Chan Chung Sing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 93-101). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ix / List of Abbreviations --- p.xvii / Chapter CHATPER ONE --- INTRODUCTION / Chapter 1.1 --- Glucose Homeostasis --- p.1 / Chapter 1.1.1 --- Function --- p.1 / Chapter 1.1.2 --- Origins and regulation of glucose --- p.2 / Chapter 1.1.3 --- Glucoregulatory factors --- p.4 / Chapter 1.1.4 --- Insulin --- p.6 / Chapter 1.1.4.1 --- Function of Insulin --- p.7 / Chapter 1.1.4.2 --- Discovery and Production of Insulin --- p.7 / Chapter 1.1.4.3 --- Insulin Signaling Pathway --- p.8 / Chapter 1.1.4.3.1 --- Insulin Receptor --- p.8 / Chapter 1.1.4.3.2 --- MAPK Pathway --- p.9 / Chapter 1.1.4.3.3 --- Phosphatidylinositol 3-kinase (PI3-K) Pathway --- p.10 / Chapter 1.1.5 --- Glucose Transporters --- p.11 / Chapter 1.1.6 --- Role of skeletal muscle in glucose homeostasis --- p.13 / Chapter 1.1.7 --- Insulin Resistance --- p.14 / Chapter 1.1.8 --- Glucose abnormality and its complications --- p.16 / Chapter 1.2 --- Actin --- p.19 / Chapter 1.2.1 --- Function of Actin --- p.20 / Chapter 1.2.2 --- Actin Accessory Protein --- p.22 / Chapter 1.2.3 --- Actin Polymerization --- p.23 / Chapter 1.3 --- "Interaction between Insulin, GLUT4 and Actin in Glucose Homeostasis" --- p.24 / Chapter 1.3.1 --- Insulin-Induced Actin Remodeling --- p.25 / Chapter 1.3.2 --- Actin Remodeling and Insulin-Induced GLUT4 Translocation --- p.26 / Chapter 1.3.3 --- Involvement of Insulin Signaling Molecules in Actin Remodeling --- p.27 / Chapter 1.3.4 --- Actin Remodeling and Insulin Resistance --- p.30 / Chapter 1.4 --- Hypothesis and Objective --- p.30 / Chapter 1.4.1 --- Rationale --- p.30 / Chapter 1.4.2 --- Hypothesis --- p.31 / Chapter 1.4.3 --- Objective --- p.31 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.33 / Chapter 2.2 --- Cell Culture --- p.36 / Chapter 2.2.1 --- Cell Culture --- p.36 / Chapter 2.2.2 --- Reagents Preparation and Incubation --- p.39 / Chapter 2.3 --- 2-Deoxyglucose Uptake --- p.39 / Chapter 2.4 --- Immunofluorescence Microscopy --- p.41 / Chapter 2.4.1 --- Permeabilized cell staining --- p.41 / Chapter 2.4.2 --- Membrane-intact cell staining --- p.43 / Chapter 2.4.3 --- The analysis of actin remodeling reduction --- p.44 / Chapter 2.5 --- Live Image Microscopy --- p.44 / Chapter 2.6 --- Transmission Electron Microscope Study --- p.44 / Chapter 2.7 --- Statistical Analysis --- p.46 / Chapter CHAPTER THREE --- RESULTS / Chapter 3.1 --- Cell Growth --- p.48 / Chapter 3.2 --- Acute Effect of Insulin on L6 myotubes --- p.48 / Chapter 3.2.1 --- Immunofluorescence Microscopy --- p.49 / Chapter 3.2.1.1 --- The time profile of insulin on actin cytoskeletonin permeabilized L6 myotubes --- p.49 / Chapter 3.2.1.2 --- The concentration effect of insulin on actin cytoskeletonin permeabilized L6 myotubes --- p.50 / Chapter 3.2.1.3 --- Relationship between actin cytoskeleton and GLUT4mycin permeabilized L6 myotubes --- p.51 / Chapter 3.2.1.4 --- Translocation of GLUT4myc in membrane-intact L6 myotubes --- p.51 / Chapter 3.2.1.5 --- "Effect of methyl-β-cyclodextrins, MeOH or EtOHin permeabilized and membrane-intact L6 myotubes" --- p.52 / Chapter 3.2.2 --- 2-Deoxyglucose Uptake --- p.52 / Chapter 3.2.2.1 --- "Effects of insulin, methyl-β-cyclodextrins, MeOH and EtOH in L6 myotubes" --- p.52 / Chapter 3.2.3 --- TEM Study --- p.53 / Chapter 3.2.3.1 --- Effects of insulin on actin cytoskeleton and GLUT4myc in L6 myotubes --- p.53 / Chapter 3.3 --- Effect of high glucose and high insulin incubation in L6 myotubes --- p.54 / Chapter 3.3.1 --- Immunofluorescence Microscopy --- p.54 / Chapter 3.3.1.1 --- High insulin and high glucose preincubation in permeabilized L6 myotubes --- p.55 / Chapter 3.3.1.2 --- Effect of high insulin and high glucose incubationin membrane-intact L6 myotubes --- p.55 / Chapter 3.3.2 --- 2-Deoxyglucose Uptake --- p.56 / Chapter 3.3.2.1 --- Effect of high insulin and high glucose incubation in L6 myotubes --- p.56 / Chapter 3.3.3 --- TEM Study --- p.57 / Chapter 3.3.3.1 --- Effect of high insulin and high glucose incubation in L6 myotubes --- p.57 / Chapter 3.4 --- Effect of FFA incubation in L6 myotubes --- p.58 / Chapter 3.4.1 --- Immunofluorescence Microscopy --- p.58 / Chapter 3.4.1.1 --- FFA preincubation in permeabilized L6 myotubes --- p.58 / Chapter 3.4.1.2 --- FFA incubation in membrane-intact L6 myotubes --- p.59 / Chapter 3.4.2 --- 2-Deoxyglucose Uptake --- p.59 / Chapter 3.4.2.1 --- FFA incubation in L6 myotubes (24 hours) --- p.60 / Chapter 3.4.3 --- TEM Study --- p.62 / Chapter 3.4.3.1 --- FFA incubation in L6 myotubes --- p.62 / Chapter 3.5 --- Effect of CHO incubation in L6 myotubes --- p.62 / Chapter 3.5.1 --- Immunofluorescence Microscopy --- p.62 / Chapter 3.5.1.1 --- CHO preincubation in permeabilized L6 myotubes --- p.63 / Chapter 3.5.1.2 --- CHO incubation in membrane-intact L6 myotubes --- p.63 / Chapter 3.5.2 --- 2-Deoxyglucose Uptake --- p.64 / Chapter 3.5.2.1 --- CHO incubation in L6 myotubes (24 hours) --- p.64 / Chapter 3.5.3 --- TEM Study --- p.65 / Chapter 3.5.3.1 --- CHO incubation in L6 myotubes --- p.65 / Chapter 3.6 --- Overall changes in glucose uptake after preincubation experiment --- p.65 / Chapter CHAPTER FOUR --- DISCUSSION / Chapter 4.1 --- Effect of insulin on L6 myotubes --- p.69 / Chapter 4.2 --- "Effect of methyl-β-cyclodextrins, MeOH and EtOH on L6 myotube" --- p.75 / Chapter 4.3 --- Effect of pretreatment of cells in conditions of insulin resistance --- p.76 / Chapter 4.3.1 --- Effect of high glucose and high insulin preincubation on L6 myotubes --- p.76 / Chapter 4.3.2 --- Effect of FFA preincubation on L6 myotubes --- p.78 / Chapter 4.3.3 --- Effect of CHO preincubation on L6 myotubes --- p.82 / Chapter 4.3.4 --- Effect of cell preincubation in conditions of insulin resistance on L6 myotubes (TEM) --- p.83 / Chapter 4.4 --- Summary of the effects of cell preincubation in conditions of insulin resistance --- p.84 / Chapter 4.5 --- Possible mechanisms involved in insulin resistance induction --- p.86 / Chapter 4.5.1 --- Possible changes in GLUT expression and activities --- p.87 / Chapter 4.5.2 --- Possible changes in insulin signaling propagation --- p.88 / Chapter 4.5.3 --- Altered functioning of various actin accessory proteins --- p.89 / Chapter 4.6 --- Limitation of the study --- p.90 / Chapter 4.7 --- Conclusion --- p.90 / Chapter 4.8 --- Future study --- p.91 / REFERENCES --- p.93 / TABLES

Indiana University-Purdue University Indianapolis (IUPUI) / Diabetes mellitus is a group of related metabolic diseases that share a common pathological mechanism: insufficient insulin signaling. Insulin is a hormone secreted from pancreatic β cells that promotes energy storage and consequently lowers blood glucose. In contrast, the hormone glucagon, released by pancreatic α cells, plays a critical complementary role in metabolic homeostasis by releasing energy stores and increasing blood glucose. Restoration of β cell mass in diabetic patients via β cell regeneration is a conceptually proven approach to finally curing diabetes. Moreover, in situ regeneration of β cells from endogenous sources would circumvent many of the obstacles encountered by surgical restoration of β cell mass via islet transplantation. Regeneration may occur both by β cell self-duplication and by neogenesis from non-β cell sources. Although the mechanisms regulating the β cell replication pathway have been highly investigated, the signals that regulate β cell neogenesis are relatively unknown. In this dissertation, I have used zebrafish as a genetic model system to investigate the process of β cell neogenesis following insulin signaling depletion by various modes. Specifically, I have found that after their ablation, β cells primarily regenerate from two discrete cellular sources: differentiation from uncommitted pancreatic progenitors and transdifferentiation from α cells. Importantly, I have found that insulin and glucagon play crucial roles in controlling β cell regeneration from both sources. As with metabolic regulation, insulin and glucagon play counter-balancing roles in directing endocrine cell fate specification. These studies have revealed that glucagon signaling promotes β cell formation by increasing differentiation of pancreas progenitors and by destabilizing α cell identity to promote α to β cell transdifferentiation. In contrast, insulin signaling maintains pancreatic progenitors in an undifferentiated state and stabilizes α cell identity. Finally, I have shown that insulin also regulates pancreatic exocrine cell development. Insufficient insulin signaling destabilized acinar cell fate and impairs exocrine pancreas development. By understanding the roles of pancreatic hormones during pancreas development and regeneration can provide new therapeutic targets for in vivo β cell regeneration to remediate the devastating consequences of diabetes.