There is ongoing controversy about the effects of insulin on protein synthesis in vivo. Human studies using isotopic techniques are in conflict with the results from in vitro and animal studies. Previous human studies have used surrogates of amino acyl-tRNA (AA-tRNA) (the obligatory precursor of protein synthesis) in the measurements of protein synthesis because of the inability to measure AA-tRNA isotopic enrichment in small tissue samples. This laboratory recently developed methodologies to measure stable isotopic enrichment in AA-tRNA from human biopsy samples taken under local anesthesia and to purify myosin from human skeletal muscle biopsy samples. These novel techniques will be used to determine the effect of insulin on fractional protein synthetic rates of mixed muscle protein and myosin in human objects in the fasted state and during amino acid replacement. Previously, measurements of protein synthesis were made only in the whole body, mixed muscle tissue, or across specific organs, not for specific proteins. Effect of intraarterial infusion of insulin on muscle proteins synthesis in healthy control subjects will be examined, first, then the effect of systemic insulin treatment will be assessed in patients with IDDM. Protein synthetic rates will be estimated using various surrogates of AA-tRNA and compared to rates determined from AA-tRNA to directly determine the extent to which currently used methods provide an accurate assessment of protein synthesis. Protein breakdown across the leg and the splanchnic region will be simultaneously measured, allowing the hypothesis that lower doses of insulin are needed to achieve insulin's maximal effect in the leg as compared to the splanchnic region to be tested. Since insulin-deprivation increases glucagon and GH levels in IDDM patients, and these hormones have opposite effects on protein metabolism, the combined effects of these hormones on protein dynamics in the whole body and across the leg and the splanchnic region will be investigated. Currently, there are no validated surrogates for AA-tRNA enrichment in the liver and the direct measurement of liver AA-tRNA is impractical in humans. Studies using an adult miniature pig model will be performed to test the hypothesis that during continuous infusion of a stable isotope, VLDL ApoB isotopic enrichment at plateau is the same as liver AA-tRNA enrichment. These studies will be performed in the fasted state and with insulin infusion and insulin plus amino acid infusion. These studies will provide new insight into the regulation of protein metabolism in diabetes and healthy humans. Hopefully, this information in turn will lead to more effective therapies for people with diabetes mellitus and other catabolic illnesses.
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