The long-term objective of this laboratory is to understand the mechanism of insulin resistance in the human liver from patients with NIDDM. This new effort is possible because: 1) a method to isolate and culture insulin-responsive hepatocytes from an open liver biopsy has been developed, and 2) ECU School of Medicine has a population of patients from whom an intraoperative liver biopsy can be obtained. Over 100 gastric bypass procedures are performed annually for the treatment of morbid obesity, of whom 27% have NIDDM, 20% impaired glucose tolerance, and the rest have normal glucose tolerance. Initial studies with isolated hepatocytes from these and other non- obese, non-diabetic patients undergoing elective surgery will establish the interrelationships between insulin action, binding, internalization, and degradation. Then, insulin receptor structure, metabolism and function, and the level and abundance of the various species of the insulin receptor mRNA will be studied before the intracellular domain can be assigned as the site for the defect in NIDDM. We will test the hypothesis that in NIDDM hyperglycemia causes nonenzymatic glycosylation of the insulin receptor and alters its function. Also, in addition to determining the activity of the insulin receptor kinase in vitro and in vivo, strategies are developed to demonstrate phosphorylation of endogenous substrates by the insulin receptor kinase. Finally, analytical and enzymatic methods will be used in isolated hepatocytes and liver homogenates to ascertain if the carbohydrate intolerance of NIDDM is due to increased glucose production, decreased glucose utilization, or a combination of both. Special effort will be given to two important regulatory loci: 1) pyruvate dehydrogenase activity, the critical link enzyme between carbohydrate and lipid metabolism, and 2) fructose 2,6- bisphosphate concentration, a key compound in the control of glucose metabolism. The significance of this proposal is enhanced by the recognition that the background knowledge from human liver at the cellular level is virtually nonexistent. It is hoped that this work will establish the cellular basis for the insulin resistance in NIDDM and help to develop a more rational therapeutic approach to the disease.