INVESTIGATION OF THE ROLES OF TWO RAC1 EFFECTORS PHOSPHATIDYLINOSITOL-5 KINASE Iα AND P21-ACTIVATED KINASE IN INSULIN-SECRETING CELLS ZHANG JIPING NATIONAL UNIVERSITY OF SINGAPORE 2008 INVESTIGATION OF THE ROLES OF TWO RAC1 EFFECTORS PHOSPHATIDYLINOSITOL-5 KINASE Iα AND P21-ACTIVATED KINASE IN INSULINSECRETING CELLS ZHANG JIPING (B.Sc., SICHUAN University) A THESIS SUBMITTED FOR THE DEGREE OF PHILOSOPHY OF DOCTORATE DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERISTY OF MEDICAL INSTITUTES NATIONAL UNIVERSITY OF SINGAPORE 2008 ACKNOWLEDGEMENTS First of all, I would like to express my sincere appreciation and thanks to my supervisor A/P Li Guodong for his invaluable guidance, support and encouragement during the course of this research This research would not have been possible without his insightful ideas Many thanks to all my fellow lab-mates, Jingsong, Xiefei, Ruihua, Heqing, Songhooi, Shiying and Michelle, in NUMI for the useful discussion sessions that helped me throughout the research My appreciation also goes out to my friends, Shugui, Xiying, Xiaowei and Chenwei who helped me tremendously all along and provided me with many useful inspirations Special thanks to my devoted parents, who offered me with never ending supports, encouragements and the very academic foundations that make everything possible Not to forget my husband who had always cared and encouraged me these years Finally, I would like to thank the National University of Singapore for facilities and generous financial support that makes this research project a success This work was supported by the grant from the National Medical Research Council of Singapore (NMRC 0803/2003) i PUBLICATION LIST Full papers: Zhang J, Luo R, Li GD Effects of knockdown of type Iα phosphatidylinositol-4phosphate 5-kinase on insulin secretion and glucose metabolism in INS-1 β-cells Endocrinology, May 2009, 150(5):2127-2135 Li GD, Luo R, Zhang J, Yeo KS, Lian Q, Xie F, Tan EKW, Caille D, Kon O.L, SaltoTellez M, Meda P, and Lim SK Generating mESC-derived insulin-producing cell lines through an intermediate lineage-restricted progenitor line Stem Cell Res, Volume 2, Issue 1, January 2009, Pages 41-55 Li GD, Luo R, Zhang J, Yeo KS, Xie F, Tan EKW, Caille D, Que J, Kon O.L, SaltoTellez M, Meda P, and Lim SK Derivation of functional insulin-producing cell lines from primary mouse embryo culture Stem Cell Res, Volume 2, Issue 1, January 2009, Pages 29-40 Li J, Luo R, Hooi S, Ruga P, Zhang J, Meda P and Li GD (2005) Ectopic expression of syncollin in INS-1 beta-cells sorts it into granules and impairs regulated secretion Biochemistry-US 44:4365-4374 Zhang J, Luo R, Li GD Knockdown of p21-activated kinase protects insulinsecreting INS-1 cells from high glucose-induced apoptosis in preparation Conference papers: Zhang J, Luo R, Li GD (2008) Attenuation of high glucose-induced INS-1 cell apoptosis by knocking down an isoform of P21-activated kinase (PAK) Diabetes 57(suppl ):A?; Poster presentation at 68th ADA Annual Scientific Sessions, 6-10 June, 2008, San Francisco, CA, USA Zhang J, Luo R, Xie F, Li GD (2007) Knockdown of a downstream effector of the small G-protein Rac by siRNA affects glucose metabolism and insulin secretion in INS-1 β-cells Diabetologia 50 (suppl 1):S88; oral presentation at 43rd Annual Meeting of the European Association for the Study of Diabetes (EASD), Amsterdam, The Netherlands, 17-21 Sep 2007 Zhang J, Teh SH, Luo RH, Xie F, Li, GD (2007) Involvement of type Iα phosphatidylinositol-4-phosphate 5-kinase in glucose-induced insulin secretion in INS1 cells Presented at 