NAD+ precursor increases aerobic performance in mice



Nicotinamide riboside (NR) acts as a potent NAD+ precursor and improves mitochondrial oxidative capacity and mitochondrial biogenesis in several organisms. However, the effects of NR supplementation on aerobic performance remain unclear. Here, we evaluated the effects of NR supplementation on the muscle metabolism and aerobic capacity of sedentary and trained mice.


Male C57BL/6 J mice were supplemented with NR (400 mg/Kg/day) over 5 and 10 weeks. The training protocol consisted of 5 weeks of treadmill aerobic exercise, for 60 min a day, 5 days a week. Bioinformatic and physiological assays were combined with biochemical and molecular assays to evaluate the experimental groups.


NR supplementation by itself did not change the aerobic performance, even though 5 weeks of NR supplementation increased NAD+ levels in the skeletal muscle. However, combining NR supplementation and aerobic training increased the aerobic performance compared to the trained group. This was accompanied by an increased protein content of NMNAT3, the rate-limiting enzyme for NAD + biosynthesis and mitochondrial proteins, including MTCO1 and ATP5a. Interestingly, the transcriptomic analysis using a large panel of isogenic strains of BXD mice confirmed that the Nmnat3 gene in the skeletal muscle is correlated with several mitochondrial markers and with different phenotypes related to physical exercise. Finally, NR supplementation during aerobic training markedly increased the amount of type I fibers in the skeletal muscle.


Taken together, our results indicate that NR may be an interesting strategy to improve mitochondrial metabolism and aerobic capacity.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. 1.

    Saltin B, Henriksson J, Nygaard E, Andersen P, Jansson E (1977) Fiber types and metabolic potentials of skeletal muscles in sedentary man and endurance runners. Ann NY Acad Sci 301:3–29.

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Hawley JA, Hargreaves M, Joyner MJ, Zierath JR (2014) Integrative biology of exercise. Cell 159:738–749

    CAS  Article  Google Scholar 

  3. 3.

    Perry CGR, Lally J, Holloway GP, Heigenhauser GJF, Bonen A, Spriet LL (2010) Repeated transient mRNA bursts precede increases in transcriptional and mitochondrial proteins during training in human skeletal muscle. J Physiol 588:4795–4810.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Calvo JA, Daniels TG, Wang X, Paul A, Lin J, Spiegelman BM, Stevenson SC, Rangwala SM (2008) Muscle-specific expression of PPARgamma coactivator-1alpha improves exercise performance and increases peak oxygen uptake. J Appl Physiol 104:1304–1312.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Pirinen E, Cantó C, Jo YS, Morato L, Zhang H, Menzies KJ, Williams EG, Mouchiroud L, Moullan N, Hagberg C, Li W, Timmers S, Imhof R, Verbeek J, Pujol A, Van Loon B, Viscomi C, Zeviani M, Schrauwen P, Sauve AA, Schoonjans K, Auwerx J (2014) Pharmacological inhibition of poly(ADP-ribose) polymerases improves fitness and mitochondrial function in skeletal muscle. Cell Metab 19:1034–1041.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Cantó C, Houtkooper RH, Pirinen E, Youn DY, Oosterveer MH, Cen Y, Fernandez-Marcos PJ, Yamamoto H, Andreux PA, Cettour-Rose P, Gademann K, Rinsch C, Schoonjans K, Sauve AA, Auwerx J (2012) The NAD + precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity. Cell Metab 15:838–847.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Zhang H, Ryu D, Wu Y, Gariani K, Wang X, Luan P, D’Amico D, Ropelle ER, Lutolf MP, Aebersold R, Schoonjans K, Menzies KJ, Auwerx J (2016) NAD + repletion improves mitochondrial and stem cell function and enhances life span in mice. Science 352:1436–1443.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Cantó C, Gerhart-Hines Z, Feige JN, Lagouge M, Noriega L, Milne JC, Elliott PJ, Puigserver P, Auwerx J (2009) AMPK regulates energy expenditure by modulating NAD + metabolism and SIRT1 activity. Nature 458:1056–1060.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Alberts B, Johnson A, Lewis J et al (2002) Molecular Biology of the Cell. Garland Science, New York

    Google Scholar 

  10. 10.

    Reeves PG, Nielsen FH, Fahey GC (1993) AIN-93 purified diets for laboratory rodents: final report of the american institute of nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 123:1939–1951

    CAS  Article  Google Scholar 

  11. 11.

