NEW ANIMAL MODEL FOR EARLY ONSET NONIMMUNE DIABETES

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The long term objective is to evaluate how changes in calcium homeostasis in the beta cell of the pancreas results in development of very early onset non-immune diabetes. Transgenic mice have been produced in which the rat insulin II gene promotor has been ligated to a chicken calmodulin minigene. Expression of this minigene is specific to the insulin-producing beta cells and results in an increased calmodulin content. Calmodulin is a calcium binding protein that is present at high concentration in beta cells and serves to regulate several enzymes involved in glucose-mediated insulin secretion. The 5X increase in calmodulin occurs early in embryonic beta cell development and results in diabetes that becomes evident within a few hours after birth. The disease is characterized by increased blood glucose, decreased insulin in both pancreas and serum as well as gradual depletion of beta cells. Except for reduced insulin content, beta cells in the late embryo appear normal whereas those of 2 day neonates are markedly abnormal. Results suggest that excess calmodulin interferes with beta cell function leading to diabetes. The diabetic state and excess calmodulin are involved in beta cell toxicity. We propose to complete analysis of both fetal and neonatal pancreas in the existing transgenic lines. Analysis will include electron microscopic examination and immunogold localization of calmodulin and the islet hormones. Pancreatic islets will be isolated and evaluated for insulin secretion in response to glucose, glucose metabolism, glucose sensitive K+ channels, Ca++ conductance and changes in intracellular Ca++. The second aim is to determine whether diabetes is specifically due to elevated calmodulin or increased Ca++ buffering capacity. We will generate new lines of transgenic mice in which the transgene will be a mutant calmodulin molecule that binds calcium normally but cannot bind to or activate calmodulin-dependent enzymes. The glycemic state of these mice will be evaluated by measurement of blood glucose and insulin. If these are also diabetic, similar experiments as described for the current transgenic lines will be performed. The final aim is to address the importance of normal levels of calmodulin on development of the endocrine pancreas and proliferation and differentiated function of beta cells. This problem will be evaluated utilizing transgenic mice that express either a calmodulin antisense RNA or a calmodulin binding protein in beta cells. The purpose is to decrease calmodulin levels and question the functional significance of this reduction. Completion of these aims will provide insight into molecular events that could produce diabetes with characteristics similar to those present in both IDDM and NIDDM in humans.