Following ingestion of food, insulin stimulates the cellular utilization of glucose and other nutrients, and failures in this process leads to diabetes mellitus. The complete biochemical pathway by which insulin acts on sugar uptake and related processes is not know, but two well-known proteins, the insulin receptor and the glucose transporter, are certain to be involved in the post- prandial clearance of blood glucose. It is the long-term goal of this application to understand the biochemical basis of insulin- activated glucose transport with specific experimental emphasis on the glucose transport protein and its cellular environment. It has been recognized for nearly ten years that a major mechanism, and probably the most important mechanism, by which insulin stimulates glucose transport is by causing the redistribution of transport proteins from and intracellular site, where they are inaccessible to the circulation, to the plasma membrane, where they can function in the cellular uptake of glucose. Recent work form our lab has demonstrated that the insulin-sensitive glucose transporter is immunologically distinct from other known forms of glucose transport proteins, a result that is also consistent with a large body of less direct evidence from any labs. Moreover, several labs have prepared partially purified intracellular membranes form insulin-responsive cells that are enriched in glucose transporters. Given this background, the specific aims of this application are to purify the insulin-sensitive glucose transporter, to sequence the protein or its proteolytically-derived peptides and to clone cDNA for this transporter as a possible means of identifying why this protein is insulin sensitive. A good possibility is that the site of insulin responsiveness resides in some protein, other than the transporter, in the intracellular membrane in which the transporters are located in resting cells. Thus, these vesicles will also be purified to homogeneity by immunoaffinity chromatography, and their protein constituents will be biochemically and immunologically characterized. Data generated in this way should also point backward towards the insulin receptor such that the biochemical steps linking movement of intracellular vesicles to receptor activation may be elucidated. Given the known information derived from the experimental approach described here will be of use in understanding certain forms of this common disease.
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