AICAR stimulates mitochondrial biogenesis and BCAA catabolic enzyme expression in C2C12 myotubes.

Type 2 diabetes is characterized by reduced insulin sensitivity, elevated blood metabolites, and reduced mitochondrial metabolism. Insulin resistant populations often exhibit reduced expression of genes governing mitochondrial metabolism such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Interestingly, PGC-1α regulates the expression of branched-chain amino acid (BCAA) metabolism, and thus, the consistently observed increased circulating levels of BCAA in diabetics may be partially explained by reduced PGC-1α expression. Conversely, PGC-1α upregulation appears to increase BCAA catabolism. PGC-1α activity is regulated by 5'-AMP-activated protein kinase (AMPK), however, only limited experimental data exists on the effect of AMPK activation in the regulation of BCAA catabolism. The present report examined the effects of the commonly used AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) on the metabolism and expression of several related targets (including BCAA catabolic enzymes) of cultured myotubes. C2C12 myotubes were treated with AICAR at 1 mM for up to 24 h. Mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Metabolic gene and protein expression were assessed via qRT-PCR and western blot, respectively. AICAR treatment significantly increased mitochondrial content and peak mitochondrial capacity. AICAR treatment also increased AMPK activation and mRNA expression of several regulators of mitochondrial biogenesis but reduced glycolytic metabolism and mRNA expression of several glycolytic enzymes. Interestingly, branched-chain alpha-keto acid dehydrogenase a (BCKDHa) protein was significantly increased following AICAR-treatment suggesting increased overall BCAA catabolic capacity in AICAR-treated cells. Together, these experiments demonstrate AICAR/AMPK activation can upregulate BCAA catabolic machinery in a model of skeletal muscle.

Stimulation of rat liver branched-chain alpha-keto acid dehydrogenase activity by low doses of bezafibrate.

Multienzyme branched-chain alpha-ketoacid dehydrogenase complex (BCKDH) catalyzes the regulatory step of oxidative catabolism of indispensable branched-chain amino acids (BCAA). The activity of the BCKDH complex is regulated by a reversible phosphorylation, end-product inhibition and by changes in the gene expression of BCKDH component enzymes. It has been shown previously that a high dose of bezafibrate (an agent added to rat chow at final concentration of 0.5%) changes mRNA levels of BCKDH-related enzymes and increases dephosphorylation of the complex leading to stimulation of liver BCKDH activity and the enhanced BCAA catabolism. The aim of the present study was to determine an in vivo effect of low, clinically relevant doses of bezafibrate on BCKDH activity in rat liver. Bezafibrate was administrated for 14 days by gastric gavage to Wistar male rats (fed low-protein chow; 8% protein) at one of the following daily doses of 5, 10 and 20mg/kgb.wt. The control group was given the vehicle (0.3% methylcellulose) only. The actual BCKDH and total BCKDH activities were assayed spectrophotometrically before and after incubation with a broad-specificity phosphatase, respectively. The mRNA levels of the selected genes (BCKDH catalytic subunits and regulatory enzymes) were quantified by means of semi-quantitative RT-PCR. Current catalytic activity of BCKDH (described as BCKDH activity state - the proportion of the BCKDH complex in its active dephosphorylated form) increased by 2.1 ± 0.2, 2.3 ± 0.2 and 2.7 ± 0.2 fold (p<0.01). Changes in BCKDH activity did not correspond with changes in mRNA levels of the complex catalytic subunits. Moreover, mRNA levels of regulatory enzymes remained unaltered. Initially bezafibrate caused a transient insignificant reduction in body weight, but it had no effect on the final body weight. The highest dose of bezafibrate induced hepatomegaly. In conclusion, these data indicate that under conditions of dietary protein restriction low, clinically relevant doses of bezafibrate have a similar adverse effect on rat liver BCKDH activity and BCAA degradation rate as the high experimental dose. Up-regulation of liver BCKDH activity by low doses of bezafibrate appears to result mainly from changes in phosphorylation status of the complex (increased dephosphorylation) and is not associated with elevations in mRNA levels of BCKDH enzymatic components.

The bkdR gene of Streptomyces coelicolor is required for morphogenesis and antibiotic production and encodes a transcriptional regulator of a branched-chain amino acid dehydrogenase complex.

