The major findings of the current grant were that the insulin resistance associated with diabetes and obesity is due to a decrease in the amount of glucose transport protein in muscle and to an inability to translocate the glucose transporters to the cell membrane in response to insulin. Exercise training overcomes insulin resistance by increasing the expression of the glucose transporter protein in muscle. In addition, acute exercise increases muscle glucose transport by inducing the translocation of glucose transporters to the cell membrane. We propose to expand these important observations with the following studies. Muscle glucose transporter (GLUT4) protein and mRNA are increased by exercise training and decreased in diabetic animals. Our hypothesis is that the changes in GLUT4 glucose transporter mRNA are a result of altered gene expression mediated through the second messenger c-AMP pathway. GLUT4 gene transcription will be assayed by nuclear run-on analysis and mRNA stability will be assessed by measuring the decline of GLUT4 mRNA in muscle perfused with actinomycin D. If transcription is regulated, footprint analysis of the GLUT4 promoter will be investigated. Agents that activate adenylate cyclase or inhibit phosphodiesterase will be used to investigate the role of c-AMP in regulation of GLUT4 gene expression. The insulin resistance in obese Zucker rats has been shown to be due to an inability to translocate glucose transporters to the cell membrane in response to insulin. Our hypothesis is that there are two intracellular compartments of glucose transporters, one recruited by insulin and the other by muscle contraction (or hypoxia), and that insulin-resistance is a consequence of glucose transporters being sequestered in a compartment from which they can only be recruited by muscle contraction (or hypoxia). This hypothesis will be investigated by measuring glucose transport and membrane distribution of GLUT4 transporters in perfused muscle of lean, sedentary- obese, and exercised-obese animals. Muscles of these rats will be perfused in the basal state (normoxic, without insulin), in the presence of insulin (10-7M), and in the hypoxic state. The combined techniques of membrane isolation and immunocytolocalization will be used to investigate the two pools of transporters.
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