Diabetes is a complex group of metabolic disorders with genetic, immunological, and environmental etiologies. Decades of diabetes research have elucidated many genetic drivers of normal islet function and dysfunction. Furthermore, genome wide associated studies (GWAS) have discovered that most diabetes susceptibility loci fall outside of coding regions, which suggests a role for noncoding elements in the development of disease. This highlights our incomplete understanding of the islet regulome and suggests the need for detailed functional analyses of noncoding genes to precisely determine their contribution to diabetes susceptibility and disease progression. Transcriptome analyses have revealed that the eukaryotic genome is pervasively transcribed. Strikingly, only a small proportion of the transcriptome is subsequently translated into protein; the majority is made up non-protein coding RNAs (ncRNAs). The most abundant class of these ncRNAs are called long noncoding RNAs (lncRNAs), defined as transcripts longer than 200 nucleotides that lack protein-coding potential. The establishment of lncRNAs, once dismissed as genomic dark matter, as essential gene regulators in many biological processes has redefined the central role for RNA in cells. While evidence suggests a role for lncRNAs in islets and diabetes, in vivo functional characterization of islet lncRNAs is lacking. For my thesis project, I sought to understand the lncRNA regulatory mechanisms that promote pancreas development and function. We conducted comparative transcriptome analyses between embryonic mouse pancreas and adult mouse islets and identified several pancreatic lncRNAs that lie in close proximity to essential pancreatic transcription factors. One of the candidate lncRNAs, Pax6 Upstream Antisense RNA (Paupar), mapped near Pax6, a gene encoding an essential pancreatic regulatory protein. We demonstrate Paupar is enriched in glucagon-producing alpha cells where it promotes the alternative splicing of Pax6 to an isoform required for activation of essential alpha cell genes. Consistently, deletion of Paupar in mice resulted in dysregulation of Pax6 alpha cell target genes and corresponding alpha cell dysfunction. These findings illustrate a distinct mechanism by which lncRNAs can contribute to cell-specific regulation of broadly expressed transcription factors to coordinate critical functions within a cell.

Metabolic syndrome defines a cluster of related comorbidities including obesity, Type 2 diabetes, fatty liver disease, and cardiovascular diseases. Increasingly prevalent in Western countries, metabolic syndrome diseases are a major focus of efforts to understand the complex genetics that underlie disease risk and severity. Immunoglobulin domain-containing receptor 2 (ILDR2) is an ER transmembrane protein first identified as a candidate genetic modifier of diabetes susceptibility in the context of obesity. Obese, leptin-deficient mice with hypomorphic Ildr2 expression had hypoinsulinemic hyperglycemia with reduced beta cell mass, suggesting that ILDR2 plays a role in maintain beta cell mass and function. Further studies proposed a role for ILDR2 in hepatic lipid metabolism as Ildr2 shRNA-mediated knockdown (KD) caused hepatic steatosis in mice. The goal of this thesis work is to clarify the role of ILDR2 in diabetes and hepatic steatosis in an effort to elucidate the specific mechanism of ILDR2. We developed a conditional Ildr2 knockout (KO) allele, enabling tissue-specific ablation in mice. Liver-specific and hepatocyte-specific KO mice did not develop hepatic steatosis. However, liver-specific KO mice treated with adenoviral Ildr2 shRNA accumulated hepatic triglycerides, suggesting off-target effects of the shRNA. Using RNA sequencing and sequence alignment, several gene candidates for shRNA off-targeting effect were identified. Future studies are proposed to elucidate role(s) of these genes in the previously described phenotype of Ildr2 KD mice. I conclude that Ildr2 ablation may contribute to the development of hepatic steatosis, but does not play a major role in hepatic lipid metabolism. We also developed beta cell-specific (RIP2-cre) and pancreas-specific (Pdx-cre) Ildr2 KO mice and characterized them for diabetic phenotypes. Pancreas-specific KO mice displayed impaired glucose tolerance, reduced insulin secretion and decreased calcium signaling in islets. These results confirm a role for ILDR2 in islet cell function. Experiments performed in RIP2-cre beta cell-specific KO mice were confounded by effects of the Cre construct, prohibiting definitive conclusions about the role of ILDR2 in the beta cell. Additionally, because Ildr2 is expressed at low levels in beta cells, we propose that ILDR2 may function in islet macrophages. Overall, this work defines the metabolic functions of ILDR2, clarifying its role in hepatic lipid metabolism, and confirming its role in islet cell function. In addition, I discuss preliminary evidence suggesting that ILDR2 may function in the brain to regulate body weight and metabolism.

