The Role of Sox17 in Normal and Pathological Beta Cell
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University of Cincinnati Date: August 23, 2012 The Role of Sox17 in Normal and Pathological Beta Cell A dissertation submitted to the Division of Graduate Studies and Research of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate Program in Molecular and Developmental Biology of the College of Medicine 2012 by Diva Jonatan Bachelor of Science, Xavier University, 2006 Committee Chair: James Wells, PhD Jeffrey Whitsett, MD, PhD Aaron Zorn, PhD Jonathan Katz, PhD Gail Deutsch, MD ABSTRACT Glucose homeostasis is a complex process involving many regulatory molecules and disruption of this process can result in diabetes. Sox17 is a transcription factor and a key regulator in various developmental and disease contexts. During endoderm development, Sox17 acts, in part, by transcriptionally regulating other important endodermal transcription factors including HNF! and Foxa2, which are also known regulators of postnatal cell function. Our data shows that Sox17 is expressed in all islet cells. Thus, we hypothesize that Sox17 is a key modulator of ! cell homeostasis. In the course of this study, we discovered a novel role for Sox17 in regulating insulin trafficking and secretion in normal and pathological cells. Loss of Sox17 throughout pancreas development in a wildtype background resulted in mice with prediabetes. These mice had higher proinsulin protein content in the ER of the islet cells and dilated secretory organelles in cells. In line with the prediabetes phenotype, these mice went on to develop additional symptoms of diabetes when placed on a high fat diet, including elevated fasting glucose levels and an inability to respond to a glucose challenge. This suggested that Sox17 affects either insulin processing and/or transit through the secretory system. To more directly investigate these possibilities, we used a tetracycline regulated transgenic system to overexpress Sox17 in mature !cells in wildtype background. Transgenic Sox17 expression resulted in rapid trafficking and secretion of improperly processed proinsulin. At the transcriptional level, Sox17 altered expression of genes involved in biological processes that regulate iii hormone transport, secretion, and cellular localization, which led to diabetes after prolonged exposure. This demonstrates that Sox17 affects insulin trafficking throughout the secretory pathway. We therefore wanted to explore the possibility that physiologic levels of Sox17 might be used to positively impact diabetic phenotypes using a genetic model of diabetes (MODY4). We did so by overexpressing Sox17 two-fold in a MODY4 mouse model (Pdx1-tTA hemizygote mice). Increased expression of Sox17 in the !-cells of MODY4 animals was sufficient to transiently normalize basal blood glucose and insulin levels as well as restore islet cell organization architecture; however, Sox17 overexpression was not able to rescue the inability of MODY4 animals to properly respond to glucose challenge. Together, these data demonstrate new and critical role for Sox17 in regulating insulin trafficking and secretion processes in the adult pancreas that are important to ensure proper glucose homeostasis. This study also suggests that modulation of Sox17-regulated pathways can be used therapeutically to improve cell function in the context of diabetes. iv v Table of Contents ABSTRACT iii TABLE OF CONTENTS 1 LIST OF FIGURES AND TABLES 4 CHAPTER 1. Introduction Glucose homeostasis, prediabetes and the different forms of diabetes 7 MODY and the Islet Transcriptional Factor Network Involved 13 Insulin Biosynthesis, Processing, and Secretion 14 Overview: Functional Roles of Sox Family Proteins 18 Overview of Sox Family of Transcription Factors 18 Sox Proteins in Endocrine Pancreas Development and Function 19 The Roles of Sox17 in Different Biological Contexts 22 References 26 Figure Legends 34 Figures 35 CHAPTER 2. The Role of Sox17 in Insulin Processing and Cell Secretory Pathway Abstract 39 Introduction 40 Materials and methods 43 Results 47 1 Discussion 59 Acknowledgements 66 Sources of Funding 66 References 67 Figure Legends 73 Figures 78 Supplementary Figure Legends 86 Supplementary Figures 89 CHAPTER 3. The Partial Rescue of MODY4 Phenotypes by Sox17 Summary 115 Introduction 116 Materials and Methods 121 Results 123 Discussion 128 Acknowledgements 130 Sources of Funding 131 References 131 Figure Legends 134 Figures 137 CHAPTER 4. Summary and Discussion Major Findings 144 2 Sox17 in normal cells 144 Sox17 in pathological cells 146 Experimental limitations and alternative approaches 147 Acknowledgements 149 Sources of funding 150 References 150 Figure Legends 152 Figures 152 3 LIST OF FIGURES AND TABLES CHAPTER 1. Figure 