67th ADA Annual Scientific Sessions, 21-26 June, 2007, Chicago, IL, USA Diabetes 56(suppl 1): A435 Li GD, Luo R, Kon OL, Salto-Tellez M, Xie F, Zhang J, Lim SK (2006) Differentiation of embryonic progenitor cells into insulin-producing cells for diabetes therapy P.67-8, Abstract Collection Oral presentation at the 5th Asian-Pacific Organization for Cell Biology (APOCB) Congress, P.R China / Beijing, 28-31 Oct 2006 ii Li GD, Luo R, Xie F, Zhang J, Kon OL, Salto-Tellez M, and Lim SK (2006) Deriving insulin-producing cells from the embryonic progenitor for treatment of Type diabetes Mol Biol Cell 17(suppl.):2102 (CD-ROM) Li GD, Luo R, Zhang J, Kon OL, Salto-Tellez M, Xie F, and Lim SK (2006) Insulinproducing Cells Derived from Embryonic Progenitor Cells Reverse Hyperglycemia in Streptozotocin-induced Diabetic Animals Diabetes 55(suppl 1): A22 Invited speaker at ASCB’s 46th annual meeting Li GD, Luo R, Zhang J, Kon OL, Salto-Tellez M, Xie F, Meda P and Lim SK (2006) Generation of insulin-producing cells from embryonic progenitor cells for transplantation in type diabetic mice In: Poster Session Abstracts book, p191 Accepted for presentation at the 4th ISSCR Annual Meeting, 29 June - July, 2006, Toronto, Ontario, Canada Zhang J, Luo R, Kon OL, Xie F, Lim SK and Li GD (2005) Generation and verification of functional insulin-producing cells derived from embryonic progenitor cells Mol Biol Cell 16(suppl.):386a (CD-ROM) Zhang J, Luo R, Kon OL, Xie F, Lim SK and Li GD (2005) Generation and characterization of insulin-producing cells derived from mouse embryonic progenitor Cells Ann Acad Med Sing 34(suppl): S213, Basic Science Poster Award at Combined Scientific Meeting 2005, Singapore 10 Li GD, Luo R, Xie F, Zhang J and Lim SK (2005) Long term propagation and functionality of insulin-producing cells derived from mouse embryos Accepted for presentation at the 3rd ISSCR Annual Meeting, 23-25 June 2005, San Francisco, CA, USA 11 Li J, Zhang J and Li GD (2005) Glucose and forskolin induced translocation of phosphatidylinositol 4-phosphate 5-kinase in insulin-secreting INS-1 cells is coupled with Rac1 activation Diabetes 54(suppl 1): A421 12 Li GD, Luo R, Zhang J, Xie F and Lim SK (2005) Insulin-producing Cells Derived from Mouse Embryo Exhibit Functional Secretory Responses to Secretagogues Diabetes 54(suppl 1): Accepted but withdrawn iii TABLE OF CONTENTS ACKNOWLEDGEMENTS i PUBLICATION LIST ii TABLE OF CONTENTS iv SUMMARY vii LIST OF TABLES AND FIGURES ix ABBREVIATIONS xii CHAPTER INTRODUCTION .1 1.1 General background .2 1.1.1 Diabetes mellitus and β-cell malfunction 1.1.2 Effect of hyperglycemia on β-cells 1.1.3 Insulin biosynthesis 1.1.4 Regulation of insulin secretion 1.1.5 Insulin granules exocytosis 1.2 Rho GTPases, cytoskeleton, insulin secretion and viability of β-cells 1.2.1 Re-organization of cytoskeleton in exocytosis 1.2.2 Regulators of cytoskeleton involved in exocytosis 1.2.3 insulin-secreting cell models for pancreatic β-cell 10 1.2.4 Role of Rho GTPases in exocytosis 11 1.2.5 Role of Rac in cell viability .12 1.3 Effectors act downstream of small G-protein Rac1 13 1.3.1 Phosphatidylinositol-4-phosphate 5-kinase (PIP5K) 14 1.3.1.1 Nomenclature of PIP5K .14 1.3.1.2 Structure and distribution of PIP5K .15 1.3.1.3 Regulation of PIP5K 17 1.3.1.4 Biological function of PIP5K and PIP2 18 1.3.1.5 Potential role of PIP5K in insulin secretion 22 1.3.2 P21-activated kinase (PAK) .23 1.3.2.1 Nomenclature of PAK 23 1.3.2.2 Structure of PAK 24 1.3.2.3 Regulation and activation of PAK 25 1.3.2.4 Downstream effectors of PAK and their biological