METABOLIC HETEROGENEITY OF CARBON SUBSTRATE UTILIZATION IN MAMMALIAN HEART - NMR DETERMINATIONS OF MITOCHONDRIAL VERSUS CYTOSOLIC COMPARTMENTATION

Carbon-13(C-13) nuclear magnetic resonance (NMR) spectroscopy can be used to target specific pathways of intermediary metabolism within intact tissues and was employed in this study to evaluate the compartmentation of pyruvate metabolism between the cytosol and mitochondrial matrix. The distribution of C-13 into the tissue alanine, lactate, and glutamate pools was evaluated during metabolism of [3-C-13]pyruvate in intact, isolated perfused rabbit hearts with and without activation of pyruvate dehydrogenase activity by dichloroacetate (5 mM). Equilibrium between the intracellular alanine and pyruvate pools was in evidence from the rapid evolution of the steady-state C-13 signal arising from the 3-carbon of alanine in intact hearts perfused with 2.5 mM 99.4% [3-C-13]pyruvate. Augmented pyruvate oxidation, in response to perfusion with dichloroacetate, was evident within C-13 NMR spectra of intact hearts as a relative increase in signal intensity of 53-62% (p < 0.05) from the 4-carbon resonance of C-13-enriched glutamate when compared to the unaffected alanine signal. The increased bulk flow of [3-C-13]pyruvate into the tricarboxylic acid cycle in response to dichloroacetate resulted in elevated fractional enrichment of glutamate from 68% in controls to 83% in the treated group (p < 0.04), via interconversion with alpha-ketoglutarate, without changes in the actual tissue content of glutamate. Evidence of metabolic heterogeneity of cytosolic and mitochondrial pyruvate pools was also obtained from analysis of tissue extracts with in vitro NMR spectroscopy. Although dichloroacetate increased the percentage of labeled acetyl CoA units entering the tricarboxylic acid cycle from 83% to 94% (p<0.03), indirect assessment from the observed enrichment levels of alanine showed the fractional enrichment of intracellular pyruvate in both groups to be on the order of only 60%. Lactate formation from labeled pyruvate, as opposed to endogenous pyruvate, was yet lower in the cytosol at 21-24%, remaining unaffected by the manipulation of pyruvate dehydrogenase activity. The results are consistent with different pools of pyruvate within the myocyte that are associated with either glycolytic metabolism within the cytosol or preferential oxidation within the mitochondrial matrix. Therefore, the metabolic fate of pyruvate is closely associated with the original carbohydrate source (endogenous versus exogenous) and the intracellular compartmentation of metabolic events. This metabolic heterogeneity was detected by virtue of communication between the cytosol and mitochondrial matrix as translated into the fractional C-13 enrichments of the primarily cytosolic alanine and glutamate pools which are then detectable by NMR spectroscopy.