Mitochondrial metabolism of valproic acid

The beta-oxidation of valproic acid (2-propylpentanoic acid), an anticonvulsant drug with hepatotoxic side effects, was studied with subcellular fractions of rat liver and with purified enzymes of beta-oxidation. 2-Propyl-2-pentenoyl-CoA, a presumed intermediate in the beta-oxidation of valproic acid, was chemically synthesized and used to demonstrate that enoyl-CoA hydratase or crotonase catalyzes its hydration to 3-hydroxy-2-propylpentanoyl-CoA. The latter compound was not acted upon by soluble L-3-hydroxyacyl-CoA dehydrogenases from mitochondria or peroxisomes but was dehydrogenated by an NAD(+)-dependent dehydrogenase associated with a mitochondrial membrane fraction. The product of the dehydrogenation, presumably 3-keto-2-propylpentanoyl-CoA, was further characterized by fast bombardment mass spectrometry. 3-Keto-2-propylpentanoyl-CoA was not cleaved thiolytically by 3-ketoacyl-CoA thiolase or a mitochondrial extract but was slowly degraded, most likely by hydrolysis. The availability of 2-propylpentanoyl-CoA (valproyl-CoA) and its beta-oxidation metabolites facilitated a study of valproate metabolism in coupled rat liver mitochondria. Mitochondrial metabolites identified by high-performance liquid chromatography were 2-propylpentanoyl-CoA, 3-keto-2-propylpentanoyl-CoA, 2-propyl-2-pentenoyl- CoA, and trace amounts of 3-hydroxy-2-propylpentanoyl-CoA. It is concluded that valproic acid enters mitochondria where it is converted to 2-propylpentanoyl-CoA, dehydrogenated to 2-propyl-2-pentenoyl-CoA by 2-methyl-branched chain acyl-CoA dehydrogenase, and hydrated by enoyl-CoA hydratase to 3-hydroxy-2-propylpentanoyl-CoA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of valproic acid on hepatic carbohydrate and lipid metabolism

Valproic acid (dipropylacetic acid), an antiepileptic agent known to be hepatotoxic in some patients, caused inhibition of lactate gluconeogenesis, fatty acid oxidation, and fatty acid synthesis by isolated hepatocytes. The latter process was the most sensitive to valproic acid, 50% inhibition occurring at ca. 125 microM with cells from meal-fed female rats. The medium-chain acyl-CoA ester fraction was increased whereas coenzyme A (CoA), acetyl-CoA, and the long chain acyl-CoA fractions were decreased by valproic acid. The increase in the medium chain acyl-CoA fraction was found by high-pressure liquid chromatography to be due to the accumulation of valproyl-CoA plus an apparent CoAester metabolite of valproyl-CoA. Salicylate inhibited valproyl-CoA formation and partially protected against valproic acid inhibition of hepatic metabolic processes. Octanoate had a similar protective effect, suggesting that activation of valproic acid in the mitosol is required for its inhibitory effects. It is proposed that either valproyl-CoA itself or the sequestration of CoA causes inhibition of metabolic processes. Valproyl-CoA formation also appears to explain valproic acid inhibition of gluconeogenesis by isolated kidney tubules. No evidence was found for the accumulation of valproyl-CoA in brain tissue, suggesting that the effects of valproic acid in the central nervous system are independent of the formation of this metabolite.     

Studies on the extra-mitochondrial CoA -ester formation of valproic and Delta4 -valproic acids

The hypothesis whether valproic acid (VPA) and its main microsomal metabolite, Delta(4)-valproic acid, can be activated to the respective CoA esters in the cell cytosol was investigated. The valproyl-CoA formation was measured in different subcellular fractions obtained by differential centrifugation of liver homogenates of rats treated with VPA (studies ex vivo) and digitonin fractionation of rat hepatocytes incubated with VPA and cofactors (studies in vitro). The results show that VPA activation may occur in the cytosol and is not restricted to the mitochondrial matrix as believed until now. Furthermore, the activation of Delta(4)-VPA is demonstrated in vitro. Valproyl-CoA and Delta(4)-valproyl-CoA were detected after in vitro incubations and the former also in the mitochondrial and cytosolic fractions obtained from liver cells of treated rats. The activation to valproyl-CoA was characterized in cytosolic fractions, optimized with respect to time and protein and the kinetic constants (K(m)(app)) were estimated for the reaction substrates. Other medium-chain fatty acids decreased the formation of valproyl-CoA suggesting a competition for both mitochondrial and extra-mitochondrial VPA activating enzymes. The present findings suggest additional mechanisms of mitochondrial dysfunction associated with VPA, and they may contribute to the further understanding of the toxic effects associated with this drug.     

