| Diabetes is a heterogeneous group of metabolic disorders characterized by hyperglycemia, with beta-cell dysfunction as a major contributing factor. Specific insulin gene mutations can cause diabetes in humans. The primary goal of my thesis research has been to increase our knowledge of how insulin gene mutations cause diabetes, by understanding how they contribute to beta-cell failure and loss.;The first section of my dissertation describes our in vitro characterization of ten human insulin gene mutations and how they contribute to beta-cell dysfunction and neonatal diabetes. MIN6 mouse insulinoma cells were used as a model system in which to study the subcellular localization, processing, and secretion of fluorescently tagged human proinsulin mutants. We found that these mutations fell into three categories, each characterized by their degree of ER retention and effect on wild-type insulin secretion. The most severe mutations were C43G, F48C, and C96Y. These proteins were not localized to secretory granules due to significant retention in the ER, and were therefore not secreted. Expression of mutant proteins was associated with attenuated secretion of co-expressed wild-type insulin. We found increased Chop expression with these mutants, indicating the activation of the unfolded protein response and impending apoptosis. These results suggest that mutant proinsulin expression impairs the beta-cell secretory response before triggering apoptosis. Furthermore, our evidence suggests that these mutations affect proinsulin production in different ways, and therefore effort should be made to understand the biology of each mutation.;The second section of my thesis describes novel methods we developed to perform glucose injections and collection of blood for downstream assays using adult zebrafish. The overall goal of this study was to enhance the utility of the zebrafish model by allowing the study of beta-cell function. We describe optimal anesthesia methods, fasting duration and technique, IP glucose injection, and small-scale blood collection and glucose measurement. These useful and reliable techniques were applied to on-going studies.;In the third section of my thesis, I sought to understand how insulin gene mutations contribute to beta-cell dysfunction in vivo, using the adult zebrafish as a model. I generated transgenic zebrafish with beta-cell specific expression of human preproinsulin, wild-type or mutant C43G, bearing GFP tags, permitting analysis of subcellular localization, processing, and secretion of the protein. The C43G mutant protein was held in the ER of beta-cells, was not trafficked to secretory granules, and was not processed from proinsulin. Despite excessive accumulation of C43G-GFP and highly irregular beta-cell morphology, hyperglycemia was not observed in these fish, and circulating C-peptide levels appeared normal. Based on these results, I hypothesize that a continuous cycle of beta-cell death and regeneration may be occurring.;The adult zebrafish is a useful model system in which to study insulin gene mutations. Their remarkable capacity for beta-cell regeneration provides an interesting opportunity to study situations that normally lead to beta-cell death and diabetes in mouse models. Future experiments will help us understand this process and could provide direction for additional research on therapeutic interventions for diabetic patients with insulin gene mutations. |
