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:

NRH salvage and conversion to NAD+ requires NRH kinase activity by adenosine kinase

Nature Metabolism volume 2, pages 364–379 (2020)Cite this article

Subjects

Abstract

Dihydronicotinamide riboside (NRH) has been suggested to act as a precursor for the synthesis of NAD+, but the biochemical pathway converting it has been unknown. Here, we show that NRH can be converted into NAD+ via a salvage pathway in which adenosine kinase (ADK, also known as AK) acts as an NRH kinase. Using isotope-labelling approaches, we demonstrate that NRH is fully incorporated into NAD+, with NMNH acting as an intermediate. We further show that AK is enriched in fractions from cell lysates with NRH kinase activity, and that AK can convert NRH into NAD+. In cultured cells and mouse liver, pharmacological or genetic inhibition of AK blocks formation of reduced nicotinamide mononucleotide (NMNH) and inhibits NRH-stimulated NAD+ biosynthesis. Finally, we confirm the presence of endogenous NRH in the liver with metabolomics. Our findings establish NRH as a natural precursor of NAD+ and reveal a new route for NAD+ biosynthesis via an NRH salvage pathway involving AK.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: NRH is converted into NMNH before being made into NAD+.
Fig. 2: Identification of the NRH kinase.
Fig. 3: Characterization of AK as an NRH kinase.
Fig. 4: Pharmacology of AK inhibitors on NRH kinase activity and on NAD+ concentration enhancement.
Fig. 5: HAP1 AK KO cells lack NRH kinase activity.
Fig. 6: NRH is incorporated into NAD+ in a manner dependent on AK activity.
Fig. 7: NMNH is a metabolite of a biosynthetic pathway that produces NADH and NAD+.
Fig. 8: AK inhibitor antagonizes NRH impacts in mouse liver.

Similar content being viewed by others

Data availability

Relevant data generated or analysed during this study are included in this article (and its Supplementary Information files). Unpublished data that support the findings of this study are available from the corresponding author upon request. Source data for Figs. 1, 3, 5 and 8 are presented with the paper.

References

  1. Yang, Y. & Sauve, A. A. NAD+ metabolism: bioenergetics, signaling and manipulation for therapy. Biochim. Biophys. Acta 1864, 1787–1800 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Rajakumar, K. Pellagra in the United States: a historical perspective. South Med. J. 93, 272–277 (2000).

    CAS  PubMed  Google Scholar 

  3. Wilder, R. M. A brief history of the enrichment of flour and bread. JAMA 162, 1539–1541 (1956).

    CAS  Google Scholar 

  4. Kornberg, A. & Lindberg, O. DPN pyrophosphatase. Fed. Proc. 7, 165 (1948).

    CAS  PubMed  Google Scholar 

  5. Kornberg, A. The participation of inorganic pyrophosphate in the reversible enzymatic synthesis of diphosphopyridine nucleotide. J. Biol. Chem. 176, 1475 (1948).

    CAS  PubMed  Google Scholar 

  6. Preiss, J. & Handler, P. Enzymatic synthesis of nicotinamide mononucleotide. J. Biol. Chem. 225, 759–770 (1957).

    CAS  PubMed  Google Scholar 

  7. Preiss, J. & Handler, P. Biosynthesis of diphosphopyridine nucleotide. II. Enzymatic aspects. J. Biol. Chem. 233, 493–500 (1958).

    CAS  PubMed  Google Scholar 

  8. Preiss, J. & Handler, P. Biosynthesis of diphosphopyridine nucleotide. I. Identification of intermediates. J. Biol. Chem. 233, 488–492 (1958)..

  9. Bogan, K. L. & Brenner, C. Nicotinic acid, nicotinamide, and nicotinamide riboside: a molecular evaluation of NAD+ precursor vitamins in human nutrition. Annu. Rev. Nutr. 28, 115–130 (2008).

    CAS  PubMed  Google Scholar 

  10. Sauve, A. A. NAD+ and vitamin B3: from metabolism to therapies. J. Pharmacol. Exp. Ther. 324, 883–893 (2008).

    CAS  PubMed  Google Scholar 

  11. Gholson, R. K., Ueda, I., Ogasawara, N. & Henderson, L. M. The enzymatic conversion of quinolinate to nicotinic acid mononucleotide in mammalian liver. J. Biol. Chem. 239, 1208–1214 (1964).

    CAS  PubMed  Google Scholar 

  12. Nishizuka, Y. & Hayaishi, O. Enzymic synthesis of niacin nucleotides from 3-hydroxyanthranilic acid in mammalian liver. J. Biol. Chem. 238, 483–485 (1963).

    CAS  PubMed  Google Scholar 

  13. Bieganowski, P. & Brenner, C. Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss-Handler independent route to NAD+ in fungi and humans. Cell 117, 495–502 (2004).