Thesis (Ph.D.) - University of Queensland, 2003. / Includes bibliography.

Lau Tung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 142-157). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Abreviations --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Pancreas and its functions --- p.1 / Chapter 1.1.1 --- Structure of pancreas --- p.1 / Chapter 1.1.2 --- Exocrine function --- p.4 / Chapter 1.1.3 --- Endocrine function --- p.7 / Chapter 1.1.3.1 --- Pancreatic islet and islet cells --- p.7 / Chapter 1.1.3.2 --- Regulation of insulin secretion --- p.10 / Chapter 1.1.3.3 --- Mechanism for glucose-stimulated insulin release --- p.14 / Chapter 1.1.3.4 --- Bi-phase response of insulin secretion --- p.16 / Chapter 1.2 --- Pancreatic Renin-Angiotensin System --- p.19 / Chapter 1.2.1 --- Circulating RAS and local RAS --- p.19 / Chapter 1.2.2 --- RAS inhibitors --- p.25 / Chapter 1.2.2.1 --- Angiotensin converting enzyme inhibitor --- p.25 / Chapter 1.2.2.2 --- Non-specific Ang II receptor blocker --- p.28 / Chapter 1.2.2.3 --- Specific AT1 receptor antagonist --- p.29 / Chapter 1.2.2.4 --- Specific AT2 receptor antagonist --- p.30 / Chapter 1.2.3 --- RAS and Pancreas --- p.30 / Chapter 1.2.3.1 --- Expression and localization of pancreatic RAS --- p.30 / Chapter 1.2.3.2 --- Regulation of pancreatic RAS and its clinical relevance --- p.32 / Chapter 1.3 --- Islet Transplantation and RAS --- p.34 / Chapter 1.3.1 --- Whole pancreas and islet transplantation --- p.34 / Chapter 1.3.2 --- Problems encountered in islet transplantation --- p.36 / Chapter 1.3.3 --- Potential role of RAS in islet transplantation --- p.38 / Chapter 1.4 --- Diabetes Mellitus and RAS --- p.40 / Chapter 1.4.1 --- Diabetes Mellitus --- p.40 / Chapter 1.4.2 --- Type 1 diabetes and its animal model --- p.42 / Chapter 1.4.3 --- Type 2 diabetes and its animal model --- p.44 / Chapter 1.4.4 --- RAS blockade in diabetes patients --- p.46 / Chapter 1.4.5 --- Potential role of RAS in Diabetes Mellitus --- p.47 / Chapter 1.5 --- Aims of Study --- p.49 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Experimental animals and mouse models --- p.50 / Chapter 2.1.1 --- Experimental animals for islet isolation and transplantation --- p.50 / Chapter 2.1.2 --- Mouse model for type 2 diabetes --- p.51 / Chapter 2.2 --- Islet isolation and transplantation --- p.52 / Chapter 2.2.1 --- Enzymatic islet isolation --- p.52 / Chapter 2.2.2 --- Islet transplantation --- p.53 / Chapter 2.3 --- Biological assay on islet functions --- p.53 / Chapter 2.3.1 --- Measurement of islet insulin release --- p.53 / Chapter 2.3.2 --- Measurement of islet glucose oxidation rate --- p.56 / Chapter 2.3.3 --- Measurement of islet (pro)insulin biosynthesis --- p.59 / Chapter 2.3.4 --- Measurement of islet total protein synthesis --- p.60 / Chapter 2.4 --- Chronic losartan treatment --- p.62 / Chapter 2.5 --- Perfusion experiment of transplanted islet graft --- p.62 / Chapter 2.6 --- Insulin content of the islet graft --- p.63 / Chapter 2.7 --- Islet graft (pro)insulin and total protein biosynthesis --- p.64 / Chapter 2.8 --- Real-time RT-PCR Analysis --- p.64 / Chapter 2.8.1 --- Design of primers and probes --- p.67 / Chapter 2.8.2 --- Use of internal control --- p.69 / Chapter 2.8.3 --- RT-PCR reaction --- p.69 / Chapter 2.8.4 --- Calculation using the comparative CT method --- p.70 / Chapter 2.9 --- Western Blot Analysis --- p.71 / Chapter 2.10 --- Immunocytochemistry --- p.72 / Chapter 2.11 --- Statistical data analysis --- p.73 / Chapter Chapter 3 --- Results / Chapter 3 .1 --- Effect of Angiotensin II and Losartan on islet insulin release --- p.74 / Chapter 3.1.1 --- Insulin release from normal islets --- p.74 / Chapter 3.2 --- "Effect of Angiotensin II and Losartan on islet glucose oxidation rate, (pro)insulin and total protein biosynthesis" --- p.77 / Chapter 3.2.1 --- Glucose oxidation rate of isolated normal islets --- p.77 / Chapter 3.2.2 --- (pro)insulin and total protein biosynthesis of isolated normal islets --- p.77 / Chapter 3.3 --- Regulation of RAS components in islet transplantation --- p.81 / Chapter 3.3.1 --- Expression