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Branched-chain alpha-ketoacid dehydrogense complex (BCKDH) is a regulatory enzyme of valine, isoleucine and leucine catabolism. Its activity is mainly regulated by covalent modification achieved by a specific BCKDH kinase (BDK) and phosphatase (BDP). The goal of our study was to investigate the effect of increasing doses of fenofibrate on BDK and BCKDH activities in rat's liver. For 14 days fenofibrate was administrated to Wistar male rats (fed chow containing 8% protein) at one of the daily doses: 5, 10, 20 and 50mg/kg. Control group was given only vehicle (0.3% methylcellulose). BDK activity as well as actual BCKDH activity and total BCKDH activity were assayed spectrophotometrically and BDK protein amount was determined by Western blotting. In rats administered fenofibrate BDK activity decreased by 61%, 64%, 66% and 89% (p<0.0001). Changes in BDK protein expression did not correspond with changes in BDK activity. BCKDH complex actual activity was 3.7+/-0.3, 4.1+/-0.1, 4.6+/-0.3 and 4.0+/-0.3fold higher (p<0.0001) and BCKDH total activity 1.3+/-0.1, 1.3+/-0.1, 1.5+/-0.1 and 1.3+/-0.1fold higher comparing to control group (p<0.001). BCKDH activity state (percentage of active, dephosphorylated form) increased 2.8+/-0.2, 3.1+/-0.1, 3.2+/-0.1 and 3.0+/-0.1fold (p<0.0001). In addition, fenofibrate prevented body weight gain starting from the dose of 10mg/kg/day and induced hepatomegaly in a dose-dependent manner. It can be concluded that fenofibrate under condition of protein restriction starting from the lowest dose inhibits BDK activity at the posttranslational level and increases BCKDH activity state. It is conceivable that fenofibrate decreases of branched-chain amino acids (BCAA) levels by stimulation of their catabolism. Since leucine plays an important role in up-regulation of protein anabolism in muscles, the reduced level of this amino acid may be one of the factors involved in pathomechanism of myopathy observed during treatment with fenofibrate.

Levels of expression of two subunits of the liver branched-chain alpha-ketoacid dehydrogenase complex in response to extremes of dietary protein intake (50% versus 0% protein diet) were determined by quantitative immunoblotting. Dietary protein deficiency decreased the amount of E1 alpha protein to a greater extent than E2 protein. The ratio of E1 alpha to E2 was below 1 in the liver of animals starved for protein and above 1 in the liver of animals fed the high-protein diet. Supplementation of the 0% protein diet with 5% leucine (but not 5% valine) had the same effect as the 50% protein diet. The extremes of dietary protein also resulted in a divergent pattern of expression of the mRNAs for the subunits of the complex. The E1 beta message showed the expected corollary of being greater in the liver of the high-protein-fed rats than the no-protein-fed rats. In contrast, the E2 message was not affected by the two extremes of dietary protein and the E1 alpha message was greater in the liver of the no-protein-fed rats than the high-protein-fed rats. Thus, coordinate regulation of gene expression of the subunits of the complex does not occur in response to dietary protein. Post-transcriptional regulatory mechanisms most likely determine the amount of the complex and the ratio of its subunits. The decrease in E1 alpha/E2 protein ratio that occurs in dietary protein deficiency may increase sensitivity of the complex to phosphorylation-mediated inhibition by branched-chain alpha-ketoacid dehydrogenase kinase.

Severe ketotic diabetes induced in rats by streptozotocin resulted in a reduction in activity of the hepatic branched-chain alpha-ketoacid dehydrogenase complex, regardless of whether activity was expressed on the basis of liver wet weight, total liver, liver protein, or liver DNA. A decrease in enzyme specific activity (units of enzyme activity per mg of enzyme protein) was found responsible for the reduction in measurable enzyme activity of the complex. Insulin treatment reversed the decrease in enzyme specific activity. Treatment of tissue extracts with phosphoprotein phosphatase had no effect, indicating that activity of the complex was decreased by some mechanism other than reversible phosphorylation. Specific protein components of the complex were also not found reduced by the diabetic state. Induction of severe ketotic diabetes in rats previously fed a low-protein diet resulted in activation of the enzyme as a consequence of dephosphorylation. Nevertheless, the specific activity of the dephosphorylated enzyme of diabetic, low-protein-fed rats was decreased relative to that of control, low-protein-fed animals. Reconstitution studies with tissue extracts fortified with the purified E1 component indicate that severe diabetes induces a defect in this component of the hepatic branched-chain alpha-ketoacid dehydrogenase complex.

Ethanol inhibited glucose synthesis from alpha-ketoisovalerate by isolated rat hepatocytes without significant inhibition of flux through the branched-chain alpha-ketoacid dehydrogenase complex. Accumulation of 3-hydroxyisobutyrate, an intermediate in the catabolism of alpha-ketoisovalerate, was increased by ethanol, indicating inhibition of flux at the level of 3-hydroxyisobutyrate dehydrogenase. 3-Hydroxybutyrate caused the same effects as ethanol, suggesting inhibition was a consequence of an increase in the mitochondrial NADH/NAD+ ratio. Flux through the 3-hydroxyisobutyrate dehydrogenase was more sensitive to regulation by the mitochondrial NADH/NAD+ ratio than flux through the branched-chain alpha-ketoacid dehydrogenase. Oleate also inhibited glucose synthesis from alpha-ketoisovalerate, but marked inhibition of flux through the branched-chain alpha-ketoacid dehydrogenase complex was caused by this substrate.

Feeding clofibric acid to rats caused little or no change in total activity of the liver branched-chain 2-oxo acid dehydrogenase complex (BCODC). No change in mass of liver BCODC was detected by immunoblot analysis in response to dietary clofibric acid. No changes in abundance of mRNAs for the BCODC E1 alpha, E1 beta and E2 subunits were detected by Northern-blot analysis. Likewise, dietary clofibric acid had no effect on the activity state of liver BCODC (percentage of enzyme in the dephosphorylated, active, form) of rats fed on a chow diet. However, dietary clofibric acid greatly increased the activity state of liver BCODC of rats fed on a diet deficient in protein. No stable change in liver BCODC kinase activity was found in response to clofibric acid in either chow-fed or low-protein-fed rats. Clofibric acid had a biphasic effect on flux through BCODC in hepatocytes prepared from low-protein-fed rats. Stimulation of BCODC flux at low concentrations was due to clofibric acid inhibition of BCODC kinase, which in turn allowed activation of BCODC by BCODC phosphatase. Inhibition of BCODC flux at high concentrations was due to direct inhibition of BCODC by clofibric acid. The results suggest that the effects of clofibric acid in vivo on branched-chain amino acid metabolism can be explained by the inhibitory effects of this drug on BCODC kinase.