Regulation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria.

The mechanism by which fatty acid addition leads to the inactivation of pyruvate dehydrogenase in intact rat liver mitochondria was investigated. In all cases the fatty acid octanoate was added to mitochondria oxidizing succinate. Addition of fatty acid caused an inactivation of pyruvate dehydrogenase in mitochondria incubated under State 3 conditions (glucose plus hexokinase), in uncoupled, oligomycin-treated mitochondria, and in rotenone-menadione-treated mitochondria, but not in uncoupled mitochondria or in mitochondria incubated under State 4 conditions. A number of metabolic conditions were found in which pyruvate dehydrogenase was inactivated concomitant with an elevation in the ATP/ADP ratio. This is consistent with the inverse relationship between the ATP/ADP ratio and the pyruvate dehydrogenase activity proposed by various laboratories. However, in several other metabolic conditions pyruvate dehydrogenase was inactivated while the ATP/ADP ratio either was unchanged or even decreased. This observation implies that there are likely other regulatory factors involved in the fatty acid-mediated inactivation of pyruvate dehydrogenase. Incubation conditions in State 3 were found in which the ATP/ADP and the acetyl-CoA/CoASH ratios remained constant and the pyruvate dehydrogenase activity was correlated inversely with the NADH/NAD+ ratio. Other State 3 conditions were found in which the ATP/ADP and the NADH/NAD+ ratios remained constant while the pyruvate dehydrogenase activity was correlated inversely with the acetyl-CoA/CoASH ratio. Further evidence supporting these experiments with intact mitochondria was the observation that the pyruvate dehydrogenase kinase activity of a mitochondrial extract was stimulated strongly by acetyl-CoA and was inhibited by NAD+ and CoASH. In contrast to acetyl-CoA, octanoyl-CoA inhibited the kinase activity. These results indicate that the inactivation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria may be mediated through effects of the NADH/NAD+ ratio and the acetyl-CoA/CoASH ratio on the interconversion of the active and inactive forms of the enzyme complex catalyzed by pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.     

Free-radical metabolism of carbon tetrachloride in rat liver mitochondria. A study of the mechanism of activation.

Alterations in liver mitochondria as consequence of rat poisoning with carbon tetrachloride (CCl4) have been reported over many years, but the mechanisms responsible for causing such damage are still largely unknown. Isolated rat liver mitochondria incubated under hypoxic conditions with succinate and ADP were found able to activate CCl4 to a free-radical species identified as trichloromethyl free radical (CCl3) by e.s.r. spectroscopy coupled with the spin-trapping technique. The incubation of mitochondria in air decreased free-radical production, indicating that a reductive reaction was involved in the activation of CCl4. However, in contrast with liver microsomes (microsomal fractions), mitochondria did not require the presence of NADPH, and the process was not significantly influenced by inhibitors of cytochrome P-450. The addition of inhibitors of the respiratory chain such as antimycin A and KCN decreased free-radical formation by only 30%, whereas rotenone displayed a greater effect (approx. 84% inhibition), but only when preincubated for 15 min with mitochondria not supplemented with succinate. These findings suggest that the mitochondrial electron-transport chain is responsible for the activation of CCl4. A conjugated-diene band was observed in the lipids extracted from mitochondria incubated with CCl4 under anaerobic conditions, indicating that stimulation of lipid peroxidation was occurring as a result of the formation of free-radical species.     

