyruvate tolerance may be due to the secondary effect of defective GLP-1 production, which is known to suppress hepatic glucose output. Overall, the Pak1-/- mice exhibit phenotypes that depict a clear picture of aberrant glucose homeostasis. Together with the known essential function of Pak1 in muscle glucose uptake and pancreatic insulin secretion, the findings of this study reiterate and also expand the important role of Pak1 in glucose homeostasis in exerting its effects in multiple organs.

180 A Peptide hormones Insulin Wnt

GPCR RTK 7-TMR

PI3K cAMP GSK3β Pak1

PKA β-cat

TCF/β-cat CREB

gcg Hepatic glucose GLP-1 production B

Glucose homeostasis

Hormone gene Hormone production expression Pak1

Hepatic glucose Glucose uptake production

Fig. 7.1 Overall summary and significance of study. (A) This study identified the role of Pak1 as a regulator of gcg expression, GLP-1 production, and as a linker in the crosstalk between insulin and Wnt signaling pathways. Ablation of Pak1 in mice led to reduced intestinal and brain gcg expression, and lower circulating postprandial GLP- 1 and insulin levels, culminating in glucose intolerance. In aged Pak1-/- mice, a defect in hepatic glucose production is associated with and implicated to be caused by reduced plasma GLP-1 levels. (B) Our characterization of the Pak1-/- mice present a combination of defects: impaired glucose tolerance, reduced circulating insulin and GLP-1 levels, and aberrant hepatic glucose production, a potential secondary phenomenon due to the defect in GLP-1. Overall, these phenotypes depict a clear picture of dysfunctional glucose homeostasis. Together with the known functions of Pak1 in muscle glucose uptake and insulin secretion, the findings of this study reiterate the important role of Pak1 in glucose metabolism and homeostasis in the physiological context. 181

7.3 Future work

7.3.1 Liver-specific Pak1 knockout mice

My interpretation of the observations made in the aged Pak1-/- mice is that reduced GLP-

1 contributes to elevated hepatic glucose production. I further demonstrated that enhancement of

GLP-1 levels, either through stimulating gcg expression, or through attenuating GLP-1 degradation, was correlated with significant amelioration of the hepatic defect in the aged Pak1-/- mice. Concurrent with the reduction in circulating GLP-1 levels, an attenuated insulin response following glucose challenge was observed in the aged Pak1-/- mice. In order to examine the direct in vivo effect of Pak1 in the liver, while circumventing the contributions of GLP-1 and insulin, the generation of liver-specific Pak1-/- (L-Pak1-/-) mice will be required. The transgenic mouse line that expresses Cre recombinase under the liver-specific albumin promoter (Alb-Cre) is commercially available, and crossing the Alb-Cre line with the Pak1-/- mouse line would produce the L-Pak1-/- mouse line required for these further studies. Based on the suppressive effect of IPA3 on glucose production in mouse primary hepatocytes (as described in Aim III), I speculate that the L-Pak1-/- mice would exhibit reduced hepatic glucose production and improved

IPPTT.

7.3.2 IPA3 as a potential glucose-lowering drug

The currently identified Pak inhibitors are classified into two major categories: ATP- competitive inhibitors and allosteric inhibitors. The ATP-competitive inhibitors were the first ones to be discovered, and consist of chemical compounds that target the ATP-binding pocket

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within the catalytic domain of all Paks (572). In recent years, a group of compounds have been identified that interact with Pak outside of its catalytic domain, and hence are non-ATP- competitive.

IPA3 was discovered as a potent, non-ATP-competitive, allosteric inhibitor that targets group I Paks (365). It is a symmetric dimer joined by a disulfide bond, which targets the PBD of

Paks. IPA3 binds covalently to the Pak1 regulatory domain and prevents its binding to activator

Cdc42, thereby directly inhibiting Cdc42-induced Pak1 auto-phosphorylation at Thr423.

However, pre-activated Pak1 neither binds to nor is inhibited by IPA3. When screened against a kinetome, IPA3 exhibited high kinase specificity, where less than 4% of the kinases were shown to be inhibited by IPA3.

