THE ROLE OF P21-ACTIVATED PROTEIN KINASE 1 IN METABOLIC HOMEOSTASIS by 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 © Copyright by Yu-ting Chiang 2014 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 non- endocrine 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 co- localized 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 ii 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. iii 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) iv 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.......................................