ARTICLE
`
`Received 30 Jan 2016 | Accepted 12 Aug 2016 | Published 10 Oct 2016
`
`OPEN
`Nicotinamide riboside is uniquely and orally
`bioavailable in mice and humans
`Samuel A.J. Trammell1, Mark S. Schmidt1, Benjamin J. Weidemann1, Philip Redpath2, Frank Jaksch3,
`Ryan W. Dellinger3, Zhonggang Li4, E Dale Abel1,4, Marie E. Migaud1,2 & Charles Brenner1,4
`
`DOI: 10.1038/ncomms12948
`
`Nicotinamide riboside (NR) is in wide use as an NADþ precursor vitamin. Here we determine
`the time and dose-dependent effects of NR on blood NADþ metabolism in humans. We
`report that human blood NADþ can rise as much as 2.7-fold with a single oral dose of NR in
`a pilot study of one individual, and that oral NR elevates mouse hepatic NADþ with distinct
`and superior pharmacokinetics to those of nicotinic acid and nicotinamide. We further show
`that single doses of 100, 300 and 1,000 mg of NR produce dose-dependent increases in the
`blood NADþ metabolome in the first clinical trial of NR pharmacokinetics in humans. We
`also report that nicotinic acid adenine dinucleotide (NAAD), which was not thought to be en
`route for the conversion of NR to NADþ , is formed from NR and discover that the rise in
`NAAD is a highly sensitive biomarker of effective NADþ repletion.
`
`1 Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA. 2 John King Laboratory, School of Pharmacy,
`Queens University Belfast, Belfast BT7 1NN, UK. 3 ChromaDex, Inc., 10005 Muirlands Blvd, Suite G, Irvine, California 92618, USA. 4 Department of Internal
`Medicine, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA. Correspondence and requests for materials should be addressed to
`C.B. (email: charles-brenner@uiowa.edu).
`
`NATURE COMMUNICATIONS | 7:12948 | DOI: 10.1038/ncomms12948 | www.nature.com/naturecommunications
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`Nicotinamide adenine dinucleotide (NADþ ), the central
`
`redox coenzyme in cellular metabolism1,2 functions as a
`hydride
`group
`acceptor,
`forming NADH with
`concomitant
`oxidation
`of metabolites
`derived
`from
`carbohydrates, amino acids and fats. The NADþ /NADH ratio
`controls the degree to which such reactions proceed in oxidative
`versus reductive directions. Whereas fuel oxidation reactions
`require NADþ as a hydride acceptor, gluconeogenesis, oxidative
`phosphorylation, ketogenesis, detoxification of reactive oxygen
`species (ROS) and lipogenesis require reduced co-factors, NADH
`and NADPH, as hydride donors (Fig. 1). In addition to its role as
`a coenzyme, NADþ is the consumed substrate of enzymes such
`as poly-ADPribose polymerases (PARPs), sirtuins and cyclic
`ADPribose synthetases1. In redox reactions,
`the biosynthetic
`structures of NADþ , NADH, NADPþ and NADPH are
`preserved. In contrast, PARP3, sirtuin4 and cyclic ADPribose
`synthetase5
`activities hydrolyze
`the
`linkage between the
`nicotinamide (Nam) and the ADPribosyl moieties of NADþ to
`signal DNA damage, alter gene expression, control post-
`translational modifications and regulate calcium signalling.
`In animals, NADþ -consuming activities and cell division
`necessitate ongoing NADþ synthesis, either through a de novo
`pathway that originates with tryptophan or via salvage pathways
`from three NAD þ precursor vitamins, Nam, nicotinic acid (NA)
`and nicotinamide riboside (NR)2. Dietary NADþ precursors,
`which include tryptophan and the three vitamins, prevent
`in milk6,7,
`pellagra. Though NR is present
`the
`cellular
`concentrations of NADþ , NADH, NADPþ and NADPH are
`much higher than those of other NADþ metabolites8,9, such that
`dietary NADþ precursor vitamins are largely derived from
`enzymatic breakdown of NADþ . Thus, although milk is a source
`of NR6,7, the more abundant sources of NR, Nam and NA are
`unprocessed foods, in which plant and animal cellular NADþ
`metabolites are broken down to these compounds. Human
`digestion and the microbiome10 play roles in the provision of
`these vitamins in ways that are not
`fully characterized. In
`addition, the conventional NADþ precursor vitamins, NA and
`Nam, have long been supplemented into human and animal diets
`to prevent pellagra and promote growth11,12. Though NR has
`been available as a GMP-produced supplement since 2013 and
`animal safety assessment indicates that it is as nontoxic as Nam13,
`no human testing has been reported.
`Different tissues maintain NAD þ levels through reliance on
`different biosynthetic routes and precursors14,15 (Fig. 1). Because
`NADþ -consuming activities frequently occur as a function of
`cellular stresses3 and produce Nam, the ability of a cell to salvage
`productive NADþ synthesis
`Nam into
`through Nam
`phosphoribosyltransferase (NAMPT) activity versus methylation
`of Nam to N-methylnicotinamide (MeNam)
`regulates
`the
`NADþ -dependent
`NADþ
`processes16.