My study demonstrated that IPA3 potently attenuated forskolin- and glucagon-stimulated glucose production in isolated mouse hepatocytes. The standard concentration of IPA3 used in this study was 10-20 μM; however, I also found that IPA3 elicited comparable responses at the much lower dose of 1 μM. A previous study showed that the IC50 of IPA3 was 2.5 μM, and that

IPA3 at a concentration of 10μM was able to inhibit ~95% of Pak1 kinase activity (365).

Therefore, the potency and selectivity of IPA3 makes it an ideal candidate for clinical use, including in the treatment of cancer or potentially metabolic diseases.

Based on my finding of the glucose-lowering ability of IPA3 on isolated hepatocytes, one would need to further assess the effect of IPA3 in vivo. Currently, there are no reports on the metabolic effects of IPA3 in vivo, although a few reports exist in the literature on the effect of

Pak inhibitors in rodent models. One study showed that administration of IPA3 reduced airway responsiveness in mice, indicating its potential use as a hyper-ventilative treatment (573).

FRAX486, another group I Pak inhibitor, rescued seizures and behavioral abnormalities when

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administered to a mental retardation mouse model (574). Another related compound, the group I

Pak inhibitor FRAX597, was found to inhibit tumorigenesis of NF2-associated schwannomas in immune-compromised mouse models (575).

In expanding my research to study the effect of IPA3 in vivo, and in order to examine liver-specific effects of IPA3, targeted delivery of IPA3 to the liver will be needed. Extensive research is underway to design liposome-based and polymer-based targeted drug delivery to the diseased liver. In mice, injection into tail vein has been used to deliver and target nucleic acid based substances to the liver (576). This approach may be used to target IPA3 to the liver in mice, and to examine whether IPA3 administration leads to reduced hepatic glucose production in vivo. In addition to IPPTT, other more sophisticated techniques would include directly assessing endogenous/hepatic glucose production using pancreatic clamp techniques. Ultimately, the clinical potential of IPA3 as a glucose-lowering drug can only be fully realized if a suitable route of administration can be found, if the effective pharmaceutical dosage can be achieved and sustained, and if the drug has tolerable toxicity in the liver and the body overall. As noted previously, there is currently very active interest in developing Pak inhibitors as anti-cancer drugs, and hence their therapeutic potential in the treatment of metabolic disorders can be investigated in parallel.

7.3.3 The role of Pak1 and Wnt signaling in adipogenesis

The role of the Wnt pathway in adipogenesis has been recognized for over a decade.

Adipogenesis is the process where preadipocytes differentiate into adipocytes, which is the major cell type for lipid storage and metabolism. The transcriptional cascade for adipogenesis has been

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well characterized, with peroxisome proliferator-activated receptor γ (PPARγ) and the

CCAAT/enhancer binding protein α (C/EBPα) being chief regulators of the adipogenic program.

PPARγ is both necessary and sufficient for adipocyte differentiation(577), while C/EBPα is implicated in regulating adipocyte insulin sensitivity (578). For a few recent reviews please refer to (191,577,579). Adipogenesis is regulated by external stimuli, one of which is the recently identified involvement of the Wnt signaling pathway. In addition to adipogenesis, the Wnt signaling has also been implicated in regulating adipocyte function (580-584).

In preadipocyte cell lines, expression of Wnt1, an activator of Wnt signaling, potently represses adipogenesis (580,584). Administration of pharmacological Wnt activators produce similar results (580,581). Conversely, inhibiting Wnt signaling in preadipocytes stimulates differentiation (580,581,585-587). Preadipocytes produce endogenous Wnts, one of them being

Wnt10b. Overexpression of Wnt10b induces cytosolic β-cat accumulation and the inhibition of adipogenesis (580). The mechanisms underlying Wnt-mediated repression of adipogenesis is not entirely understood; however, it has been suggested that β-cat is the central mechanism for inhibiting adipogenesis. For example, Cby, a nuclear β-cat antagonist, was found to be expressed in adipose tissues. Ectopic Cby expression led to induced differentiation of the preadipocyte cell line 3T3-L1, while Cby depletion resulted in enhanced β-cat activity and inhibition of 3T3-L1 cell differentiation (585). Similarly, expression of another inhibitor of Wnt/β-cat signaling,

Dickkopf-1, promoted 3T3-L1 cell differentiation (587). The in vivo role of Wnt signaling is beginning to be elucidated. Overexpression of Wnt10b in mice leads to about 50% reduction in adiposity, and these mice are resistant to diet-induced and genetic obesity (582,583). Mice expressing the Wnt10b transgene also exhibit improved glucose homeostasis and enhanced insulin sensitivity (582,583).

