De-Souza, Evandro A

Ferreira, Laís SS

Apollo (Cambridge)

J Physiol 599.10 (2021) pp 2525–2526 2525TheJournalofPhysiologyJOURNAL CLUBOf mice and men: opposingeffects of nicotinamide ribosideon skeletal muscle physiology atrest and during exerciseLaís S. S. Ferreira1,2and Evandro A. De-Souza31Institute of Neuropathology, UniversityMedical Centre Freiburg, Freiburg,Germany2Faculty of Biology, University of Freiburg,Freiburg, Germany3Neurobiology Division, MRC Laboratoryof Molecular Biology, Cambridge, UKEmail: esouza@mrc-lmb.cam.ac.ukEdited by: Scott Powers & Bruno GrassiLinked articles: This Journal Club articlehighlights an article by Stocks et al. Toread this article, visit https://doi.org/10.1113/JP280825.BackgroundPhysical exercise is one of the most efficientand researched strategies available forimproving human health span. As away of sustaining muscle contractionduring exercise, cellular mechanisms driveresources to support ATP production. Thebalance between the oxidized and reducedforms of NAD is important for sustainingenergy-producing metabolic reactions.NAD+ is a coenzyme involved in pleiotropiccellular processes, including the regulationof various metabolic enzymes but alsochromatin remodelling and DNA repair.In rodents, NAD+ levels have been shownto decrease with ageing due to a rise in theactivity of the CD38 NAD glycohydrolaseenzyme driving age-related phenotypes,such as the decay of mitochondrial function(Yoshino et al. 2018). Thus, because of itspossible therapeutic applications, NAD+dietary supplement precursors such asnicotinamide riboside (NR) gained alot of attention (Yoshino et al. 2018).Although the findings are promising, theyare primarily based on studies performedin invertebrates and mammalian models,while a subset of recent studies indicatesthat the effects of NAD supplementation inhumans might be much more subtle thanwhatwas observed in the preclinical studies.For example, dietary NR supplementationfailed to alter mitochondrial physiologyin the skeletal muscle of obese individuals(Dollerup et al. 2020), conflicting with thepositive findings in mice.Nicotinamide riboside supplementation hasno effect on the mitochondrial physiologyof human muscleIn a recent study published in The Journalof Physiology, Stocks and colleaguesinvestigated the effects of 1 g NR per day onskeletal muscle physiology in eight youngmale individuals at rest or during exercise(Stocks et al. 2021). The authors’ hypothesiswas that prior to acute exercise, NRtreatment would increase NAD pools and,subsequently, boost post-exercise signallingresponses. Based on previous researchon the effects of NR on the availability ofhuman skeletal muscle NAD+, a 1-weeksupplementationwithNRwas chosen. Afterthat, participants were subjected to exerciseto exhaustion on a bicycle ergometer. Theparticipants had their physical enduranceevaluated and skeletal muscle biopsies werecollected to determine the consequencesof NR supplementation on post-exercisesignalling responses, including severalmetabolic and mitochondrial parameters.The authors found no changes betweengroups in cardio-respiratory performanceparameters after 1 week of supplementationwith NR compared to the group thatreceived a cellulose placebo. In addition, thelevels of different metabolites commonlymodified during physical exercise, such asplasma non-esterified fatty acid, glucose,glycerol and lactate, were unaffected at restor with exercise.Alterations in mitochondrial physiologyand biogenesis are amongst the remarkableeffects of NR supplementation in pre-clinical studies. Therefore, the authorsinvestigated if NR would be able to promoteany mitochondrial physiological alterationswith biopsies of the skeletal muscle. Neitherexercise nor NR had significant effects onmitochondrial oxygen consumption noron the levels of different mitochondrialproteins. The group also monitoredwhether NR treatment for 7 days couldmodify the levels of sirtuin (SIRT1 andSIRT3) in human skeletal muscle, asthese are highly modulated by NAD+dietary supplements in rodents, as wellas mediators of the beneficial outcomesof these molecules in the rodent models.SIRT1 and SIRT3 activity were accessedby measuring the levels of acetylation oftheir targets (p53Lys382 and MnSODK122,respectively) along with the global levelsof