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.

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

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.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

References

  1. Voet, D. & Voet, J. Biochemistry 3rd edn. (Wiley, 2004).

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

    CAS  Article  Google Scholar 

  3. 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).

    CAS  Article  Google Scholar 

  4. WHO Working Group. Glucose-6-phosphate dehydrogenase deficiency. Bull. World Health Organ. 67, 601–611 (1989).

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  7. Nguyen, P. et al. Liver lipid metabolism. J. Anim. Physiol. Anim. Nutr. (Berl.) 92, 272–283 (2008).

    CAS  Article  Google Scholar 

  8. Young, J.W., Shrago, E. & Lardy, H.A. Metabolic control of enzymes involved in lipogenesis and gluconeogenesis. Biochemistry 3, 1687–1692 (1964).

    CAS  Article  Google Scholar 

  9. Wise, E.M. Jr. & Ball, E.G. Malic enzyme and lipogenesis. Proc. Natl. Acad. Sci. USA 52, 1255–1263 (1964).

    CAS  Article  Google Scholar 

  10. Wise, L.S., Sul, H.S. & Rubin, C.S. Coordinate regulation of the biosynthesis of ATP-citrate lyase and malic enzyme during adipocyte differentiation. Studies on 3T3-L1 cells. J. Biol. Chem. 259, 4827–4832 (1984).

    CAS  PubMed  Google Scholar 

  11. Si, Y., Yoon, J. & Lee, K. Flux profile and modularity analysis of time-dependent metabolic changes of de novo adipocyte formation. Am. J. Physiol. Endocrinol. Metab. 292, E1637–E1646 (2007).

    CAS  Article  Google Scholar 

  12. Katz, J. & Rognstad, R. The metabolism of tritiated glucose by rat adipose tissue. J. Biol. Chem. 241, 3600–3610 (1966).

    CAS  PubMed  Google Scholar 

  13. Kather, H., Rivera, M. & Brand, K. Interrelationship and control of glucose metabolism and lipogenesis in isolated fat cells. Control of pentose phosphate-cycle activity by cellular requirement for reduced nicotinamide adenine dinucleotide phosphate. Biochem. J. 128, 1097–1102 (1972).

    CAS  Article  Google Scholar 

  14. 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 on the metabolism of adipose. J. Biol. Chem. 239, 675–685 (1964).

    CAS  PubMed  Google Scholar 

  15. Green, H. & Meuth, M. An established pre-adipose cell line and its differentiation in culture. Cell 3, 127–133 (1974).

    CAS  Article  Google Scholar 

  16. Rosen, E.D. & MacDougald, O.A. Adipocyte differentiation from the inside out. Nat. Rev. Mol. Cell Biol. 7, 885–896 (2006).

    CAS  Article  Google Scholar 

  17. 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  Google Scholar 

  18. Yang, X.M. & MacKenzie, R.E. NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is the mammalian homolog of the mitochondrial enzyme encoded by the yeast MIS1 gene. Biochemistry 32, 11118–11123 (1993).

    CAS  Article  Google Scholar 

  19. Ochoa, S., Mehler, A.H. & Kornberg, A. Biosynthesis of dicarboxylic acids by carbon dioxide fixation; isolation and properties of an enzyme from pigeon liver catalyzing the reversible oxidative decarboxylation of 1-malic acid. J. Biol. Chem. 174, 979–1000 (1948).

    CAS  PubMed  Google Scholar 

  20. Jitrapakdee, S. et al. Structure, mechanism and regulation of pyruvate carboxylase. Biochem. J. 413, 369–387 (2008).

    CAS  Article  Google Scholar 

  21. Rutter, W.J. & Lardy, H.A. Purification and properties of pigeon liver malic enzyme. J. Biol. Chem. 233, 374–382 (1958).

    CAS  PubMed  Google Scholar 

  22. DeBerardinis, R.J. et al. Beyond aerobic glycolysis: transformed cells can engage in glutamine metabolism that exceeds the requirement for protein and nucleotide synthesis. Proc. Natl. Acad. Sci. USA 104, 19345–19350 (2007).

    CAS  Article  Google Scholar 

  23. Yuan, Z. & Hammes, G.G. Elementary steps in the reaction mechanism of chicken liver fatty acid synthase. pH dependence of NADPH binding and isotope rate effect for beta-ketoacyl reductase. J. Biol. Chem. 259, 6748–6751 (1984).

    CAS  Google Scholar 

  24. 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).

    CAS  Article  Google Scholar 

  25. Si, Y., Shi, H. & Lee, K. Impact of perturbed pyruvate metabolism on adipocyte triglyceride accumulation. Metab. Eng. 11, 382–390 (2009).

    CAS  Article  Google Scholar 

  26. Kim, J.W., Tchernyshyov, I., Semenza, G.L. & Dang, C.V. HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab. 3, 177–185 (2006).