of RAS components in endogenous islets and transplanted islets --- p.81 / Chapter 3.3.2 --- Localization of AT1-receptor in endogenous islets --- p.87 / Chapter 3.3.3 --- Expression of AT1-receptor protein in endogenous and transplanted islets --- p.89 / Chapter 3.3.4 --- Relative abundance of RAS components in kidney and liver --- p.91 / Chapter 3.3.5 --- Insulin release from perfused transplanted islet graft --- p.93 / Chapter 3.3.5 --- (pro)insulin and total protein biosynthesis of transplanted islet graft --- p.96 / Chapter 3.4 --- Effect of Angiotensin II and losartan on diabetic islets --- p.99 / Chapter 3.4.1 --- Expression of RAS components in diabetic pancreas --- p.99 / Chapter 3.4.2 --- Localization of AT1 receptors in diabetic pancreas --- p.105 / Chapter 3.4.3 --- Insulin release from islets of type 2 diabetic mice --- p.107 / Chapter 3.4.4 --- (pro)insulin and total protein biosynthesis of islets from type 2 diabetic mice --- p.112 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Effect of angiotensin II and losartan on islet insulin release --- p.116 / Chapter 4.2 --- Existence of local RAS in pancreatic islets --- p.119 / Chapter 4.3 --- Regulation of islet RAS components in transplanted islets --- p.122 / Chapter 4.4 --- Clinical relevance of islet RAS in transplantation --- p.125 / Chapter 4.5 --- Regulation of islet RAS by type 2 diabetes --- p.126 / Chapter 4.6 --- Clinical relevance of islet RAS in type 2 diabetes --- p.134 / Chapter 4.7 --- Conclusion --- p.140 / Chapter 4.8 --- Further studies --- p.141 / Chapter Chapter 5 --- Bibliography --- p.142

Thesis (M.Sc.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Experimental Surgery, Department of Surgery, University of Alberta. Title from pdf file main screen (viewed on July 28, 2009). Includes bibliographical references.

Thesis (Ph.D.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Experimental Surgery, Department of Surgery. Title from pdf file main screen (viewed on August 29, 2009). Includes bibliographical references.

Two major problems preventing the clinical application of pancreatic islet transplantation were investigated. The problem of allograft rejection was studied in rats, made diabetic by streptozotocin treatment. It was shown that DA rats given LEW renal allografts and treated with cyclosporine accepted their grafts, and subsequently developed a strain-specific unresponsive state that allowed successful transplantation of LEW islets without further immunosuppression, whilst BN islets were rejected normally. The effect was demonstrated to be independent of the site of islet transplantation, and, once an islet allograft had been accepted, it was possible to remove the original renal allograft without affecting the transplanted islets. The effect was shown to apply to another strain combination (LEW into PVG), and also to animals made unresponsive to renal allografts by another method (donor-specific blood transfusion). The problem of separation of adequate numbers of viable islets from the pancreas was studied in the rat, dog, pig and human. To aid the investigation, supravital staining techniques were developed, using neutral red to identify the islets, and fluorescein diacetate and ethidium bromide to assess islet viability. A variety of islet isolation techniques were investigated, and a new technique for isolation of islets from the dog pancreas, yielding up to 160,000 islets from 1 pancreas with a maximum purity of 80%, was developed. The structural integrity and in vitro function of the isolated islets was demonstrated, but it was not possible to prevent diabetes by autotransplantation of islets to the portal vein of pancreatectomised dogs. A method for isolation of islets from the human pancreas was developed from that used in the dog, yielding up to 80,000 islets from a whole pancreas, with a maximum purity of 40%. The technique was shown to be both simple and reliable. The structural integrity and in vitro function of the isolated islets was demonstrated, and the viability of the islets proven by successful transplantation under the kidney capsule of nude mice.

