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 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 Pak1ꢀThr423ꢀandꢀβ-cat

Ser675ꢀphosphorylation.ꢀTheꢀstimulationꢀofꢀinsulinꢀonꢀβ-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

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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 10^th 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) 

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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 1.1.2.2 1.1.2.3 1.1.2.4 1.1.2.5 1.1.2.6 1.1.2.7
Hormones................................................................................................................................................................................................ 4 The pancreas and islets of Langerhans..................................................................................................................................... 5 Insulin....................................................................................................................................................................................................... 6 Glucagon ................................................................................................................................................................................................11 Somatostatin........................................................................................................................................................................................13 Pancreatic polypeptide...................................................................................................................................................................15 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 1.4.1.2 1.4.1.3
The discovery of Paks 1-3.............................................................................................................................................................48 The discovery of Paks 4-6.............................................................................................................................................................52 Structural features, activation mechanisms, and upstream regulators of Paks .................................................53

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1.4.1.3.1 1.4.1.3.2 1.4.1.3.3
Structural features of Paks.....................................................................................................................................................53 Activation mechanism of group I Paks.............................................................................................................................55 Positive regulators of Pak1....................................................................................................................................................58
Negative regulators of Pak1.........................................................................................................................................................59 Substrate specificity.........................................................................................................................................................................62 The role of Paks in tumorigenesis and cancer ....................................................................................................................62 The functions of Paks ......................................................................................................................................................................64
Cell cycle progression...............................................................................................................................................................64 Cell survival and apoptosis....................................................................................................................................................66 Cytoskeleton remodeling........................................................................................................................................................67 Host-pathogen response.........................................................................................................................................................69 Gene transcription and mRNA splicing............................................................................................................................70 Endothelial and vascular biology........................................................................................................................................72 Metabolic homeostasis.............................................................................................................................................................73
1.4.1.4 1.4.1.5 1.4.1.6 1.4.1.7
1.4.1.7.1 1.4.1.7.2 1.4.1.7.3 1.4.1.7.4 1.4.1.7.5 1.4.1.7.6 1.4.1.7.7

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 pathway s . ...................86

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RATIONALE, HYPOTHESIS, AND RESEARCH AIMS............................................................................. 88

2.1 2.2
RATIONALE.................................................................................................................................................................89 HYPOTHESIS AND RESEARCH AIMS .........................................................................................................................89

GENERAL MATERIALS AND METHODS .................................................................................................. 91

• 3.1
• CHEMICALS AND ANTIBODIES..................................................................................................................................92

WESTERN BLOTTING.................................................................................................................................................92

RNA EXTRACTION AND REAL-TIME QUANTITATIVE REVERSE-TRANSCRIPTASE PCR...................................93

EXPERIMENTAL ANIMALS, MAINTENANCE, AND GENOTYPING...........................................................................94 MOUSE ORGAN WEIGHT MEASUREMENTS .............................................................................................................95 IMMUNOHISTOCHEMISTRY OF MOUSE INTESTINE AND PANCREAS ...................................................................95 STATISTICAL ANALYSES AND DENSITOMETRY ANALYSIS....................................................................................95
3.2 3.3 3.4 3.5 3.6 3.7

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P21-ACTIVATED PROTEIN KINASE 1 MEDIATES THE CROSSTALK BETWEEN INSULINꢀANDꢀΒETA-
CATENIN ON REGULATING PROGLUCAGON GENE EXPRESSION IN THE GUT ................................................. 96

4.1 4.2 4.3
ABSTRACT...................................................................................................................................................................97 INTRODUCTION ..........................................................................................................................................................97 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

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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- stimulatedꢀβ -cat Ser675 phosphorylation.............................. 104

4.5 4.6
DISCUSSION .............................................................................................................................................................111 ACKNOWLEDGEMENTS ..........................................................................................................................................113

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ABLATION OF P21-ACTIVATED PROTEIN KINASE 1 PERTURBS GLUCOSE HOMEOSTASIS ..............114

• 5.1
• ABSTRACT................................................................................................................................................................115

INTRODUCTION .......................................................................................................................................................115 MATERIALS AND METHODS...................................................................................................................................116
5.2 5.3

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 leve l . ......................................................................................................................................................... 119

5.4.3 Pak1^−/− mice have reduced brainstem gcg expression level ................................................................. 121 5.4.4 Pak1^−/− mouse brain neurons show a bolishedꢀresponseꢀtoꢀinsulinꢀonꢀβ -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 5.6
DISCUSSION .............................................................................................................................................................131 ACKNOWLEDGEMENTS ..........................................................................................................................................136