| Malonyl-CoA, an intermediate of fatty acid synthesis in lipogenic organs, modulates the activity of carnitine palmitoyl transferase I in most tissues. The tissue concentration of malonyl-CoA is regulated by the activities of acetyl-CoA carboxylase and malonyl-CoA decarboxylase, which are each a subject to complex regulation.; The research presented here was aimed at measuring the kinetics of malonyl-CoA and at assessing the origin of its acetyl moiety in the heart.; First, the turnover of malonyl-CoA was measured from the kinetics of its labeling in isolated rat hearts perfused with 40% enriched NaH 13CO3. The turnover of malonyl-CoA represents the rate of acetyl-CoA carboxylation in the intact heart. Conditions were selected to result in stable malonyl-CoA concentrations. The tissue content of malonyl-CoA was found to be directly proportional to its turnover rate. The activities of acetyl-CoA carboxylase and malonyl-CoA decarboxylase showed no correlation with the malonyl-CoA turnover rates. Second, the carbon sources of the acetyl moiety of malonyl-CoA were determined in isolated rat hearts perfused with glucose, lactate, pyruvate, and fatty acids. Various combinations of 13C-labeled substrates were used to generate singly- and doubly-labeled malonyl-CoA and mitochondrial acetyl-CoA. The mass isotopomer distribution of malonyl-CoA revealed that fatty acids are a major source of its acetyl moiety. Comparison of the MID of malonyl-CoA and of the acetyl moiety of citrate, showed that peroxisomal beta-oxidation provides a large fraction of the fatty acid-derived acetyl moiety of malonyl-CoA. By supplying acetyl-CoA to acetyl-CoA carboxylase, peroxisomal beta-oxidation may control the rate of mitochondrial fatty acid oxidation in the heart.; Finally, an attempt at utilizing stable isotopes to probe malonyl-CoA compartmentation led to the quantitative assessment of the reversibility of the reactions of the propionyl-CoA pathway in intact rat hearts and livers. The MID of propionyl-CoA, methylmalonyl-CoA and succinyl-CoA from intact hearts and livers perfused with 13C-tracers (NaH13CO 3, [U-13C3]propionate, or [U- 13C3](lactate + pyruvate)) showed that (i) the propionyl-CoA carboxylase reaction is slightly reversible only at low propionyl-CoA flux, (ii) the methylmalonyl-CoA enantiomers are in isotopic equilibrium under all conditions tested and (iii) the methylmalonyl-CoA mutase reaction is reversible, but its reversibility decreases as the flux of propionyl-CoA increases. |
