title: Quantitative flux analysis reveals folate-dependent NADPH production.
abstract: ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important. Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP(+) to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP(+) and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.
authors.affiliation: 1] Department of Chemistry and Lewis Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08540, USA [2].
authors.forename: Jing
authors.initials: J
authors.lastname: Fan
authors.affiliation: 1] Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA [2].
authors.forename: Jiangbin
authors.initials: J
authors.lastname: Ye
authors.affiliation: Department of Chemistry and Lewis Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08540, USA.
authors.forename: Jurre J
authors.initials: JJ
authors.lastname: Kamphorst
authors.affiliation: 1] Department of Chemistry and Lewis Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08540, USA [2] Department of Computer Science, Technion - Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Tomer
authors.initials: T
authors.lastname: Shlomi
authors.affiliation: Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.
authors.forename: Craig B
authors.initials: CB
authors.lastname: Thompson
authors.affiliation: Department of Chemistry and Lewis Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08540, USA.
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Rabinowitz
mesh_terms: D000818:Animals; D002244:Carbon / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D005492:Folic Acid / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007527:Isoenzymes / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D002955:Leucovorin / Q000031:analogs & derivatives / Q000378:metabolism; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism; D009249:NADP / Q000096:biosynthesis* / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010427:Pentose Phosphate Pathway; D012694:Serine / Q000378:metabolism; D013763:Tetrahydrofolates / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; C010161:10-formyltetrahydropteroylglutamic acid; D012694:Serine; D009249:NADP; D002244:Carbon; D005492:Folic Acid; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D005978:Glutathione; D002955:Leucovorin; D005998:Glycine