    CAS  PubMed  Google Scholar 

  14. Gomes, A. P. et al. Declining NAD+ induces a pseudohypoxic state disrupting nuclear-mitochondrial communication during aging. Cell 155, 1624–1638 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Katsyuba, E. & Auwerx, J. Modulating NAD+ metabolism, from bench to bedside. EMBO J. 36, 2670–2683 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Canto, C. et al. The NAD+ precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity. Cell Metab. 15, 838–847 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Gariani, K. et al. Eliciting the mitochondrial unfolded protein response by nicotinamide adenine dinucleotide repletion reverses fatty liver disease in mice. Hepatology 63, 1190–1204 (2016).

    CAS  PubMed  Google Scholar 

  18. Zhang, H. et al. NAD+ repletion improves mitochondrial and stem cell function and enhances life span in mice. Science 352, 1436–1443 (2016).

    CAS  PubMed  Google Scholar 

  19. Barbosa, M. T. et al. The enzyme CD38 (a NAD glycohydrolase, EC 3.2.2.5) is necessary for the development of diet-induced obesity. FASEB J. 21, 3629–3639 (2007).

    CAS  PubMed  Google Scholar 

  20. Bai, P. et al. PARP-1 inhibition increases mitochondrial metabolism through SIRT1 activation. Cell Metab. 13, 461–468 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Khan, N. A. et al. Effective treatment of mitochondrial myopathy by nicotinamide riboside, a vitamin B3. EMBO Mol. Med. 6, 721–731 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Cerutti, R. et al. NAD+-dependent activation of Sirt1 corrects the phenotype in a mouse model of mitochondrial disease. Cell Metab. 19, 1042–1049 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Gong, B. et al. Nicotinamide riboside restores cognition through an upregulation of proliferator-activated receptor-gamma coactivator 1alpha regulated beta-secretase 1 degradation and mitochondrial gene expression in Alzheimer’s mouse models. Neurobiol. Aging 34, 1581–1588 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Hou, Y. et al. NAD(+) supplementation normalizes key Alzheimer’s features and DNA damage responses in a new AD mouse model with introduced DNA repair deficiency. Proc. Natl Acad. Sci. USA 115, E1876–E1885 (2018).

    CAS  PubMed  Google Scholar 

  25. Sorrentino, V. et al. Enhancing mitochondrial proteostasis reduces amyloid-beta proteotoxicity. Nature 552, 187–193 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Xu, W. et al. Lethal cardiomyopathy in mice lacking transferrin receptor in the heart. Cell Rep. 13, 533–545 (2015).

    PubMed  PubMed Central  Google Scholar 

  27. Zhang, R. et al. Short-term administration of nicotinamide mononucleotide preserves cardiac mitochondrial homeostasis and prevents heart failure. J. Mol. Cell Cardiol. 112, 64–73 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Tummala, K. S. et al. Inhibition of de novo NAD+ synthesis by oncogenic URI causes liver tumorigenesis through DNA damage. Cancer Cell 26, 826–839 (2014).

    CAS  PubMed  Google Scholar 

  29. Das, A. et al. Impairment of an endothelial NAD+-H2S signaling network is a reversible cause of vascular aging. Cell 173, 74–89 e20 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Trammell, S. A. et al. Nicotinamide riboside is uniquely and orally bioavailable in mice and humans. Nat. Commun. 7, 12948 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Dellinger, R. W. et al. Repeat dose NRPT (nicotinamide riboside and pterostilbene) increases NAD+ levels in humans safely and sustainably: a randomized, double-blind, placebo-controlled study. NPJ Aging Mech. Dis. 3, 17 (2017).

    PubMed  PubMed Central  Google Scholar 

  32. Dollerup, O. L. et al. A randomized placebo-controlled clinical trial of nicotinamide riboside in obese men: safety, insulin-sensitivity, and lipid-mobilizing effects. Am. J. Clin. Nutr. 108, 343–353 (2018).

    PubMed  Google Scholar 

  33. Airhart, S. E. et al. An open-label, non-randomized study of the pharmacokinetics of the nutritional supplement nicotinamide riboside (NR) and its effects on blood NAD+ levels in healthy volunteers. PLoS One 12, e0186459 (2017).

    PubMed  PubMed Central  Google Scholar 

  34. Martens, C. R. et al. Chronic nicotinamide riboside supplementation is well-tolerated and elevates NAD+ in healthy middle-aged and older adults. Nat Commun. 9, 1286 (2018).

    PubMed  PubMed Central  Google Scholar 

  35. Yang, Y., Mohammed, F. S., Zhang, N. & Sauve, A. A. Dihydronicotinamide riboside is a potent NAD+ concentration enhancer in vitro and in vivo. J. Biol. Chem. 294, 9295–9307 (2019).

    CAS  PubMed  Google Scholar 

  36. Megarity, C. F. et al. The two common polymorphic forms of human NRH-quinone oxidoreductase 2 (NQO2) have different biochemical properties. FEBS Lett. 588, 1666–1672 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Miller, R. L. et al. Adenosine kinase from rabbit liver. II. Substrate and inhibitor specificity. J. Biol. Chem. 254, 2346–2352 (1979).

