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Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Effects of the free acids and their carnitine esters on coenzyme A-dependent oxidations in rat liver mitochondria.

ABSTRACT: 1. The synthesis of pent-4-enoyl-l-carnitine, cyclopropanecarbonyl-l-carnitine and cyclobutanecarbonyl-l-carnitine is described. 2. Pent-4-enoate strongly inhibits palmitoyl-l-carnitine oxidation in coupled but not in uncoupled mitochondria. Pent-4-enoyl-l-carnitine strongly inhibits palmitoyl-l-carnitine oxidation in uncoupled mitochondria. Prior intramitochondrial formation of pent-4-enoyl-CoA is therefore necessary for inhibition. 3. There was a small self-limiting pulse of oxidation of pent-4-enoyl-l-carnitine during which the ability to inhibit the oxidation of subsequently added palmitoyl-l-carnitine developed. 4. Pent-4-enoate and pent-4-enoyl-l-carnitine are equally effective inhibitors of the oxidation of all even-chain acylcarnitines of chain length C(4)-C(16). Pent-4-enoyl-l-carnitine also inhibits the oxidation of pyruvate and of 2-oxoglutarate. 5. Pent-4-enoate strongly inhibits the oxidation of palmitate but not that of octanoate. This is presumably due to competition between octanoate and pent-4-enoate for medium-chain acyl-CoA ligase. 6. There was less inhibition of the oxidation of pyruvate by pent-4-enoyl-l-carnitine, and of palmitoyl-l-carnitine by cyclopropanecarbonyl-l-carnitine, after pre-incubation with 10mm-arsenate. This suggests that these inhibitions were caused either by depletion of free CoA or by increase of acyl-CoA concentrations, since arsenate deacylates intramitochondrial acyl-CoA. There was little effect on the inhibition of palmitoyl-l-carnitine oxidation by pent-4-enoyl-l-carnitine. 7. Penta-2,4-dienoate strongly inhibited palmitoyl-l-carnitine oxidation in coupled mitochondria; acrylate only inhibited slightly. 8. Pent-4-enoate (0.1mm) caused a rapid and almost complete decrease in free CoA and a large increase in acid-soluble acyl-CoA when incubated with coupled mitochondria. Cyclopropanecarboxylate caused a similar decrease in CoA, with an equivalent rise in acid-soluble acyl-CoA concentrations. n-Pentanoate caused extensive lowering of CoA and a large increase in acid-soluble acyl-CoA and acetyl-CoA concentrations. Octanoate caused a 50% lowering of CoA and an increase in acid-soluble acyl-CoA and acetyl-CoA concentrations. 9. Cyclopropanecarboxylate and n-pentanoate were less potent inhibitors of palmitate oxidation than was pent-4-enoate. 10. It is concluded that pent-4-enoate causes a specific inhibition of beta-oxidation after the formation intramitochondrially of its metabolites.

SUBMITTER: Holland PC

PROVIDER: S-EPMC1165935 | biostudies-other | 1973 Sep

REPOSITORIES: biostudies-other

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Project description:1. Pent-4-enoyl-CoA and its metabolites penta-2,4-dienoyl-CoA and acryloyl-CoA, as well as n-pentanoyl-CoA, cyclopropanecarbonyl-CoA and cyclobutanecarbonyl-CoA, were examined as substrates or inhibitors of purified enzymes of beta-oxidation in an investigation to locate the site of inhibition of fatty acid oxidation by pent-4-enoate. 2. The reactions of various acyl-CoA derivatives with l-carnitine and of various acyl-l-carnitine derivatives with CoA, catalysed by carnitine acetyltransferase, were investigated and V(max.) and K(m) values were determined. Pent-4-enoyl-CoA and n-pentanoyl-CoA were good substrates, whereas cyclobutanecarbonyl-CoA, cyclopropanecarbonyl-CoA and acryloyl-CoA reacted more slowly. A very slow rate with penta-2,4-dienoyl-CoA was detected. Pent-4-enoyl-l-carnitine, n-pentanoyl-l-carnitine and cyclobutanecarbonyl-l-carnitine were good substrates and cyclopropanecarbonyl-l-carnitine reacted more slowly. 3. Pent-4-enoyl-CoA and n-pentanoyl-CoA were substrates for butyryl-CoA dehydrogenase and for octanoyl-CoA dehydrogenase, and both compounds were equally effective competitive inhibitors of these enzymes with butyryl-CoA or palmitoyl-CoA respectively as substrates. V(max.), K(m) and K(i) values were determined. 4. None of the acyl-CoA derivatives inhibited enoyl-CoA hydratase or 3-hydroxybutyryl-CoA dehydrogenase. Penta-2,4-dienoyl-CoA was a substrate for enoyl-CoA hydratase when the reaction was coupled to that catalysed by 3-hydroxybutyryl-CoA dehydrogenase. 5. In a reconstituted sequence with purified enzymes crotonoyl-CoA was largely converted into acetyl-CoA, and pent-2-enoyl-CoA into acetyl-CoA and propionyl-CoA. Penta-2,4-dienoyl-CoA was slowly converted into acetyl-CoA and acryloyl-CoA. 6. Penta-2,4-dienoyl-CoA, a unique metabolite of pent-4-enoate, was the only compound that specifically inhibited an enzyme of the beta-oxidation sequence, 3-oxoacyl-CoA thiolase. The formation of penta-2,4-dienoyl-CoA could explain the strong inhibition of fatty acid oxidation in intact mitochondria by pent-4-enoate.

Project description:1. The total acid-soluble carnitine concentrations of four tissues from Merino sheep showed a wide variation not reported for other species. The concentrations were 134, 538, 3510 and 12900nmol/g wet wt. for liver, kidney cortex, heart and skeletal muscle (M. biceps femoris) respectively. 2. The concentration of acetyl-CoA was approximately equal to the concentration of free CoA in all four tissues and the concentration of acid-soluble CoA (free CoA plus acetyl-CoA) decreased in the order liver>kidney cortex>heart>skeletal muscle. 3. The total amount of acid-soluble carnitine in skeletal muscle of lambs was 40% of that in the adult sheep, whereas the concentration of acid-soluble CoA was 2.5 times as much. A similar inverse relationship between carnitine and CoA concentrations was observed when different muscles in the adult sheep were compared. 4. Carnitine was confined to the cytosol in all four tissues examined, whereas CoA was equally distributed between the mitochondria and cytosol in liver, approx. 25% was present in the cytosol in kidney cortex and virtually none in this fraction in heart and skeletal muscle. 5. Carnitine acetyltransferase (EC 2.3.1.7) was confined to the mitochondria in all four tissues and at least 90% of the activity was latent. 6. Acetate thiokinase (EC 6.2.1.1) was predominantly (90%) present in the cytosol in liver, but less than 10% was present in this fraction in heart and skeletal muscle. 7. In alloxan-diabetes, the concentration of acetylcarnitine was increased in all four tissues examined, but the total acid-soluble carnitine concentration was increased sevenfold in the liver and twofold in kidney cortex. 8. The concentration of acetyl-CoA was approximately equal to that of free CoA in the four tissues of the alloxan diabetic sheep, but the concentration of acid-soluble CoA in liver increased approximately twofold in alloxan-diabetes. 9. The relationship between CoA and carnitine and the role of carnitine acetyltransferase in the various tissues is discussed. The quantitative importance of carnitine in ruminant metabolism is also emphasized.

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Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Effects of the free acids and their carnitine esters on coenzyme A-dependent oxidations in rat liver mitochondria.

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