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

Abstract

The intracellular compartmentation of pyruvate in primary cultures of cortical neurons was investigated by high resolution (13)C NMR using mixtures of different pyruvate precursors conveniently labeled with (13)C or unlabeled. Cells were incubated with 1-5 mM (1-(13)C, 1,2-(13)C(2) or U-(13)C(6)) glucose only or with mixtures containing 1.5 mM (1-(13)C or U-(13)C(6)) glucose, 0.25-2.5 mM (2-(13)C or 3-(13)C) pyruvate and 1 mM malate. Extracts from cells and incubation media were analyzed by (13)C NMR to determine the relative contributions of the different precursors to the intracellular pyruvate pool. When ((13)C) glucose was used as the sole substrate fractional (13)C enrichments and (13)C isotopomer populations in lactate and glutamate carbons were compatible with a unique intracellular pool of pyruvate. When mixtures of ((13)C) glucose, ((13)C) pyruvate and malate were used, however, the fractional (13)C 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 (13)C isotopomer distribution. Moreover, neurons incubated with 1 mM (1,2-(13)C(2)) glucose and 0.25-5 mM (3-(13)C) pyruvate produced exclusively (3-(13)C) 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.

The malate–aspartate NADH shuttle components are novel metabolic longevity regulators required for calorie restriction-mediated life span extension in yeast

“…The concomitant increase in respiration is necessary and sufficient for the activation of Sir2-mediated silencing and extension in life span (Lin et al 2002). The fact that respiration produces NAD from NADH (Bakker et al 2001;Cruz et al 2001), as well as the finding that NADH can function as a competitive inhibitor of Sir2 activity (Lin et al 2004), reinforce the idea that an increase in the NAD/NADH ratio activates Sir2 during CR. A link between CR and increased mitochondrial metabolism has also been reported in higher eukaryotes including mammals (Nisoli et al 2005;Bishop and Guarente 2007).…”

Ex Vivo Analysis of Lactate and Glucose Metabolism in the Rat Brain under Different States of Depressed Activity

“…As the result of the lactate consumption in neurons, the lactate gradient between neurons and astrocytes would be maintained, which would constitute the driving force for the move of lactate from astrocytes to neurons, thus making possible the regeneration of NADϩ to fulfill the astrocytic energy demand from glycolysis. On the other hand, the increased neuronal use of blood and astrocytic glycolytic lactate as neuronal oxidative substrates with brain activity emphasizes the possibility of the redox switch in neurons as proposed (33). Indeed, these authors evidenced that the two pyruvate species, the glycolytic pyruvate and the pyruvate synthesized from exogenous lactate by lactate dehydrogenase, were compartmentalized and that the production of the first was inhibited when the second was used preferentially as an oxidative substrate.…”

Pyruvate prevents aging of mouse oocytes

“…Dumollard et al (2007) proposed the presence of discrete pools of pyruvate inside the oocyte: one from the culture medium, which is rapidly metabolized by the mitochondria, while a second pool derived from lactate is poorly used by the mitochondria. Such intracellular compartmentation of pyruvate pools has also been described in neuronal and glial cells (Cruz et al 2001, Zwingmann et al 2001. According to Dumollard et al (2007), the lactate-derived pyruvate may be preferentially converted in the cytosol to alanine by alanine aminotransferase (ALT), because bovine oocytes contain strong ALT activities and bovine and porcine embryos produce large amounts of alanine (Gopichandran & Leese 2003, Humpherson et al 2005.…”