Mitochondrial metabolism of 3-mercaptopropionic acid. Chemical synthesis of 3-mercaptopropionyl coenzyme A and some of its S-acyl derivatives

The metabolism of 3-mercaptopropionic acid in mitochondria was studied by use of purified mitochondrial enzymes and rat heart mitochondria. Metabolites of 3-mercaptopropionic acid were separated by high performance liquid chromatography and identified by comparing them with chemically synthesized derivatives of 3-mercaptopropionic acid. The initial step in the metabolism of 3-mercaptopropionic acid is its conversion to a CoA thioester, most likely catalyzed by medium-chain acyl-CoA synthetase. The resulting 3-mercaptopropionyl-CoA is a poor substrate of acyl-CoA dehydrogenase but substitutes effectively for CoASH in reactions catalyzed by 3-ketoacyl-CoA thiolase and acetoacetyl-CoA thiolase. S-Acyl-3-mercaptopropionyl-CoA thioesters formed in the thiolase-catalyzed reactions are not at all or only poorly acted upon by acyl-CoA dehydrogenases. However, they are hydrolyzed by thioesterase(s) to CoASH and S-acyl-3-mercaptopropionic acid. The hydrolysis of S-acyl-3-mercaptopropionyl-CoA thioesters proceeds more rapidly than the hydrolysis of fatty acyl-CoA thioesters of comparable chain lengths. Free CoASH is also regenerated from S-acetyl-3-mercaptopropionyl-CoA and more rapidly from 3-mercaptopropionyl-CoA as a result of their reactions with carnitine catalyzed by carnitine acetyltransferase. These findings lead to the suggestion that the major mitochondrial CoA-containing metabolites of 3-mercaptopropionic acid are S-acyl-3-mercaptopropionyl-CoA thioesters.     

Pyruvate uptake is inhibited by valproic acid and metabolites in mitochondrial membranes

The pyruvate uptake rate in inverted submitochondrial vesicles prepared from rat liver was optimized and further characterized; the potential inhibitory effects of the anticonvulsive drug valproic acid or 2-n-propyl-pentanoic acid (VPA), Delta4-valproic acid or 2-n-propyl-4-pentenoic acid and the respective coenzyme A (CoA) conjugates were studied in the presence of a proton gradient. All tested VPA metabolites inhibited the pyruvate uptake, but the CoA esters were stronger inhibitors (40% and 60% inhibition, respectively, for valproyl-CoA and Delta4-valproyl-CoA, at 1mM). At the same concentration, the specific inhibitor 2-cyano-4-hydroxycinnamate decreased the pyruvate uptake rate by 70%. The reported inhibition of the mitochondrial pyruvate uptake may explain the significant impairment of the pyruvate-driven oxidative phosphorylation induced by VPA.     

Metabolism of valproic acid by hepatic microsomal cytochrome P-450

Incubation of valproic acid with rat liver microsomes led to the formation of 3-, 4- and 5-hydroxy-valproic acid. The latter two metabolites, which have been characterized previously from in vivo studies, may be regarded as products of fatty acid ω-1 and ω hydroxylation, respectively. 3-Hydroxy-valproic acid, however, had been thought to derive from the β-oxidation pathway in mitochondria. Conversion of valproic acid to all three metabolites in microsomes required NADPH (NADH was less effective), utilized molecular oxygen, was suppressed by inhibitors of cytochrome P-450 and was stimulated (notably at C-3 and C-4) by phenobarbital pretreatment of the rats. It is concluded that rat liver microsomal cytochrome P-450 catalyzes ω-2 hydroxylation of valproic acid, a reaction not detected previously with fatty acids in mammalian systems, and that the product, 3-hydroxyvalproic acid, should not be used to assess in vivo metabolism of valproate via the β-oxidation pathway.     

Phosphatidic acid synthesis in mitochondria. Topography of formation and transmembrane migration.

The topography of formation and migration of phosphatidic acid (PA) in the transverse plane of rat liver mitochondrial outer membrane (MOM) were investigated. Isolated mitochondria and microsomes, incubated with sn-glycerol 3-phosphate and an immobilized substrate palmitoyl-CoA-agarose, synthesized both lyso-PA and PA. The mitochondrial and microsomal acylation of glycerophosphate with palmitoyl-CoA-agarose was 80-100% of the values obtained in the presence of free palmitoyl-CoA. In another series of experiments, both free polymyxin B and polymyxin B-agarose stimulated mitochondrial glycerophosphate acyltransferase activity approximately 2-fold. When PA loaded mitochondria were treated with liver fatty acid binding protein, a fifth of the phospholipid left the mitochondria. The amount of exportable PA reduced with the increase in the time of incubation. In another approach, PA-loaded mitochondria were treated with phospholipase A(2). The amount of phospholipase A(2)-sensitive PA reduced when the incubation time was increased. Taken together, the results suggest that lysophosphatidic acid (LPA) and PA are synthesized on the outer surface of the MOM and that PA moves to the inner membrane presumably for cardiolipin formation.     

