Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

NADPH production by the oxidative pentose-phosphate pathway supports folate metabolism

Abstract

NADPH donates high-energy electrons for antioxidant defence and reductive biosynthesis. Cytosolic NADP is recycled to NADPH by the oxidative pentose-phosphate pathway (oxPPP), malic enzyme 1 (ME1) and isocitrate dehydrogenase 1 (IDH1). Here we show that any one of these routes can support cell growth, but the oxPPP is uniquely required to maintain a normal NADPH/NADP ratio, mammalian dihydrofolate reductase (DHFR) activity and folate metabolism. These findings are based on CRISPR deletions of glucose-6-phosphate dehydrogenase (G6PD, the committed oxPPP enzyme), ME1, IDH1 and combinations thereof in HCT116 colon cancer cells. Loss of G6PD results in high NADP, which induces compensatory increases in ME1 and IDH1 flux. But the high NADP inhibits DHFR, resulting in impaired folate-mediated biosynthesis, which is reversed by recombinant expression of Escherichia coli DHFR. Across different cancer cell lines, G6PD deletion produced consistent changes in folate-related metabolites, suggesting a general requirement for the oxPPP to support folate metabolism.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: G6PD is required to maintain cell growth, NADPH/NADP ratio and redox defence.
Fig. 2: CRISPR-based genetic screen identifies no cytosolic NADPH producers beyond oxPPP, ME1 and IDH1.
Fig. 3: Loss of G6PD induces ME1 and IDH1 flux without altering their enzyme activities.
Fig. 4: Fatty acid synthesis is maintained in HCT116 G6PD knockout cells.
Fig. 5: G6PD knockout cells are defective in folate metabolism due to impaired DHFR activity.
Fig. 6: Across cell lines, G6PD knockout consistently causes folate deficiency.

Similar content being viewed by others

Data availability

Source data used to generate Fig. 2a–c and Supplementary Fig. 2b–d are provided as Supplementary Data 1. Uncropped versions of blots are provided in Supplementary Figs. 813. Other data that support the findings of this study are available from the authors upon request. Matlab code used for matrix deconvolution for NADPH redox hydride is provided as Supplementary Note 1. R code and R package used for natural abundance correction are publicly available from GitHub (https://github.com/XiaoyangSu/Isotope-Natural-Abundance-Correction and https://github.com/lparsons/accucor)41.

References

  1. Lewis, C. A. et al. Tracing compartmentalized NADPH metabolism in the cytosol and mitochondria of mammalian cells. Mol. Cell 55, 253–263 (2014).

    Article  CAS  Google Scholar 

  2. Lunt, S. Y. & Heiden, M. G. V. Aerobic glycolysis: meeting the metabolic requirements of cell proliferation. Annu. Rev. Cell Dev. Biol. 27, 441–464 (2011).

    Article  CAS  Google Scholar 

  3. Jiang, P. et al. p53 regulates biosynthesis through direct inactivation of glucose-6-phosphate dehydrogenase. Nat. Cell Biol. 13, 310–316 (2011).

    Article  CAS  Google Scholar 

  4. Jiang, P., Du, W., Mancuso, A., Wellen, K. E. & Yang, X. Reciprocal regulation of p53 and malic enzymes modulates metabolism and senescence. Nature 493, 689–693 (2013).

    Article  CAS  Google Scholar 

  5. Cappellini, M. & Fiorelli, G. Glucose-6-phosphate dehydrogenase deficiency. Lancet 371, 64–74 (2008).

    Article  CAS  Google Scholar 

  6. Howes, R. E. et al. Spatial distribution of G6PD deficiency variants across malaria-endemic regions. Malar. J. 12, 418 (2013).

    Article  Google Scholar 

  7. Al-Dwairi, A., Pabona, J. M. P., Simmen, R. C. M. & Simmen, F. A. Cytosolic malic enzyme 1 (ME1) mediates high fat diet-induced adiposity, endocrine profile, and gastrointestinal tract proliferation-associated biomarkers in male mice. PLoS ONE 7, e46716 (2012).

    Article  CAS  Google Scholar 

  8. Itsumi, M. et al. Idh1 protects murine hepatocytes from endotoxin-induced oxidative stress by regulating the intracellular NADP+/NADPH ratio. Cell Death Differ. 22, 1837–1845 (2015).

    Article  CAS  Google Scholar 

  9. Nicol, C. J., Zielenski, J., Tsui, L.-C. & Wells, P. G. An embryoprotective role for glucose-6-phosphate dehydrogenase in developmental oxidative stress and chemical teratogenesis. FASEB J. 14, 111–127 (2000).