    Gariani K, Menzies KJ, Ryu D, Wegner CJ, Wang X, Ropelle ER, Moullan N, Zhang H, Perino A, Lemos V, Kim B, Park Y-K, Piersigilli A, Pham TX, Yang Y, Ku CS, Koo SI, Fomitchova A, Cantó C, Schoonjans K, Sauve AA, Lee J-Y, Auwerx J (2016) Eliciting the mitochondrial unfolded protein response by nicotinamide adenine dinucleotide repletion reverses fatty liver disease in mice. Hepatology 63:1190–1204.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Ryu D, Zhang H, Ropelle ER, Sorrentino V, Mázala DAG, Marshall PL, Campbell MD, Ali AS, Knowels GM, Bellemin S, Iyer SR, Wang X, Gariani K, Sauve AA, Cantó C, Conley KE, Walter L, Lovering RM, Chin ER, Jasmin BJ, Marcinek DJ, Menzies KJ, Auwerx J (2016) NAD + repletion improves muscle function in muscular dystrophy and counters global PARylation. Sci Transl Med 8:361ra139.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Ferreira JC, Rolim NP, Bartholomeu JB, Gobatto CA, Kokubun E, Brum PC (2007) Maximal Lactate Steady State in Running Mice: effect of Exercise Training. Clin Exp Pharmacol Physiol 34:760–765.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    da Rocha AL, Pereira BC, Pauli JR, Cintra DE, de Souza CT, Ropelle ER, da Silva AS (2015) Downhill running-based overtraining protocol improves hepatic insulin signaling pathway without concomitant decrease of inflammatory proteins. PLoS One 10:e0140020

    Article  Google Scholar 

  15. 15.

    Walker JM (1994) The bicinchoninic acid (BCA) assay for protein quantitation BT–basic protein and peptide protocols. In: Walker JM (ed) Humana Press. Totowa, NJ, pp 5–8

    Google Scholar 

  16. 16.

    Gilda JE, Gomes AV (2013) Stain free total protein staining is a superior loading control to β-actin for western blots. Anal Biochem.

    Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Fortes MAS, Marzuca-Nassr GN, Vitzel KF, da Justa Pinheiro CH, Newsholme P, Curi R (2016) Housekeeping proteins: how useful are they in skeletal muscle diabetes studies and muscle hypertrophy models? Anal Biochem 504:38–40.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Baptista IL, Leal ML, Artioli GG, Aoki MS, Fiamoncini J, Turri AO, Curi R, Miyabara EH, Moriscot AS (2010) Leucine attenuates skeletal muscle wasting via inhibition of ubiquitin ligases. Muscle Nerve 41:800–808.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Williams EG, Mouchiroud L, Frochaux M, Pandey A, Andreux PA, Deplancke B, Auwerx J (2014) An evolutionarily conserved role for the aryl hydrocarbon receptor in the regulation of movement. PLoS Genet 10:1–9.

    CAS  Article  Google Scholar 

  20. 20.

    Williams EG, Wu Y, Jha P, Dubuis S, Blattmann P, Argmann CA, Houten SM, Amariuta T, Wolski W, Zamboni N, Aebersold R, Auwerx J (2016) Systems proteomics of liver mitochondria function. Science.

    Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

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

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Nikiforov A, Dölle C, Niere M, Ziegler M (2011) Pathways and subcellular compartmentation of NAD biosynthesis in human cells: from entry of extracellular precursors to mitochondrial NAD generation. J Biol Chem 286:21767–21778.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Shoshan-Barmatz V, De Pinto V, Zweckstetter M, Raviv Z, Keinan N, Arbel N (2010) VDAC, a multi-functional mitochondrial protein regulating cell life and death. Mol Aspects Med 31:227–285.

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Memme JM, Oliveira AN, Hood DA (2016) Chronology of UPR activation in skeletal muscle adaptations to chronic contractile activity. Am J Physiol Cell Physiol 310:1024–1036.

    Article  Google Scholar 

  25. 25.

    Cerutti R, Pirinen E, Lamperti C, Marchet S, Sauve AA, Li W, Leoni V, Schon EA, Dantzer F, Auwerx J, Viscomi C, Zeviani M (2014) NAD(+)-dependent activation of Sirt1 corrects the phenotype in a mouse model of mitochondrial disease. Cell Metab 19:1042–1049.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Khan NA, Auranen M, Paetau I, Pirinen E, Euro L, Forsström S, Pasila L, Velagapudi V, Carroll CJ, Auwerx J, Suomalainen A (2014) Effective treatment of mitochondrial myopathy by nicotinamide riboside, a vitamin B3. EMBO Mol Med 6:721–731.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Kourtzidis IA, Stoupas AT, Gioris IS, Veskoukis AS, Margaritelis NV, Tsantarliotou M, Taitzoglou I, Vrabas IS, Paschalis V, Kyparos A, Nikolaidis MG (2016) The NAD(+) precursor nicotinamide riboside decreases exercise performance in rats. J Int Soc Sports Nutr 13:32.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Dolopikou CF, Kourtzidis IA, Margaritelis NV, Vrabas IS, Koidou I, Kyparos A, Theodorou AA, Paschalis V, Nikolaidis MG (2019) Acute nicotinamide riboside supplementation improves redox homeostasis and exercise performance in old individuals: a double-blind cross-over study. Eur J Nutr.

