Studies on the influence of fatty acids on pyruvate dehydrogenase interconversion in rat-liver mitochondria.
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1. The effect of fatty acids on the interconversion of pyruvate dehydrogenase between its active (nonphosphorylated) and inactive (phosphorylated) forms was measured in rat liver mitochondria respiring in state 3 with pyruvate plus malate and 2-oxoglutarate plus malate and during state 4 to state 3 transition in the presence of different substrates. The content of intramitochondrial adenine nucleotides was determined in the parallel experiments. 2. Decrease of the intramitochondrial ATP/ADP ratio with propionate and its increase with palmitoyl-L-carnitine in state 3 is accompanied by a shift of the steady-state of the pyruvate dehydrogenase system towards the active or the inactive form, respectively. 3. Transition from the high energy state (state4) to the active respiration (state3) in mitochondria oxidizing 2-oxoglutarate or plamitoyl-L-carnitine causes an increase of the amount of the active form of pyruvate dehydrogenase due to the decrease of ATP/ADP ratio in the matrix. 4. No change in ATP/ADP ratio can be observed in the presence of octanoate in mitochondria oxidizing pyruvate or 2-oxoglutarate in state 3 or during state 4 to state 3 transition. Simultanelusly, no significant change in phosphorylation state of pyruvate dehydrogenase occurs and a low amount of the enzyme in the active form is present with octanoate or octanoate plus 2-oxoglutarate. Pyruvate abolishes this effect of octanoate and shifts the steady-state of pyruvate dehydrogenase system towards the active form. 5. These results indicate that fatty acids influence the interconversion of pyruvate dehydrogenase mainly by changing intramitochondrial ATP/ADP ratio. However, the comparison of the steady-state level of the pyruvate dehydrogenase system in the presence of different substrates in various metabolic conditions provides some evidence that accumulation of acetyl-CoA and high level of NADH may promote the phosphorylation of pyruvate dehydrogenase. 6. Pyruvate exerts its protective effect against phosphorylation of pyruvate dehydrogenase in the presence of fatty acids of short, medium or long chain in a manner which depends on its concentration. It is suggested that in isolated mitochondria pyruvate counteracts the effect of acetyl-CoA and NADH on pyruvate dehydrogenase kinase. (+info)
Perfluorooctanoic acid, a peroxisome-proliferating hypolipidemic agent, dissociates apolipoprotein B48 from lipoprotein particles and decreases secretion of very low density lipoproteins by cultured rat hepatocytes.
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The hypolipidemic effect is evoked by various peroxisome proliferators. Modulation of gene transcription via peroxisome proliferator-activated receptor (PPAR) is generally responsible for this effect. In addition, we have found a PPAR-independent mechanism in which fibrates, known peroxisome proliferators, decrease hepatic secretion of very low density lipoproteins (VLDL) through inhibition of phosphatidylcholine synthesis via methylation of phosphatidylethanolamine (PE) (T. Nishimaki-Mogami et al., Biochim. Biophys. Acta 1304 (1996) 21-31). In the present study, we show a novel mechanism by which perfluorooctanoic acid (PFOA), a potent peroxisome proliferator and inhibitor of PE methylation, exerts its hypolipidemic effect. PFOA (100 microM) added to the medium rapidly decreased the secretion of triglyceride by cultured rat hepatocytes, which was independent of the activity of cellular PE methylation. Analysis of the density of apoB secreted into the medium showed that PFOA decreased apoB48 in VLDL, but increased apoB48 in the bottom d>1.21 fraction. This lipid-poor apoB48 was also generated by incubating medium that had been harvested from control cells with PFOA, indicating that PFOA has the ability to dissociate apoB48 from lipoprotein particles. Exposure of cells to PFOA for 2 h prior to the experiment was sufficient to generate lipid-poor apoB48, indicating that PFOA exerted its effect intracellularly. Taken together, the data suggest that a strong interaction of PFOA with apoB48 disturbs the association of apoB48 with lipids in the process of intracellular VLDL assembly, thereby inhibiting VLDL secretion. This study shows that the mechanisms of hypolipidemic effect caused by various classes of peroxisome proliferators are diverse. (+info)
Inhibition and stimulation of long-chain fatty acid oxidation by chloroacetaldehyde and methylene blue in rats.