    Article  Google Scholar 

  27. Lu, C.W., Lin, S.C., Chen, K.F., Lai, Y.Y. & Tsai, S.J. Induction of pyruvate dehydrogenase kinase-3 by hypoxia-inducible factor-1 promotes metabolic switch and drug resistance. J. Biol. Chem. 283, 28106–28114 (2008).

    CAS  Article  Google Scholar 

  28. Hosogai, N. et al. Adipose tissue hypoxia in obesity and its impact on adipocytokine dysregulation. Diabetes 56, 901–911 (2007).

    CAS  Article  Google Scholar 

  29. Trayhurn, P. Hypoxia and adipose tissue function and dysfunction in obesity. Physiol. Rev. 93, 1–21 (2013).

    CAS  Article  Google Scholar 

  30. Mullen, A.R. et al. Reductive carboxylation supports growth in tumour cells with defective mitochondria. Nature 481, 385–388 (2012).

    CAS  Article  Google Scholar 

  31. Kamphorst, J.J., Chung, M.K., Fan, J. & Rabinowitz, J.D. Quantitative analysis of acetyl-CoA production in hypoxic cancer cells reveals substantial contribution from acetate. Cancer Metab. 2, 23 (2014).

    Article  Google Scholar 

  32. Price, N.E. & Cook, P.F. Kinetic and chemical mechanisms of the sheep liver 6-phosphogluconate dehydrogenase. Arch. Biochem. Biophys. 336, 215–223 (1996).

    CAS  Article  Google Scholar 

  33. Hermes, J.D., Roeske, C.A., O'Leary, M.H. & Cleland, W.W. Use of multiple isotope effects to determine enzyme mechanisms and intrinsic isotope effects. Malic enzyme and glucose-6-phosphate dehydrogenase. Biochemistry 21, 5106–5114 (1982).

    CAS  Article  Google Scholar 

  34. 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).

    CAS  Article  Google Scholar 

  35. Lee, C.Y., Lee, S.M., Lewis, S. & Johnson, F.M. Identification and biochemical analysis of mouse mutants deficient in cytoplasmic malic enzyme. Biochemistry 19, 5098–5103 (1980).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  37. Wellen, K.E. et al. ATP-citrate lyase links cellular metabolism to histone acetylation. Science 324, 1076–1080 (2009).

    CAS  Article  Google Scholar 

  38. Boucher, A. et al. Biochemical mechanism of lipid-induced impairment of glucose-stimulated insulin secretion and reversal with a malate analogue. J. Biol. Chem. 279, 27263–27271 (2004).

    CAS  Article  Google Scholar 

  39. Munger, J. et al. Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy. Nat. Biotechnol. 26, 1179–1186 (2008).

    CAS  Article  Google Scholar 

  40. Lu, W. et al. Metabolomic analysis via reversed-phase ion-pairing liquid chromatography coupled to a stand alone orbitrap mass spectrometer. Anal. Chem. 82, 3212–3221 (2010).

    CAS  Article  Google Scholar 

  41. Sutterlin, H.A., Zhang, S. & Silhavy, T.J. Accumulation of phosphatidic acid increases vancomycin resistance in Escherichia coli. J. Bacteriol. 196, 3214–3220 (2014).

    Article  Google Scholar 

  42. Edens, W.A., Urbauer, J.L. & Cleland, W.W. Determination of the chemical mechanism of malic enzyme by isotope effects. Biochemistry 36, 1141–1147 (1997).

    CAS  Article  Google Scholar 

  43. Seyama, Y. et al. Identification of sources of hydrogen atoms in fatty acids synthesized using deuterated water and stereospecifically deuterium labelled NADPH by gas chromatographic mass spectrometric analysis. Biomed. Mass Spectrom. 5, 357–361 (1978).

    CAS  Article  Google Scholar 

  44. Wiechert, W., Möllney, M., Isermann, N., Wurzel, M. & de Graaf, A.A. Bidirectional reaction steps in metabolic networks: III. Explicit solution and analysis of isotopomer labeling systems. Biotechnol. Bioeng. 66, 69–85 (1999).

    CAS  Article  Google Scholar 

  45. Weitzel, M. et al. 13CFLUX2—high-performance software suite for 13C metabolic flux analysis. Bioinformatics 29, 143–145 (2013).

    CAS  Article  Google Scholar 

  46. Waltz, R.A., Morales, J.L., Nocedal, J. & Orban, D. An interior algorithm for nonlinear optimization that combines line search and trust region steps. Math. Program. 107, 391–408 (2006).

    Article  Google Scholar 

  47. Antoniewicz, M.R., Kelleher, J.K. & Stephanopoulos, G. Determination of confidence intervals of metabolic fluxes estimated from stable isotope measurements. Metab. Eng. 8, 324–337 (2006).

    CAS  Article  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Joshua D Rabinowitz.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–13 and Supplementary Tables 1 and 2. (PDF 10153 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2047

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing