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.

A Novel mechanism for inhibition of β‐oxidation by methylenecyclopropylacetyl‐CoA, a metabolite of hypoglycin

“…Palmitoyl-carnitine oxidation was unimpaired in mitochondria uncoupled by addition of 0.2/aM trifluoromethoxycarbonylcyanide phenylhydrazone, 10 mM arsenate and valinomycin (0.2 mg/ml) even after prolonged incubation with 1.0 mM MCPA, indicating that prior conversion of MCPA to MCPA-CoA by butyryl-CoA (medium chain acyl-CoA) synthetase (EC 6.2.1.2) in the matrix is necessary for inhibition (see [8] ). We have failed to prepare MCPA-CoA, either chemically or enzymically using purified butyryl-CoA synthetase [ 13], suggesting that MCPA-CoA is very unstable.…”

“…Oxygen uptake by mitochondria in state 3 [9] was recorded polarographically in 3.0 ml of medium at 30°C containing 100 mM KC1, 20 mM 3-(N-morpholino) propanesulphonate, 2.5 mM phosphate, 5 mM MgC12,1.0 mM EDTA, 10 mM malonate, 2.0 mM ADP and 9 m~ defatted bovine serum albumin, pH 7.2. With these conditions acyl groups are quantitatively converted to acetoacetate and the oxygen uptake is a direct measure of the flux through/3-oxidation [8]. Extracts of acetone-dried rat liver mitochondria (2 g) were made by stirring with 10 ml of 10 mM potassium phosphate, pH 7.2 at 0°C, followed by centrifugation (4.5 X 10 6 g min) and passage through a column of Sephadex G25 pre-equilibrated with phosphate buffer.…”

“…Extracts of acetone-dried rat liver mitochondria (2 g) were made by stirring with 10 ml of 10 mM potassium phosphate, pH 7.2 at 0°C, followed by centrifugation (4.5 X 10 6 g min) and passage through a column of Sephadex G25 pre-equilibrated with phosphate buffer. Protein was measured by a biuret method [8]. Acyl-CoA dehydrogenase activities (EC 1.3.99.-) were assayed at 20°C and pH 7.2 with 60 taM acylCoA as substrate [10].…”

“…Hypoglycin and MCPA were prepared essentially by published methods [5,7] and acyl-carnitine and acylCoA esters were synthesised and characterised as described previously [8,9]. Rat liver mitochondria were isolated in 0.3 mM mannitol 2.5 mM 2(N-hvdroxyethylpiperazin-N'-yl) ethanesulphonate, 0.1 mM ethyleneglycol-2-(aminoethyl)-tetraacetate, pH 7.2 [8].…”

“…Rat liver mitochondria were isolated in 0.3 mM mannitol 2.5 mM 2(N-hvdroxyethylpiperazin-N'-yl) ethanesulphonate, 0.1 mM ethyleneglycol-2-(aminoethyl)-tetraacetate, pH 7.2 [8]. Oxygen uptake by mitochondria in state 3 [9] was recorded polarographically in 3.0 ml of medium at 30°C containing 100 mM KC1, 20 mM 3-(N-morpholino) propanesulphonate, 2.5 mM phosphate, 5 mM MgC12,1.0 mM EDTA, 10 mM malonate, 2.0 mM ADP and 9 m~ defatted bovine serum albumin, pH 7.2.…”