1. MODY genes islet transcriptional network 35 Figure 2. Insulin biosynthesis in pancreatic cells 36 Figure 3. Glucose sensing and glucose-stimulated insulin release pathway 37 CHAPTER 2. Figure 1. Sox17 is not required for cell development 78 Figure 2. Sox17-paLOF results in elevated proinsulin protein in the islets 79 Figure 3. Loss of Sox17 in the pancreas causes accumulation of proinsulin in the ER and structural changes in secretory organelles 80 Figure 4. Sox17-paLOF mice are prediabetic and prone to high fat diet-induced stress of ! cells 81 Figure 5. Sox17 overexpression for 24 hours is sufficient to alter proinsulin:total insulin protein ratio and proinsulin secretion in vivo, followed by accumulation of proinsulin in the plasma, leading to diabetes after prolonged exposure of Sox17 82 Figure 6. 24 hours of Sox17 overexpression alters proinsulin trafficking through the secretory organelle machinery 84 Figure 7. Sox17 regulates pathways involved in insulin transport and secretion 85 Supplementary Figure 1. Sox17 immunostaining in Sox17pa-LOF, wildtype, and Sox17- GOF islets 89 4 Supplementary Figure 2. Percent colocalizations between proinsulin and organelle markers, and their total regional areas 89 Supplementary Figure 3. Plasma proinsulin, total insulin, and their ratio in Sox17-paLOF mice on high fat diet 90 Supplementary Figure 4. Transcriptional insulin processing enzymes level 91 Supplementary Figure 5. Plasma proinsulin and total insulin levels in Sox17-GOF mice 91 Supplementary Figure 6. Percent colocalization and total regional areas of proinsulin and various organelle markers 92 Supplementary Figure 7. Microarray validation of altered genes in Sox17-GOF islets 93 Supplementary Table 1. Primary and Secondary Antibodies 95 Supplementary Table 2. Sox17-GOF islet microarray gene list 97 CHAPTER 3. Figure 1. Sox17 overepression in the context of MODY4 background rescued resting hyperglycemia 135 Figure 2. Sox17 overexpression rescued MODY4 disrupted islet architecture 138 Figure 3. Sox17 altered the distribution of islet sizes and cell-cell adhesion contacts of the MODY4 mice 140 CHAPTER 4. Figure 1. The role of Sox17 in regulating insulin trafficking 5 Chapter 1. Introduction 6 Glucose homeostasis, prediabetes and the different forms of diabetes. “Disease does not occur unexpectedly, it is the result of constant violation of Nature’s laws.”- Hippocrates Glucose is the body’s primary source of energy and maintenance of blood glucose homeostasis is achieved by a complex endocrine regulatory network. Central to this network is the hormone insulin, which is secreted in response to elevated glucose levels and acts on peripheral tissues in several ways. In the liver, insulin promotes conversion of glucose to glycogen for storage. In other tissues like skeletal muscle, insulin activates the insulin signaling receptor, which interfaces with several downstream effector pathways, such as the insulin receptor substrates/phosphatidylinositol 3-kinase pathway (IRS/PI3-K) and the Ras/mitogen-activated protein kinase (MAPK) pathway, to mediate glucose uptake and metabolism (reviewed in1,2). Insulin protein is regulated at several levels: insulin biosynthesis, processing, secretion, cell uptake, and its breakdown in the body. A defect in any of these stages of insulin regulation, or loss of cell mass, can lead to elevated blood glucose levels (hyperglycemia), and over the long term, this can result in diabetes. The World Health Organization Diabetes Fact Sheet 2011 suggests that there are 346 millions of people with diabetes worldwide. According to the 2011 National Diabetes Fact Sheet, released by the American Diabetes Association, there is a total 25.8 million of diabetic children and adults in the United States, which is 8.3% of the population. In addition, 79 million people are considered to be prediabetic, meaning these people are exhibiting one or more 7 symptoms of glucose dysregulation, including impaired fasting blood glucose, elevated ratio of plasma proinsulin:insulin, and impaired glucose tolerance3-8.In terms of the rate of mortality, it is estimated that diabetes causes more deaths per year than breast cancer and AIDS combined. There are several different types of diabetes, such as the polygenic forms of diabetes: type 1 diabetes and type 2 diabetes, and the monogenic forms of diabetes: mature onset diabetes of the young (MODY) and neonatal diabetes. Type 1 diabetes (~5% of diabetes cases, according to the National Diabetes Information Clearinghouse - NIDC) is a result of autoimmune-mediated destruction of pancreatic cells, leading to loss of insulin production. It is usually diagnosed in children and young adults. Type 2 diabetes is the most common form of diabetes (90-95% of diabetes cases) and is associated