`efficiency
`of
`biosynthetic genes are also under circadian control17,18. Both
`NAMPT expression and NADþ levels decline in a number of
`tissues as a function of aging and overnutrition19–22.
`High-dose NA but not high-dose Nam is prescribed to treat
`and prevent dyslipidemias, although its use is limited by painful
`flushing23,24. Whereas it takes only B15 mg per day of NA or
`Nam to prevent pellagra, pharmacological doses of NA can be as
`high as 2–4 g. Despite the 4100-fold difference in effective dose
`between pellagra prevention and dyslipidemia treatment, we
`proposed that the beneficial effects of NA on plasma lipids might
`simply depend on function of NA as an NADþ boosting
`compound1. According to this view, sirtuin activation would
`likely be part of the mechanism because Nam is an NADþ
`precursor in most cells14,15 but inhibits sirtuins at high doses25.
`On the basis of the ability of NR to elevate NADþ synthesis,
`increase sirtuin activity and extend lifespan in yeast6,26, NR has
`
`NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12948
`
`been employed in mice to elevate NADþ metabolism and
`improve health in models of metabolic stress. Notably, NR allows
`mice to resist weight gain on high-fat diet27, prevent noise-
`induced hearing loss28 and maintain the regenerative potential of
`stem cells in aging mice, providing a longevity advantage29. In
`addition, the hepatic NADþ metabolome has been interrogated
`as a function of prediabetic and type 2 diabetic mouse models.
`The data indicate that levels of liver NADPþ and NADPH,
`which are required for resistance to ROS, are severely challenged
`by diet-induced obesity, and that diabetes and the NADþ
`metabolome
`can be
`partially
`controlled while
`diabetic
`neuropathy can be blocked by oral NR30.
`Data indicate that NR is a mitochondrially favoured NADþ
`precursor31 and in vivo activities of NR have been interpreted as
`depending on mitochondrial sirtuin activities27,28, though not to
`targets32,33.
`the
`exclusion
`of
`nucleocytosolic
`Similarly,
`nicotinamide mononucleotide (NMN), the phosphorylated form
`of NR, has been used to treat declining NADþ in mouse models
`of overnutrition and aging19,20. Beneficial effects of NMN have
`been shown to depend on SIRT120. However, because of the
`abundance of NADþ -dependent processes, the effects of NR and
`NMN may depend on multiple targets including sirtuins, PARP
`family members, cADPribose synthetases, NADþ -dependent
`oxidoreductases and NADPH-dependent ROS detoxification
`enzymes30.
`it is necessary to
`To translate NR technologies to people,
`determine NR oral availability and utilization in different tissues.
`Here we began with targeted quantitative NADþ metabolomics
`of blood and urine in a pilot experiment in which a healthy 52-
`year-old man took 1,000 mg of NR daily for 7 days. These data
`indicate that blood cellular NADþ rose 2.7-fold after one dose of
`NR and that NA adenine dinucleotide (NAAD) unexpectedly
`increased 45-fold. We then performed a detailed analysis of 128
`mice comparing oral NR, Nam and NA in a manner that
`eliminated the possibility of circadian artefacts. These data
`indicate that NR boosts hepatic NADþ and NADþ - consuming
`activities
`to a greater degree than Nam or NA. Further
`experiments clarified that NR is a direct precursor of NAAD
`and that NAAD sensitively reports on increased NADþ
`metabolism in mouse liver and heart. Finally, we performed a
`clinical study with 12 healthy human subjects at three single doses
`of NR. We demonstrated that NR supplementation safely
`increases NADþ metabolism at all doses and validated elevated
`NAAD as an unexpected, sensitive biomarker of boosting
`NADþ . The unique oral bioavailability of NR in mice and
`people and methods established herein enable clinical translation
`of NR to improve wellness and treat human diseases.
`
`Results
`Oral NR increases human blood NADþ with elevation of NAAD.
`GMP-synthesized NR showed no activity as a mutagen or toxin13.
`Despite use as an over-the-counter supplement, no data addressing
`human availability were
`available. A healthy 52-year-old
`male (65 kg) contributed blood and urine before seven days of
`orally self-administered NR (1,000 mg per morning). Blood was
`taken an additional nine times during the first day and at 24 h after
`the first and last dose. Blood was separated into peripheral blood
`mononuclear cells (PBMC) and plasma before quantitative NADþ
`metabolomics by liquid chromatography (LC)-mass spectrometry
`(MS)9, which was expanded to quantify methylated and oxidized
`metabolites of Nam30. As shown in Supplementary Table 1 and
`Fig. 2, the PBMC NADþ metabolome was unaffected by NR for
`the first 2.7 h. In six measurements from time zero through 2.7 h,
`NADþ had a mean concentration of 18.5 mM; while Nam had a
`mean concentration of 4.1 mM and the methylated and oxidized
`
`2
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`Nam metabolite, N-methyl-2-pyridone-5-carboxamide (Me2PY),
`had a mean concentration of 2.6 mM. However, at 4.1 h post
`ingestion, PBMC NADþ and Me2PY increased by factors of 2.3
`and 4.2, respectively.