    Article  CAS  Google Scholar 

  10. Buescher, J. M. et al. A roadmap for interpreting 13C metabolite labeling patterns from cells. Curr. Opin. Biotechnol. 34, 189–201 (2015).

    Article  CAS  Google Scholar 

  11. Katz, J. & Rognstad, R. The labeling of pentose phosphate from glucose-14C and estimation of the rates of transaldolase, transketolase, the contribution of the pentose cycle, and ribose phosphate synthesis. Biochemistry 6, 2227–2247 (1967).

    Article  CAS  Google Scholar 

  12. Metallo, C. M., Walther, J. L. & Stephanopoulos, G. Evaluation of 13C isotopic tracers for metabolic flux analysis in mammalian cells. J. Biotechnol. 144, 167–174 (2009).

    Article  CAS  Google Scholar 

  13. Fan, J. et al. Quantitative flux analysis reveals folate-dependent NADPH production. Nature 510, 298–302 (2014).

    Article  CAS  Google Scholar 

  14. Liu, L. et al. Malic enzyme tracers reveal hypoxia-induced switch in adipocyte NADPH pathway usage. Nat. Chem. Biol. 12, 345–352 (2016).

    Article  CAS  Google Scholar 

  15. Zhang, Z., Chen, L., Liu, L., Su, X. & Rabinowitz, J. D. Chemical basis for deuterium labeling of fat and NADPH. J. Am. Chem. Soc. 139, 14368–14371 (2017).

    Article  CAS  Google Scholar 

  16. Shreve, D. S. & Levy, H. R. Kinetic mechanism of glucose-6-phosphate dehydrogenase from the lactating rat mammary gland. Implications for regulation. J. Biol. Chem. 255, 2670–2677 (1980).

    CAS  PubMed  Google Scholar 

  17. Ducker, G. S. & Rabinowitz, J. D. One-carbon metabolism in health and disease. Cell Metab. 25, 27–42 (2017).

    Article  CAS  Google Scholar 

  18. Locasale, J. W. Serine, glycine and one-carbon units: cancer metabolism in full circle. Nat. Rev. Cancer 13, 572–583 (2013).

    Article  CAS  Google Scholar 

  19. Tibbetts, A. S. & Appling, D. R. Compartmentalization of mammalian folate-mediated one-carbon metabolism. Annu. Rev. Nutr. 30, 57–81 (2010).

    Article  CAS  Google Scholar 

  20. Yang, M. & Vousden, K. H. Serine and one-carbon metabolism in cancer. Nat. Rev. Cancer 16, 650–662 (2016).

    Article  CAS  Google Scholar 

  21. Ducker, G. S. et al. Reversal of cytosolic one-carbon flux compensates for loss of the mitochondrial folate pathway. Cell Metab. 23, 1140–1153 (2016).

    Article  CAS  Google Scholar 

  22. Ye, J. et al. Serine catabolism regulates mitochondrial redox control during hypoxia. Cancer Discov. 4, 1406–1417 (2014).

    Article  CAS  Google Scholar 

  23. Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  Google Scholar 

  24. Appleman, J. R. et al. Unusual transient- and steady-state kinetic behavior is predicted by the kinetic scheme operational for recombinant human dihydrofolate reductase. J. Biol. Chem. 265, 2740–2748 (1990).

    CAS  PubMed  Google Scholar 

  25. Nemkov, T. et al. Metabolism of citrate and other carboxylic acids in erythrocytes as a function of oxygen saturation and refrigerated storage. Front. Med. (Lausanne) 4, 175 (2017).

    Article  Google Scholar 

  26. Flatt, J. P. & Ball, E. G. Studies on the metabolism of adipose tissue XV. An evaluation of the major pathways of glucose catabolism as influenced by insulin and epinephrine. J. Biol. Chem. 239, 675–685 (1964).

    CAS  PubMed  Google Scholar 

  27. Koh, H.-J. et al. Cytosolic NADP+-dependent isocitrate dehydrogenase plays a key role in lipid metabolism. J. Biol. Chem. 279, 39968–39974 (2004).

    Article  CAS  Google Scholar 

  28. Jiang, L. et al. Reductive carboxylation supports redox homeostasis during anchorage-independent growth. Nature 532, 255–258 (2016).

