Etomoxir, sodium 2-[6-(4-chlorophenoxy)hexyl] oxirane-2-carboxylate, inhibits triacylglycerol depletion in hepatocytes and lipolysis in adipocytes.

The effects of etomoxir, an inhibitor of mitochondrial long-chain fatty acid oxidation, on triacylglycerol metabolism in rat hepatocytes and adipocytes were investigated. Etomoxir inhibited the depletion of triacylglycerol stores in hepatocytes incubated without exogenous fatty acids and inhibited lipolysis in adipocytes. The effects on hepatocytes could be attributed to two mechanisms. At low concentrations (1-10 microM) R-etomoxir increased fatty acid esterification by inhibition of beta-oxidation. This effect was specific for the R-enantiomer and was associated with increased triacylglycerol secretion. At higher concentrations (50-100 microM) RS-etomoxir inhibited lipolysis and triacylglycerol secretion, independently of inhibition of carnitine palmitoyl-transferase I. These effects of RS-etomoxir on triacylglycerol metabolism and lipolysis may contribute to the chronic hypolipidaemic effects of etomoxir in vivo.

The measurement of mitochondrial beta-oxidation by release of 3H2O from [9,10-3H]hexadecanoate: application to skeletal muscle and the use of inhibitors as models of metabolic disease.

We describe the use of a simple assay for beta-oxidation which depends on the release of 3H2O from [9,10-3H]hexadecanoate. This was compared with the use of [1-14C]hexadecanoate which gave comparable results when all the products of beta-oxidation were measured. The prediction that 75% of the tritium is released as 3H2O and 25% as [2-3H]acetyl units was confirmed. The assay was used successfully to demonstrate impaired beta-oxidation in tissue preparations from rats treated with etomoxir and methylenecyclopropylpyruvate which are known inhibitors of beta-oxidation. Abnormalities of beta-oxidation were also detected in skeletal muscle from patients with defects of mitochondrial oxidation.

Stereospecificity of the inhibition by etomoxir of fatty acid and cholesterol synthesis in isolated rat hepatocytes.

The racemates of substituted 2-oxiranecarboxylates are potent inhibitors of fatty acid oxidation and fatty acid and cholesterol synthesis. We show in the accompanying paper [Agius L, Peak M and Sherratt HSA, Biochem Pharmacol 42: 1711-1715, 1991] that only the R-enantiomer of etomoxir, a potent hypoglycaemic compound, inhibits fatty acid oxidation in hepatocytes. We demonstrate in this paper that although the R-enantiomer of etomoxir is esterified to its CoA-ester more readily than the S-enantiomer, both the R- and S-enantiomers are equally potent inhibitors of fatty acid and cholesterol synthesis from acetate in rat hepatocytes. The inhibition of fatty acid synthesis is not due to direct inhibition of fatty acid synthetase and the inhibition of cholesterol synthesis occurs at a site proximal to formation of mevalonate. Since the S-enantiomer inhibits fatty acid and cholesterol synthesis but not fatty acid oxidation the inhibition of the biosynthetic pathways is not coupled to inhibition of fatty acid oxidation.

Metabolic consequences of methylenecyclopropylglycine poisoning in rats.

We describe the effects of methylenecyclopropylglycine in fasted rats. A 75% decrease in the blood glucose concentration and an increase of lactate and pyruvate were observed 6 h after administration of 100 mg of this amino acid/kg. By contrast with the effects reported for hypoglycin [Williamson & Wilson (1965) Biochem. J. 94, 19c-21c], the plasma concentrations of ketone bodies decreased after administration of methylenecyclopropylglycine and the concentrations of branched-chain amino acids in the plasma were increased 6-fold. The oxidation of decanoylcarnitine or of palmitate was nearly completely inhibited in rat liver mitochondria from methylenecyclopropylglycine-poisoned rats. The activities of acetoacetyl-CoA and of 3-oxoacyl-CoA thiolase were decreased to 25% and less than 10% of the controls. There was a pronounced aciduria, due to the excretion of dicarboxylic acids and of oxidation products of branched-chain amino acids. The accumulation of the toxic metabolite methylenecyclopropylformyl-CoA in the mitochondrial matrix was detected after administration of methylenecyclopropylglycine. Similarly we confirmed experimentally that methylenecyclopropylacetyl-CoA accumulates in mitochondria incubated with methylenecyclopropylpyruvate.