`In yeast, deletion of NR kinase 1 (NRK1) does not eliminate
`utilization of NR26. As
`shown in Fig. 1, NR can be
`phosphorylyzed to Nam by purine nucleoside phosphorylase
`to NADþ synthesis
`and still
`contribute
`through Nam
`salvage26,34. However, as shown in Fig. 2, Nam concentration
`in the human subject’s PBMCs merely fluctuated in a range of 2.6
`to 7.1 mM throughout all 11 observations. The 4.2-fold increase in
`Me2PY concentration at
`the 4.1 h time point suggests that
`increased cellular NADþ accumulation is accompanied by
`increased NADþ -consuming activities
`linked to increased
`methylation and oxidation of the Nam product.
`In the subject’s PBMCs at 7.7 and 8.1 h post ingestion, NADþ
`and Me2PY peaked, increasing above baseline concentrations by
`2.7-fold and 8.4-fold, respectively. At these times, unexpectedly,
`glutamine-dependent NADþ
`NAAD,
`the
`substrate
`of
`
`synthetase35,36, which is only expected to be produced in
`biosynthesis of NADþ from tryptophan and NA2, was elevated
`from less than 20 nM to as high as 0.91 mM. Whereas NAAD
`lagged the rise in PBMC NADþ by one time point, the relative
`rise in PBMC NADþ was not as pronounced as the spike in
`NAAD, which was at least 45-fold above the baseline level.
`Although contrary to expectations, these data suggested that NR
`might be incorporated into NAAD after formation of NADþ and
`chased back to the NADþ peak as NADþ declines.
`Complete NADþ metabolomic data from the human subject’s
`PBMCs, plasma and urine are provided in Figs 2–4 and
`Supplementary Tables 1–3. These data show that all of the
`phosphorylated compounds—NAMN, NAAD, NADþ , NADPþ ,
`NMN and ADPR—are found exclusively in blood cells and are
`not found in plasma or urine. Notably, the peak of NADPþ ,
`which represents cellular NADPþ plus NADPH oxidized in
`extraction, and the peak of ADPR, which signals an increase in
`NADþ -consuming activities, co-occur with peak NADþ . Using
`methods optimized for recovery of nucleotides, NR was not
`
`O
`
`NH
`2
`
`N+
`
`O
`
`OH
`OH
`NMN
`
`O
`
`PO
`
`O–
`
`O–
`
`NADK
`NADK2
`
`H
`
`O
`
`NH
`2
`
`N
`
`2’P-ADPR
`
`NADH
`
`NMNAT1-3
`
`H
`
`O
`
`NH
`2
`
`N
`
`2’P-ADPR
`
`NADPH
`
`O
`
`NH
`2
`
`NADK
`NADK2
`
`O
`
`NH
`2
`
`N+
`
`O
`
`O
`
`OH OH
`
`NH
`2
`
`O
`
`P
`
`O
`
`N+
`
`2’P-ADPR
`
`NADP+
`
`O
`
`O–
`
`ADP
`
`H
`
`+
`N
`
`O
`
`NADSYN1
`
`O–
`
`OH
`
`OH
`
`Tryptophan
`
`De
`novo
`O
`
`O–
`
`NMNAT1-3
`
`+
`N
`
`O
`
`OH OH
`NAMN
`
`O
`
`PO
`
`O–
`
`O–
`
`NAAD
`
`N
`
`N
`
`O
`
`N
`
`N
`
`O–
`
`O
`
`P
`
`O
`
`NAMPT
`
`NRK1,2
`
`O
`
`NH2
`
`N+
`
`O
`
`HO
`
`OH OH
`NR
`
`OH OH
`
`NAD+
`
`NAD+
`consuming enzymes
`
`O
`
`NH
`2
`
`PNP
`
`N
`
`Nam
`
`+
`
`NNMT
`
`ADPR
`products
`
`Mammalian enzymatic step
`
`Bacterial enzymatic step
`
`O
`
`NH
`2
`
`N+
`
`CH
`3
`MeNam
`
`pncA
`
`O
`
`NH
`2
`
`O
`
`N
`
`CH
`3
`
`Me2PY
`
`O
`
`O
`
`AOX1
`
`NH2
`
`N
`
`NRK1,2
`
`NAPRT
`
`O
`
`O–
`
`PNP
`
`O
`
`O–
`
`N
`
`NA
`
`N+
`
`O
`
`HO
`
`OH OH
`NAR
`
`CH
`3
`Me4PY
`Figure 1 | The NADþ metabolome. NADþ is synthesized by salvage of the vitamin precursors, NA, Nam and NR, or from tryptophan in the de novo
`pathway. NADþ can be reduced to NADH, phosphorylated to NADPþ or consumed to Nam. Nam can also be methylated and oxidized to waste products.
`NAAD was not thought to be a precursor of NADþ from NR.
