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

TLDR

Mammalian cells normally produce citrate by mitochondrial pyruvate decarboxylation, but under hypoxia or mitochondrial dysfunction they rely on NADPH‑dependent reductive carboxylation of α‑ketoglutarate to generate citrate. The study aimed to determine how cells supply the reducing equivalents required for this reductive carboxylation pathway when mitochondrial function is impaired. The authors found that reductive carboxylation is coupled to concurrent α‑ketoglutarate oxidation, and that blocking AKG oxidation or disrupting NADH‑to‑NADPH transfer reduces reducing equivalents and suppresses the pathway, demonstrating that bidirectional AKG metabolism is essential for supplying the reducing power needed for reverse IDH activity.

Abstract

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.