This proposal is directed at the development of molecular engineering that can be used to improve the performance of insulin secreting cells lines. Available cell lines do not always adequately release insulin in response to glucose and other physiological stimuli and the threshold for glucose stimulated insulin release is often markedly decreased. It is hypothesized that this is due to an elevated hexokinase:glucokinase ratio. In the first specific aim, gene transfection studies with adenovirus vectors will be used in an attempt to normalize the hexokinase:glucokinase ratio. The importance of the subcellular localization of glucokinase using glucokinase:hexokinase chimera molecules will also be determined along with an analysis why overexpression of glucokinase in beta cells does not lead to major metabolic effects. It is hypothesized that this is due to a glucokinase-derived metabolite and attempts will be made to isolate this unknown metabolite. The second specific aim will address the question why cell lines and also in vitro incubated beta cells show a poor response to glucose. The magnitude of insulin release is often far from that observed in the perfused pancreas or in freshly isolated islets. It is hypothesized that the poor glucose response is due to a deficient expression of certain phospholipase C isoforms and a failure to increase inositol phosphate synthesis in response to glucose. It will be tested that delivery of the gene(s) for these enzymes will restore glucose sensing. The importance of these studies is to develop cell lines for possible insulin replacement in IDDM and to understand beta cell dysfunction in NIDDM.
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