The purpose of this project is to investigate the mechanisms through which insulin, sulfonylureas, and glucose regulate glucose uptake and the glucose transporter in skeletal muscle. Although the research will include studies on rapid regulatory responses, it will emphasize more gradually-developing, long-term regulation of glucose transport. These long-term regulatory events are thought to be of great importance in diabetes mellitus and other disease states, but have not been well defined. The specific goals are: (1) To determine the mechanisms of long-term regulation of glucose transport by insulin in the L6 rat skeletal muscle cell line. The mechanism through which insulin increases the total number of glucose transporters in L6 cells will be studied with biosynthetic labeling methods. Specific probes for the transporter will include an anti-transporter monoclonal antibody, cytochalasin B, and transporter cDNA. (2) To determine the molecular basis for the generation by sulfonylureas of nonfunctional or hypofunctional transporters in L6 cells that can be activated by insulin. This will be investigated with the same techniques and also with surface-labeling and other cell fractionation methods. (3) To determine whether sulfonylureas have rapid as well as long- term effects on L6 cells glucose transport and to determine whether a rapid response to sulfonylureas requires the presence of insulin, as is the case for long-term sulfonylurea effects. (4) To study the long-term and short-term regulation of glucose uptake and of the glucose transporter in normal and streptozotocin diabetic rats in vivo. Chronic, indwelling jugular venous catheters will be used to independently control the concentrations of insulin, glucose, and sulfonylareas for periods varying from minutes to several days. Regulation of glucose transport will then be assessed by determinations of insulin-stimulated glucose uptake in isolated muscles and by determining the total quantity, subcellular distribution, and biochemical characteristics of the glucose transporter. The experiments in this project should provide a basis for understanding the regulation of glucose transport in muscle in general. It also should provide important new information about the processes that lead to insulin resistance in diabetes mellitus and other human diseases.