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The effects of chloroacetaldehyde (CAA) and methylene blue, both alone and together, on mitochondrial metabolism, hepatic glutathione content, and bile flow were investigated in rats. Oxidation of [1-14C]palmitic acid, [1-14C]octanoic acid, and [1,4-14C]succinic acid allowed for the differentiation between carnitine-dependent long-chain fatty acid metabolism, medium chain fatty acid oxidation, and citric acid cycle activity, respectively. CAA, a metabolite of the anticancer drug ifosfamide, which may be responsible for ifosfamide-induced encephalopathy, inhibited palmitic acid metabolism but not octanoic or succinic acid oxidation, depleted hepatic glutathione, and stimulated bile flow. Methylene blue, which is clinically used to either prevent or reverse ifosfamide-associated encephalopathy, markedly stimulated palmitic acid oxidation either in the presence or absence of CAA, but did not affect the oxidation of octanoic and succinic acid or hepatic glutathione. Taken together, this study demonstrates that CAA inhibits palmitic acid metabolism. Methylene blue stimulates long-chain fatty acid oxidation, most likely by facilitating the translocation of fatty acids into mitochondria, and compensates for the CAA effect in vivo. (+info)
Adipose differentiation related protein (ADRP) expressed in transfected COS-7 cells selectively stimulates long chain fatty acid uptake.
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Adipose differentiation related protein (ADRP) is a 50-kDa novel protein cloned from a mouse 1246 adipocyte cDNA library, rapidly induced during adipocyte differentiation. We have examined ADRP function, and we show here that ADRP facilitates fatty acid uptake in COS cells transfected with ADRP cDNA. We demonstrate that uptake of long chain fatty acids was significantly stimulated in a time-dependent fashion in ADRP-expressing COS-7 cells compared with empty vector-transfected control cells. Oleic acid uptake velocity increased significantly in a dose-dependent manner in ADRP-expressing COS-7 cells compared with control cells. The transport Km was 0.051 microM, and Vmax was 57.97 pmol/10(5) cells/min in ADRP-expressing cells, and Km was 0.093 microM and Vmax was 20.13 pmol/10(5) cells/min in control cells. The oleate uptake measured at 4 degrees C was only 10% that at 37 degrees C. ADRP also stimulated uptake of palmitate and arachidonate but had no effect on uptake of medium chain fatty acid such as octanoic acid and glucose. These data suggest that ADRP specifically enhances uptake of long chain fatty acids by increasing the initial rate of uptake and provide novel information about ADRP function as a saturable transport component for long chain fatty acids. (+info)
Novel method for evaluation of the oligomeric structure of membrane proteins.
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Assessment of the quaternary structure of membrane proteins by PAGE has been problematic owing to their relatively poor solubility in non-dissociative detergents. Here we report that several membrane proteins can be readily solubilized in their native quaternary structure with the use of the detergent perfluoro-octanoic acid (PFO). Further, PFO can be used with PAGE, thereby providing a novel, accessible tool with which to assess the molecular mass of homo-multimeric protein complexes. (+info)
Mitochondrial metabolism of pyruvate is required for its enhancement of cardiac function and energetics.
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Pyruvate augmentation of contractile function and cytosolic free energy of ATP hydrolysis in myocardium could result from pyruvate catabolism in the mitochondria or from increased ratio of the cytosolic NAD-/NADH redox couple via the lactate dehydrogenase equilibrium. OBJECTIVE: To test the hypothesis that cytosolic oxidation by pyruvate is sufficient to increase cardiac function and energetics. METHODS: Isolated working guinea-pig hearts received 0.2 mM octanoate +/- 2.5 mM pyruvate as fuels. alpha-Cyano-3-hydroxycinnamate (COHC, 0.6 mM) was administered to selectively inhibit mitochondrial pyruvate uptake without inhibiting pyruvate's cytosolic redox effects or octanoate oxidation. The effects of pyruvate and COHC on sarcoplasmic reticular- Ca2+ handling were examined in 45Ca-loaded hearts. RESULTS: Pyruvate increased left ventricular stroke work and power 40%, mechanical efficiency 29%, and cytosolic ATP phosphorylation potential nearly fourfold. 14CO2 formation from [1-14C]pyruvate was inhibited 65% by COHC, and octanoate oxidation, i.e. 14CO2 formation from [1-14C]octanoate, concomitantly increased threefold. COHC prevented pyruvate enhancement of left ventricular function, mechanical efficiency and cytosolic phosphorylation potential, but did not alter respective levels in pyruvate-free control hearts and augmented cytosolic oxidation by pyruvate. Pyruvate increased sarcoplasmic reticular Ca2+ turnover, i.e. Ca2+ uptake and release, as indicated by 62% decrease in caffeine-induced 45Ca release following 40 min 45Ca washout (P < 0.01). In presence of COHC, pyruvate did not lower caffeine-induced 45Ca release; thus. COHC abrogated pyruvate enhancement of Ca2+ turnover (P < 0.001). CONCLUSION: Pyruvate oxidation of cytosolic redox state is not sufficient to increase cardiac function, cytosolic energetics and sarcoplasmic reticular Ca2+ turnover when mitochondrial pyruvate transport is disabled; thus, mitochondrial metabolism of pyruvate is essential for its metabolic inotropism. (+info)
Novel biodegradable aromatic plastics from a bacterial source. Genetic and biochemical studies on a route of the phenylacetyl-coa catabolon.
