REGULATION OF GENE EXPRESSION OF BCKDH AND ITS KINASE

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Background: Branched-chain keto acid (BCKA) dehydrogenase is the key enzyme regulating oxidation of branched-chain amino acids (BCAA). It is generally believed that the activity of this enzyme is regulated by phosphorylation-dephosphorylation of the enzyme protein. However, our recent study in primary cultured - hepatocytes showed that dexamethasone and cyclic AMP increase the enzyme activity by increasing the expression of BCKA dehydrogenase subunit genes. These results have prompted questions on the effect of other hormones and nutrients on the expression of these genes. Hypothesis: We hypothesize that a) certain nutrients and hormones alter the activity of BCKA dehydrogenase by regulating the gene expression of the enzyme subunits, and b) the importance of this mechanism versus regulation by modulation of activity of BCKA dehydrogenase kinase (the specific phosphorylation enzyme) varies by different effectors and in different tissues. Specific Aim #1: To determine whether the flux through BCKA dehydrogenase in primary cultured cells or cell lines derived from liver and skeletal muscle is altered by branched-chain amino and keto acids, pyruvate and glucose, fatty acids, ethanol acetaldehyde, insulin, cyclic AMP. Previous studies have shown that these agents cause alterations in oxidation of BCAA in tissues and their concentrations are also altered in diabetes and chronic ethanol consumption. Specific Aims #2 and #3: To investigate whether the effect of each of the above agents includes alteration in gene expression of either BCKA dehydrogenase or BCKA dehydrogenase kinase. Specific Aim #4: To investigate the integrated effect of the above agents by determining the mechanism of alteration in BCAA oxidation in the liver and skeletal muscle of diabetic and ethanol-fed rats. Methods: The measurements will include the flux through BCKA dehydrogenase in cultured cells (liver and muscle) incubated for 6-24 h with a range of concentrations of each of the above agents; basal and total activities of BCKA dehydrogenase; activities of BCKA dehydrogenase kinase and phosphatase; Western analysis of the protein mass of E1alpha, E1beta, and E2 subunits of BCKA dehydrogenase; protein mass of BCKA dehydrogenase kinase; Northern analysis of the relative abundance of mRNAs encoding BCKA dehydrogenase and its specific kinase; rates of gene transcription, and rates of synthesis of BCKA dehydrogenase and its associated kinase. Clinical Significance: Oxidation of BCAA is altered in a wide variety of common clinical conditions, such as diabetes and alcoholism. The up- and down-regulation of BCKA dehydrogenase activity serves to protect the body from toxic effects of BCKA, as seen in Maple Syrup urine Disease, and to conserve body protein as seen during undernutrition.