The overall objective of this research is to continue our work in the characterization of the hormonal and substrate factors which regulate branched chain amino acid (BCAA) and protein metabolism in normal and diabetic man. Using single isotope dilution methods employing both radioactive and stable isotopes of leucine, an open two pool model of leucine metabolism was developed. This model utilizes simultaneous infusions of [4,5-3H] leucine and [1-14C] Alpha-ketoisocaproate (KIC), the Alpha-ketoacid of leucine. From the plasma [3H] and [14C] specific activities of both leucine and KIC, estimates of the rates of entry and exit from the leucine and KIC pools can be made which we believe provide estimate of whole body proteolysis, protein synthesis and leucine oxidation.
The specific aims of this proposal are: a) to evaluate the responsiveness and attempt to validate this dual isotope model; b) to investigate the effects of glucose, insulin, leucine, isoleucine and threonine on the whole body metabolism of leucine and protein; c) to determine the effects of insulin and dietary leucine during meal absorption on protein synthesis, proteolysis and leucine oxidation. The isotope model will be evaluated by determining its responsiveness to changes in rates of leucine metabolism and by comparing rate of oxidation of leucine estimate from the model and from expired [14C]CO2 and plasma [1-14C]KIC specific activity. In addition, the effects of stable isotope tracer infusion rates on leucine and protein metabolism will be determined. Completion of these studies will permit a critical evaluation of our dual isotope model of leucine metabolism in man. A full understanding of the strengths and of the limitations of this and other methods presently available for measuring rates of leucine oxidatin used in determining rates of protein synthesis is necessary. Validation of the two-pool model will permit abandonment of the laborious, potentially inaccurate collections of expired CO2. Evaluations and application of stable isotopes to this model will permit investigation of leucine and protein metabolism in infants, children and adults without breath or urine collections, will avoid the high rates of stable isotope infusion and will eliminate exposure of subjects to radiolabeled isotopes.
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