Regulation of the branched-chain 2-oxo acid dehydrogenase complex in hepatocytes isolated from rats fed on a low-protein diet

Abstract

Hepatocytes isolated from rats fed on a chow diet or a low-protein (8 % ) diet were used to study the effects of various factors on flux through the branched-chain 2-oxo acid dehydrogenase complex. The activity of this complex was also determined in cell-free extracts of the hepatocytes. Hepatocytes isolated from chow-fed rats had greater flux rates (decarboxylation rates of 3-methyl-2-oxobutanoate and 4-methyl-2-oxopentanoate) than did hepatocytes isolated from rats fed on the low-protein diet. Oxidizable substrates tended to inhibit flux through the branched-chain 2-oxo acid dehydrogenase, but inhibition was greater with hepatocytes isolated from rats fed on the low-protein diet. 2-Chloro-4-methylpentanoate (inhibitor of branched-chain 2-oxo acid dehydrogenase kinase), dichloroacetate (inhibitor of both pyruvate dehydrogenase kinase and branched-chain 2-oxo acid dehydrogenase kinase) and dibutyryl cyclic AMP (inhibitor of glycolysis) were effective stimulators of branched-chain oxo acid decarboxylation with hepatocytes from rats fed on a low-protein diet, but had little effect with hepatocytes from rats fed on chow diet. Activity measurements indicated that the branched-chain 2-oxo acid dehydrogenase complex was mainly (96%) in the active (dephosphorylated) state in hepatocytes from chow-fed rats, but only partially (50%) in the active state in hepatocytes from rats fed on a low-protein diet. Oxidizable substrates markedly decreased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had much less effect in hepatocytes from chow-fed rats. 2-Chloro-4-methylpentanoate and dichloroacetate increased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had no effect on the activity state of the enzyme in hepatocytes from chow-fed rats. The results indicate that protein starvation greatly increases the sensitivity of the hepatic branched-chain 2-oxo acid dehydrogenase complex to regulation by covalent modification.

Modulation of rat skeletal muscle branched-chain alpha-keto acid dehydrogenase in vivo. Effects of dietary protein and meal consumption.

“…As others have shown diet-mediated changes in BCKAD activities from liver, kidney, and heart (12,13,17,19,20), we first sought to optimize the assay for BCKAD isolated from muscles of rats fed diffiering levels of protein. Rats were meal-fed 0,25 (normal), or 50% (high) casein diets for 2 wk.…”

“…BCKAD activity is thought to be the major regulator of BCAA oxidation in vivo (14). As liver BCKAD is fully active in normally fed rats (13,(17)(18)(19), activation of extrahepatic BCKAD would be expected to result in enhanced oxidation of BCAA in vivo. In the present study, skeletal muscle BCKAD was only 1-2% active during the postabsorptive period in rats fed 25% casein (Figs.…”

“…In normally fed rats, BCKAD from liver is essentially fully active (i.e., dephosphorylated) (13,(17)(18)(19). In contrast, < 20% of skeletal muscle BCKAD is in the active form (12,13,18,20,21).…”

Down-regulation of rat mitochondrial branched-chain 2-oxoacid dehydrogenase kinase gene expression by glucocorticoids

“…The activity of the BCOD complex varies between tissues, and is regulated by nutritional and hormonal stimuli [5][6][7][8][9]. This provides an effective mechanism by which to modulate the flux of branched-chain amino acids through the BCOD complex.…”

Structural and Biochemical Characterization of Human Mitochondrial Branched-chain α-Ketoacid Dehydrogenase Phosphatase

“…Rats fed low-protein diets show increased phosphorylation of the hepatic BCKDC (5). Starvation and diabetes increase BCKDC activity in skeletal muscle by decreasing phosphorylation of the enzyme complex (6).…”