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`NATURE COMMUNICATIONS | 7:12948 | DOI: 10.1038/ncomms12948 | www.nature.com/naturecommunications
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`NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12948
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`0 5 10 15 20 25
`Time (h)
`
`150
`
`200
`
`0 5 10 15 20 25
`Time (h)
`
`150
`
`200
`
`01234
`
`02468
`
`150
`
`200
`
`012345
`
`a
`
`NMN (µM)
`
`d
`
`Nam (µM)
`
`g
`
`Me4PY (µM)
`
`0 5 10 15 20 25
`0 5 1015 20 25
`0 5 10 15 20 25
`Time (h)
`Time (h)
`Time (h)
`Figure 2 | Elevation of the PBMC NADþ metabolome by oral NR in a 52 year-old male. A healthy 52-year-old male ingested 1,000 mg NC Cl daily for 1
`week. PBMCs were prepared from blood collected before the first dose (0 h), at seven time points after the first dose (0.6, 1, 1.4, 2.7, 4.1, 7.7 and 8.1 h),
`before second dose (23.8 h) and 24 h after the seventh dose (167.6 h). Concentrations (a, NMN; b, NADþ ; c, NADPþ ; d, Nam; e, MeNam; f, Me2PY;
`g, Me4PY; h, NAAD; i, ADPR) are with respect to whole blood volumes. The data indicate that the NADþ metabolome—with the exception of Nam and
`NAAD—is elevated by the 4.1 time point post ingestion. Whereas PBMC Nam was never elevated by NR, NAAD was elevated by the next time point.
`NADþ and NAAD remained elevated 24 h after the last dose.
`
`0 5 10 15 20 25
`Time (h)
`
`150
`
`200
`
`0 5 10 15 20 25
`Time (h)
`
`150
`
`200
`
`40
`
`30
`
`20
`
`10
`
`0
`
`25
`20
`15
`10
`
`05
`
`c
`
`NADP+ (µM)
`
`f
`
`Me2PY (µM)
`
`150 200
`
`01234
`
`i
`
`ADPR (µM)
`
`5 10 15 20 25
`Time (h)
`
`150
`
`200
`
`60
`
`40
`
`20
`
`b
`
`NAD+ (µM)
`
`0
`
`0
`
`0 5 10 15 20 25
`Time (h)
`
`150
`
`200
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`0.0
`
`e
`
`MeNam (µM)
`
`1.0
`0.8
`0.6
`0.4
`0.2
`0.0
`
`h
`
`NAAD (µM)
`
`150
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`200
`
`recovered. As shown in Figs 3 and 4, the major time-dependent
`waste metabolite in plasma and urine was Me2PY, which rose
`B10-fold from pre-dose to time points after NADþ peaked in
`PBMCs.
`
`NR is the superior hepatic NADþ precursor vitamin. On the
`basis of known NADþ biosynthetic pathways31, it was difficult to
`understand how levels of NAAD rose in human PBMCs after an
`oral dose of NR. Though NR did not elevate Nam in blood
`samples at any time during the n¼ 1 experiment, it remained
`possible that NR was partially converted to Nam before salvage
`synthesis to NADþ . Such conversion to Nam might allow
`bacterial hydrolysis of Nam to NA by pncA gene products—
`potentially in the gut10—and subsequent conversion to NADþ
`through an NAAD intermediate. NAAD was reported in mouse
`liver when 500 mg kg 1 of
`radioactive Nam was
`injected
`intraperitoneally (IP) into the body cavity of mice37. However,
`NAAD was observed in kidneys, ovaries, lung, heart and brain in
`addition to liver in mice IP-injected with 500 mg kg 1 of NA but
`not Nam38. Moreover, careful analysis of mouse liver perfused
`with radioactive NA and Nam indicated that NAAD is produced
`from NA but not Nam at physiological concentrations39. To our
`knowledge,
`formation of blood or tissue NAAD from oral
`administration of Nam or NR has never been observed.
`Although some mouse experiments have been done with IP
`administered NR at dosages of 1,000 mg kg 1 twice per day28,
`NR is active as an oral agent at a daily dose of 400 mg kg 1 by
`supplementation into food27,29,30 and demonstrated potent
`NADþ boosting activity in the n¼ 1 human experiment at
`
`15 mg kg 1 (Fig. 2). On the basis of weight/surface area, the
`conversion between human adult dose and mouse dose is a factor
`of 12.3 (ref. 40), suggesting that mice should be administered
`185 mg kg 1 to achieve comparable levels of supplementation
`with the human pilot experiment. We therefore designed a
`reverse translational experiment in which mice were administered
`185 mg kg 1 of NR or the mole equivalent doses of Nam and NA
`by oral gavage. To ascertain the timecourse by which these
`vitamins boost the hepatic NADþ metabolome without the
`complication of circadian oscillation of NADþ metabolism17,18,
`we euthanized all mice at B2 pm. Thus, gavage was performed at
`0.25, 1, 2, 4, 6, 8 and 12 h before tissue harvest. To stop
`metabolism synchronously, mouse livers were harvested by
`freeze-clamping. As shown in Fig. 5, we additionally performed
`saline gavages at all
`time points and euthanized mice for
`quantitative NADþ metabolomic analysis to ensure that animal
`handling does not alter levels of NADþ metabolites. The flat
`timecourses of
`saline
`gavages
`established methodological
`levels of hepatic NADþ metabolites
`soundness. Baseline
`(pmol mg 1) at 2 pm were 1,000±35 for NADþ , 230±29 for
`Nam, 210±20 for NADPþ , 66±13 for ADPR and o15 for all
`other NADþ metabolites. Hepatic levels of NA, NAR, NAMN,
`NAAD have baselines of o4. As a point of orientation to
`quantitative metabolomics in tissue samples, 1,000 pmol mg 1 is
`B1 mM, 200 is B200 mM and 10 is B10 mM.
