Oxidation of Alpha-Ketoglutarate Is Required for Reductive Carboxylation in Cancer Cells with Mitochondrial Defects

Mammalian cells generate citrate by decarboxylating pyruvate in the mitochondria to supply the tricarboxylic acid (TCA) cycle. In contrast, hypoxia and other impairments of mitochondrial function induce an alternative pathway that produces citrate by reductively carboxylating α-ketoglutarate (AKG) via NADPH-dependent isocitrate dehydrogenase (IDH). It is unknown how cells generate reducing equivalents necessary to supply reductive carboxylation in the setting of mitochondrial impairment. Here, we identified shared metabolic features in cells using reductive carboxylation. Paradoxically, reductive carboxylation was accompanied by concomitant AKG oxidation in the TCA cycle. Inhibiting AKG oxidation decreased reducing equivalent availability and suppressed reductive carboxylation. Interrupting transfer of reducing equivalents from NADH to NADPH by nicotinamide nucleotide transhydrogenase increased NADH abundance and decreased NADPH abundance while suppressing reductive carboxylation. The data demonstrate that reductive carboxylation requires bidirectional AKG metabolism along oxidative and reductive pathways, with the oxidative pathway producing reducing equivalents used to operate IDH in reverse. [Display omitted] •Cells with mitochondrial defects use bidirectional metabolism of the TCA cycle•Glutamine supplies the succinate pool through oxidative and reductive metabolism•Oxidative TCA cycle metabolism is required for reductive citrate formation•Oxidative metabolism produces reducing equivalents for reductive carboxylation Cancer cells with mitochondrial defects use an unconventional and poorly understood metabolic pathway known as reductive carboxylation. Here, Mullen et al. have uncovered a redox relay system that enables cancer cells to engage this pathway. The authors show that this system requires oxidative metabolism of α-ketoglutarate, which generates reducing equivalents that are used to support the reductive carboxylation pathway, providing a mechanism for the reductive carboxylation pathway's regulation in cancer cells with mitochondrial defects.