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.

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.

A case of carnitine palmitoyltransferase II deficiency in human skeletal muscle.

A 20-year-old man was shown to have a deficiency of carnitine palmitoyltransferase (CPT) II in skeletal muscle. The evidence was: (i) there was no significant oxidation of [9,10-3H]-palmitate or of [1-14C]palmitate in mitochondrial fractions from fresh skeletal muscle from the patient; (ii) all the CPT activity in a homogenate of fresh muscle from the patient was overt (CPT I) with no increase in activity after the inner membrane was disrupted; (iii) all the CPT activity in the patient's muscle was inhibited by malonyl-CoA; and (iv) an immunoreactive peptide of 67 kDa corresponding to CPT II, present in mitochondria from controls, was absent in those from the patient.

Glucose kinetics during acute and chronic treatment of rats with 2[6(4-chloro-phenoxy)hexyl]oxirane-2-carboxylate, etomoxir.

(1) The effects of 2[6(4-chlorophenoxy)hexyl]oxirane-2-carboxylate (etomoxir), a candidate antiketonaemic and antidiabetic drug, on glucose turnover and recycling of glucose carbon in rats were determined using [3-3H, U-14C]glucose. (2) Etomoxir (Na salt) was infused continuously at a rate of 2 mg/hr in fasted male Wistar ab Boots rats (250-280 g) that had been maintained on a standard diet, or on a diet containing 0.1% of etomoxir for 10 days. (3) In rats treated acutely with etomoxir, plasma glucose concentrations were decreased by about 1 mM, glucose turnover was decreased by 14%, and recycling of glucose carbon by 30% compared with the controls infused with 0.14 M NaCl. (4) Infusion of etomoxir in rats chronically pretreated with etomoxir had little effect on plasma glucose concentrations, but increased glucose turnover and recycling of glucose carbon by 40%. (5) Acute infusion of etomoxir caused dramatic lowering of blood 3-hydroxybutyrate concentrations from 1 mM to about 0.03 mM with little change in other intermediary metabolites. (6) In rats chronically fed etomoxir, the proportion of pyruvate dehydrogenase in quadriceps muscle in the active form was 31% compared with 15% in the controls. (7) It was concluded that etomoxir in the acute dose given had only moderate effects on glucose turnover and that chronic administration of etomoxir caused increased glucose turnover and glucose recycling in the rat.

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.

Effects of 2[5(4-chlorphenyl)pentyl]oxirane-2-carboxylate on fatty acid synthesis and fatty acid oxidation in isolated rat hepatocytes.

The effects of the hypoketonaemic and hypoglycaemic compound 2[5(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA) on fatty acid synthesis and fatty acid oxidation in rat hepatocytes were examined. Two microM-POCA caused a small stimulation of fatty acid synthesis which might be due to an increased flux through pyruvate dehydrogenase. Ten to one hundred microM-POCA inhibited (40-70%) fatty acid synthesis. At low concentrations (less than or equal to 5 microM) POCA was a more powerful inhibitor of fatty acid oxidation than of synthesis, but at higher concentrations (10-100 microM) the inhibition of synthesis and oxidation was similar. One hundred microM POCA-CoA inhibited acetyl-CoA carboxylase by about 22% and 100 microM-palmitoyl-CoA by about 33%. Since POCA was a more potent inhibitor of fatty acid synthesis than palmitate, but POCA-CoA did not inhibit acetyl-CoA carboxylase more strongly than palmitoyl-CoA, it is suggested that POCA-CoA may inhibit fatty acid synthase directly.

The effects of 2[5(4-chlorophenyl)pentyl]oxirane-2-carbonyl-Co-A on mitochondrial oxidations.