`Targeted NADþ metabolomics8,9
`simultaneous
`allows
`assessment of
`functionally important metabolites
`such as
`NADþ and NADPþ along with metabolites that could serve
`as biomarkers of biosynthetic processes, such as NA, NAR,
`NAMN, NR, NMN and NAAD. In addition, quantification of
`
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`2.5
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`
`b
`
`MeNam (µM)
`
`0
`
`5 10 15 20 25
`Time (h)
`
`150
`
`200
`
`0 5 10 15 20 25
`Time (h)
`
`150
`
`200
`
`012345
`
`d
`
`Me4PY (µM)
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`2.0
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`1.5
`
`1.0
`
`0.5
`
`0.0
`
`20
`
`15
`
`10
`
`05
`
`a
`
`Nam (µM)
`
`c
`
`Me2PY (µM)
`
`0
`
`150
`
`200
`
`5 10 15 20 25
`0 5 10 15 20 25
`Time (h)
`Time (h)
`Figure 3 | Elevation of the plasma NADþ metabolome by oral NR in a 52-year-old male. A healthy 52-year-old male ingested 1,000 mg NC Cl daily for 1
`week. Plasma samples were prepared from blood collected before the first dose (0 h), at seven time points after the first dose (0.6, 1, 1.4, 2.7, 4.1, 7.7 and 8.1 h),
`before second dose (23.8 h) and 24 h after the seventh dose (167.6 h). Concentrations (a, Nam; b, MeNam; c, Me2PY; d, Me4PY) are with respect to whole blood
`volumes. The data indicate that plasma MeNam, Me4PY and Me2PY are strongly elevated by oral NR. No phosphorylated species were found in plasma.
`
`150
`
`200
`
`increases in ADPR, Nam, MeNam, Me2PY and N-methyl-4-
`pyridone-5-carboxamide (Me4PY) on a common absolute scale
`with NADþ permits assessment of increased NADþ -consuming
`with NADþ
`activities
`associated
`precursor
`vitamin
`supplementation.
`Hepatic concentrations of 13 NADþ metabolites were
`quantified in three to four mice at seven time points after gavage
`of saline and each vitamin. In addition, on each experimental day,
`three mice were gavaged with saline and euthanized to serve as
`time zero samples. Each vitamin produced a temporally distinct
`pattern of hepatic NADþ metabolites. Consistent with rapid
`phosphorylation of NR and NAR by NR kinases41, the only
`NADþ metabolites that do not produce hepatic peaks as a
`function of gavage of NADþ precursor vitamins are NR and
`NAR (Supplementary Fig. 1a,b). The accumulation curves of
`some metabolites as a function of each vitamin are strikingly
`similar. For example, the accumulation of NMN (Fig. 5a) is nearly
`identical to that of NADþ (Fig. 5b) and NADPþ (Fig. 5c),
`though at a scale of B1:400:40, respectively. In addition, the
`accumulation of Me4PY (Fig. 5f) is nearly identical to that of
`Me2PY (Supplementary Fig. 1c).
`As shown in Fig. 5b, NA produced the least increase in hepatic
`NADþ and also was 4–6 h faster than NR and Nam in kinetics of
`hepatic NADþ accumulation. When NA was provided by oral
`liver NA peaked (340±30 pmol mg 1)
`gavage,
`in 15 min
`(Fig. 5g). Hepatic NA appearance was followed by an expected
`peak of 220±29 NAAD at 1 h post gavage (Fig. 5i) and a rise in
`hepatic NADþ from 990±25 baseline to 2,200±150 at 2 h
`(Fig. 5b). Hepatic NADPþ due to NA (Fig. 5c) rose in parallel to
`that of hepatic NADþ . In the hours after gavage of NA, as
`hepatic NADþ and NADPþ fell, there was clear evidence of
`enhanced NADþ -consuming activities with significant rises in
`ADPR (Fig. 5j), Nam (Fig. 5d), MeNam (Fig. 5e), Me2PY
`(Supplementary Fig. 1c) and Me4PY (Fig. 5f). Thus, oral
`administration of NA doubled hepatic NADþ from B1 to
`B2 mM through expected intermediates and produced an
`increase in NADþ consumption and methylated products,
`MeNam, Me2PY and Me4PY. Net conversion by the liver of
`
`NA to Nam has been documented for decades30,37,38. Essentially,
`the liver transiently elevates NADþ biosynthetic capacity so long
`as NA is available while increasing NADþ -consuming activities,
`thereby making Nam available to other tissues. Expression of
`hepatic NNMT results in net production of MeNam from NADþ
`precursors, which stabilizes SIRT1 protein in liver and is
`associated with better lipid parameters in mice and some
`human populations42,43.
`As
`shown in Fig. 5g and consistent with radioactive
`experiments39, oral Nam was not used by the liver as NA
`because it did not produce a peak of NA at any time after gavage.
