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

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 glutamine-derived α-ketoglutarate even in normoxic conditions. These data support a role for glutamine carboxylation in maintaining citrate synthesis and cell growth under hypoxic conditions.

[1]  H. Brunengraber,et al.  Investigations by mass isotopomer analysis of the formation of D‐2‐hydroxyglutarate by cultured lymphoblasts from two patients with D‐2‐hydroxyglutaric aciduria , 2004, FEBS letters.

[2]  R. Deberardinis,et al.  Beyond aerobic glycolysis: Transformed cells can engage in glutamine metabolism that exceeds the requirement for protein and nucleotide synthesis , 2007, Proceedings of the National Academy of Sciences.

[3]  Daniel E Bauer,et al.  ATP citrate lyase inhibition can suppress tumor cell growth. , 2005, Cancer cell.

[4]  G. Semenza,et al.  HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. , 2006, Cell metabolism.

[5]  Anthony Mancuso,et al.  Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction , 2008, Proceedings of the National Academy of Sciences.

[6]  G. Kalna,et al.  Metabolic Profiling of Hypoxic Cells Revealed a Catabolic Signature Required for Cell Survival , 2011, PloS one.

[7]  Gregory Stephanopoulos,et al.  Evaluation of 13C isotopic tracers for metabolic flux analysis in mammalian cells. , 2009, Journal of biotechnology.

[8]  L. Cantley,et al.  Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation , 2009, Science.

[9]  W. Craigen,et al.  Mutations in the D-2-hydroxyglutarate dehydrogenase gene cause D-2-hydroxyglutaric aciduria. , 2005, American journal of human genetics.

[10]  Elizabeth L. Johnson,et al.  Quiescent Fibroblasts Exhibit High Metabolic Activity , 2010, PLoS biology.

[11]  W. Wheaton,et al.  Hypoxia. 2. Hypoxia regulates cellular metabolism. , 2011, American journal of physiology. Cell physiology.

[12]  N. Denko,et al.  HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption. , 2006, Cell metabolism.

[13]  M. Celeste Simon,et al.  The impact of O2 availability on human cancer , 2008, Nature Reviews Cancer.

[14]  M. Keating,et al.  Metabolic Alterations in Highly Tumorigenic Glioblastoma Cells , 2011, The Journal of Biological Chemistry.

[15]  K. Sharp,et al.  Identification of additional IDH mutations associated with oncometabolite R(−)-2-hydroxyglutarate production , 2012, Oncogene.

[16]  N. Chandel,et al.  Hypoxic but not anoxic stabilization of HIF-1alpha requires mitochondrial reactive oxygen species. , 2002, American journal of physiology. Lung cellular and molecular physiology.

[17]  R. Deberardinis,et al.  The transcription factor HIF-1alpha plays a critical role in the growth factor-dependent regulation of both aerobic and anaerobic glycolysis. , 2007, Genes & development.

[18]  Omar Abdel-Wahab,et al.  The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. , 2010, Cancer cell.

[19]  J. Rydström.  Mitochondrial NADPH, transhydrogenase and disease. , 2006, Biochimica et biophysica acta.

[20]  B. Chance,et al.  Localization and kinetics of reduced pyridine nucleotide in living cells by microfluorometry. , 1959, The Journal of biological chemistry.

[21]  Justin R. Cross,et al.  ATP-Citrate Lyase Links Cellular Metabolism to Histone Acetylation , 2009, Science.

[22]  L. Liau,et al.  Cancer-associated IDH1 mutations produce 2-hydroxyglutarate , 2009, Nature.

[23]  C. Wykoff,et al.  The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis , 1999, Nature.

[24]  Tsung-Cheng Chang,et al.  c-Myc suppression of miR-23 enhances mitochondrial glutaminase and glutamine metabolism , 2009, Nature.

[25]  Gary Fiskum,et al.  Glutamine metabolism in AS-30D hepatoma cells. Evidence for its conversion into lipids via reductive carboxylation , 1995, Molecular and Cellular Biochemistry.