2[5(4-Chlorophenyl)pentyl]oxirane-2-carbonyl-CoA (POCA-CoA) was prepared 2[a5(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA) and characterised chromatographically. POCA-CoA does not inhibit citrate cycle oxidations or effect oxidative phosphorylation by rat liver mitochondria. POCA-CoA at low (microM) concentrations, but not free POCA-, specifically inhibits palmitoyl-CoA oxidation at the stage of carnitine palmitoyltransferase I (CPT I) situated on the outer face of the inner mitochondria membrane. Palmitoyl-carnitine oxidation was not inhibited by POCA-CoA. POCA-CoA inhibits palmitoyl-CoA oxidation in liver mitochondria from fed rats more strongly than it does in mitochondria from fasted rats, similarly to the inhibition by malonyl-CoA [E.D. Saggerson and C.A. Carpenter, FEBS Lett. 129, 225 (1981)]. Palmitoyl-CoA, by contrast with palmitoylcarnitine, is not quantitatively oxidised to acetoacetate by liver mitochondrial fractions, presumably due to competing palmitoyl-CoA hydrolase activity. In the presence of POCA-CoA the amount oxidised is decreased further because the slower rate of oxidation allows more palmitoyl-CoA to be hydrolysed to palmitate. The oxidation of palmitoyl-CoA, but not that of palmitoyl-carnitine, was strongly decreased in washed liver and muscle mitochondrial fractions from POCA-fed animals. POCA- inhibited the oxidation of [U-14C]palmitate in cultured human fibroblasts, and caused small increases in 14CO2 production from [1-14C]pyruvate and [U-14C]glucose. Inhibition of beta-oxidation at the stage of CPT I by POCA-CoA can explain the powerful hypoketonaemic and hypoglycaemic effects of POCA in fasted normal and fasted diabetic animals [H.P.O. Wolf, K. Eistetter and G. Ludwig, Diabetologia 22, 456 (1982)].

The effects of valproate on intermediary metabolism in isolated rat hepatocytes and intact rats.

Valproate is a valuable anticonvulsant which is associated with hepatotoxicity in some patients. In concentrations in the range found in man during valproate therapy (0.1-1.0 mM), it inhibited pyruvate and palmitate oxidation, urea synthesis and gluconeogenesis by 30-50% in isolated rat hepatocytes. Valproate (100 mg/kg body weight) is also hypoglycaemic and hypoketonaemic in fasted rats. All these inhibitions can be explained in terms of the accumulation of valproyl-CoA and its further metabolites in the matrix of hepatic mitochondria. Although these inhibitions are only partial, and normally well tolerated, they could significantly impair liver function when there is an additional insult, such as may occur with multiple drug therapy or if there is already an inborn error of metabolism. Such an association with inborn errors may explain the higher incidence of valproate-associated toxicity in children. It may be of more value to measure blood urea and ammonia concentrations routinely shortly after starting valproate therapy than to do conventional liver function tests.

Metabolic changes in fed rats caused by chronic administration of ethyl 2[5(4-chlorophenyl)pentyl]oxirane-2-carboxylate, a new hypoglycaemic compound.

Ethyl 2[5(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA) is strongly hypoglycaemic in fasted normal and diabetic rats [H. P. O. Wolf, K. Eistetter and G. Ludwig, Diabetologia 22, 456 (1982)]. POCA was fed for 12 weeks to rats on a standard low-fat (3%) diet at levels of 0.05% and 0.2% to give daily intakes of about 50 and 200 mg/per kg body-wt respectively. This is much more than effective hypoglycaemic doses in fasted rats (5-10 mg/kg body-wt). The animals appeared healthy but they had slightly decreased rates of weight gain compared with the controls. POCA caused a 15% increase in the weight of the myocardium and accumulation of lipid in the liver. Chronic administration of POCA did not cause any large changes in water-soluble blood metabolite concentrations, although VLDL-triacylglycerol and both VLDL and HDL cholesterol concentrations were lowered. There were only small changes in some metabolites of the glycolytic and gluconeogenic pathways and the citrate cycle in liver and skeletal muscle. ATP concentrations were maintained in all groups. There were 2- to 3-fold increases in the total content of CoA and of carnitine and their acylated forms. POCA-feeding caused small decreases in LPL activities in heart and had variable effects in adipose tissue. POCA was also fed to a few rats on a high fat (30%) diet for 4 weeks. Only small changes in blood, liver and muscle metabolite concentrations were found, except for large increases in the liver CoA and carnitine contents. It was concluded that POCA does not cause large perturbations of glucose homeostasis, or acute toxic effects, during 12 weeks administration to normal animals at high dose levels. The very-long term importance of accumulation of lipid in liver; increase in myocardial weight; and also of hepatic peroxisomal proliferation [A. J. Bone, H. S. A. Sherratt, D. M. Turnbull and H. Osmundsen, Biochem. biophys. Res. Commun. 104, 708 (1982)] cannot yet be determined. The possible use of POCA and related compounds in the chemotherapy of diabetes merits further investigation.