Effects of valproic acid on cardiac metabolism

We investigated whether the antiepileptic valproic acid (VPA) might interfere with oxidative metabolism in heart, as it does in liver. We administered VPA to working rat hearts perfused with radiolabeled carbohydrate and fatty acid fuels. Measurements included oxidation rates of (i) glucose, pyruvate, or lactate in the presence of palmitate and (ii) palmitate, octanoate, or butyrate in the presence of glucose. Oxidation rates were quantified as the rate of appearance of 14CO2 or 3H2O from 14C- or 3H-labeled substrates. In hearts perfused with palmitate, VPA (1 mmol/L) strongly inhibited the oxidation of pyruvate and lactate but slightly stimulated the oxidation of glucose. VPA also inhibited lactate or pyruvate uptake into erythrocytes in vitro. In hearts perfused with glucose, VPA strongly inhibited the oxidation of palmitate and octanoate but had no effect on butyrate oxidation. The absence of valproate CoA ligase activity in cell-free homogenates indicated that the inhibition of fatty acid oxidation by VPA did not require prior activation to valproyl-CoA. The results are consistent with the hypothesis that VPA selectively interferes with myocardial fuel oxidation by mechanisms that are independent of conversion to the CoA thioester.     

Regulation of fatty acid beta-oxidation in rat heart mitochondria

In an attempt to elucidate the mechanism by which the rate of fatty acid oxidation is tuned to the energy demand of the heart, the effects of changing intramitochondrial ratios of [acetyl-CoA]/[CoASH] and [NADH]/[NAD+] on the rate of beta-oxidation were studied. When 10 mM L-carnitine was added to coupled rat heart mitochondria to lower the ratio of [acetyl-CoA]/[CoASH], the rate of palmitoylcarnitine beta-oxidation, as measured by the formation of acid-soluble products, was stimulated more than fourfold at state 4 respiration while beta-oxidation at state 3 respiration was hardly affected. Neither oxaloacetate nor acetoacetate, added to mitochondria to lower the [NADH]/[NAD+] ratio, stimulated beta-oxidation. Rates of respiration at states 3 and 4 were unchanged by additions of L-carnitine, oxaloacetate, or acetoacetate. Determinations of intramitochondrial ratios of [acetyl-CoA]/[CoASH] by high performance liquid chromatography yielded values close to 10 for palmitoylcarnitine-supported respiration at state 4 and 2.5 at state 3 respiration. Addition of 10 mM L-carnitine caused a dramatic decrease of these ratios to less than 0.2 at both respiration states. Studies with purified or partially purified enzymes revealed strong inhibitions of 3-ketoacyl-CoA thiolase by acetyl-CoA and of L-3-hydroxyacyl-CoA dehydrogenase by NADH. Moreover, the activity of 3-ketoacyl-CoA thiolase at concentrations of acetyl-CoA and CoASH prevailing at state 3 respiration was 4 times higher than its activity in the presence of acetyl-CoA and CoASH observed at state 4. Altogether, this study leads to the conclusion that the rate of beta-oxidation in heart can be regulated by the intramitochondrial ratio of [acetyl-CoA]/[CoASH] which reflects the energy demand of the tissue. The thiolytic cleavage catalyzed by 3-ketoacyl-CoA thiolase may be the site at which beta-oxidation is controlled by the [acetyl-CoA]/[CoASH] ratio.     