`Though there was a clear increase in hepatic NADþ 2 h after
`Nam gavage, the Nam gavage drove increased hepatic NADþ
`accumulation from 2 to 8 h with a peak at 8 h (Fig. 5b). Nam
`gavage produced two peaks of Nam in the liver (Fig. 5d), the first
`at 15 min, consistent with simple transport of Nam to liver. The
`second broad peak was coincident with elevation of NADþ and
`NADPþ (Fig. 5c,d) and elevation of the NAD þ -consuming
`metabolomic signature of ADPR (Fig. 5j), MeNam, Me4PY and
`Me2PY (Fig. 5e,f and Supplementary Fig. 1c).
`Of the metabolites associated with NADþ -consuming activ-
`ities, ADPR is the only one that must be formed from NADþ
`because Nam, MeNam and the oxidized forms of MeNam could
`appear in liver from the gavaged Nam without conversion to
`NADþ .
`three NADþ precursor vitamins
`Interestingly, of
`provided in bolus at equivalent oral doses, Nam provided the
`least increase in ADPR (Fig. 5j). Whereas the area under the
`curve (AUC) of the Nam-driven rise in hepatic NADþ indicated
`a B50% advantage of Nam over NA (Fig. 5b), there was a 450%
`deficit in Nam-driven ADPR accumulation versus NA (Fig. 5j).
`This is consistent with the idea that high-dose NA, though not an
`ideal hepatic NADþ precursor, is effective as a cholesterol agent
`whereas Nam is not44 because high-dose Nam inhibits sirtuins1.
`Notably, NR is active as a cholesterol-lowering agent in overfed
`mice30.
`As shown in Fig. 1, Nam is expected to proceed through NMN
`but not NR, NAR, NaMN or NAAD en route to forming NADþ .
`Though there was no elevation of hepatic NR or NAR with oral
`
`NATURE COMMUNICATIONS | 7:12948 | DOI: 10.1038/ncomms12948 | www.nature.com/naturecommunications
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`NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12948
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`40
`
`30
`
`20
`
`10
`
`0
`
`b
`
`MeNam
`
`(µmol mmol–1 creatinine)
`
`Pre first dose
`Pre second dose
`8.1–12
`
`0–4.14.1–7.7
`
`Pre fifth dose
`
`Pre fifth dose
`
`01234
`
`(µmol mmol–1 creatinine)
`
`Nam
`
`a
`
`Pre first dose
`8.1–12
`4.1–7.7
`0–4.1
`
`Pre second dose
`
`Time (h)
`
`Time (h)
`
`40
`
`30
`
`20
`
`10
`
`0
`
`d
`
`Me4PY
`
`(µmol mmol–1 creatinine)
`
`Pre first dose
`0–4.1
`8.1–12
`4.1–7.7
`
`Pre second dose
`
`Pre fifth dose
`
`200
`
`150
`
`100
`
`50
`
`0
`
`c
`
`Me2PY
`
`(µmol mmol–1 creatinine)
`
`Pre first dose
`4.1–7.7
`0–4.1
`8.1–12
`
`Pre second dose
`
`Pre fifth dose
`
`Time (h)
`Time (h)
`Figure 4 | Elevation of the urinary NADþ metabolome by oral NR in a 52-year-old male. A healthy 52-year-old male ingested 1,000 mg NC Cl daily for 1
`week. Urine was collected before the first dose, in three collection fractions in the first 12 h and before the second and fifth daily dose. Concentrations
`(a, Nam; b, MeNam; c, Me2PY; d, Me4PY) are normalized to urinary creatinine. The data indicate that urinary MeNam, Me4PY and Me2PY are the
`dominant metabolites elevated by oral NR. No phosphorylated species were found in urine.
`
`Nam,
`there was also little elevation of hepatic NMN—this
`metabolite never reached a mean value of 5 pmol mg 1 at any
`time after Nam administration (Fig. 5a). Surprisingly, as shown in
`Fig. 5i, 2–4 h after oral Nam, NAAD was elevated to nearly 200
`from a baseline of o2 pmol mg 1. Elevated NAAD occurred
`during the broad peak of elevated hepatic NADþ and NADPþ
`(Fig. 5b,c). These data suggest that the rise in NAAD is a
`biomarker of increased NADþ synthesis and does not depend on
`the conventionally described precursors of NAAD, namely NA
`and tryptophan.
`As shown in Fig. 5b, NR elevated hepatic NADþ by more than
`fourfold with a peak at 6 h post gavage. NR also produced the
`greatest elevation of NMN (Fig. 5a), NADPþ (Fig. 5c), Nam
`(Fig. 5d), NAMN (Fig. 5h), NAAD (Fig. 5i) and ADPR (Fig. 5j) in
`terms of peak height and AUC. Importantly, although gavage of
`Nam produces a peak of Nam in the liver at 15 min, the peak of
`Nam from NR gavage corresponds to the peak of NAD þ , NMN,
`NADPþ and ADPR. These data establish that oral NR has clearly
`different hepatic pharmacokinetics than oral Nam. More NADþ
`and NADPþ were produced from NR than from Nam. In
`addition, there was three times as much accumulation of ADPR,
`indicating that NR drives greater NADþ -consuming activities in
`liver than mole equivalent doses of Nam and NA. Though it has
`been speculated that NR would be a more potent NADþ and
`sirtuin-boosting vitamin than conventional niacins45, these are
`the first in vivo data in support of this hypothesis.
