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

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 sim20.85 parts per million (ppm; -0.119 mumol/mg protein/hr) derived from the methyl carbon of [3-13C]lactate; and (2) an exponentially increasing shifted singlet at sim20.74 ppm (0.227 mumol/ 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. © 2004 Wiley-Liss, Inc.