The relationship between mitochondrial activation and toxicity of some substituted carboxylic acids

The activation of 4-bromocrotonic acid, 4-bromo-2-octenoic acid, valproic acid, and 3-methylglycidic acid by conversion to their CoA thioesters and the effects of these carboxylic acids on palmitoylcarnitine-supported respiration were studied with rat liver and rat heart mitochondria. 4-Bromocrotonic acid was activated by both liver and heart mitochondria, whereas 4-bromo-2-octenoic acid and valproic acid were only activated by liver mitochondria. 3-Methylglycidic acid was not a substrate of mitochondrial activation. All of the carboxylic acids that were activated also inhibited palmitoylcarnitine-supported respiration. 3-Methylglycidoyl-CoA was found to irreversibly inhibit 3-ketoacyl-CoA thiolase in a concentration-dependent and time-dependent manner. Together, these results lead to the conclusion that substituted medium-chain carboxylic acids, which enter mitochondria directly, may inhibit β-oxidation as long as they are activated and perhaps further metabolized in the mitochondrial matrix to compounds that sequester CoA and/or inhibit β-oxidation enzymes. Liver is more susceptible to inhibition by such xenobiotic carboxylic acids due to the broader substrate specificity of its mitochondrial medium-chain acyl-CoA synthetase (EC 6.2.1.2).     

Hepatic hydrolysis of octanoyl-CoA and valproyl-CoA in control and valproate-fed animals

1. Medium-chain acyl-CoA hydrolase activities were determined in liver from control and valproate-fed rats and rabbits. 2. Octanoyl-CoA was readily hydrolyzed by all control liver preparations. 3. Animals that had been fed diets containing 0.5 or 1.0% (w/w) valproic acid had significantly elevated octanoyl-CoA hydrolase activities. 4. The CoA ester of valproic acid, a branched-chain isomer of octanoic acid, was poorly hydrolyzed by liver preparations from both control and valproate-fed animals. 5. Livers from the valproate-fed animals contained high levels of medium-chain acyl-CoA; total CoA content was also increased. 6. The inefficiency of hepatic valproyl-CoA hydrolysis may play a role in the toxicity of valproic acid.     

The inhibition by valproic acid of the mitochondrial oxidation of monocarboxylic and omega-hydroxymonocarboxylic acids: possible implications for the metabolism of gamma-aminobutyric acid

The interactions of 1-5 mM valproic acid with the hepatic fatty acid oxidation are here described. Valproic acid was not substrate for hepatic peroxisomal fatty acid oxidation. Its activation outside the mitochondrial matrix compartment was poor when compared to that of octanoic acid, a fatty acid containing the same number of carbones. Valproic acid did not inhibit the fatty acyl-CoA oxidase nor the cyanide-insensitive acyl-CoA oxidation. Valproic acid inhibited the mitochondrial oxidations of both long-chain monocarboxylyl-CoAs and omega-hydroxymonocarboxylyl-CoAs. Valproic acid prevented the oxidation by coupled mitochondria of decanoic and 10-hydroxydecanoic acids. Both butyric and 4-hydroxybutyric acids were oxidized by coupled mitochondria. These activities were abolished by preincubating the enzyme source with valproic acid. Administration to rats of 0.5% (w/w)- or 1% (w/w)-valproate containing diets were efficient in producing increased liver peroxisomal population and beta-oxidation. Preliminary investigations on the effects of valproic acid on mitochondrial fatty acid oxidation as a function of the animal used for the experiments pointed out an association of the protection of the mitochondrial process against the toxicity of the drug with enhanced carnitine acyltransferase and acyl-CoA hydrolase activities.     

A radioisotopic assay of picomolar concentrations of coenzyme A in liver tissue

A single-step enzyme assay using [14C]palmitic acid and bacterial acyl-coenzyme A synthetase (EC 6.2.1.3) is described for the determination of reduced coenzyme A (CoASH) levels in liver samples. Use of this technique provides a rapid and accurate determination of CoASH in the range 1-250 pmol. Application of the method to the quantitation of CoASH in samples of human liver tissue and rat liver homogenate, isolated hepatocytes, and mitochondria is described.     

Metabolic activation of unsaturated derivatives of valproic acid. Identification of novel glutathione adducts formed through coenzyme A-dependent and -independent processes

