Quantitative flux analysis reveals folate-dependent NADPH production

A metabolomics quantification of NADPH production and consumption fluxes in proliferating mammalian cells reveals that, in addition to canonical pathways such as the oxidative pentose phosphate pathway, NADPH can also be produced by a folate metabolism pathway, a discovery providing new insights into the metabolism of cell growth. NADPH is a coenzyme that is involved in many redox processes in cells, including lipogenesis, oxidative stress and tumour growth. The most direct route for the production of NADPH from glucose is via the oxidative pentose phosphate pathway. In this manuscript, the authors use various metabolomics methodologies to quantify NADPH production and consumption fluxes in proliferating mammalian cells, and show that NADPH can also be produced when methylene tetrahydrofolate is oxidized to 10-formyl-tetrahydrofolate. This is unexpected — folate metabolism was not previously recognized as an important source of NADPH — and is particularly interesting in light of the importance of serine and glycine, the major carbon sources of this folate-dependent pathway, in cancer growth. 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 biosynthesis1. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important2,3. 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.