    Article  CAS  Google Scholar 

  29. Metallo, C. M. et al. Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia. Nature 481, 380 (2011).

    Article  Google Scholar 

  30. Wise, D. R. et al. Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of α-ketoglutarate to citrate to support cell growth and viability. Proc. Natl Acad. Sci. USA 108, 19611–19616 (2011).

    Article  CAS  Google Scholar 

  31. Liu, C. T. et al. Functional significance of evolving protein sequence in dihydrofolate reductase from bacteria to humans. Proc. Natl Acad. Sci. USA 110, 10159–10164 (2013).

    Article  CAS  Google Scholar 

  32. Kwon, Y. K. et al. A domino effect in antifolate drug action in Escherichia coli. Nat. Chem. Biol. 4, 602–608 (2008).

    Article  CAS  Google Scholar 

  33. Field, M. S., Kamynina, E., Watkins, D., Rosenblatt, D. S. & Stover, P. J. Human mutations in methylenetetrahydrofolate dehydrogenase 1 impair nuclear de novo thymidylate biosynthesis. Proc. Natl Acad. Sci. USA 112, 400–405 (2015).

    Article  CAS  Google Scholar 

  34. Piskounova, E. et al. Oxidative stress inhibits distant metastasis by human melanoma cells. Nature 527, 186–191 (2015).

    Article  CAS  Google Scholar 

  35. Zheng, Y. et al. Mitochondrial one-carbon pathway supports cytosolic folate integrity in cancer cells. Cell 175, 1546–1560.e17 (2018).

    Article  CAS  Google Scholar 

  36. Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).

    Article  CAS  Google Scholar 

  37. Wang, Y.-P. et al. Regulation of G6PD acetylation by SIRT2 and KAT9 modulates NADPH homeostasis and cell survival during oxidative stress. EMBO J. 33, 1304–1320 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Hui, S. et al. Glucose feeds the TCA cycle via circulating lactate. Nature 551, 115–118 (2017).

    Article  Google Scholar 

  39. Melamud, E., Vastag, L. & Rabinowitz, J. D. Metabolomic analysis and visualization engine for LC−MS data. Anal. Chem. 82, 9818–9826 (2010).

    Article  CAS  Google Scholar 

  40. Chen, L., Ducker, G. S., Lu, W., Teng, X. & Rabinowitz, J. D. An LC-MS chemical derivatization method for the measurement of five different one-carbon states of cellular tetrahydrofolate. Anal. Bioanal. Chem. 409, 5955–5964 (2017).

    Article  CAS  Google Scholar 

  41. Su, X., Lu, W. & Rabinowitz, J. D. Metabolite spectral accuracy on orbitraps. Anal. Chem. 89, 5940–5948 (2017).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank G. Ducker, J. Ghergurovich and X. Teng for scientific discussion and help. This work was supported by funding from the US National Institutes of Health (grant nos. R01CA163591, P30CA072720, DP1DK113643, P30DK019525, R01DK107667 and R01DK114103) and Department of Energy (grant nos. DE-SC0018420 and DE-SC0018260).

Author information

Authors and Affiliations

Authors

Contributions

L.C. and J.D.R. came up with the general approach. L.C., Z.Z and H.D.Z. generated and characterized different cell lines used in this studies. L.C., A.H., M.M, Z.A. and J.D.R. designed and performed CRISPR libraries screening. L.C. and Z.Z. designed and performed isotope tracing studies. L.C. performed metabolomics studies. L.C. and J.D.R. wrote the paper with help from all authors.

Corresponding author

Correspondence to Joshua D. Rabinowitz.

Ethics declarations

Competing interests

J.D.R. is a founder of Raze Therapeutics and advisor to L.E.A.F. Pharmaceuticals. J.D.R. is a co-inventor on patent application owned by Princeton University covering diagnostics and therapeutics related to NADPH production by the 10-formyl-THF pathway (US20170000769).

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Supplementary Information

Supplementary Figures 1–13, Supplementary Tables 1–3 and Supplementary Note 1

Reporting Summary

Supplementary Data 1

CRISPR-based genetic screen scores for three comparisons: HCT116 WT versus ΔG6PD cells, ΔG6PD versus ΔG6PD/ΔIDH1 cells and WT versus ΔG6PD/ΔIDH1 cells.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, L., Zhang, Z., Hoshino, A. et al. NADPH production by the oxidative pentose-phosphate pathway supports folate metabolism. Nat Metab 1, 404–415 (2019). https://doi.org/10.1038/s42255-019-0043-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s42255-019-0043-x

This article is cited by

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research