Figure 1. L-Lactate Lowers the NAD⁺:NADH Ratio and Induces a GAPDH Block.

(A and B) Murine CD4⁺CD25⁻ T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE), co-stimulated for 3 days with anti-CD3ε/CD28-coated beads, and exposed to NaCl, Na L-lactate, HCl, and L-lactic acid at the indicated doses in low-glucose media (30 mg × dL⁻¹). The pH of the HCl condition was adjusted to match the L-lactic-acid-containing media. Teff proliferation was analyzed by CFSE dilution through flow cytometry. (A) shows representative and (B) pooled data from 11 independent experiments (two-way ANOVA with multiple comparison of treatment condition and dose).

(C and D) Phenformin (70 mg kg⁻¹ day⁻¹) was administered to C57BL/6J mice.

(C) 2 h after phenformin injection, CG4⁺ venous blood gas showed increased L-lactate (6/grp).

(D) C57BL/6J mice received an major histocompatibility complex (MHC)-mismatched BALB/c heart transplant and were treated with phenformin (70 mg kg⁻¹ day⁻¹) for 14 days or DMSO vehicle control (3/grp, log rank [Mantel-Cox] test).

(E) L-lactate derivative analysis from modified media with 20 mM [¹³C₃] L-lactate and 1.11 mM [¹²C₆] D-glucose shows that 16 h anti-CD3ε/CD28 stimulated Tconvs oxidize L-lactate into pyruvate and metabolize it in the Krebs cycle. MID denotes mass isotopomer distribution, and M0–M6 denote the number of [¹³C] per indicated molecule. Data are pooled from three independent experiments.

(F) Hypothesis: L-lactate-mediated reduction of NAD⁺ to NADH blocks the GAPDH forward reaction.

(G) Tconvs were isolated from C57BL/6J mice and co-stimulated for 16 h with CD3ε/CD28 mAb-coated beads and 20 mM NaCl, Na lactate, or Na pyruvate. NAD:NADH was measured by cycling. Pyruvate increased although lactate decreased the proportion of oxidized NAD⁺. Data are pooled from seven independent experiments (paired one-way ANOVA)

(H) Tconvs were stimulated with CD3ε/CD28 mAb-coated beads and cultured in low-glucose medium (30 mg × dL⁻¹) for 48 h ± 20 mM NaCl, Na L-lactate, or Na pyruvate and 1 μM heptelidic acid (GAPDHi) or vehicle control (H₂O). Metabolites were extracted and analyzed via LC-MS. The heatmap shows increased (red) and decreased (blue) glycolytic intermediates ion count normalized to the NaCl and vehicle control (dotted line indicates GAPDH reaction). Addition of Na pyruvate led to a depletion of pre-GAPDH triose phosphate, although Na L-lactate (NADH reduction) led to a decline in post-GAPDH metabolites, similar to direct GAPDH inhibition.

(I) 10⁶ CD8⁺ T cells were co-stimulated and cultured with ±20 mM NaCl, Na L-lactate, GAPDHi, or Na pyruvate, and glucose was measured in the supernatant. Data are pooled from seven independent experiments (one-way ANOVA). *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars indicate SEM.