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Novel biodegradable bacterial plastics, made up of units of 3-hydroxy-n-phenylalkanoic acids, are accumulated intracellularly by Pseudomonas putida U due to the existence in this bacterium of (i) an acyl-CoA synthetase (encoded by the fadD gene) that activates the aryl-precursors; (ii) a beta-oxidation pathway that affords 3-OH-aryl-CoAs, and (iii) a polymerization-depolymerization system (encoded in the pha locus) integrated by two polymerases (PhaC1 and PhaC2) and a depolymerase (PhaZ). The complete assimilation of these compounds requires two additional routes that specifically catabolize the phenylacetyl-CoA or the benzoyl-CoA generated from these polyesters through beta-oxidation. Genetic studies have allowed the cloning, sequencing, and disruption of the genes included in the pha locus (phaC1, phaC2, and phaZ) as well as those related to the biosynthesis of precursors (fadD) or to the catabolism of their derivatives (acuA, fadA, and paa genes). Additional experiments showed that the blockade of either fadD or phaC1 hindered the synthesis and accumulation of plastic polymers. Disruption of phaC2 reduced the quantity of stored polymers by two-thirds. The blockade of phaZ hampered the mobilization of the polymer and decreased its production. Mutations in the paa genes, encoding the phenylacetic acid catabolic enzymes, did not affect the synthesis or catabolism of polymers containing either 3-hydroxyaliphatic acids or 3-hydroxy-n-phenylalkanoic acids with an odd number of carbon atoms as monomers, whereas the production of polyesters containing units of 3-hydroxy-n-phenylalkanoic acids with an even number of carbon atoms was greatly reduced in these bacteria. Yield-improving studies revealed that mutants defective in the glyoxylic acid cycle (isocitrate lyase(-)) or in the beta-oxidation pathway (fadA), stored a higher amount of plastic polymers (1.4- and 2-fold, respectively), suggesting that genetic manipulation of these pathways could be useful for isolating overproducer strains. The analysis of the organization and function of the pha locus and its relationship with the core of the phenylacetyl-CoA catabolon is reported and discussed. (+info)
Effects of the rodent peroxisome proliferator and hepatocarcinogen, perfluorooctanoic acid, on apoptosis in human hepatoma HepG2 cells.
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The effects of perfluorooctanoic acid (PFOA), a potent hepatocarcinogen and peroxisome proliferator in rodents, on human cells have not yet been examined. In the present study we demonstrate that treatment of human hepatoblastoma HepG2 cells with PFOA induces apoptosis, as well as perturbs the cell cycle. This apoptosis was characterized by electron microscopy, which revealed typical nucleosomal fragmentation (also observed as a 'DNA ladder' upon electrophoresis on agarose) and was quantitated using propidium iodide staining of cellular DNA and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. This process was dose- and time-dependent: apoptosis became manifest with 200 microM and maximal (45% of the cells) upon exposure to 450 microM PFOA for 24 h. Electrophoresis of the DNA from HepG2 cells exposed to 500 microM PFOA for 24 h or to 400 microM PFOA for 48 h revealed a smear typical of non-specific degradation. These findings indicate that in the presence of high concentrations of PFOA for long times, HepG2 cells undergo primary and secondary necrosis. Quantitation of trypan blue exclusion supported this conclusion. Flow cytometric analysis revealed that the cell cycle of HepG2 cells was perturbed by exposure to 50-150 microM PFOA. A 50 microM concentration resulted in a significant increase in the proportion of G(2)/M cells and, simultaneously, a decrease in the number of cells in the S phase, whereas treatment with 100 or 150 microM PFOA increased the proportion of cells in the G(0)/G(1) phase and decreased the number of cells in the G(2)/M and S phases. Simultaneous flow cytometric analysis of apoptosis-associated DNA strand breaks using the TUNEL procedure and of propidium iodide staining of cellular DNA revealed DNA breaks in HepG2 cells exposed to 150 microM PFOA, prior to nuclear fragmentation. (+info)