Intracellular compartmentation of pyruvate in primary cultures of corticalneurons as detected by C-13 NMR spectroscopy with multiple C-13 labels

The intracellular compartmentation of pyruvate in primary cultures of corti cal neurons was investigated by high resolution C-13 NMR using mixtures of different pyruvate precursors conveniently labeled with C-13 or unlabeled. Cells were incubated with 1-5 MM (1-C-13, 1,2-C-13(2) or U-C-13(6)) glucose only or with mixtures containing 1.5 mM (1-C-13 or U-C-13(6)) glucose, 0.2 5-2.5 mM (2-C-13 or 3-C-13) pyruvate and 1 mM malate. Extracts from cells a nd incubation media were analyzed by C-13 NMR to determine the relative con tributions of the different precursors to the intracellular pyruvate pool. When (C-13) glucose was used as the sole substrate fractional C-13 enrichme nts and C-13 lsotopomer populations in lactate and glutamate carbons were c ompatible with a unique intracellular pool of pyruvate. When mixtures of (C -13) glucose, (C-13) pyruvate and malate were used, however, the fractional C-13 enrichments of the C2 and C3 carbons of lactate were higher than thos e of the C2 and C3 carbons of alanine and depicted a different C-13 isotopo mer distribution. Moreover, neurons incubated with 1 mM (1,2-C-13(2)) gluco se and 0.25-5 mM (3-C-13) pyruvate produced exclusively (3-C-13) lactate, r evealing that extracellular pyruvate is the unique precursor of lactate und er these conditions. These results reveal the presence of two different poo ls of intracellular pyruvate; one derived from extracellular pyruvate, used mainly for lactate and alanine production and one derived from glucose use d primarily for oxidation. A red-ox switch using the cytosolic NAD(+)/NADH ratio is proposed to modulate glycolytic flux, controlling which one of the two pyruvate pools is metabolized in the tricarboxylic acid cycle when sub strates more oxidized or reduced than glucose are used. (C) 2001 Wiley-Liss , Inc.