Wong, Mei Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 207-251). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / PUBLICATIONS BASED ON WORK IN THIS THESIS --- p.vii / ABBREVIATIONS --- p.viii / TABLE OF CONTENTS --- p.x / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Diabetes Mellitus --- p.1 / Chapter 1.2 --- Structure and Functions of the Pancreas --- p.2 / Chapter 1.2.1 --- Size of Pancreatic β-Cells --- p.4 / Chapter 1.2.2 --- Signaling Pathways of Insulin Secretion from Pancreatic β-Cells --- p.4 / Chapter 1.3 --- Classification of Diabetes --- p.6 / Chapter 1.3.1 --- Type 1 Diabetes --- p.6 / Chapter 1.3.2 --- Type 2 Diabetes --- p.8 / Chapter 1.4 --- Pathologies of Type 2 Diabetes --- p.9 / Chapter 1.4.1 --- Hyperglycemia --- p.9 / Chapter 1.4.1.1 --- A dvanced Glycosylation End Products --- p.11 / Chapter 1.4.1.2 --- Protein Kinase C Activation --- p.13 / Chapter 1.4.1.3 --- The Glucosamine Pathway --- p.14 / Chapter 1.4.1.4 --- Oxidative Stress --- p.15 / Chapter 1.4.2 --- Insulin Resistance --- p.15 / Chapter 1.4.3 --- Loss of β-Cell Mass and β-Cell Dysfunction --- p.18 / Chapter 1.5 --- Complications of Diabetes Mellitus --- p.21 / Chapter 1.5.1 --- Cardiovascular Diseases --- p.21 / Chapter 1.5.2 --- Diabetic Retinopathy --- p.22 / Chapter 1.5.3 --- Diabetic Nephropathy --- p.23 / Chapter 1.5.4 --- Neuropathy --- p.24 / Chapter 1.6 --- Anti-Diabetic Drugs for Type 2 Diabetes Mellitus --- p.25 / Chapter 1.6.1 --- Secretagogues --- p.25 / Chapter 1.6.2 --- Sensitizers --- p.26 / Chapter 1.6.3 --- Alpha-Glucosidase Inhibitors --- p.27 / Chapter 1.6.4 --- Peptide Analogs --- p.27 / Chapter 1.6.4.1 --- Incretin Mimetics --- p.27 / Chapter 1.6.4.2 --- Dipeptidyl Peptidase-4 Inhibitors --- p.28 / Chapter 1.7 --- Insights of Porcine Islets in Treatment of Diabetics --- p.28 / Chapter 1.8 3 --- -Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMG CoA Reductase) --- p.31 / Chapter 1.8.1 --- Statins --- p.32 / Chapter 1.8.2 --- Pleiotropic Effects of Statins --- p.36 / Chapter 1.8.2.1 --- Statins and Isoprenylated Proteins --- p.36 / Chapter 1.8.2.2 --- Statins and Endothelial Functions --- p.38 / Chapter 1.8.2.3 --- Statins and Platelet Functions --- p.39 / Chapter 1.8.2.4 --- Statins and Plaque Stability --- p.39 / Chapter 1.8.2.5 --- Statins and Vascular Inflammation --- p.40 / Chapter 1.9 --- Clinical Studies of Statins on Diabetics --- p.41 / Chapter 1.10 --- Possible Factors Involved in Simvastatin-Regulated Insulin Secretion --- p.44 / Chapter 1.10.1 --- AMP-Activated Protein Kinase --- p.44 / Chapter 1.10.2 --- Caveolin-1 --- p.46 / Chapter 1.10.3 --- Sterol-Regulatory Elementary Binding Protein --- p.50 / Chapter 1.10.4 --- Protein Phosphatase 2A --- p.52 / Chapter 1.10.5 --- Calcium Sensing Receptor --- p.55 / Chapter 1.11 --- Objectives of Study --- p.59 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.60 / Chapter 2.1 --- Materials --- p.60 / Chapter 2.1.1 --- Solutions --- p.60 / Chapter 2.1.2 --- Antibodies --- p.63 / Chapter 2.2 --- Methods --- p.64 / Chapter 2.2.1 --- Maintenance of Pancreas Function --- p.64 / Chapter 2.2.2 --- Islet Isolation --- p.65 / Chapter 2.2.3 --- Hematoxylin and Eosin (H&E) Staining --- p.65 / Chapter 2.2.4 --- Simvastatin and Simvastatin-Na+ --- p.66 / Chapter 2.2.5 --- AICAR --- p.67 / Chapter 2.2.6 --- Compound C --- p.67 / Chapter 2.2.7 --- Incubation of Islets --- p.67 / Chapter 2.2.8 --- Western