`As was seen in the n¼ 1 human blood experiment, at time
`points in which the abundant NADþ metabolites, NADþ and
`NADPþ , were elevated by NR by Btwofold or more, NAAD rose
`from undetectable levels to B10% of the level of NADþ , thereby
`increased NADþ
`becoming a highly sensitive biomarker of
`metabolism. Though compounds such as MeNam, Me2PY and
`
`Me4PY are also correlated with increased NADþ synthesis, they
`are waste products that can be produced without NADþ synthesis,
`whereas NAAD is functional NADþ precursor.
`To test whether NAAD is also elevated in other tissues and
`through other routes of administration, we euthanized mice after
`6 days of NR or saline by IP administration and analysed hepatic
`and cardiac NAD þ metabolomes. As shown in Fig. 6, steady-
`state levels of hepatic NADþ and NADPþ are much more
`responsive to NR than are steady state levels of cardiac NADþ
`and NADPþ . However, cardiac NADþ metabolism was clearly
`elevated on the basis of statistically significant elevation of NMN,
`Nam, MeNam and Me4PY. Among these metabolites, only NMN,
`which was elevated in the heart by approximately twofold, could
`increased NADþ formation.
`be considered diagnostic for
`In addition, NAMN and NAAD were increased by about
`B100-fold in heart and liver with NAAD rising to B10% of
`the concentration of heart and liver NADþ in supplemented
`animals. These data validate NAAD as a metabolite that
`sensitively and reliably marks increased NADþ metabolism even
`in tissues in which steady-state levels of NADþ are little changed.
`
`NR is incorporated into NAAD. Appearance of hepatic NAAD
`after gavage of Nam or NR, and of hepatic NAMN after gavage of
`NR suggested that there is an NADþ and/or NMN deamidating
`activity when NADþ and NADPþ levels are high. Alternatively,
`high levels of NADþ metabolites might
`inhibit glutamine-
`dependent NADþ synthetase, thereby resulting in accumulation
`of NAMN and NAAD derived from tryptophan. To test whether
`NR is incorporated into the peak of NAAD that appears after NR
`gavage, we synthesized NR with incorporation of deuterium at
`the ribosyl C2 and 13C into the carbonyl of the Nam moiety. This
`double-labelled NR was provided to 15 mice by oral gavage at an
`
`6
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`NATURE COMMUNICATIONS | 7:12948 | DOI: 10.1038/ncomms12948 | www.nature.com/naturecommunications
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`*
`
`N R
`
`N A
`
`Na m
`
`20,000
`
`15,000
`
`10,000
`
`5,000
`
`0
`
`AUC (pmol h mg–1)
`
`###
`†††
`
`###
`‡‡
`
`15
`
`5T
`
`10
`ime (h)
`
`6,000
`
`4,000
`
`2,000
`
`0
`
`0
`
`#
`
`1,500
`
`†††
`‡‡‡
`
`###
`†††
`‡‡
`
`###
`†††
`‡‡‡
`
`8,000
`
`***
`
`***
`
`***
`
`NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12948
`
`b
`
`NAD+ (pmol mg–1)
`
`d
`
`***
`
`***
`
`N R
`
`N A
`
`Na m
`
`50
`40
`30
`20
`10
`0
`
`AUC (pmol h mg–1)
`
`2,000
`
`###
`†††
`
`###
`†
`
`15
`
`5T
`
`10
`ime (h)
`
`15
`
`10
`
`0
`
`05
`
`a
`
`NMN (pmol mg–1)
`
`c
`
`600
`
`N R
`
`N A
`
`Na m
`
`**
`
`***
`
`6,000
`
`4,000
`
`2,000
`
`0
`
`AUC (pmol h mg–1)
`
`###
`†††
`‡
`
`###
`‡‡‡
`‡‡
`
`‡‡
`
`15
`
`5T
`
`10
`ime (h)
`
`Nam (pmol mg–1)
`
`1,000
`
`500
`
`0
`
`0
`
`N R
`
`N A
`
`Na m
`
`1,500
`
`1,000
`
`500
`
`0
`
`AUC (pmol h mg–1)
`
`###
`†
`
`400
`
`###
`
`#
`
`†
`
`200
`
`0
`
`0
`
`5
`10
`Time (h)
`
`15
`
`††
`‡‡‡
`
`††
`‡‡
`
`‡
`
`30
`
`*
`
`*
`
`***
`
`f
`
`40
`
`30
`
`‡‡‡
`
`††
`‡‡‡
`
`†††
`‡‡‡
`
`‡‡‡
`
`NADP+ (pmol mg–1)
`
`e
`
`Na m
`
`N R
`
`N A
`
`*
`
`***
`
`250
`200
`150
`100
`50
`0
`
`AUC (pmol h mg–1)
`
`#
`
`#
`
`5
`10
`Time (h)
`
`15
`
`30
`
`20
`
`10
`
`0
`
`AUC (pmol h mg–1)
`
`15
`
`###
`†††
`
`20
`
`10
`
`0
`
`0
`
`20
`15
