DIFFERENTIALLY EXPRESSED PROTEINS IN THE PANCREAS OF DIABETIC MICE A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Linghua Qiu June 2005 This dissertation entitled DIFFERENTIALLY EXPRESSED PROTEINS IN THE PANCREAS OF DIABETIC MICE by LINGHUA QIU has been approved for the Department of Biological Sciences and the College of Arts and Sciences by John J. Kopchick Goll-Ohio Professor of Molecular Biology Leslie A. Flemming Dean, College of Arts and Sciences QIU, LINGHUA. Ph.D. June 2005. Biological Sciences Differentially Expressed Proteins in the Pancreas of Diabetic Mice. (222pp.) Director of Dissertation: John J. Kopchick C57BL/6J male mice fed a high-fat diet become obese and develop type 2 diabetes (T2DM), which serves as a model of obesity-associated diabetes in humans. In this study, proteins were extracted from the pancreas of diabetic and control mice and were resolved by 2 dimensional gel electrophoresis (2-DE). The pancreatic protein profiles were compared between control and diabetic mice. Eleven protein spots were differentially expressed and 10 of them were identified by mass spectrometry (MS) analyses. REG1 and REG2 proteins, which may be involved in the regeneration of pancreatic β-cells, were up-regulated very early in the progression of obese mice to T2DM. Up-regulation of Reg1 and Reg2 may reflect the effort by the pancreas to ameliorate the hyperglycemic condition by stimulating the proliferation of pancreatic β-cells resulting in subsequent insulin secretion. Rho GDP-dissociation inhibitor 1 (GDI-1), 1-Cys peroxiredoxin protein, and pancreatic elastase 3B were also up-regulated in the pancreas of diabetic mice relative to control. However, how these expression levels are related to the diabetic condition is unclear. Glutathione peroxidase (Gpx1), which functions in the clearance of reactive oxidative species (ROS), was down-regulated in diabetic mice. The down- regulation of glutathione peroxidase in pancreas could contribute to the progressive deterioration of β-cell function, which may be related to the hyperglycemia induced oxidative stress. Finally, the protein levels of the receptor of activated protein kinase C1, which function in many signaling cascades, was decreased in diabetic mice when compared with normal controls. Because of their potential importance to T2DM, Reg 2 and Gpx1 were selected as potential targets of further investigation. Generation of transgenic mice that over-express Reg2 or Gpx1 can provide valuable information to understand the biological function of Reg2 and Gpx1 during the development of diabetes in mice. As a part of this strategy, expression vectors for Reg2 and Gpx1 were constructed and stable mammalian cell lines which express the genes were established. Expression was confirmed at the mRNA and protein levels for the two genes. Transgenic mice with the Reg2 gene under the control of mouse metallothionien I promoter were generated. These studies will provide a means to study the physiological function of these proteins in association with diabetes and β-cell function. Approved: John J. Kopchick Goll-Ohio Professor of Molecular Biology Acknowledgments I wish to express sincere appreciation to the many individuals who have offered support, inspiration, and encouragement throughout my studies and research endeavors at Ohio University. My special gratitude is extended to Dr. John J. Kopchick, my doctoral advisor, for his guidance and encouragement throughout the course of my graduate study, which has resulted in the research presented in this dissertation. I will always feel inspired by his boundless enthusiasm, dedication to excellence, careful attention to detail, and infinite patience. I feel privileged to have had the opportunity to study under his guidance. I also would like to thank the other members of my committee; Drs. Susan Evans, Peter Coschigano, and Calvin James, for their valuable suggestions and assistance. I would like to acknowledge the contributions of my colleagues—Dr. Bruce Kelder for performing cell transfection to produce stable cell lines; Ms. Gayle Matheny for managing mice and obtaining physiological parameters of the mice; Dr. Shigeru Okada for collecting several of the mouse tissues; Dr. Karen Coschigano for sharing pancreas mRNA samples. I also would like to thank Debbie Holman and Dr. Maria Lozykowski for generating the transgenic mice and for the care of mice. Additionally, I would like to thank our group members for their suggestions and technical assistance. I also would like to thank Dr. Peter Coschigano, Dr. Joan Cunningham, Dr. Mary Chamberlin, and Dr. Tomohiko Sugiyama for the training provided to me as a teaching assistant. Finally, I would like to extend my special thanks to Dr. Robert Colvin, Chairman of the Molecular and Cellular Biology Program, for his support and friendly advice. I would like to acknowledge the following for the financial support provided to me during the course of my graduate work: The Department of Biological Sciences, Molecular and Cellular Biology Program, DiAthegen LLC, the State of Ohio’s Eminent Scholar Program that includes a gift from Milton and Lawrence Goll, and Ohio University SEA program. Finally, I am grateful to my parents, and to my wife -- Guili Xie, for their constant support and encouragement throughout my life. This dissertation is dedicated to them and to my two children, Fuming and John. John was born in Athens and the two will always call themselves Athenias and/or Bobcats. 