The ability of 2-n-propyl-4-pentenoic acid (Δ⁴-VPA) and 2-n-propyl-2(E)-pentenoic acid ([E]-Δ²-VPA), two unsaturated metabolites of valproic acid (VPA), to form reactive intermediates, deplete hepatic glutathione (GSH) and cause accumulation of liver triglycerides was investigated in the rat. With the aid of ionspray liquid chromatography-tandem mass spectrometry (LC-MS/MS), three GSH adducts were detected in the bile of Δ⁴-VPA-treated animals and were identified as 4-hydroxy-5-glutathion-S-yl-VPA-γ-lactone, 5-glutathion-S-yl-(E)-Δ³-VPA and 3-oxo-5-glutathion-S-yl-VPA. A fourth conjugate was identified tentatively as 4-glutathion-S-yl-5-hydroxy-VPA. Quantitative analysis of the corresponding N-acetylcysteine (NAC) conjugates in urine indicated that metabolism of Δ⁴-VPA via the GSH-dependent pathways accounted for approximately 20% of an acute dose (100 mg kg⁻¹ i.p.). In contrast, when rats were given an equivalent dose of (E)-Δ²-VPA, only one GSH adduct (5-glutathion-S-yl-(E)-Δ³-VPA) was detected at low concentrations in bile. In vitro experiments with rat liver mitochondria demonstrated that Δ⁴-VPA undergoes coenzyme A- and ATP-dependent metabolic activation in this organelle via the β-oxidation pathway to intermediates which bind covalently to proteins. When liver homogenates and hepatic mitochondria from rats injected with Δ⁴-VPA, (E)-Δ²-VPA or VPA were analyzed for GSH content, it was found that only Δ⁴-VPA depleted GSH pools significantly. Treatment of rats with Δ⁴-VPA and (to a lesser extent) VPA led to an accumulation of liver triglycerides, whereas (E)-Δ²-VPA had no measurable effect. It is concluded that Δ⁴-VPA undergoes metabolic activation by both microsomal cytochrome P-450-dependent and mitochondrial coenzyme A-dependent processes, and that the resulting electrophilic intermediates, which are trapped in part by GSH, may mediate the hepatotoxic effects of this compound. In contrast, (E)-Δ²-VPA is not transformed to any appreciable extent to reactive metabolites, which thus accounts for the apparent lack of hepatotoxicity of this positional isomer in the rat.     

4-Bromo-2-octenoic acid specifically inactivates 3-ketoacyl-CoA thiolase and thereby fatty acid oxidation in rat liver mitochondria

In an attempt to develop a compound which would specifically inhibit 3-ketoacyl-CoA thiolase (EC 2.3.1.16) in whole mitochondria, 4-bromo-2-octenoic acid was synthesized and studied. After rat liver mitochondria were preincubated with 4-bromo-2-octenoic acid for 3 min, respiration supported by either palmitoylcarnitine or pyruvate was completely abolished, whereas no inhibition was observed with rat heart mitochondria. Addition of carnitine stimulated respiration supported by pyruvate without relieving inhibition of palmitoylcarnitine-dependent respiration. Hence, this compound seems to be a specific inhibitor of beta-oxidation. When the enzymes of beta-oxidation were assayed in a soluble extract prepared from mitochondria preincubated with 4-bromo-2-octenoic acid, only 3-ketoacyl-CoA thiolase was found to be inactivated. 4-Bromo-2-octenoic acid is metabolized by mitochondrial beta-oxidation enzymes to 3-keto-4-bromooctanoyl-CoA which effectively and irreversibly inhibits 3-ketoacyl-CoA thiolase but not acetoacetyl-CoA thiolase (EC 2.3.1.9). Even though 3-keto-4-bromooctanoyl-CoA inhibits the latter enzyme reversibly, 4-bromo-2-octenoic acid does not inhibit ketogenesis in rat liver mitochondria with acetylcarnitine as a substrate. It is concluded that 4-bromo-2-octenoic acid specifically inhibits mitochondrial fatty acid oxidation by inactivating 3-ketoacyl-CoA thiolase in rat liver mitochondria.     

Regulation of palmitoyl-CoA inhibition of mitochondrial adenine nucleotide transport by cytosolic fatty acid binding protein.

Defatted liver fatty acid binding protein (FABP) reverses the inhibitory effect of palmitoyl-CoA on adenine nucleotide transport in rat liver mitochondria; addition of titrating amounts of FABP to mitochondria pretreated with palmitoyl-CoA stimulates nucleotide transport and that activation parallels the removal of the inhibitor from mitochondria. This effect is specific only for FABP; all other cytosolic proteins which do not bind fatty acids do not influence nucleotide transport activity. Addition of free fatty acids (which can compete for ligand binding sites on FABP) to mitochondria pretreated with palmitoyl-CoA interferes with the reversal activity of FABP. Adding FABP alone to freshly isolated mitochondria also activates nucleotide transport activity suggesting that the originally submaximal activity is probably due to the presence of endogenous long-chain acyl-CoA esters in the mitochondrial preparation. Because FABP is present in relatively high concentration in most mammalian cells, these observations offer a likely explanation of why the potent inhibitory effects of long-chain acyl-CoA esters on adenine nucleotide transport in isolated mitochondria are not seen in the intact cell.     

