Studies on Lactate Transport Pre-incubation with 10 nM Inhibitor 1 completely abolished the intracellular acidification (Fig. 9). To determine the selectivity of the new inhibitory compounds, L-lactate transport mediated by MCT2, MCT3 and MCT4 were investigated. Substantially higher rates of L-lactate transport for MCT3 and MCT4 were observed, with no observable L-lactate inhibition upon pre-incubation with 100 nM Inhibitor 1 (Fig. 10). Competition binding studies showed that labeled Inhibitor 3 was selective for MCT1, relative to MCT2. Western blotting, using an antibody to hMCT1, demonstrated that MCT1 levels were greater in activated T cells than in resting cells. The ability of T cells to transport L-lactate across the membrane was studied using BCECF. L-lactate was not transported in resting T cells, whereas L-lactate transport was observed in activated T cells.

The Role of MCT1 in Lactate Transport To elucidate the role of MCT1 in lactate transport, the effects of MCT1 inhibition on the intracellular lactate level and lactate production was studied. Activated T cells were incubated with Inhibitor 1 for 4 hours which substantially increased intracellular lactate (Fig. 11a). Incubation with Inhibitor 1 also decreased the amount of lactate in the media by approximately 30% and reduced DNA synthesis by 50% (Fig. 11b). These findings suggest that MCT1, rather than MCT4, is involved in lactate efflux in T cells. These results also suggest that a direct correlation exists between the blockade of lactate efflux via MCT1 and inhibition of proliferation.

Figure 9. (a-d) Changes in pHi due to L-lactate uptake. In (a) no inhibitor, (b) 10 nM Inhibitor 1, (c) 10 nM Inhibitor 2 and (d) 10 nM Inhibitors 2-4. The immunomodulatory compounds demonstrate specific binding in cells expressing hMCT1 (Murray et al, 2005).

Metabolic Consequences The increase in glycolytic rate observed in activated T cells is essential both for providing ATP for ATP-consuming processes and for supplying metabolic intermediates channeled through the pentose phosphate pathway for nucleotide biosynthesis. These findings suggest that the small but significant reduction in glycolytic flux resulting from MCT1 blockage suppresses cellular metabolism such that active lymphocyte proliferation cannot be sustained (Murray et al, 2005).

Figure 10. (a,b) Changes in pHi due to L-lactate uptake in cells expressing (a) hMCT3 and (b) hMCT4 in the presence of 100 nM Inhibitor 1. (c) The determined Kd (0.7 +/- 0.1 nM) for hMCT1 for L-lactate from saturation binding studies (Murray et al, 2005).

Conclusions In the past, many inhibitory compounds were unable to discriminate between the monocarboxylaye isoforms. The development of a potent and specific class of inhibitory compounds by Murray et al, 2005 appears to remedy past conflicts. The newly developed MCT1 inhibitors may provide one approach for experimentally exploring the roles of aerobic glycolyis in cell proliferation (Broer, S., 2005). By intricately controlling lactate efflux the MCT1 inhibitors may assist in the elucidation of the mechanism by which aerobic glycolysis occurs. The new inhibitory compounds may also illuminate the mechanism of the monocarboxylate transporters. Additionally, the characterized MCT1 inhibitors are promising immunosuppressant compounds. The inhibition of T lymphocyte proliferation in vivo strongly suggests the MCT1 inhibitors may be powerful therapeutic agents.

Figure 11. In (a) are the effects of increasing concentrations of Inhibitor 1 on intracellular lactate levels. The effects of lactate production on DNA synthesis are shown are shown in (b) (Murray et al, 2005).