Mitochondria: structure and function.

Mitochondria are the main site of ATP synthesis in aerobic cells, using the free energy of the oxidation of metabolic fuels by oxygen. They have a matrix space containing the enzymes of the citrate cycle and beta-oxidation, enclosed by an inner membrane containing the 4 complexes of the electron transport chain, ATP synthase and specific carriers for metabolites. Mitochondria also have a relatively permeable outer membrane and an intermembrane space. ATP synthesis (oxidative phosphorylation) is critically dependent on the structural integrity of the mitochondrion. Electrons from substrate oxidations feed into the electron transport chain at complex I or complex II, and then successively flow to complex III, complex IV and finally to oxygen. Complexes I, III and IV are redox pumps and electron transport causes extrusion of protons from the matrix generating an electrochemical proton gradient (proton motive force) across the inner membrane. Protons return to the matrix 'through' ATP synthase driving the synthesis of ATP. The stoichiometry of proton extrusion and the yield of ATP are still uncertain. Mitochondria have genetic continuity and are inherited maternally. They possess a small amount of DNA which codes for some, but not all, of the subunits of complexes I, III, IV of ATP synthase. mtDNA also codes for mitochondrial ribosomal and messenger RNAs involved in the synthesis of mitochondrially coded subunits. All other mitochondrial peptides are synthesised on cytosolic ribosomes and are imported and targeted to their specific intramitochondrial locations, often after proteolytic removal of leader sequences.

Impaired mitochondrial beta-oxidation in a patient with an abnormality of the respiratory chain. Studies in skeletal muscle mitochondria.

Defects of complex I of the mitochondrial respiratory chain are important causes of neurological disease. We report studies that demonstrate a severe deficiency of complex I activity with less severe abnormalities of complexes III and IV (less than 5, 63, and 30% of control values, respectively) in a skeletal muscle mitochondrial fraction from a 22-yr-old female with weakness, lactic acidemia, and the deposition of intramuscular neutral lipid. The observation that lipid accumulates in this and other patients with complex I deficiency suggests impaired mitochondrial fatty acid oxidation. To investigate this mechanism we have shown impaired flux through beta-oxidation [( U-14C]hexadecanoate oxidation was 66% of control rate) and accumulation of specific acyl-CoA ester intermediates. The changes in fatty acid metabolism in complex I deficiency are secondary to the reduced state within the mitochondrial matrix with low NAD+/NADH ratios.

Altered acyl-CoA metabolism in riboflavin deficiency.

We have recently described the effects of riboflavin deficiency on the metabolism of dicarboxylic acids (Draye et al. (1988) Eur. J. Biochem. 178, 183-189). As both mitochondria and peroxisomes are thought to be involved, we have examined the activities of various enzymes in these organelles in the livers of riboflavin-deficient rats. Mitochondrial beta-oxidation of fatty acids was severely depressed due to loss of activity of the three fatty acyl-CoA dehydrogenases, whereas there was an enhancement of peroxisomal beta-oxidation due to an increased activity of the FAD-dependent fatty acyl-CoA oxidase, although the activities of other peroxisomal flavoproteins, D-amino acid oxidase and glycolate oxidase, were lowered. Hepatocyte morphometry revealed an increase in the numbers of peroxisomes, indicating a proliferation induced by the deficiency. The mitochondrial acyl-CoA dehydrogenases involved in branched-chain amino acid metabolism were also severely decreased leading to characteristic organic acidurias. There was some loss of activity of the flavin-dependent sections of the electron transport chain (complexes I and II), but these were probably not sufficient to affect normal function in vivo. The specificity of these effects allows the use of the riboflavin-deficient rat as a model for the study of dicarboxylate metabolism.