7 Table of Contents Page Abstract 3 Acknowledgments 5 List of Tables 9 List of Figures 10 List of Abbreviations 12 Introduction to Type 2 Diabetes Mellitus 15 Definition 16 The developmental history of T2DM 19 Insulin Secretion and Action 21 Biphasic Insulin Secretion 22 24-Hour Insulin Secretion Pattern 24 Regulation of Insulin Secretion 26 Regulation of Blood Glucose 31 Causes of T2DM 36 The Primary Cause: β-Cell Dysfunction versus Insulin Resistance 37 Obesity-Related Genes and Substances 42 MODY: A Monogenic Case 48 Genes Involved in the Insulin Signaling Pathway 54 Synergistic Effect of Insulin Receptor and Insulin Receptor Substrates 63 Other Susceptibility Genes 67 Summary of Causes 72 Biology of the Pancreas and Pancreatic β-cells 75 Introduction 76 β-cell Dysfunction and Structural Damage in T2DM 79 The Decompensation Model for β-Cell Dysfunction in T2DM 80 Regulatory/Transcriptional Factors in Developmental Stages 81 Research Objectives 89 Proteomic Analysis of the Pancreas of Type 2 Diabetic Mice 92 Abstract 93 Introduction 95 Materials and Methods 98 8 Results 103 Discussion 111 Differentially Expressed Proteins in the Pancreas of Diet-Induced Obese and Diabetic Mice 115 Abstract 116 Introduction 117 Materials and Methods 120 Results 127 Discussion 133 Cloning and Expression of Reg2 and Glutathione Peroxidase in Mouse L Cells and Production of Reg2 Transgenic Mice 139 Abstract 140 Introduction 141 Materials and Methods 147 Results 162 Discussion 175 General Summary and Conclusion 176 Working Model of Type 2 Diabetes 179 Future Work 181 References 183 Appendix A: Gel Images of the Pancreas from A Control and A Diabetic Mouse 218 Appendix B: Close-Up View of Differentially Expressed Protein Spots 220 9 List of Tables Table Page 1. The Subtypes of MODY and the Related Genetic Defects 50 2. The relative frequency and distribution of cell types in a pancreatic islet 78 3. Identification of protein spots by mass spectrometry 108 4. Quantitative analysis of differentially expressed protein spots 110 5. Identification of protein spots by mass spectrometry 130 6. Quantitative analysis of differentially expressed protein spots 131 7. Proteins or peptides identified by MS and MS/MS analyses from band A and B 172 8. Glutathione peroxidase activity in Gpx1 cell lines and control L cells 173 10 List of Figures Figure Page 1. Pathophysiology of T2DM 18 2. Developmental history of obesity-related T2DM 20 3. Biphasic insulin secretion in response to glucose 23 4. Mean 24 h insulin profile in normal and obese subjects 25 5. Model for coupling of glucose metabolism to insulin secretion in pancreatic β-cells 28 6. The suggested model for the genesis and post-transcriptional suppression of microRNAs and small interfering RNAs 30 7. The relationship between estimated hepatic sinusoidal insulin levels and tracer-determined glucose production in overnight fasted humans 33 8. The relationship between arterial insulin levels and whole body glucose utilization in overnight fasted humans 34 9. Insulin signal transduction pathway 55 10. The conversion of proinsulin to insulin 58 11. The distribution of islets in the human pancreas 77 12. Expression patterns of different transcription factors and their indispensable roles during the distinct developmental stages of β-cells 82 13. Body weight profiles for the two groups of C57BL/6J mice on a low-fat normal chaw (LF), and a high-fat diet (HF) 104 14. Fasting blood glucose levels at different ages for the two groups of mice on low-fat (LF) and high-fat (HF) diets 105 11 15. Fasting serum insulin levels at different ages for the two groups of mice on low-fat (LF) and high-fat (HF) diets 106 16. 2-DE image of the pancreas of C57BL/6J mouse fed on normal diet for 8 weeks 109 17. 2-DE image of the pancreas of C57BL/6J mouse fed on LF for 8 weeks 129 18. Northern blot analysis of Reg2 and Gpx1 132 19. Cloning strategy for the construction of pMet-Reg2-bGH expression vector 150 20. Gpx1 expression vector pCMV•SPORT6 152 21. The sequence of the Reg2 gene in the vector pMet-Reg2-bGH 163 22. Slot blot analysis for Reg2 cell lines 164 23. Slot blot analysis for Gpx1 cell lines 165 24 Northern blot analysis for Reg2 in cell lines Rg-6, -9, -13, and -15 166 25. Northern analysis for Gpx1 in cell lines Gp-1, -2, -12, and -14 168 26.