H. Stanley A. Sherratt

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

ABSTRACT: 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 HI 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.

Mechanisms of the metabolic disturbances caused by hypoglycin and by pent-4-enoic acid in vivo studies

Abstract 1. In the presence of the hypoglycin metabolites methylenecyclopropylpyruvate (MCPP) and methylenecyclopropylacetate (MCPA), rat liver mitochondria oxidized palmitoyl-carnitine only as far as butyrate and at a decreased rate. Although pent-4-enoic acid (pent-4-enoate) inhibited the rate of β-oxidation of palmitoyl-carnitine by mitochondria, the oxygen uptake was consistent with the complete oxidation of the substrate. 2. The inhibition of β-oxidation by pent-4-enoate was partially reversed by very high concentrations of l -carnitine. Conditions were also defined for the sustained oxidation of pent-4-enoate by mitochondria. 3. Pent-4-enoate inhibited pyruvate oxidation, but only at concentrations much higher than those needed to inhibit β-oxidation. MCPA had no effect on either pyruvate or 2-oxoglutarate oxidation in mitochondria. 4. Soluble extracts of rat or ox liver mitochondria completely oxidized pent-4-enoyl-CoA and the oxidation of butyryl-CoA added subsequently was unaffected. Incubation of soluble extracts with pent-4-enoyl-CoA in the absence of cofactors caused inhibition of acetoacetyl-CoA thiolase activity. However, this enzyme was not inhibited in intact mitochondria by pent-4-enoate. 5. MCPA specifically inhibited butyryl-CoA dehydrogenase in both intact mitochondria and in soluble extracts supplemented with ATP and CoASH. 6. Both MCPP and MCPA (1 mM) caused a rapid decrease in CoASH concentrations in mitochondria; acetyl-CoA concentrations were unaffected. Concentrations of pent-4-enoate (20 μM) sufficient to inhibit β-oxidation caused only a slight decrease in CoASH whereas higher concentrations (0.1–1.0 mM) caused a more extensive depletion of CoASH. However, evidence is presented to suggest that CoASH depletion is not the mechanism by which these compounds inhibit β-oxidation. 7. Pent-4-enoate and MCPA were substrates for butyryl-CoA synthetase. K m and V max values for several unusual, straight and branched chain fatty acids were determined. 8. Some short-chain acyl-CoA esters were substrates for an acyl-CoA hydrolase located in the mitochondrial matrix. 9. Some short-chain acyl-CoA esters competitively inhibited the activation of pyruvate carboxylase by acetyl-CoA. 10. The possible mechanisms by which hypoglycin and pent-4-enoate cause inhibition of β-oxidation and hypoglycaemia in vivo are discussed.

The effects of 2{5(4-chlorophenyl) pentyl}oxirane-2-carbonyl-CoA 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)].

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.

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.

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.

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 patients 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.

Increased activity of peroxisomal β-oxidation in rat liver caused by ethyl 2{5(4-chlorophenyl)pentyl}-oxiran-2-carboxylate: An inhibitor of mitochondrial β-oxidation

Abstract Ethyl 2{5(4-chlorophenyl)pentyl}oxiran-2-carboxylate (POCA) is a new hypoglycaemic compound. The POCA-CoA ester strongly inhibits β-oxidation at carnitine palmitoyltransferase I. Chronic administration of POCA to rats decreases plasma concentrations of cholesterol and triacylglycerol and increases the number of hepatic peroxisomes similarly to hypolipidaemic drugs related to clofibrate. Peroxisomal fractions from rats fed a diet containing 0.2% of POCA for 4 weeks were prepared on self-generated Percoll gradients. POCA induced a 4-fold increase in catalase activity and peroxisomal β-oxidation, agreeing with the morphological data. The increase in peroxisomal β-oxidation caused by POCA feeding does not prevent accumulation of lipid following the inhibition of mitochondrial β-oxidation.

Differences between human, rat and guinea pig hepatocyte cultures: A comparative study of their rates of β-oxidation and esterification of palmitate and their sensitivity to R-etomoxir

Rat hepatocyte cultures have higher rates of beta-oxidation of palmitate and lower rates of esterification to glycerolipid than human or guinea pig hepatocytes. The R-enantiomer of etomoxir (sodium 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate), a hypoglycaemic compound and inhibitor of carnitine palmitoyltransferase I, inhibited palmitate beta-oxidation in all three species, but the sensitivity to inhibition was highest in human hepatocytes and lowest in rat hepatocytes. The concentration causing half-maximal inhibition was approximately: 0.1 microM in human; 1 microM in guinea pig and 10 microM in rat hepatocytes. In human and in guinea pig hepatocytes the inhibition of beta-oxidation by R-etomoxir was associated with an increase in the esterification of palmitate but in rat hepatocytes R-etomoxir lowered the total rate of palmitate metabolism. The S-enantiomer of etomoxir had no significant effect on beta-oxidation or esterification of palmitate in any of the three species. It is concluded that there are significant differences between human, rat and guinea pig hepatocytes, not only in the relative partitioning of palmitate between beta-oxidation and esterification, but also in the sensitivity to an inhibitor of carnitine palmitoyltransferase I.

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

© 1997 Federation of European Biochemical Societies.