Hamilton, Christian; (1999) Malonyl-CoA metabolism in cardiac myocytes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Subcellular fractions of rat heart homogenates were prepared and the pathways of malonyl-CoA utilisation studied. The presence of fatty acyl-CoA elongation (FACE) activity was not detected in any subcellular fraction but an appreciable amount of malonyl-CoA decarboxylase activity was. Approximately 70% of the decarboxylase activity was localised in the mitochondrial fraction. Intact mitochondrial studies suggested the presence of malonyl-CoA decarboxylase activity overt to the inner mitochondrial membrane. Oxygen electrode studies showed that 5mM acetyl-carnitine (in the presence of 3mM malate) was a good respiratory substrate for intact rat heart mitochondria, while ImM malonyl-CoA was not. Cardiac myocytes were used to study levels of malonyl-CoA under various physiological conditions. In the presence of 5mM glucose, malonyl-CoA content was measured at 6.5-7.5 nmol/g dry wt in triacylglycerol (TAG)-loaded and non-TAG-loaded myocytes. lOnM insulin increased this by 25% in non-TAG-loaded myocytes but not in TAG-loaded myocytes. 0.5mM palmitate lowered malonyl-CoA levels by 35% and 39% in non-TAG-loaded and TAG-loaded myocytes respectively, while increasing glucose concentration (0-5mM), resulted in a parallel increase in malonyl-CoA in both. Adrenaline (0-2μM), isoprenaline (0-1μM) and phenylephrine (0-1μM) had no significant effect on the level of malonyl-CoA in insulin treated, non-TAG-loaded myocytes. Long-chain ester levels were measured in TAG-loaded and non-TAG-loaded myocytes. 0.5mM palmitate increased long-chain acyl-carnitine levels by 23% in TAG-loaded only, while 10nM insulin decreased levels by ~14% in both. Increasing glucose concentration (0-5mM) increased long-chain acyl-CoA and long-chain acylcarnitine levels by 27% and 46% respectively in non-TAG-loaded myocytes. Levels of long-chain esters were measured in freeze-clamped non-working perfused rat hearts and were 2-2.7 fold lower than in myocytes. The rate of malonyl-CoA synthesis was estimated in glucose depleted myocytes. Readdition of 5mM glucose increased malonyl-CoA levels from 3.0 to 5.0 nmol/g dry wt. within 12 min, with a half time of ~1.8 min. 5LIM isoprenaline increased lipolysis in non-TAG-loaded myocytes by 94%. 0.5mM palmitate decreased lipolysis by 54% and 37% in TAG-loaded and non-TAG-loaded myocytes respectively.

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