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Malic enzyme tracers reveal hypoxia-induced switch in adipocyte NADPH pathway usage

Nature Chemical Biology volume 12, pages 345–352 (2016)Cite this article

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Abstract

The critical cellular hydride donor NADPH is produced through various means, including the oxidative pentose phosphate pathway (oxPPP), folate metabolism and malic enzyme. In growing cells, it is efficient to produce NADPH via the oxPPP and folate metabolism, which also make nucleotide precursors. In nonproliferating adipocytes, a metabolic cycle involving malic enzyme holds the potential to make both NADPH and two-carbon units for fat synthesis. Recently developed deuterium (2H) tracer methods have enabled direct measurement of NADPH production by the oxPPP and folate metabolism. Here we enable tracking of NADPH production by malic enzyme with [2,2,3,3-2H]dimethyl-succinate and [4-2H]glucose. Using these tracers, we show that most NADPH in differentiating 3T3-L1 mouse adipocytes is made by malic enzyme. The associated metabolic cycle is disrupted by hypoxia, which switches the main adipocyte NADPH source to the oxPPP. Thus, 2H-labeled tracers enable dissection of NADPH production routes across cell types and environmental conditions.

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Figure 1: NADPH consumption during adipogenesis.
Figure 2: NADPH production by the oxPPP.
Figure 3: NADPH production by folate metabolism.
Figure 4: Carbon flux through malic enzyme.
Figure 5: Tracing hydride flux through malic enzyme.
Figure 6: Hypoxia increases adipocyte NADPH production by the oxPPP and blocks that by malic enzyme.

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Acknowledgements

We thank C. Thompson and M. Birnbaum for helpful discussions. This work was supported by US National Institutes of Health grants R01CA163591 (J.D.R.), R01AI097382 (J.D.R.) and P30DK019525 (to the University of Pennsylvania Diabetes Research Center). J.F. was supported by a Howard Hughes Medical Institute fellowship. K.E.W. is supported by American Diabetes Association grant 7-12-JF-59. S.S. is supported by postdoctoral fellowship 5T32CA009140-40.

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Authors and Affiliations

  1. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey, USA

    Ling Liu, Jing Fan, Junyoung O Park & Joshua D Rabinowitz

  2. Department of Chemistry, Princeton University, Princeton, New Jersey, USA

    Ling Liu, Jing Fan & Joshua D Rabinowitz

  3. Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, USA

    Junyoung O Park

  4. Department of Cancer Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Supriya Shah & Kathryn E Wellen

  5. Diabetes Research Center, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Ling Liu, Supriya Shah, Kathryn E Wellen & Joshua D Rabinowitz

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Contributions

J.D.R., K.E.W., L.L. and J.F. conceived the project. L.L. performed and analyzed most experiments. L.L., J.O.P., J.F. and J.D.R. conducted the flux analysis. S.S. performed electroporation experiments and analyzed the data. J.D.R. and L.L. wrote the manuscript.

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Correspondence to Joshua D Rabinowitz.

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Liu, L., Shah, S., Fan, J. et al. Malic enzyme tracers reveal hypoxia-induced switch in adipocyte NADPH pathway usage. Nat Chem Biol 12, 345–352 (2016). https://doi.org/10.1038/nchembio.2047

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