A C-13 MASS ISOTOPOMER STUDY OF ANAPLEROTIC PYRUVATE CARBOXYLATION INPERFUSED RAT HEARTS

Anaplerotic pyruvate carboxylation was examined in hearts perfused wit h physiological concentrations of glucose, [U-C-13(3)]lactate, and [U- C-13(3)]pyruvate. Also, a fatty acid, [1-C-13]octanoate, or ketone bod ies were added at concentrations providing acetyl-CoA at a rate result ing in either low or substantial pyruvate decarboxylation. Relative co ntributions of pyruvate and fatty acids to citrate synthesis were dete rmined from the C-13 labeling pattern of effluent citrate by gas chrom atography-mass spectrometry (see companion article, Comte, B., Vincent , G., Bouchard, B., and Des Rosiers, C. (1997) J. Biol. Chem, 272, 261 17-26124). Precision on flux measurements of anaplerotic pyruvate carb oxylation depended on the mix of substrates supplied to the heart. Ana plerotic fluxes were precisely determined under conditions where acety l-CoA was predominantly supplied by beta-oxidation, as it occurred wit h 0.2 or 1 mM octanoate, Then, anaplerotic pyruvate carboxylation prov ided 3-8% of the OAA moiety of citrate and was modulated by concentrat ions of lactate and pyruvate in the physiological range, Also, the con tribution of pyruvate to citrate formation through carboxylation was e qual to or greater than through decarboxylation. Furthermore, C-13 lab eling data on tissue citric acid cycle intermediates and pyruvate sugg est that (i) anaplerosis occurs also at succinate and (ii) catapleroti c malate decarboxylation is low, Rather, the presence of citrate in th e effluent perfusate of hearts perfused with physiological concentrati ons of glucose, lactate, and pyruvate and concentrations of octanoate leading to maximal oxidative rates suggests a cataplerotic citrate eff lux from mitochondria to cytosol. Taken altogether, our data raise the possibility of a link between pyruvate carboxylation and mitochondria l citrate efflux. In view of the proposed feedback regulation of glyco lysis by cytosolic citrate, such a link would support a role of anaple rosis and cataplerosis in metabolic signal transmission between mitoch ondria and cytosol in the normoxic heart.