Substituted 2-oxiranecarboxylic acids: a new group of candidate hypoglycaemic drugs.

Drugs to treat diabetes that can be taken orally have long been sought, although the successful management of insulin-dependent diabetes mellitus by simple chemotherapy may be an unachievable goal. The only drugs currently used for the treatment of non-insulin-dependent diabetes have limited effectiveness. In this article Peter Selby and Stanley Sherratt describe the development of a new group of candidate hypoglycaemic drugs, esters of substituted 2-oxiranecarboxylic acids, which merit full clinical evaluation. These drugs are hydrolysed to the free acids which are then converted to their coenzyme A esters in cells. The CoA esters inactivate carnitine palmitoyltransferase I in the outer mitochondrial membrane, thus preventing the excessive oxidation of long-chain fatty acids that occurs in diabetes. This causes a secondary decrease in hepatic gluconeogenesis and an increase in peripheral glucose utilization leading to improved glucose tolerance.

Measurement of the acyl-CoA intermediates of beta-oxidation by h.p.l.c. with on-line radiochemical and photodiode-array detection. Application to the study of [U-14C]hexadecanoate oxidation by intact rat liver mitochondria.

The quantitative isolation of acyl-CoA esters of chain length C2-C17 from mitochondrial incubations and their analysis by reverse-phase radio-h.p.l.c. is described. Photodiode-array detection was used to characterize 2-enoyl-CoA esters. The chromatographic behaviour of all 27 intermediates of the beta-oxidation of hexadecanoyl-CoA is documented. Only C16, C14 and C12 intermediates were detected in uncoupled mitochondria oxidizing [U-14C]hexadecanoyl-CoA in the presence of fluorocitrate and carnitine, providing evidence for some organization of the enzymes of beta-oxidation [Garland, Shepherd & Yates (1965) Biochem. J. 97, 587-594; Sumegi & Srere (1984) J. Biol. Chem. 259, 8748-8752]. Rotenone increased concentrations of 3-hydroxyacyl-CoA and 2-enoyl-CoA esters and inhibited flux. These experiments provide the first direct unambiguous measurements of acyl-CoA esters in intact respiring rat liver mitochondrial fractions.

Fatal lactic acidosis in infancy with a defect of complex III of the respiratory chain.

We report our studies on the metabolic defects which caused a newborn infant to present with a severe lactic acidemia (25 mM) and to die on the 3rd d after birth despite intensive supportive measures. The mitochondrial fractions prepared from skeletal muscle and liver oxidised NAD+-linked substrates and succinate slowly. Spectrophotometric assays for complexes I, II, and III of the respiratory chain demonstrate a specific defect of complex III in the skeletal muscle and liver mitochondrial fractions. The concentrations of cytochrome b were 75% lower in the skeletal muscle and heart mitochondria than in control preparations. The amount of non-heme iron sulphur protein of complex III was low in skeletal muscle, liver, and heart. This case differs from previous reports of complex III deficiency in three respects: the patient presented in the neonatal period, the defect was expressed in several tissues, and it was fatal.

Tissue specific defect of complex I of the mitochondrial respiratory chain.

Deficiency of complex I is one of the most commonly reported defects of the mitochondrial respiratory chain in man. Clinical evidence of tissue specific expression of complex I deficiency has not previously been confirmed biochemically. We report here slow oxidation of NAD+-linked substrates, low activity of complex I and low amounts of immunoreactive complex I peptides in skeletal muscle mitochondria from a patient with muscle weakness and lactic acidosis. In liver mitochondria complex I activity was normal and all the immunoreactive subunits of complex I were present in normal amounts.