Blot --- p.68 / Chapter 2.2.9 --- Enzyme-Linked Immunosorbent Assay (ELISA) --- p.69 / Chapter 2.2.10 --- Statistical Analysis --- p.71 / Chapter CHAPTER 3 --- HISTOLOGY OF PORCINE PANCREATIC ISLETS OF LANGERHANS --- p.72 / Chapter 3.1 --- Comparison of Sizes of Porcine Pancreatic Islets in Histological Sections of Pancreas --- p.72 / Chapter CHAPTER 4 --- PROTEIN EXPRESSION OF HMG COA REDUCTASE --- p.75 / Chapter 4.1 --- Effect of Incubation Time on HMG CoA Reductase Expression --- p.75 / Chapter 4.2 --- Short-Term Effect of Simvastatin on HMG CoA Reductase Expression --- p.78 / Chapter 4.3 --- Long-Term Effect of Simvastatin on HMG CoA Reductase Expression --- p.81 / Chapter 4.4 --- Effect of Osmolality on HMG CoA Reductase Expression --- p.83 / Chapter 4.5 --- Effect of Simvastatin on Ser871 p-HMG CoA Reductase Expression --- p.87 / Chapter CHAPTER 5 --- EVALUATION OF THE ROLE OF SIMVASTATIN IN INSULIN SECRETION VIA HMG CO A REDUCTASE REGULATION --- p.90 / Chapter 5.1 --- Effect of Simvastatin on Insulin Secretion --- p.90 / Chapter 5.2 --- Effect of Different Concentrations of Simvastatin on Insulin Secretion --- p.94 / Chapter 5.3 --- Effect of Simvastatin on Insulin Content --- p.96 / Chapter CHAPTER 6 --- ROLE OF AMPK EXPRESSION IN INSULIN SECRETION PATHWAY --- p.100 / Chapter 6.1 --- Effect of Simvastatin on Thr172 p-AMPK α and AMPK α1 Expressions --- p.100 / Chapter 6.2 --- Evaluation of the Role of Simvastatin in AMPK Regulation --- p.104 / Chapter 6.3 --- Evaluation of the Role of PP2A in AMPK Regulation --- p.108 / Chapter 6.4 --- Evaluation of the Role of Simvastatin on Insulin Secretion via AMPK Regulation --- p.111 / Chapter 6.4.1 --- AMPK Regulation on Releasable Insulin Secretion --- p.111 / Chapter 6.4.2 --- AMPK Regulation on Non-Releasable Insulin Content and Total Insulin Content --- p.112 / Chapter CHAPTER 7 --- EFFECT OF SIMVASTATIN ON THE EXPRESSION OF REGULATORY PROTEINS INVOLVED IN INSULIN SECRETION --- p.119 / Chapter 7.1 --- Effect of Simvastatin on SREBP-2 Expression --- p.119 / Chapter 7.2 --- Effect of Simvastatin on Caveolin-1 Expression --- p.121 / Chapter 7.3 --- Effect of Simvastatin on Calcium Sensing Receptor Expression --- p.123 / Chapter CHAPTER 8 --- EFFECT OF SIMVASTATIN-NA+ ON INSULIN SECRETION --- p.126 / Chapter 8.1 --- Effect of Simvastatin-Na+ on HMG CoA Reductase Expression --- p.126 / Chapter 8.2 --- Effect of Simvastatin-Na+ on Insulin Secretion --- p.128 / Chapter 8.3 --- Effect of Different Concentrations of Simvastatin-Na+ on Insulin Secretion --- p.130 / Chapter 8.4 --- Effect of Simvastatin-Na+ on Insulin Content --- p.132 / Chapter CHAPTER 9 --- EFFECT OF PRAVASTATIN ON INSULIN SECRETION --- p.136 / Chapter 9.1 --- Effect of Pravastatin on Insulin Secretion --- p.136 / Chapter 9.2 --- Effect of Pravastatin on Insulin Content --- p.138 / Chapter CHAPTER 10 --- EFFECT OF METHYL-B-CYCLODEXTRIN ON INSULIN SECRETION --- p.142 / Chapter 10.1 --- Effect of Methyl-β-cyclodextrin on Insulin Secretion --- p.142 / Chapter 10.2 --- Effect of Methyl-β-cyclodextrin on Insulin Content --- p.144 / Chapter CHAPTER 11 --- DISCUSSION --- p.149 / Chapter 11.1 --- Importance of Studying Porcine Pancreatic Islets and Islet Distribution --- p.150 / Chapter 11.2 --- Screening of Concentration and Incubation Time of Simvastatin on Porcine Pancreatic Islets --- p.152 / Chapter 11.3 --- Glucose-Independent Effect of Simvastatin on Protein Expression