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

We report a novel (C-13, 2 H) 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-C-13]lactate in Krebs-Henseleit Buffer containing 50% (H2O)-H-2 (vol/vol) for up to 30 hr. C-13 NMR analysis of aliquots progressively taken from the medium, showed: (1) a linearly decreasing singlet at similar to20.85 parts per million (ppm; -0.119 mumol/mg protein/hr) derived from the methyl carbon of [3-C-13]lactate; and (2) an exponentially increasing shifted singlet at similar to20.74 ppm (0.227 mumol/ mg protein/hr) from the methyl carbon of [3-C-13 2-H-2]Iactate. The shifted singlet appears because during its transit through the cytosol, [3-13C]Iactate generates [3-C-13, 2-H-2]lactate in the lactate dehydrogenase (LDH) equilibrium, which may return to the incubation medium through the reversible monocarboxylate carrier. The methyl group of [3-C-13, 2-H-2]lactate is shifted -0.11 ppm with respect to that of [3-C-13] lactate, making it possible to distinguish between both molecules by 13C NMR. During incubations with 2.5 mM [1-C-13]glucose and 3.98 mM [U-C-13(3)]lactate or with 2.5 mM [1-C-13]glucose and 3.93 mM [2-C-13]pyruvate, C2-deuterated lactate was produced only from [1-C-13]glucose or[U-C-13(3)]-lactate, revealing that this deuteration rocess is redox sensitive. When [1-C-13]glucose and [U-C-13]lactate were used as substrates, no significant [3-C-13]lactate production from [1-C-13]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. (C) 2004 Wiley-Liss, Inc.