A partial deficiency of cytochrome c oxidase in chronic progressive external ophthalmoplegia.

A partial deficiency of cytochrome oxidase has been found in 7 patients with chronic progressive external ophthalmoplegia and proximal myopathy or craniosomatic abnormalities. Muscle biopsies from all these patients showed morphological mitochondrial abnormalities ("ragged red" fibres) and cytochemical assay of cytochrome oxidase showed that these fibres contained no demonstrable enzyme activity. The incidence of cytochrome oxidase-negative fibres was greater than that of "ragged-red" fibres suggesting that the enzyme defect preceded the development of morphological mitochondrial changes. Biochemical analysis of skeletal muscle mitochondrial fractions from 3 patients revealed in 1 case a significantly lower concentration of cytochrome aa3 and a decreased ratio of cytochrome oxidase/succinate-cytochrome c reductase. Fasting blood metabolites were elevated in 2 patients. We suggest that partial cytochrome oxidase deficiency is the underlying defect in mitochondrial myopathy associated with the oculocraniosomatic syndromes.

Partial cytochrome oxidase deficiency without subsarcolemmal accumulation of mitochondria in chronic progressive external ophthalmoplegia.

Chronic progressive external ophthalmoplegia (CPEO) associated with proximal myopathy and/or craniosomatic abnormalities is a rare syndrome in which morphological mitochondrial changes have been found in some fibres (subsarcolemmal accumulation of mitochondria or "ragged red" fibres). We report a 14-year-old boy with CPEO and a mild proximal myopathy without these characteristic "ragged red" fibres. Histochemistry of skeletal muscle showed a mosaic of fibres without detectable cytochrome oxidase activity, while other mitochondrial enzymes were normal. The total cytochrome oxidase activity and cytochrome aa3 concentration in muscle mitochondrial fractions were only 40% of normal. This case is unique in that a biochemical defect was not accompanied by morphological abnormalities and may represent an early stage of CPEO before the development of morphological changes, or alternatively, a new variant of the disease.

Cytochrome oxidase deficiency: immunological studies of skeletal muscle mitochondrial fractions.

We report a 2-year-old girl who presented with delayed development, weakness and persistent vomiting. She had a demyelinating peripheral neuropathy. The activity of cytochrome oxidase in skeletal muscle from the patient was 10% of controls. Immunochemical studies using antibodies to holo-cytochrome oxidase and the individual subunits showed a low concentration of all detectable subunits.

Fatal infantile mitochondrial myopathy due to cytochrome c oxidase deficiency.

A case of cytochrome c oxidase deficiency primarily affecting skeletal muscle is described. The child was admitted at 4 weeks due to failure to thrive and examination at that time revealed weakness and hypotonia. His condition deteriorated until at 11 weeks respiratory arrest necessitated artificial ventilation and death occurred at 14 weeks. Biochemical investigation showed lactic acidaemia and generalised aminoaciduria. Histochemical examination of muscle obtained at biopsy showed strong reactions for some oxidative enzymes, but by contrast cytochrome c oxidase could not be detected. Cytochrome c oxidase activity was less than 5% of control values in an extract of fresh muscle. The reduced-minus oxidised absorption spectra of muscle mitochondrial fractions prepared from post-mortem tissue showed an absence of cytochrome aa3 and a partial deficiency of cytochrome b. Ultra-structural examination showed abnormal mitochondria with loss of cristae and an abnormal granular matrix. The family history suggests autosomal recessive inheritance.

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.