The irreversible degradation of branched-chain amino acids (BCAAs) by branched-chain ketoacid dehydrogenase (BCKAD) is regulated downward when dietary protein is limited as in the cases of anorexia or the use of low- protein diets to treat the symptoms of or slow progression of uremia. Increased BCKAD activity causing accelerated degradation of BCAA could blunt or block this nutritional response and limit the availability of BCAA for protein synthesis. Besides protein intake, acidosis accelerates BCAA catabolism; correction of the acidosis of kidney failure normalizes whole-body leucine oxidation. Glucocorticoids (GC) are involved in this process because: 1) GC are increased in acidosis and uremia; and 2) GC can modulate the activation of BCKAD. Preliminary experiments suggest that BCKAD activity and subunit mRNA levels are increased in muscles of adrenalectomized (ADX) rats fed a low-protein diet only when they are acidotic and given GC. In sharp contrast, BCKAD activity and subunit mRNA levels are decreased in liver. Our objectives are 1) to determine if conditions causing malnutrition change BCAA metabolism by varying the activity of BCKAD at both genetic and biochemical levels; and 2) to define the role of GC in these responses.
Specific Aim 1 : to elucidate the role of GC in regulating BCKAD enzyme activity and/or mRNA abundance in liver and muscle. Our hypothesis is that GC play a pivotal role in changing BCKAD activity and mRNA levels in different tissues. BCKAD enzyme activity, as well as subunit mRNA abundance and transcription rates will be measured in muscle and liver of ADX acidotic rats plus/minus GC supplements. To identify the role of GC in these responses, BCKAD activity and mRNA abundance will be measured in LLC-PK1 cells which lack GC receptors and in cells transfected with the GC receptor gene; we will also study BC3H1 myocytes which express GC receptors.
Specific Aim 2 : to determine if acute uremia, a condition characterized by loss of lean body mass, affects BCKAD activity in muscle and liver, independently of GC and/or acidosis. The hypotheses is that uremia may have independent effects on BCKAD activity, protein abundance, and mRNA levels will be measured in ADX rats with ARF, plus/minus GC supplements and/or plus/minus bicarbonate supplements.
Specific Aim 3 : to determine the molecular basis for two BCKAD result from different forms of BCKAD E1alpha mRNAs expressed in tissues. Moreover, this could be the basis for the observation that the percentage of total BCKAD enzyme in the active state varies dramatically in different tissues.
Specific Aim 4 : to characterize elements in the rat BCKAD E1alpha gene promotor. The hypothesis is that the abundance of BCKAD E1alpha mRNA is regulated by altering transcription. Results from these studies should elucidate mechanisms that influence how amino acid metabolism can change even when dietary protein restriction should suppress BCAA oxidation.
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