Increasing evidence supports that proliferating cancer cells use lactate as a source of energy and a carbon source, however, determining whether lactate is oxidized in the cytosol or mitochondria is an experimental challenge. Here, we apply three independent strategies to confirm that lactate is oxidized in mitochondria: (i) isotope tracer analysis, (ii) transport-exclusion pharmacology, and (iii) genetic manipulation of putative proteins. (i) Oxidation of 2-2H-lactate by lactate dehydrogenase (LDH) produces unlabeled pyruvate and 2H-NADH, which cannot directly cross the inner mitochondrial membrane. The metabolism of proliferating cells given 2-2H-lactate was quenched and their mitochondria isolated. Mass spectrometry analysis showed 2-2H-lactate in mitochondria, supporting direct import. (ii) LDH has a binding site for pyruvate/lactate and a binding site for NADH/NAD+. The enzyme can therefore be inhibited by chemical mimetics of either pyruvate or NADH. We exploited a chemical mimetic of pyruvate able to cross the inner mitochondrial membrane and a chemical mimetic of NADH that could not. Interestingly, the inhibitors show different activities, supporting the presence of LDH in a compartment (e.g., mitochondria) that is inaccessible to a chemical mimetic of NADH. (iii) We found that LDHB uniquely localized to mitochondria of proliferating cells. After knocking out LDHB, we found that isolated mitochondria from these same cells could no longer readily oxidize lactate.

Evidence that lactate is oxidized in the mitochondria of proliferating cells

ACS Fall 2021

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Evidence that lactate is oxidized in the mitochondria of proliferating cells </i>