Glucose homeostasis in mammals is regulated by insulin, a hormone whose circulating levels increase in response to food intake. Insulin interacts with its receptor in target cells such as adipocytes and muscle to mobilize glucose transporter molecules from an intracellular, vesicular storage pool to the cell surface where they function to clear blood glucose. This process of transporter mobilization or recruitment utilizes a unique glucose transporter isoform, GLUT4, whose expression is restricted to certain insulin target cells. Experiments in the Principal Investigators own laboratory and elsewhere have shown that the ectopic expression of GLUT4 in a variety of cell types possessing insulin receptors is not sufficient to confer insulin-sensitive glucose uptake. Therefore, some number of additional, as yet unidentified, gene products must be expressed for cells to exhibit insulin-activated GLUT4 translocation. It is the goal of this application to identify such genes and the functions) of their protein products. The intracellular vesicular GLUT4 pool is an apparently unique compartment in that many of its constituent proteins are prevented from residing at the cell surface except when cells are exposed to insulin. Thus, the first specific aim of the application is to follow up on the recent discovery that the GLUT4 vesicles contain three large glycoproteins whose translocation in response to insulin is identical to that of GLUT4. The Principal Investigator believes that their protein sequence(s) may reveal clues as to the origin and biochemistry conferring insulin-responsiveness to these vesicles. Moreover, their genes may reveal the elements regulating the tissue-specific expression of the insulin-dependent glucose transport response. The previous studies have identified other vesicle proteins that may function more generally in membrane trafficking, a topic of much current interest to cell biology, and the Principal Investigator will continue to search for other protein constituents of the GLUT4-rich vesicles that may help elucidate the way in which cells mediate the necessary movement between membrane compartments. There must be communication between the insulin receptor and the target vesicles, and as yet, little or nothing is known about this step. The Principal Investigator proposes in the second specific aim to identify cellular components possibly mediating this step by a PCR-based strategy called differential mRNA display. Treatment of cultured adipocytes with Tumor Necrosis Factor alpha (TNFalpha) results in the complete down-regulation of GLUT4 and the development of insulin resistance. The differential display techniques allows the facile cloning of genes that are differentially expressed in both the TNF-treated and untreated cells, and the Principal Investigator has already identified two such novel genes. The Principal Investigator proposes to characterize these and identify additional differentially expressed genes. The most common form of diabetes is type II or maturity onset diabetes which is typically associated with """"""""inulin resistance"""""""", the failure of target tissue to respond normally to insulin. By identifying novel genes involved in mediating the insulin response, the Principal Investigator will gain a better understanding of this process which may, in turn, lead to treatments and/or cures for this common and serious disease.
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