`10
`
`05
`
`Me4PY (pmol mg–1)
`
`h
`
`N R
`
`N A
`
`Na m
`
`***
`
`***
`
`20
`
`10
`
`0
`
`AUC (pmol h mg–1)
`
`400
`
`300
`
`200
`
`100
`
`0
`
`AUC (pmol h mg–1)
`
`0
`
`5
`10
`Time (h)
`
`15
`
`012345
`
`MeNam (pmol mg–1)
`
`###
`‡‡‡
`
`###
`‡‡‡
`
`400
`
`300
`
`200
`
`100
`
`0
`
`g
`
`NA (pmol mg–1)
`
`–5
`
`5
`10
`Time (h)
`
`N R
`
`N A
`
`Na m
`
`NAMN (pmol mg–1)
`
`0
`
`5
`10
`Time (h)
`
`15
`
`N R
`
`N A
`
`Na m
`
`**
`
`*
`
`2,000
`
`1,500
`
`1,000
`
`500
`
`0
`
`N R
`
`N A
`
`Na m
`
`AUC (pmol h mg–1)
`
`#‡
`
`††
`
`##
`†††
`‡‡‡
`
`###
`‡‡‡
`
`###
`‡‡‡
`
`###
`‡‡‡
`
`#‡
`
`0
`
`5
`10
`Time (h)
`
`15
`
`500
`400
`300
`200
`100
`0
`
`j
`
`ADPR (pmol mg–1)
`
`10
`5
`Time (h)
`Saline
`NR
`Nam
`NA
`Figure 5 | NR elevates hepatic NADþ metabolism distinctly with respect to other vitamins. Either saline (orange, n¼ 3 per time point) or equivalent
`moles of NR (black, n¼ 3 per time point), NA (blue, n¼ 4 per time point) and Nam (green, n¼ 4 per time point) were administered to male C57Bl6/J mice
`by gavage. To control for circadian effects, gavage was performed at indicated times before a common B2 pm tissue collection. In the left panels, the
`hepatic concentrations (pmol per mg of wet tissue weight) of each metabolite (a, NMN; b, NADþ ; c, NADPþ ; d, Nam; e, MeNam; f, Me4PY; g, NA; h,
`NAMN; i, NAAD; and j, ADPR) are shown as a function of the four gavages. The excursion of each metabolite as a function of saline gavage is shown in
`orange; as a function of NR in black; as function of Nam in green; and NA in blue. In the right panels, the baseline-subtracted 12-hour AUCs are shown.
`(left) zP valueo0.05; zzP valueo0.01; zzzP value o0.001 Nam versus NA;
`w
`ww
`www
`P valueo0.05;
`P valueo0.01;
`P valueo0.001 Nam versus NR;
`#P valueo0.05; ##P valueo0.01; ###P valueo0.05 NA versus NR; (right) *P valueo0.05; **P valueo0.01; ***P value o0.001. The data indicate
`that NR produces greater increases in NADþ metabolism than Nam or NA with distinctive kinetics, that Nam is disadvantaged in stimulation of
`NADþ -consuming activities, and that NAAD is surprisingly produced after oral NR administration.
`
`N R
`
`N A
`
`Na m
`
`2,500
`2,000
`1,500
`1,000
`500
`0
`
`AUC (pmol h mg–1)
`
`15
`
`###
`†††
`‡
`
`###
`†††
`
`600
`
`400
`
`200
`
`###
`
`0
`
`0
`
`i
`
`NAAD (pmol mg–1)
`
`NATURE COMMUNICATIONS | 7:12948 | DOI: 10.1038/ncomms12948 | www.nature.com/naturecommunications
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`NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12948
`
`Saline
`NR
`
`***
`
`Heart
`
`***
`
`Heart
`
`Liver
`
`***
`
`Liver
`
`***
`
`500
`
`400
`
`300
`
`200
`
`100
`
`0
`
`c
`
`NADP+ (pmol mg–1)
`
`40
`35
`30
`25
`20
`15
`
`02
`
`f
`
`Me4PY (pmo mg–1)
`
`250
`
`200
`
`150
`
`100
`
`50
`
`i
`
`ADPR (pmol mg–1)
`
`***
`
`Liver
`
`***
`
`Liver
`
`***
`
`Heart
`
`***
`
`Heart
`
`**
`
`0
`
`5,000
`
`4,000
`
`3,000
`
`2,000
`
`1,000
`
`0
`
`b
`
`NAD+ (pmol mg–1)
`
`15
`
`10
`
`5
`
`0.5
`
`0.0
`
`MeNam (pmol mg–1)
`
`e
`
`450
`350
`250
`150
`
`50
`
`01
`
`h
`
`NAAD (pmol mg–1)
`
`Liver
`Heart
`Liver
`Heart
`Liver
`Heart
`Figure 6 | Rising NAAD is a more sensitive biomarker of elevated tissue NADþ metabolism than is NADþ . Male C57Bl6/J mice were intraperitoneally
`injected either saline (n¼ 8) or NR Cl (500 mg kg 1 body weight) (n¼ 6) for 6 days. Livers and hearts were freeze-clamped and prepared for metabolomic
`analysis. Concentrations of NADþ metabolites (a, NMN; b, NADþ ; c, NADPþ ; d, Nam; e, MeNam; f, Me4PY; g, NAMN; h, NAAD; i, ADPR) in
`heart and liver are presented in pmol mg 1 of wet tissue weight. Data were analysed using a two-way analysis of variance followed by a Holm–Sidak
`multiple comparisons