Intracellular compartmentation of pyruvate in primary cultures of cortical neurons as detected by (13)C NMR spectroscopy with multiple (13)C labels.
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.
Cruz
F
F
Instituto de Investigaciones Biomédicas Alberto Sols, C.S.I.C./U.A.M., c/ Arturo Duperier 4, Madrid 28029, Spain.
Villalba
M
M
García-Espinosa
M A
MA
Ballesteros
P
P
Bogónez
E
E
Satrústegui
J
J
Cerdán
S
S
D000818:Animals; D001921:Brain / Q000166:cytology / Q000000981:diagnostic imaging*; D002250:Carbon Radioisotopes / Q000378:metabolism; D002451:Cell Compartmentation / Q000502:physiology*; D002478:Cells, Cultured; D002540:Cerebral Cortex; D002952:Citric Acid Cycle / Q000502:physiology*; D005333:Fetus; D005947:Glucose / Q000378:metabolism; D006019:Glycolysis / Q000502:physiology*; D007424:Intracellular Fluid / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008293:Malates / Q000378:metabolism; D008954:Models, Biological; D009474:Neurons / Q000000981:diagnostic imaging*; D010084:Oxidation-Reduction; D010085:Oxidative Phosphorylation; D019289:Pyruvic Acid / Q000378:metabolism*; D011877:Radionuclide Imaging; D051381:Rats
D002250:Carbon Radioisotopes; D008293:Malates; C030298:malic acid; D019289:Pyruvic Acid; D005947:Glucose