Quantitative flux analysis reveals folate-dependent NADPH production

摘要

NADPH是一种辅酶，参与细胞中的很多氧化还原过程，其中包括脂肪生成、氧化应激和肿瘤生长。从葡萄糖生成NADPH的最直接路径是经由氧化性的磷酸戊糖通道。在这篇文章中，作者利用各种不同的代谢组学方法来量化増殖的哺乳动物细胞中的NADPH生成和消耗通量，发现当亚甲基四氢叶酸被氧化为10-甲酰基-四氢叶酸时NADPH也可以生成。这是出乎意料的（叶酸代谢过去并没有被发现是NADPH的一个重要来源），同时鉴于丝氨酸和甘氨酸（这一叶酸依赖性通道的主要碳来源)在癌症生长中的重要性，这一点尤为令人感兴趣。%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 phos-phorylation 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 mi-tochondrial 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 glu-tathione 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.