Products from the degradation of the branched-chain amino acids valine, leucine, and isoleucine contribute to the production of a number of important cellular metabolites, including branched-chain fatty acids, ATP and other energy production, cell-cell signaling for morphological development, and the synthesis of precursors for polyketide antibiotics. The first nonreversible reactions in the degradation of all three amino acids are catalyzed by the same branched-chain alpha-keto acid dehydrogenase (BCDH) complex. Actinomycetes are apparently unique among bacteria in that they contain two separate gene clusters, each of which encodes a BCDH enzyme complex. Here, we show that one of these clusters in Streptomyces coelicolor is regulated, at least in part, at the level of transcription by the product of the bkdR gene. The predicted product of this gene is a protein with similarity to a family of proteins that respond to leucine and serve to activate transcription of amino acid utilization operons. Unlike most other members of this class, however, the S. coelicolor bkdR gene product serves to repress transcription, suggesting that the branched-chain amino acids act as inducers rather than coactivators of transcription. BkdR likely responds to the presence of branched-chain amino acids. Its role in transcriptional regulation may be rationalized by the fact that transition from vegetative growth to aerial mycelium production, the first stage of morphological development in these complex bacteria, is coincident with extensive cellular lysis generating abundant amounts of protein that likely serve as the predominant source of carbon and nitrogen for metabolism. We suggest that bkdR plays a key role in the ability of Streptomyces species to sense nutrient availability and redirect metabolism for the utilization of branched-chain amino acids for energy, carbon, and perhaps even morphogen synthesis. A null mutant of bkdR is itself defective in morphogenesis and antibiotic production, suggesting that the role of the bkdR gene product may be more global than specific nutrient utilization.

The human malaria parasite Plasmodium falciparum possesses two distinct dihydrolipoamide dehydrogenases.

The Plasmodium falciparum genome contains genes encoding three alpha-ketoacid dehydrogenase multienzyme complexes (KADHs) that have central metabolic functions. The parasites possess two distinct genes encoding dihydrolipoamide dehydrogenases (LipDH), which are indispensable subunits of KADHs. This situation is reminiscent of that in plants, where two distinct LipDHs are found in mitochondria and chloroplasts, respectively, that are part of the organelle-specific KADHs. In this study, we show by reverse transcription polymerase chain reaction (RT-PCR) that the genes encoding subunits of all three KADHs, including both LipDHs, are transcribed during the erythrocytic development of P. falciparum. Protein expression of mitochondrial LipDH and mitochondrial branched chain alpha-ketoacid dihydrolipoamide transacylase in these parasite stages was confirmed by Western blotting. The localization of the two LipDHs to the parasite's apicoplast and mitochondrion, respectively, was shown by expressing the LipDH N-terminal presequences fused to green fluorescent protein in erythrocytic stages of P. falciparum and by immunofluorescent colocalization with organelle-specific markers. Biochemical characterization of recombinantly expressed mitochondrial LipDH revealed that the protein has kinetic and physicochemical characteristics typical of these flavo disulphide oxidoreductases. We propose that the mitochondrial LipDH is part of the mitochondrial alpha-ketoglutarate dehydrogenase and branched chain alpha-ketoacid dehydrogenase complexes and that the apicoplast LipDH is an integral part of the pyruvate dehydrogenase complex which occurs only in the apicoplast in P. falciparum.

Markers of inflammation, proteolysis, and apoptosis in ESRD.

BACKGROUND: Hemodialysis (HD) is associated with protein catabolism and augmented apoptosis. Although the effect of metabolic acidosis and inflammatory cytokines on activation of the ubiquitin-proteasome pathway and branched-chain keto acid dehydrogenase (BCKAD) is well known, the effect of HD on these pathways remains unexplored. METHODS: Twelve patients with end-stage renal disease were studied before and during HD. Eight controls also were studied. Plasma levels of complement components and cytokines, interleukin-1 (IL-1), IL-6, and tumor necrosis factor-alpha (TNF-alpha) were measured. Messenger RNA (mRNA) levels of caspase-3, a mediator of apoptosis; ubiquitin, a marker of proteolysis; and BCKAD-E2, an enzyme regulating branched-chain amino acid oxidation, were estimated in skeletal muscle biopsy specimens by means of reverse-transcriptase polymerase chain reaction. Annexin-V expression was quantified by DNA array. Before the study, participants were placed on a 1.2-g/kg/d protein diet, and metabolic acidosis was corrected. RESULTS: During HD, plasma IL-6 levels increased from 7.54 +/- 2.24 to 27.86 +/- 4.94 pg/dL (P < 0.001). Complement component, IL-1, and TNF-alpha levels did not change significantly during HD. mRNA levels of caspase-3 (0.50 +/- 0.01 versus 0.81 +/- 0.04), annexin-V (0.94 +/- 0.06 versus 1.48 +/- 0.05; P < 0.001), ubiquitin (1.10 +/- 0.03 versus 1.44 +/- 0.03), and BCKAD-E2 (0.47 +/- 0.01 versus 0.81 +/- 0.04) increased in muscle during HD compared with pre-HD values (P < 0.001). mRNA levels of ubiquitin (0.62 +/- 0.03) and BCKAD-E2 (0.58 +/- 0.02) were greater in controls than pre-HD values (P < 0.05). There were significant positive correlations between plasma IL-6 levels and expression of caspase-3, ubiquitin, and BCKAD-E2 genes. CONCLUSION: HD causes activation of cytokines, which may mediate the increase in gene expression of caspase-3, ubiquitin, and BCKAD-E2 in skeletal muscles.