functions .26 1.3.2.5 Glucotoxicity-induced pancreatic β-cell death 30 1.3.2.6 Potential role of PAK in glucotoxicity-induced β cell death 34 1.4 Aim and significance 35 CHAPTER MATERIALS AND METHODS 37 2.1 Materials .38 2 Methods 42 2.2.1 INS-1 cell culture and storage 42 2.2.2 Molecular biology 43 iv 2.2.2.1 E.Coli transformation 43 2.2.2.2 Plasmid DNA preparation 43 2.2.2.3 RNA purification 45 2.2.2.4 Reverse transcription, polymerase chains reaction and Real-time PCR 46 2.2.2.5 Transfection 48 2.2.2.5.1 Reverse transfection of siRNA duplexes 48 2.2.2.5.2 Transient transfection of GFP-PLC plasmid and PIP2 distribution assay .50 2.2.2.6 Measurement of DNA content .51 2.2.3 Protein assay 51 2.2.3.1 Protein extraction 51 2.2.3.2 Measurement of protein concentrations .52 2.2.3.3 Western Blotting 53 2.2.3.4 Phospho-JNK ELISA 54 2.2.4 Measurement of insulin secretion 55 2.2.5 Observation of cell morphology and assessment of F-actin filaments 56 2.2.6 Measurement of membrane potential 57 2.2.7 Measurement of intracellular Ca 2+ concentration at basal and upon glucose stimulation 58 2.2.8 Glucose metabolism 59 2.2.8.1 Assessment of glucose metabolism by MTS assay 59 2.2.8.2 Glucose oxidation 59 2.2.9 IP3 formation assay 60 2.2.10 Examination of cell growth and death 61 2.2.11 Caspase activity assay 62 2.2.12 ROS assay 63 2.2.13 Statistical analysis 64 CHAPTER RESULTS 65 3.1 The role of PIP5K-Iα in insulin secretion 66 3.1.1 Knockdown of PIP5K-Iα at mRNA and protein level .66 3.1.2 Knockdown of PIP5K-Iα induces changes in cell morphology and cytoskeleton 69 3.1.3 PIP5K-Iα knockdown reduces PIP2 in the plasma membrane and abolishes PIP2 redistribution during glucose stimulation, but has no effect on IP3 formation .73 3.1.4 Knockdown of PIP5K-Iα affects insulin secretion 75 3.1.4.1 PIP5K-Iα knockdown inhibits stimulated insulin secretion but augments basal insulin release .75 3.1.4.2 PIP5K-Iα knockdown inhibits both the early and late phase of insulin secretion .78 3.1.5 Knockdown of PIP5K-Iα affects glucose metabolism .79 3.1.6 Knockdown of PIP5K-Iα depolarizes the basal membrane potential 81 3.1.7 Knockdown of PIP5K-Iα affects intracellular [Ca2+]i 83 3.1.8 Knockdown of PIP5K-Iα does not affect INS-1 cell growth and death 84 3.2 The role of PAK1 in glucotoxicity-induced cell death 86 3.2.1 Reverse transfection of siRNA duplexes knocks down PAK1 86 v 3.2.2 PAK1 knockdown does not affect insulin secretion and actin cytoskeleton in INS-1 cell 87 3.2.3 Knockdown of PAK1 does not affect cell cycle at normal culture 89 3.2.4 Glucotoxicity induces INS-1 cell apoptosis .90 3.2.5 Chronic high glucose treatment increases PAK1 activation 95 3.2.6 Knockdown of PAK1 inhibits glucotoxicity-induced INS-1 cell death 96 3.2.7 PAK1 knockdown blocks high glucose induced activation of p38 MAPK 100 3.2.8 Inhibitors of p38 MAPK and JNK protects INS-1 cells from glucotoxicity-induced apoptosis 103 3.2.9 Expression of a dominant negative Rac1 mutant aggravates high glucose induced cell apoptosis 105 3.2.10 PAK1 knockdown has no effect on glucotoxicity induced oxidative stress 106 CHAPTER DISCUSSION .107 4.1 Roles of PIP5K-Iα in insulin-secreting cells 109 4.1.1 Involvement of PIP5K-Iα in cell morphology and actin cytoskeleton organization in INS-1 cells 110 4.1.2 Role of PIP5K-Iα in insulin secretion in INS-1 cells 111 4.1.3 Implication of PIP5K-Iα in PIP2 production in INS-1 cells 115 4.1.4 Role of PIP5K-Iα in glucose metabolism and membrane potential 118 4.2 The role of PAK1 in glucotoxicity-induced β-cell apoptosis 121 4.2.1 PAK1 knockdown has no effect on