Subcellular Metabolite Transport and Carbon Isotope Kinetics in the Intramyocardial Glutamate Pool

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Abstract
The pathophysiological state of the cell must be translated into the mitochondria to meet the demands for oxidative energy production. Metabolite exchange across the mitochondrial membrane provides this communication and was observed with 13C NMR spectroscopy of hearts oxidizing [2-13C]-butyrate at normal or high cytosolic redox state. Previous NMR observations of 13C turnover within the glutamate pool of intact tissues have indicated its relationship with metabolic flux through the tricarboxylic acid (TCA) cycle, but the direct influence of isotope exchange between the TCA cycle intermediates in the mitochondria and the cytosolic glutamate pool has been much less considered. This current study was designed to determine whether the physical transport of metabolites across the mitochondrial membrane of intact heart tissues could be discerned as a rate determinant for isotope turnover in the NMR-detectable glutamate pool. 13C entry into glutamate provided measures of TCA cycle flux and the interconversion between mitochondrial intermediates and cytosolic glutamate. The influence of the malate-aspartate shuttle activity was examined by comparing two groups of hearts: one group oxidizing 2.5 mM [2-13C]-butyrate (n = 5) and the other oxidizing 2.5 mM [2-13C]butyrate in the presence of a lactate (2.5 mM)-induced elevation in the cytosolic redox to stimulate shuttle activity (n = 5). High redox state did not affect TCA cycle flux but increased the rate of interconversion between alpha-ketoglutarate and glutamate from 3.1 +/- 0.2 mumol min-1 (g dry)-1 to 14.3 +/- 2.0. High resolution 13C NMR spectra of tissue extracts confirmed that the exogenous lactate did not contribute as a carbon source for the formation of either the TCA cycle intermediates or glutamate. In both groups, over 95% of the acetyl-CoA was derived from the short-chain fatty acid butyrate, irrespective of the presence of lactate. Additional hearts perfused with unlabeled butyrate and [3-13C]lactate showed no label entry into glutamate, but rather the formation of [3-13C]alanine, indicating the net reverse flux through lactate dehydrogenase to increase NADH production. Thus, the addition of lactate served only to augment cytosolic redox state to drive the malate-aspartate shuttle. The dynamic-mode acquisition of 13C NMR data from intact hearts, oxidizing [2-13C]-butyrate with or without additional lactate, demonstrated the influence of malate-aspartate shuttle activity on the 13C enrichment rates within glutamate. These data indicate metabolic communication between the mitochondria and cytosol in response to the physiological state of intact tissues.