of HMG CoA Reductase --- p.154 / Chapter 11.4 --- Role of AMPK in HMG CoA Reductase-Modulated Insulin Secretion --- p.159 / Chapter 11.5 --- Role of SREBP-2 in Simvastatin-Modulated Regulation --- p.174 / Chapter 11.6 --- Role of Calcium Sensing Receptor in Simvastatin-Modulated Regulation --- p.175 / Chapter 11.7 --- Role of Caveolin-1 in Simvastatin-Modulated Regulation --- p.179 / Chapter 11.8 --- "Effects of Simvastatin-Na+， Pravastatin and Methyl-β-cyclodextrin, and Importance of Endoplasmic Reticulum in Insulin Secretion" --- p.183 / Chapter CHAPTER 12 --- CONCLUSIONS AND FURTHER STUDIES --- p.197 / Chapter 12.1 --- Conclusions --- p.197 / Chapter 12.2 --- Further Studies --- p.203 / REFERENCES --- p.207

Pancreatic beta cell specification is a complex process, requiring proper function of numerous transcription factors. Nkx2.2 is a transcription factor that is crucial for beta cell formation, and is expressed early and throughout pancreatic development. Nkx2.2-/- mice display complete loss of the beta cell lineage and defects in the specification of other endocrine cell types, demonstrating the importance of Nkx2.2 in establishing proper endocrine cell ratios. Recent studies have also demonstrated a role for Nkx2.2 within the mature beta cell to maintain identity and function. This thesis work investigated the timing of pancreatic beta cell specification and the mechanism of this process. In these studies, Nkx2.2 was ablated specifically within the Ngn3-expressing endocrine progenitor population in vivo. These mice displayed defects similar to Nkx2.2-/- mice. Surprisingly, the disruption of endocrine cell specification did not require loss of expression of multiple essential transcription factors known to function downstream of Nkx2.2, including Ngn3, Rfx6, and NeuroD1. While these factors are all necessary for beta cell specification, their preserved expression did not rescue beta cell formation. ChIP-Seq analyses also revealed co-occupancy of Nkx2.2, Rfx6, and NeuroD1 near endocrine-related genes, suggesting Nkx2.2 may cooperate with its downstream targets to regulate beta cell fate. These results have revealed a unique requirement for Nkx2.2 during a critical window of beta cell development. In addition, the role of a conserved domain of Nkx2.2, the specific domain (SD), was assessed using Nkx2.2SDmutant mice. Transcriptional profiling of Nkx2.2SDmutant endocrine progenitors revealed a critical role for the SD domain in regulating the transcription of endocrine fate genes early in the process of endocrine differentiation. In addition, beta cell-specific deletion of the Nkx2.2 SD domain resulted in hyperglycemia, glucose intolerance and dysregulation of beta cell functional genes. This suggests the SD domain is important for mediating Nkx2.2 function within the beta cell to maintain glucose homeostasis. Together, these results have elucidated a critical developmental window for beta cell specification and demonstrated an essential role for Nkx2.2 and specifically its SD domain in this process. Furthermore, these studies suggest that beta cell transcription factors may also regulate endocrine fate in a combinatorial manner, and exert changes within the endocrine progenitor lineage. These findings have provided us with a better understanding of in vivo pancreatic development, and will improve current research efforts to differentiate beta cells in vitro from hPSCs.