Fatty acid metabolism in liver of rats treated with hypolipidemic sulphur-substituted fatty acid analogues

The purpose of this study was to investigate early biochemical changes and possible mechanisms via which alkyl(C12)thioacetic acid (CMTTD, blocked for beta-oxidation), alkyl(C12)thiopropionic acid (CETTD, undergo one cycle of beta-oxidation) and a 3-thiadicarboxylic acid (BCMTD, blocked for both omega- (and beta-oxidation) influence the peroxisomal beta-oxidation in liver of rats. Treatment of rats with CMTTD caused a stimulation of the palmitoyl-CoA synthetase activity accompanied with increased concentration of hepatic acid-insoluble CoA. This effect was already established during 12-24 h of feeding. From 2 days of feeding, the cellular level of acid-insoluble CoA began to decrease, whereas free CoASH content increased. Stimulation of [1-14C]palmitoyl-CoA oxidation in the presence of KCN, palmitoyl-CoA-dependent dehydrogenase (termed peroxisomal beta-oxidation) and palmitoyl-CoA hydrolase activities were revealed after 36-48 h of CMTTD-feeding. Administration of BCMTD affected the enzymatic activities and altered the distribution of CoA between acid-insoluble and free forms comparable to what was observed in CMTTD-treated rats. It is evident that treatment of peroxisome proliferators (BCMTD and CMTTD), the level of acyl-CoA esters and the enzyme activity involved in their formation precede the increase in peroxisomal and palmitoyl-CoA hydrolase activities. In CMTTD-fed animals the activity of cyanide-insensitive fatty acid oxidation remained unchanged when the mitochondrial beta-oxidation and carnitine palmitoyltransferase operated at maximum rates. The sequence and redistribution of CoA and enzyme changes were interpreted as support for the hypothesis that substrate supply is an important factor in the regulation of peroxisomal fatty acid metabolism, i.e., the fatty acyl-CoA species appear to be catabolized by peroxisomes at high rates only when uptake into mitochondria is saturated. Administration of CETTD led to an inhibition of mitochondrial fatty acid oxidation accompanied with a rise in the concentration of acyl-CoA esters in the liver. Consequently, fatty liver developed. The peroxisomal beta-oxidation was marginally affected. Whether inhibition of mitochondrial beta-oxidation may be involved in regulation of peroxisomal fatty acid metabolism and in development of fatty liver should be considered.     

Valproic acid metabolites inhibit dihydrolipoyl dehydrogenase activity leading to impaired 2-oxoglutarate-driven oxidative phosphorylation

The effect of the antiepileptic drug valproic acid (VPA) on mitochondrial oxidative phosphorylation (OXPHOS) was investigated in vitro. Two experimental approaches were used, in the presence of selected respiratory-chain substrates: (1) formation of ATP in digitonin permeabilized rat hepatocytes and (2) measurement of the rate of oxygen consumption by polarography in rat liver mitochondria. VPA (0.1-1.0 mM) was found to inhibit oxygen consumption and ATP synthesis under state 3 conditions with glutamate and 2-oxoglutarate as respiratory substrates. No inhibitory effect on OXPHOS was observed when succinate (plus rotenone) was used as substrate. We tested the hypothesis that dihydrolipoyl dehydrogenase (DLDH) might be a direct target of VPA, especially its acyl-CoA intermediates. Valproyl-CoA (0.5-1.0 mM) and valproyl-dephosphoCoA (0.5-1.0 mM) both inhibited the DLDH activity, acting apparently by different mechanisms. The decreased activity of DLDH induced by VPA metabolites may, at least in part, account for the impaired rate of oxygen consumption and ATP synthesis in mitochondria if 2-oxoglutarate or glutamate were used as respiratory substrates, thus limiting the flux of these substrates through the citric acid cycle.