Regulation of hepatic expression of branched-chain alpha-ketoacid dehydrogenase in mice

Abstract

Branched-chain a-ketoacid dehydrogenase (BCKAD) catalyzes the oxidative decarboxylation of the branched-chain a-ketoacids derived from the three essential amino acids leucine, isoleucine and valine. BCKAD is a multimeric enzyme complex located on the inner mitochondrial membrane and composed of an a-ketoacid decarboxylase (E1, EC 1.2.4.4), a dihydrolipoamide acyltransferase (E2, no EC number) and a dihydrolipoamide reductase (E3, EC 1.8.1.4). The E1 decarboxylase is composed of two different polypeptides, E1{dollar}\alpha{dollar} and E1{dollar}\beta{dollar}. A kinase and a phosphatase are also associated with the complex and are responsible for the inactivation (phosphorylation)-activation (dephosphorylation) of the compiex. In order to elucidate the molecular basis of branched-chain amino acid regulation, the steady state levels of BCKAD subunit RNAs and proteins were studied under varying dietary and hormonal conditions, and the promoter region of the gene encoding the E2 subunit was analyzed. A positive correlation between dietary protein intake and mRNA levels of the E1{dollar}\beta{dollar} and E2 BCKAD subunits was found in murine livers, but not in other tissues examined. The mRNA levels of the E1{dollar}\alpha{dollar} subunit, however, did not vary in response to changes in the levels of dietary protein. In contrast, starvation produced a coordinated increase in the mRNA levels of all three BCKAD specific subunits, E1{dollar}\alpha{dollar}, E1{dollar}\beta{dollar} and E2. The regulation of the expression of the BCKAD subunit genes was further examined in the rat hepatoma cells line H4IIEC3 to determine the basis of the observed pre-translational effects induced by changes in diet. Insulin was shown to negatively influence the E1{dollar}\alpha{dollar}, E1{dollar}\beta{dollar} and E2 subunit expression through both transcriptional and post-transcriptional mechanisms. Dexamethasone and dibutyryl-cAMP had positive regulatory effects on the expression of the E2 subunit. The regulatory sequences of the E2 promoter region were isolated and characterized by deletional analysis, site-directed mutagenesis and transfection into H4IIEC3 cells. A glucocorticoid responsive unit was defined which appeared to respond to multiple interacting factors. This region appears to consist of several individual sites which interact to regulate E2 promoter activity in both positive and negative directions. The existence of single regulatory regions responding to both positive and negative hormonal and dietary stimuli may be a common mechanism by which key steps in metabolism are controlled.