protein acetylation, but none of theseparameters were affected by NR during restor exercise.It is known that physical exerciseinduces a series of signalling eventsin the skeletal muscle, especially innutrient sensing pathways. Individualspresented a significant increase in thephosphorylation levels of AMPKThr172and ACCSer79 after exercise. The samedegree of induction was observed withindividuals treated with NR. There was alsonot observed any differential activation byNR on the mRNA levels of PPARGC1A,which encodes the transcriptional factorperoxisome proliferator-activated receptorγ coactivator 1α, a master regulator ofmitochondrial metabolism. In other words,different from what is observed in rodentstudies, NR does not seem to mimic orboost the effects of physical exercise on theactivation of different nutrient signallingpathways in skeletal muscle.A possibility to explain the absenceof effects of NR would be that thechosen dosage was not sufficient topromote significant alterations in themetabolism of NAD+ in skeletal muscle.To test this possibility, the skeletal muscleNAD-metabolome was evaluated by liquidchromatography–mass spectrometry.Surprisingly, there were no significantdifferences in the levels of NR or NAD+between the groups that might explainwhy the authors were unable to observedifferences in the activity of SIRT1 andSIRT3. One possibility is that the regulationof NAD+ levels and NR recycling is moreseverely controlled in humans and thatother concentrations or strategies will berequired to modify these metabolite levels.Nonetheless, they were able to detect arise in nicotinic acid mononucleotide,Nar, Me2PY and Me4Py levels, stronglyindicating that 1 week of NR treatmentwas sufficient to alter the human skeletalNAD metabolome. In future studies, it willbe interesting to include the measurement© 2021 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society DOI: 10.1113/JP281428This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distributionand reproduction in any medium, provided the original work is properly cited.2526 Journal Club J Physiol 599.10of NADH levels. Although the total levelsof NAD+ are unchanged, one possibilityis that the NAD+/NADH balance couldbe influenced by NR treatment. Finally,the authors measured the transcriptionallevel of some genes of the NAD+ synthesisand salvage pathway. They were able toidentify that physical exercise increases theexpression of the gene for nicotinamideN-methyltransferase (NNMT) – amethyltransferase of nicotinamide – andthat this activation is blunted by treatmentwith NR. Further studies with NR inhumans should validate this finding in othercohorts and investigate the physiologicalrelevance of the differential modulationof the expression of NNMT by NR. Inter-estingly, in rats, the regulation of NNMTactivity during exercise has already beenshown to influence the performance ofanimals during anaerobic exercise (Zhouet al. 2018). Finally, another point to beconsidered is that S-adenosyl methionineis a substrate for the NNMT reaction andit could also be modulated within NRtreatment.Final considerations and future questionsIn concordance with Stocks et al. (2021), acontemporary study using the same dosageof NR for 21 days in aged individualsat rest also did not reveal alterations inmitochondrial physiology (Elhassan et al.2019). Conversely, the NAD+ metabolomewas also found to be augmented in elderlyby NR. However, RNA sequencingof human skeletal muscle detecteddownregulation in genes of glycolytic,tricarboxylic acid and even mitochondrialpathways after NR supplementation, butsurprisingly, no key changes in NAD+metabolism genes were found. It is possiblethat subtle transcriptional changes couldbe found in the study of Stocks et al.