either F-actin cytoskeleton or insulin secretion 121 4.2.2 The protective role of PAK1 knockdown from glucotoxicity induced βcell death 123 4.2.3 PAK1 knockdown attenuated glucotoxicity-induced caspase-3 activation 125 4.2.4 The role of PAK1 activators in glucotoxicity 126 4.2.5 PAK1 knockdown blocked p38MAPK activation upon prolonged exposure to high glucose 127 4.3 Conclusions .131 4.4 Future work .132 4.4.1 Future work for the study of PIP5K-Iα 132 4.4.2 Future work for the investigation of PAK1 .134 REFERENCE LIST…………………………………………………………… 135 vi SUMMARY Insulin plays an essential role in the maintenance of homeostasis of blood glucose, which relies on an adequate mass and functional insulin secretion in pancreatic βcells It has been proven that the small G-protein Rac1 participates in glucose- and cAMP-induced insulin secretion This effect is accomplished probably through maintaining a functional actin structure for recruitment of insulin granules Both phosphatidylinositol-4-phosphate 5-kinase Iα (PIP5K-Iα) and p21-activated kinase (PAK1), the downstream effectors of Rac1, are suggested to be involved in mediating the action of Rac1 on actin cytoskeleton remodeling In this thesis study, their potential roles in insulin secretion and cell survival were investigated in INS-1 cell line, a widely-used pancreatic β-cell model Using RNA interference technique, effective knockdown of PIP5K-Iα and PAK1 was achieved by reverse transfection of the targeting siRNA duplexes PIP5K-Iα knockdown disrupted F-actin structure and caused changes in cell morphology In addition, the content of its product, phosphatidylinositol-4,5bisphosphate (PIP2), on plasma membrane was reduced and the glucose effect of PIP2 hydrolysis was abolished by PIP5K-Iα knockdown Although total insulin secretion in response to glucose and other stimuli was increased in PIP5K-Iα knockdown cells, the incremental insulin release over basal (2.8 mM glucose) stimulated by high glucose and forskolin was inhibited However, at resting status, PIP5K-Iα knockdown increased glucose metabolism, depolarized membrane potential, raised cytoplasmic free Ca2+ levels ([Ca2+]i), and doubled insulin secretion In contrast, metabolism and [Ca2+]i rises at high glucose were diminished These results indicate that PIP5K-Iα may play a complex role in both the proximal and vii distal steps of signaling cascades towards insulin secretion in β-cells, besides mediating the effect of Rac1 on actin cytoskeleton organization On the other hand, PAK1 knockdown had no apparent effect on both F-actin cytoskeleton and insulin secretion by various stimuli However, PAK1 knockdown attenuated INS-1 cell apoptosis and caspase-3 activation due to prolonged exposure to high (20 or 30 mM) glucose (glucotoxicity) In addition, glucotoxicity also led to activation of caspase-8 and -9, suggesting the involvement of both extrinsic and intrinsic apoptotic pathways Prolonged exposure to high glucose activated PAK1 and several mitogen-activation protein kinases (MAPKs), including p44/42 MAPK, p38 MAPK and c-jun-N-terminal kinase (JNK) However, it appeared that only p38 MAPK was activated downstream 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PIP5K-Iα in insulin secretion in INS -1 cells 11 1 4 .1. 3 Implication of PIP5K-Iα in PIP2 production in INS -1 cells 1 15 4 .1. 4 Role of PIP5K-Iα in glucose metabolism and membrane potential 11 8... .10 7 4 .1 Roles of PIP5K-Iα in insulin- secreting cells 10 9 4 .1. 1 Involvement of PIP5K-Iα in cell morphology and actin cytoskeleton organization in INS -1 cells 11 0 4 .1. 2 Role of