    CAS  Google Scholar 

  38. Ugarkar, B. G. et al. Adenosine kinase inhibitors. 1. Synthesis, enzyme inhibition, and antiseizure activity of 5-iodotubercidin analogues. J. Med. Chem. 43, 2883–2893 (2000).

    CAS  PubMed  Google Scholar 

  39. Jarvis, M. F. et al. ABT-702 (4-amino-5-(3-bromophenyl)-7-(6-morpholinopyridin-3-yl)pyrido[2, 3-d]pyrimidine), a novel orally effective adenosine kinase inhibitor with analgesic and anti-inflammatory properties: I. In vitro characterization and acute antinociceptive effects in the mouse. J. Pharmacol. Exp. Ther. 295, 1156–1164 (2000).

    CAS  PubMed  Google Scholar 

  40. Berger, F., Lau, C., Dahlmann, M. & Ziegler, M. Subcellular compartmentation and differential catalytic properties of the three human nicotinamide mononucleotide adenylyltransferase isoforms. J. Biol. Chem. 280, 36334–36341 (2005).

    CAS  PubMed  Google Scholar 

  41. Grozio, A. et al. Slc12a8 is a nicotinamide mononucleotide transporter. Nat. Metab. 1, 47–57 (2019).

    PubMed  PubMed Central  Google Scholar 

  42. Giroud-Gerbetant, J. et al. A reduced form of nicotinamide riboside defines a new path for NAD+ biosynthesis and acts as an orally bioavailable NAD+ precursor. Mol. Metab. 30, 192–202 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Boison, D. & Yegutkin, G. G. Adenosine metabolism: emerging concepts for cancer therapy. Cancer Cell 36, 582–596 (2019).

    CAS  PubMed  Google Scholar 

  44. Yang, T., Chan, N. Y. & Sauve, A. A. Syntheses of nicotinamide riboside and derivatives: effective agents for increasing nicotinamide adenine dinucleotide concentrations in mammalian cells. J. Med. Chem. 50, 6458–6461 (2007).

    CAS  PubMed  Google Scholar 

  45. Li, W. & Sauve, A. A. NAD+ content and its role in mitochondria. Methods Mol. Biol. 1241, 39–48 (2015).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was funded in part by NIH grant GM R01-106072 (A.A.S.) and from NY Spinal Cord Research Board Contract C32098GG (A.A.S.).

Author information

Authors and Affiliations

  1. Department of Pharmacology, Weill Cornell Medical College, New York, NY, USA

    Yue Yang, Ning Zhang, Guoan Zhang & Anthony A. Sauve

Authors
  1. Yue Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ning Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Guoan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anthony A. Sauve
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.Y. and A.A.S. developed the concept and study design; Y.Y. investigation; Y.Y. and A.A.S. writing, original draft; Y.Y. and A.A.S. writing, review and editing; N.Z., Y.Y. and A.A.S., synthesis and isotope methodology; G.Z. and Y.Y., proteomics and metabolomics analyses; A.A.S., obtaining funding.

Corresponding author

Correspondence to Anthony A. Sauve.

Ethics declarations

Competing interests

The authors A.S. and Y.Y. have filed a published PCT patent WO 2017/011788 Al on aspects of NRH activities in conjunction with Cornell University. A.S. is a co-inventor of issued US patent 8,106,184, that is related to methods to produce NR and derivatives. This patent is licensed by Chromadex Inc of Irvine California. A.S. is a consultant and a co-founder of Metro MidAtlantic Biotech and Metro International Biotech. The authors N.Z. and G.Z. declare no competing interests.

Additional information

Peer review information Primary handling editor: Christoph Schmitt.

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

Extended data

Extended Data Fig. 1 Known NAD+ biosynthetic pathways in mammalian cells.

The currently known NAD+ biosynthetic pathways in mammalian cells.

Extended Data Fig. 2 Structures of inhibitors and pathways of NRH metabolism.

a) Structures of AK inhibitors. b) The “NRH salvage pathway”, where NRH is transported across cell membrane, phosphorylated into NMNH by NRH kinase, then adenylated into NADH which is then oxidized to produce NAD+. Additional pathways such as nicotinamide salvage, dependent upon Nampt (FK866 sensitive) and Nrk1 dependent pathways are also depicted in the unshaded part of the Scheme. Modified from Scheme published in reference 35.

Supplementary information

Supplementary Information

Supplementary Figures 1–4 and Table 1

Reporting Summary

Source data

Source Data Fig. 2

Unprocessed silver staining gel

Source Data Fig. 3

Unprocessed Western Blots

Source Data Fig. 5

Unprocessed Western Blots

Source Data Fig. 8

Unprocessed Western Blots and gel

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, Y., Zhang, N., Zhang, G. et al. NRH salvage and conversion to NAD+ requires NRH kinase activity by adenosine kinase. Nat Metab 2, 364–379 (2020). https://doi.org/10.1038/s42255-020-0194-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s42255-020-0194-9

This article is cited by

Search

Advanced 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