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In line with the observations that modulators of β-cat are integral in regulating adipogenesis, and considering my previous studies on identifying Pak1 as a regulator of β-cat in multiple cell types and organs, it is probable that Pak1/β-cat signaling is involved in regulating adipocygenesis. In my previous studies, organ weight measurements showed that aged Pak1-/- mice have reduced epididymal fat pad weights (Fig. 6.8D), suggesting that ablation of Pak1 may lead to abnormal fat pad distribution.

To determine whole-body adiposity, I performed magnetic resonance imaging (MRI) in the same animals used for organ weight measurements. Appendix 1A (page 242) shows the whole-body fat volume measurements of the two groups of mice, where the aged Pak1-/- mice show about 25% reduction in whole-body fat volume. Appendix 1B (page 242) depicts representative MRI serial scans of one wild-type mouse (Mouse 3, top) and one knockout mouse

(Mouse 4, bottom). The reduction of epididymal fat pad and whole-body fat in the aged Pak1-/- mice would implicate that Pak1 is a positive regulator of adipogenesis.

In order to specifically examine the levels of intra-hepatic fat, spectral analysis was performed during the MRI scanning. Appendix 2 (page 243) shows that the hepatic fat content was comparable between the aged Pak1-/- mice and wild-type control mice. These preliminary data suggest that Pak1 ablation primarily causes manifestations in abdominal and subcutaneous fat, while having no substantial effect on visceral fat depots.

Based on the current literature, Wnt signaling is commonly viewed as a negative regulator of adipogenesis. As Pak1 has been identified as a stimulator of β-cat activity in multiple cell types and organs by our group and others, one would predict that Pak1 ablation would lead to an increased adiposity in mice. Potentially, our observation that the aged Pak1-/-

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mice have reduced and not elevated adiposity could be due to the defective gcg expression and

GLP-1 production.

GLP-1 has been reported to function as an intestinal signal in adipocyte biology.

Treatment with GLP-1 or its long-lasting analog Liraglutide were shown to dose-dependently activate GLP-1R/Erk/PKC/Akt in 3T3-L1 cells, and injection of Liraglutide in mice led to increased adipocyte numbers (588). Conversely, GLP-1R depletion resulted in reduced adipogenesis (588). In another study, GLP-1 treatment stimulated PPARg protein levels and

C/EBPa gene transcription, accompanied by increased numbers of small adipocytes in 3T3-L1 cells, suggesting that GLP-1 promotes adipogenesis (589). Hence, defective gut gcg expression resulting in reduced circulating GLP-1 levels in the Pak1-/- mice could lead to impaired adipogenesis.

In order to assess the direct effect of Pak1 inhibition or ablation on adipogenesis, further studies will need to be conducted. The use of the Pak inhibitor IPA3 in preadipocyte cell lines may elucidate the direct effect of Pak on adipocyte formation and differentiation, including the examinations of the effect of IPA3 on adipogenic gene expression and on PPARγ and C/EBPα expression and activity. Further assessment of the Pak1-/- mouse model, utilizing GLP-1 enhancing agents such as sitagliptin or direct GLP-1 analog administration, would further confirm whether the observed phenotypes of reduced epididymal fat pad and whole-body adiposity can be rescued and hence are indeed due to the defect in GLP-1. Due to the scope of my current study and its limitations, these experiments will be treated as future directions.

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7.3.4 GLP-2 as an intestinotrophic factor and as a treatment for intestinal diseases

GLP-2 was initially identified as a trophic factor of the intestine based on case reports of patients with PDGF-secreting tumors with associated small bowl mucosal hypertrophy

(120,590,591). GLP-2 is a 33 aa single-chain polypeptide, produced by PC2-mediated cleavage of proglucagon in intestinal L cells. To date, the major reported physiological function of GLP-2 is the stimulation of intestinal crypt cell proliferation (592,593), although GLP-2 has also been shown to regulate intestinal lipoprotein metabolism (594). The intestinotropic effect of GLP-2 was demonstrated in rodent species (595), and elevated expression of GLP-2 receptor was observed in gastrointestinal tumors such as Crohn’s disease (596). Treatment with GLP-2 in mice resulted in tropic and anti-apoptotic effects, as observed through increased cellularity of the intestinal epithelial layer and intestinal weight (120,597). The effect of GLP-2 on gut lipoprotein metabolism was demonstrated in a hamster model, where intravenous administration of GLP-2 led to reduced apolipoprotein B48 and TG levels, while administration of GLP-1 resulted in opposite effects, implicating the two glucagon-like peptides as opposing factors in regulating intestinal lipoprotein production (594).

Teduglutide is a GLP-2 analog with a glycine-to-alanine substitution at position 2, rendering it more resistant to DPP-IV mediated degradation and hence possessing a longer half- life than native GLP-2 (598). Teduglutide has been regarded as a promising treatment for short bowel syndrome, a type of malabsorption disorder caused by inflammatory bowel disease or intestinal resections (598,599). Several studies have examined the role of GLP-2 in cancer initiation or progression, and have suggested that the proliferative function of GLP-2 involves

Wnt signaling and the PI3K/Akt pathways (593,600,601).

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In my study, the aged Pak1-/- mice have reduced distal ileum weight; importantly, this was associated with lower circulating GLP-2 levels (Appendix 3, page 244). This finding implicates the causative relationship between GLP-2 levels and intestinal growth in the aged

Pak1-/- mice. To further verify reduced GLP-2 levels, one can quantify intestinal GLP-2 content using immunostaining techniques. To further identify the intestinal defects, one can examine morphological abnormalities of the intestinal tissues, measure crypt-villus height of the IECs, and quantify cell proliferation within the various intestinal cell layers using markers such as Ki67 staining.

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9 Appendices

240

9.1 Aged Pak1-/- mice exhibit reduced whole body fat

This is described in Appendix 1, on page 242.

9.2 Aged Pak1-/- mice have comparable hepatic fat content

This is described in Appendix 2, on page 243.

9.3 Aged Pak1-/- mice have reduced circulating GLP-2 levels

This is described in Appendix 3, on page 244.

241 A

10000 *

8000

6000

4000 body fat volume fatvolume body (mm^3) - 2000

Whole 0 Wild-type Pak1-KO

type) - Mouse 3 (wild Mouse

KO) - Mouse 4 (Pak1 Mouse

Appendix 1 Aged Pak1-/- mice exhibit reduced whole body fat. (A) Magnetic resonance imaging (MRI) was performed in aged mice. Adipose tissue was measured and was used to calculate whole-body fat volume. The Pak1-/- (KO) mice have moderately reduced whole-body fat volume (B) Serial MRI scans of one representative wild-type (Mouse 3, top) and one Pak1-/-animal (Mouse 4, bottom) are shown. WT, n=4. KO, n=5.

242 A B Mouse 3 (wild-type)

3E7 2E5 MRI hepatic fat 3 0 2E7 2.5 -2E5 1E7 1.5 1 0.5 2 Frequency (ppm) 1.5 0 1 10 7.5 5 2.5 0 0.5 Frequency (ppm)

Hepaticfatcontent(%) 0 Wild-type Pak1-KO Mouse 4 (Pak1-KO)

2E7 3E5 1.5E5 0 1E7 1.5 1 0.5 Frequency (ppm) 0 10 7.5 5 2.5 0 Frequency (ppm) C Liver triglyceride

5 protein)

ug 3 per 2 Triglyceride concentration dL 1 mg/ ( 0 Wild-type Pak1-KO

Appendix 2 Aged Pak1-/- mice have comparable hepatic fat content. Magnetic resonance imaging (MRI) was performed in aged mice, followed by spectral analysis of hepatic fat content and liver triglyceride measurement. (A) Hepatic fat content of aged wild- type (WT) and Pak1-/- (KO) mice, (B) Representative spectral analyses of one WT mouse (Mouse 3, top) and one KO mouse (Mouse 4, bottom), (C) Liver triglyceride content. WT, n=4. KO, n=5.

243 1.5 *

Wild-type /mL) -/- ng 1.0 Pak1 2 ( - GLP 0.5 Plasma Plasma

0.0

Appendix 3 Aged Pak1-/- mice have reduced circulating GLP-2 levels. Fasting plasma GLP-2 levels were measured using Yanaihara ELISA kit (YK142). Wild-type, n=10; Pak1-/-, n=12.

244