by Hinny Shuk-Yee Lam. / Year shown on spine: 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 92-117). / Declaration --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / Table of Contents --- p.v / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Pancreas and Islets of Langerhans --- p.1 / Chapter 1.1.1 --- Islet Hormones and Glucose Balance --- p.3 / Chapter 1.1.2 --- Glucagon and Its Derived Peptides --- p.4 / Chapter A. --- Tissue-specific Post-translational Processing --- p.4 / Chapter B. --- Features of Proglucagon Gene --- p.6 / Chapter 1.1.3 --- Insulin and Features of Its Gene --- p.9 / Chapter 1.2 --- Regulation of Islet Hormone Secretion --- p.12 / Chapter 1.2.1 --- Endocrine Control --- p.12 / Chapter A --- GIP --- p.13 / Chapter B. --- Truncated GLP-1 --- p.13 / Chapter 1.2.2 --- Paracrine Control --- p.14 / Chapter 1.2.3 --- Neuroendocrine Control --- p.15 / Chapter 1.3 --- Neuropeptide Y --- p.16 / Chapter 1.3.1 --- NPY in Central Nervous System --- p.17 / Chapter 1.3.2 --- NPY in Pancreas --- p.17 / Chapter 1.3.3 --- NPY and Islet Hormones --- p.18 / Chapter 1.4 --- Synthesis and Secretion --- p.19 / Chapter 1.5 --- Objectives of Study --- p.23 / Chapter Chapter 2 --- Materials and Methods --- p.26 / Chapter 2.1 --- Effects of NPY on Islet Gene Expression --- p.26 / Chapter 2.1.1 --- Tissue Culture --- p.26 / Chapter A. --- Materials --- p.26 / Chapter B. --- Maintenance and Passage --- p.26 / Chapter C. --- Experimental Protocol --- p.28 / Chapter 2.1.2 --- Total RNA Isolation --- p.28 / Chapter A. --- Materials --- p.28 / Chapter B. --- Extraction Using FastPrep System --- p.29 / Chapter C. --- Quantification of RNA --- p.30 / Chapter D. --- Preparation of Reagents --- p.30 / Chapter 2.1.3 --- Northern Blot Analysis --- p.31 / Chapter A. --- Materials --- p.31 / Chapter B. --- Formaldehyde Gel Electrophoresis --- p.32 / Chapter C. --- Transfer onto Nylon Membrane --- p.33 / Chapter D. --- Labeling of cDNA Probes --- p.34 / Chapter E. --- Hybridization and Autoradiography --- p.35 / Chapter F. --- Preparation of Reagents --- p.36 / Chapter 2.1.4 --- Preparation of cDNA Probe --- p.37 / Chapter A. --- Materials --- p.37 / Chapter B. --- Preparation of Competent Cells --- p.37 / Chapter C. --- Transformation --- p.38 / Chapter D. --- Plasmid DNA Isolation --- p.39 / Chapter E. --- Restriction Enzyme Digestion --- p.41 / Chapter F. --- Agarose Gel Electrophoresis --- p.42 / Chapter G. --- Isolation of DNA Fragments --- p.42 / Chapter H. --- Preparation of Reagents --- p.43 / Chapter 2.1.5 --- Data Analysis --- p.46 / Chapter 2.2 --- Effects of NPY on Cytosolic Calcium --- p.46 / Chapter 2.2.1 --- Tissue Culture --- p.47 / Chapter 2.2.2 --- Confocal Laser Scanning Microscopy --- p.47 / Chapter A. --- Materials --- p.47 / Chapter B. --- Loading of Dye --- p.48 / Chapter C. --- Cytosolic Calcium Measurement --- p.49 / Chapter D. --- Preparation of Reagents --- p.49 / Chapter Chapter 3 --- Results --- p.51 / Chapter 3.1 --- Studies on Islet Gene Expression --- p.51 / Chapter 3.1.1 --- Effect of NPY on Proglucagon Expression --- p.51 / Chapter A. --- Effect at 11 mM Glucose --- p.51 / Chapter B. --- Effect at 5 mM Glucose --- p.52 / Chapter 3.1.2 --- Effect of NPY on Proinsulin Expression --- p.52 / Chapter 3.1.3 --- "Effect of PYY, PP and FSK on Proglucagon Expression" --- p.53 / Chapter 3.2 --- Studies on Cytosolic Calcium --- p.65 / Chapter 3.2.1 --- Features of InRlG9 Cells --- p.65 / Chapter 3.2.2 --- Effect of NPY on Cellular Calcium Level --- p.66 / Chapter Chapter 4 --- Discussion --- p.77 / Chapter Chapter 5 --- References --- p.92