Futile cycling of lactate through the plasma membrane of C6 glioma cells as detected by (13C, 2H) NMR

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Abstract
We report a novel (13C, 2H) nuclear magnetic resonance (NMR) procedure to investigate lactate recycling through the monocarboxylate transporter of the plasma membrane of cells in culture. C6 glioma cells were incubated with [3‐13C]lactate in Krebs‐Henseleit Buffer containing 50% 2H2O (vol/vol) for up to 30 hr. 13C NMR analysis of aliquots progressively taken from the medium, showed: (1) a linearly decreasing singlet at ∼20.85 parts per million (ppm; −0.119 μmol/mg protein/hr) derived from the methyl carbon of [3‐13C]lactate; and (2) an exponentially increasing shifted singlet at ∼20.74 ppm (0.227 μmol/ mg protein/hr) from the methyl carbon of [3‐13C, 2‐2H]lactate. The shifted singlet appears because during its transit through the cytosol, [3‐13C]lactate generates [3‐13C, 2‐2H]lactate in the lactate dehydrogenase (LDH) equilibrium, which may return to the incubation medium through the reversible monocarboxylate carrier. The methyl group of [3‐13C, 2‐2H]lactate is shifted −0.11 ppm with respect to that of [3‐13C]lactate, making it possible to distinguish between both molecules by 13C NMR. During incubations with 2.5 mM [1‐13C]glucose and 3.98 mM [U‐13C3]lactate or with 2.5 mM [1‐13C]glucose and 3.93 mM [2‐13C]pyruvate, C2‐deuterated lactate was produced only from [1‐13C]glucose or [U‐13C3]lactate, revealing that this deuteration process is redox sensitive. When [1‐13C]glucose and [U‐13C3]lactate were used as substrates, no significant [3‐13C]lactate production from [1‐13C]glucose was detected, suggesting that glycolytic lactate production may be stopped under the high lactate concentrations prevailing under mild hypoxic or ischemic episodes or during cerebral activation.
Funding Information
  • Spanish Ministry of Education and Science (SAF 2001-2245)
  • Institute of Health Carlos III (FISss C03/08, G03/155, C03/10)
  • Community of Madrid (Strategic Group Grant 2000-3)
  • JUSTESA IMAGEN SA (Institutional support grant 2003-2007)
  • Fundação para a Ciência e Tecnologia (SFRH/BD/5407/2001)
  • Howard Hughes Bravo Program of the University of Arizona

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