    Article  PubMed  Google Scholar 

  29. 29.

    Lynch NA, Nicklas BJ, Berman DM, Dennis KE, Goldberg AP (2001) Reductions in visceral fat during weight loss and walking are associated with improvements inV˙o 2 max. J Appl Physiol 90:99–104.

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Levin BE, Dunn-Meynell AA (2004) Chronic exercise lowers the defended body weight gain and adiposity in diet-induced obese rats. Am J Physiol Integr Comp Physiol 286:R771–R778.

    CAS  Article  Google Scholar 

  31. 31.

    Thong FSL, Hudson R, Ross R, Janssen I, Graham TE (2000) Plasma leptin in moderately obese men: independent effects of weight loss and aerobic exercise. Am J Physiol Metab 279:E307–E313.

    CAS  Article  Google Scholar 

  32. 32.

    Crisol BM, Veiga CB, Lenhare L, Braga RR, Silva VRR, Adelino SR, Cintra DE, Moura LP, Pauli JR (2018) Nicotinamide riboside induces a thermogenic response in lean mice. Life Sci 211:1–7.

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Yamamoto M, Hikosaka K, Mahmood A, Tobe K, Shojaku H, Inohara H, Nakagawa T (2016) Nmnat3 is dispensable in mitochondrial NAD level maintenance in vivo. PLoS One 11:1–16.

    CAS  Article  Google Scholar 

  34. 34.

    Gulshan M, Yaku K, Okabe K, Mahmood A, Sasaki T, Yamamoto M, Hikosaka K, Usui I, Kitamura T, Tobe K, Nakagawa T (2018) Overexpression of Nmnat3 efficiently increases NAD and NGD levels and ameliorates age-associated insulin resistance. Aging Cell 17:e12798–e12798.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Chalkiadaki A, Igarashi M, Nasamu AS, Knezevic J, Guarente L (2014) Muscle-specific SIRT1 gain-of-function increases slow- twitch fibers and ameliorates pathophysiology in a mouse model of duchenne muscular dystrophy. PLoS Genet 10:1–12.

    CAS  Article  Google Scholar 

  36. 36.

    Egan B, Zierath JR (2013) Exercise metabolism and the molecular regulation of skeletal muscle adaptation. Cell Metab 17:162–184

    CAS  Article  Google Scholar 

Download references


This work was supported by grants from the São Paulo Research Foundation (FAPESP–2016/05499-1, 2016/01089-3, and 2018/07634-9) and was financed in part by the National Council for Scientific and Technological Development (CNPq) (case numbers 304771/2017-1 and 401189/2016-3) and the Coordination for the Improvement of Higher Education Personnel (CAPES)–Brazil–Finance Code 001.

Author information




BMC performed all the experiments and prepared the figures. BMC and CBV performed the diet production, animal experiments, tissue extraction, and Western blotting analyses. RRB performed the diet production, animal experiments, tissue extraction, and sample preparation. BMC and LL performed the Western blotting analyses. ILB and BMC performed the fiber type experiments. BMC, RCG, VRM, and AVC performed the performance test, exercise training, and tissue extraction. BMC performed the NAD+ quantification and mRNA analyses. ERR performed the bioinformatics analysis. LPM, ASRS, DEC, and JRP provided the laboratory support, discussion, and reviewed the manuscript. BMC and ERR wrote the manuscript. ERR was responsible for the financial support.

Corresponding author

Correspondence to Eduardo R. Ropelle.

Ethics declarations

Conflict of interests

There is no conflict of interest involving the authors and the results presented in this study.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 579 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Crisol, B.M., Veiga, C.B., Braga, R.R. et al. NAD+ precursor increases aerobic performance in mice. Eur J Nutr 59, 2427–2437 (2020).

Download citation


  • Nicotinamide riboside
  • Exercise
  • Training
  • Mitochondrial markers
  • Skeletal muscle
  • Fiber type