(2021) by employing high throughputtechniques. Based on these observations,it will be interesting to understand themechanisms of how nicotinamide ribosidecan modulate the gene expression signatureof human skeletal muscle cells, at least foraged tissues, if it is not affecting NAD+ ormitochondrial physiology. In future studies,one key point to be considered is whetherNR treatment would benefit young healthyindividuals, since they have normal levels ofNAD+. Knowing exercise partially restoresthe health of aged tissues and that NRmight have positive responses in elderly, apromising next approach for a clinical trialwould be to evaluate the combined effectsof exercise and NR supplementation duringageing (Custodero et al. 2020).Finally, this study raises a series ofquestions for the field. For instance, why isNR supplementation not so successful inaltering mitochondrial function in humanskeletal muscle when compared to rodentstudies? Studies with larger and morediverse cohorts and with longer periodsof treatment are required to enlightenthis issue. A promising venue for futureresearch would be to explore the impactof NR on other aspects of physical activity,such as fat mobilization and mitochondrialfunction in other tissues. It is also possiblethat species-specific optimal strategies toincrease NAD+ levels with external sourcesexist. Future studies could investigatethe role of other NAD+ dietary sources,such as dihydronicotinamide riboside,which is successful in boosting NAD+ onrodents through the action of adenosinekinase.ConclusionTaken together, the study of Stocks andcolleagues showed that 1 week of NRsupplementation has no identifiable effectson human skeletal muscle during restor exercise. Nevertheless, further studiesusing other demographic populations andtreatment for longer periods of time arenecessary to establish the real potential ofNR for modifying human physiology.ReferencesCustodero C, Saini SK, Shin MJ, JeonYK, Christou DD, McDermott MM,Leeuwenburgh C, Anton SD & MankowskiRT (2020). Nicotinamide riboside—Amissing piece in the puzzle of exercisetherapy for older adults? Exp Gerontol 137,110972.Dollerup OL, Chubanava S, Agerholm M,Søndergård SD, Altıntaş A, Møller AB, HøyerKF, Ringgaard S, Stødkilde-Jørgensen H,Lavery GG, Barrès R, Larsen S, Prats C,Jessen N & Treebak JT (2020). Nicotinamideriboside does not alter mitochondrialrespiration, content or morphology in skeletalmuscle from obese and insulin-resistant men.J Physiol 598, 731–754.Elhassan YS, Kluckova K, Fletcher RS, SchmidtMS, Garten A, Doig CL, Cartwright DM,Oakey L, Burley CV, Jenkinson N, WilsonM, Lucas SJE, Akerman I, Seabright A, LaiY-C, Tennant DA, Nightingale P, Wallis GA,Manolopoulos KN, Brenner C, Philp A &Lavery GG (2019). Nicotinamide ribosideaugments the aged human skeletal muscleNAD+ metabolome and induces trans-criptomic and anti-inflammatory signatures.Cell Rep 28, 1717–1728.e6.Stocks B, Ashcroft SP, Joanisse S, DansereauLC, Koay YC, Elhassan YS, Lavery GG,Quek L, O’Sullivan JF, Philp AM, WallisGA & Philp A (2021). Nicotinamide ribosidesupplementation does not alter whole-bodyor skeletal muscle metabolic responses to asingle bout of endurance exercise. J Physiol599, 1513–1531.Yoshino J, Baur JA & Imai S (2018). NAD+intermediates: The biology and therapeuticpotential of NMN and NR. Cell Metab 27,513–528.Zhou Q, Huang Z-G, Zhu X-J, Xie Z-H, YaoT-F, Wang Y-H & Li J-H (2018). Effects ofnicotinamide N-methyltransferase (NNMT)inhibition on the aerobic and the anaerobicendurance exercise capacity. Sci Sports 33,e159–e165.Additional informationCompeting interestsNo competing interests declared.Author contributionsBoth authors have read and approved the finalversion of this manuscript and agree to beaccountable for all aspects of the work inensuring that questions related to the accuracy orintegrity of any part of the work are appropriatelyinvestigated and resolved. All persons designatedas authors qualify for authorship, and all thosewho qualify for authorship are listed.FundingL.S.S.F. has received funding from the EuropeanUnion’s Horizon 2020 research and innovationprogramme under the Marie Sklodowska-Curiegrant agreement no. 813294. E.A.deS. hasreceived funding from the European Union’sHorizon 2020 research and innovationprogramme under the Marie Skłodowska-Curiegrant agreement number 894039.Keywordsexercise, human physiology, metabolism,nicotinamide riboside, skeletal muscle© 2021 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society

Of mice and men: opposing effects of nicotinamide riboside on skeletal muscle physiology at rest and during exercise.

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Of mice and men: opposing effects of nicotinamide riboside on skeletal muscle physiology at rest and during exercise.

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