The major objectives of this proposal are to gain a better understanding of the physiological action of insulin as well as to further characterize how certain factors can directly alter insulin action in fat cells. Previous studies suggest that growth hormone, glucocorticoids and a factor present in serum from patients with chronic renal disease inhibit either basal and/or insulin-stimulated glucose transport. Sulfonylureas, oral hypoglycemic agents, potentiate insulin-stimulated transport. Their mechanism of action will be further explored by measuring glucose """"""""transporters"""""""" in plasma membranes and low density microsomes before and after insulin exposure to the intact cell. These studies will investigate the possibility that these factors act by influencing the translocation of glucose """"""""transporters"""""""" in response to insulin. The mechanism of the insulin resistance associated with chronic renal disease will be extensively evaluated. There is strong evidence that this state is caused by a circulating factor. With the availability of a bio-assay for this uremic factor, it is anticipated that purification to homogeneity can be accomplished through the use of high-pressure liquid chromatography. It is also anticipated that the amino acid composition will be determined. If these aims are achieved, then further studies concerning the source of the factor and mechanism of action will be explored. The third major area of investigation is concerned with the role of the carbohydrate moiety of the insulin receptor in insulin binding, receptor function and stimulation of post receptor events. Although this area is largely unexplored, it is known that sialic acid residues are important for effecting an insulin response and that removal of terminal galactose residues results in a loss of high affinity binding. The effects of removal of sugar moieties by appropriate glycosidase digestion on several aspects of receptor function will be evaluated. These include receptor internalization, down regulation, and autophosphorylation. The latter has been touted as the post-binding initiator of insulin's cellular effects. The effects of insulin mimickers, counter-regulatory hormones and sulfonylureas on the phosphorylation or the insulin receptor will also be studied. It is anticipated that the proposed studies will contribute to our basic knowledge of the early steps of insulin action (receptor and glucose transport) as well as to our understanding of the pathophysiology of altered insulin responsiveness in certain disease states.
