The essential branched-chain amino acids (BCAA) play critical roles in maintaining normal protein homeostasis and they influence critical intracellular signaling pathways that regulate metabolic functions. In normal individuals, nutritional adaptations to a reduced dietary protein intake (e.g., fasting, a low protein diet prescription) decrease the irreversible degradation of BCAA. Catabolic conditions like chronic renal failure (CRF) or acute diabetes impair these adaptive responses that preserve protein mass, thus contributing to the loss of lean body mass. The goals of Dr. Price and colleagues are to understand the mechanisms that regulate the activity of branched-chain alpha-ketoacid dehydrogenase (BCKAD), the rate-limiting enzyme in BCAA degradation, in the major tissues where BCAA are catabolized, and to determine if there are common signals in different catabolic states that regulate BCKAD activity, and hence, BCAA levels. To address these goals, the investigators will evaluate three hypotheses: 1) Acidification and glucocorticoids influence transcription of BCKAD subunit genes through specific cis-acting response elements. The investigators will identify specific DNA promoter elements in the BCKAD E2 gene that confer responses to acidification and glucocorticoids. 2) Abnormalities in BCAA utilization in rats with CRF result from tissue-specific alterations in BCKAD activity at both genetic and biochemical levels. The investigators will define how CRF influences the activities of BCKAD and BCKAD kinase, a unique kinase that inhibits BCKAD activity, in muscle, liver and kidney in a well-established rat model. They will measure BCKAD activity, BCKAD subunit and kinase proteins and amounts of subunit and kinase mRNAs 3) Insulin modulates BCKAD and/or BCKAD activities in different tissues by a mechanism requiring the critical signaling enzyme phosphatidylinositol 3-kinase. The investigators will determine the biochemical mechanism(s) that increase BCKAD activity in rat muscle, liver and kidney in response to acute diabetes mellitus (i.e., insulin insufficiency) and then examine the signaling mechanisms by which insulin regulates BCKAD and BCKAD kinase in cultured L6 muscle cells. The investigators findings will define cellular mechanisms regulating BCAA degradation in uremia, acute diabetes and other catabolic conditions.
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