Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of α-ketoglutarate to citrate to support cell growth and viability

Abstract

Citrate is a critical metabolite required to support both mitochondrial bioenergetics and cytosolic macromolecular synthesis. When cells proliferate under normoxic conditions, glucose provides the acetyl-CoA that condenses with oxaloacetate to support citrate production. Tricarboxylic acid (TCA) cycle anaplerosis is maintained primarily by glutamine. Here we report that some hypoxic cells are able to maintain cell proliferation despite a profound reduction in glucose-dependent citrate production. In these hypoxic cells, glutamine becomes a major source of citrate. Glutamine-derived α-ketoglutarate is reductively carboxylated by the NADPH-linked mitochondrial isocitrate dehydrogenase (IDH2) to form isocitrate, which can then be isomerized to citrate. The increased IDH2-dependent carboxylation of glutamine-derived α-ketoglutarate in hypoxia is associated with a concomitant increased synthesis of 2-hydroxyglutarate (2HG) in cells with wild-type IDH1 and IDH2. When either starved of glutamine or rendered IDH2-deficient by RNAi, hypoxic cells are unable to proliferate. The reductive carboxylation of glutamine is part of the metabolic reprogramming associated with hypoxia-inducible factor 1 (HIF1), as constitutive activation of HIF1 recapitulates the preferential reductive metabolism of glutaminederived α-ketoglutarate even in normoxic conditions. These data support a role for glutamine carboxylation in maintaining citrate synthesis and cell growth under hypoxic conditions. C itrate plays a critical role at the center of cancer cell metabolism. It provides the cell with a source of carbon for fatty acid and cholesterol synthesis (1). The breakdown of citrate by ATP-citrate lyase is a primary source of acetyl-CoA for protein acetylation (2). Metabolism of cytosolic citrate by aconitase and IDH1 can also provide the cell with a source of NADPH for redox regulation and anabolic synthesis. Mammalian cells depend on the catabolism of glucose and glutamine to fuel proliferation (3). In cancer cells cultured at atmospheric oxygen tension (21% O 2 ), glucose and glutamine have both been shown to contribute to the cellular citrate pool, with glutamine providing the major source of the four-carbon molecule oxaloacetate and glucose providing the major source of the two-carbon molecule acetyl-CoA (4, 5). The condensation of oxaloacetate and acetyl-CoA via citrate synthase generates the 6 carbon citrate molecule. However, both the conversion of glucose-derived pyruvate to acetyl-CoA by pyruvate dehydrogenase (PDH) and the conversion of glutamine to oxaloacetate through the TCA cycle depend on NAD + , which can be compromised under hypoxic conditions. This raises the question of how cells that can proliferate in hypoxia continue to synthesize the citrate required for macromolecular synthesis.This question is particularly important given that many cancers and stem/progenitor cells can continue proliferating in the setting of limited oxygen availability (6, 7). Louis Pasteur first highlighted the impact of hypoxia on nutrient metabol...

Glutamine at focus: versatile roles in cancer

“…For instance, recent findings showed an unknown role of glutamine in a panel of human tumor cell lines with defective mitochondria or under a hypoxia condition. In particular, these cell lines produce citrate by reductively carboxylating α-KG via NADPH-dependent isocitrate dehydrogenase (IDH), supporting lipogenesis [37,38]. In agreement with this study, some somatic mutations in the IDH-1 and IDH-2 genes have been found in gliomas and acute myelogenous leukemia (AML).…”

L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH

“…1a) including an increased NADH/NAD + ratio 18,22 , an increased intracellular concentration of αKG 18,23 , and acidification of the extracellular and intracellular environments 24,25 . Prior reports implicated lactate dehydrogenase A (LDH) and malate dehydrogenase 1 and 2 (MDH) as contributing to hypoxia-induced L-2HG in cells (Fig.…”

“…1a) 18,23–25 , but it is unclear to what extent these conditions might be sufficient to induce production of L-2HG. Therefore, we tested the effects of acidified medium and/or exogenous αKG on cells cultured in normoxia (Fig.…”

Mitochondrial metabolic remodeling in response to genetic and environmental perturbations

“…Both IDH1 and 2 have potential roles in hypoxia‐induced reductive carboxylation,29, 31 suggesting that this pathway may be perturbed in IDH1‐mutated gliomas. Given that the cytosolic pathway has been shown to be important in hypoxic reductive carboxylation for lipogenesis,30, 32, 33, 34 and that gliomas appear to ‘favor’ IDH1 mutations, there may be a requirement for IDH2 wild‐type activity in hypoxic IDH1 mutant gliomas. However, a recent paper also showed that IDH1 ‐mutated cells retained more of their oxidative TCA cycle metabolism than their wild‐type counterparts 117.…”