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

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Abstract
The intracellular compartmentation of pyruvate in primary cultures of cortical neurons was investigated by high resolution 13C NMR using mixtures of different pyruvate precursors conveniently labeled with 13C or unlabeled. Cells were incubated with 1–5 mM (1‐13C, 1,2‐13C2 or U‐13C6) glucose only or with mixtures containing 1.5 mM (1‐13C or U‐13C6) glucose, 0.25–2.5 mM (2‐13C or 3‐13C) pyruvate and 1 mM malate. Extracts from cells and incubation media were analyzed by 13C NMR to determine the relative contributions of the different precursors to the intracellular pyruvate pool. When (13C) glucose was used as the sole substrate fractional 13C enrichments and 13C isotopomer populations in lactate and glutamate carbons were compatible with a unique intracellular pool of pyruvate. When mixtures of (13C) glucose, (13C) pyruvate and malate were used, however, the fractional 13C enrichments of the C2 and C3 carbons of lactate were higher than those of the C2 and C3 carbons of alanine and depicted a different 13C isotopomer distribution. Moreover, neurons incubated with 1 mM (1,2‐13C2) glucose and 0.25–5 mM (3‐13C) pyruvate produced exclusively (3‐13C) lactate, revealing that extracellular pyruvate is the unique precursor of lactate under these conditions. These results reveal the presence of two different pools of intracellular pyruvate; one derived from extracellular pyruvate, used mainly for lactate and alanine production and one derived from glucose used 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 substrates more oxidized or reduced than glucose are used.
Funding Information
  • Spanish Ministry of Science and Technology (MCT PB96-0864-C02)
  • Community of Madrid (CAM 97/105, 08.1/0023/97, 08.1/0046/98, 2000-3)
  • Fundación Ramón Areces