Cell Metabolism
`
`Article
`
`The NAD+ Precursor Nicotinamide Riboside
`Enhances Oxidative Metabolism
`and Protects against High-Fat Diet-Induced Obesity
`
`Carles Canto´ ,1,6,7 Riekelt H. Houtkooper,1,6,8 Eija Pirinen,1,2 Dou Y. Youn,3 Maaike H. Oosterveer,1 Yana Cen,3
`Pablo J. Fernandez-Marcos,1 Hiroyasu Yamamoto,1 Pe´ ne´ lope A. Andreux,1 Philippe Cettour-Rose,1 Karl Gademann,4
`Chris Rinsch,5 Kristina Schoonjans,1 Anthony A. Sauve,3 and Johan Auwerx1,*
`1E´ cole Polytechnique Fe´ de´ rale de Lausanne, CH-1015 Lausanne, Switzerland
`2Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, Biocenter Kuopio, University of Eastern Finland,
`70210 Kuopio, Finland
`3Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA
`4Department of Chemistry, University of Basel, CH-4056 Basel, Switzerland
`5Amazentis, Quartier de L’innovation EPFL, CH-1015 Lausanne, Switzerland
`6These authors contributed equally to this work
`7Present address: Nestle´ Institute of Health Sciences, CH-1015 Lausanne, Switzerland
`8Present address: Laboratory Genetic Metabolic Diseases, Academic Medical Center, 1105 AZ Amsterdam, The Netherlands
`*Correspondence: admin.auwerx@epfl.ch
`DOI 10.1016/j.cmet.2012.04.022
`
`SUMMARY
`
`As NAD+ is a rate-limiting cosubstrate for the sirtuin
`enzymes, its modulation is emerging as a valuable
`tool to regulate sirtuin function and, consequently,
`oxidative metabolism.
`In line with this premise,
`decreased activity of PARP-1 or CD38—both NAD+
`consumers—increases NAD+ bioavailability, result-
`ing in SIRT1 activation and protection against meta-
`bolic disease. Here we evaluated whether similar
`effects could be achieved by increasing the supply
`of nicotinamide riboside (NR), a recently described
`natural NAD+ precursor with the ability to increase
`NAD+ levels, Sir2-dependent gene silencing, and
`replicative life span in yeast. We show that NR
`supplementation in mammalian cells and mouse
`tissues increases NAD+ levels and activates SIRT1
`and SIRT3, culminating in enhanced oxidative
`metabolism and protection against high-fat diet-
`induced metabolic abnormalities. Consequently,
`our results indicate that the natural vitamin NR could
`be used as a nutritional supplement to ameliorate
`metabolic and age-related disorders characterized
`by defective mitochondrial function.
`
`INTRODUCTION
`
`The administration of NAD+ precursors, mostly in the form of
`nicotinic acid (NA), has long been known to promote beneficial
`effects on blood lipid and cholesterol profiles and even to induce
`short-term improvement of type 2 diabetes (Karpe and Frayn,
`2004). Unfortunately, NA treatment often leads to severe flush-
`ing, resulting in poor patient compliance (Bogan and Brenner,
`2008). These side effects are mediated by the binding of NA to
`
`838 Cell Metabolism 15, 838–847, June 6, 2012 ª2012 Elsevier Inc.
`
`the GPR109A receptor (Benyo et al., 2005). We hence became
`interested in the possible therapeutic use of alternative NAD+
`precursors that do not activate GPR109A.
`NR was recently identified as a NAD+ precursor, with
`conserved metabolism from yeast to mammals (Bieganowski
`and Brenner, 2004). Importantly, NR is found in milk (Bieganow-
`ski and Brenner, 2004), constituting a dietary source for NAD+
`production. Once it enters the cell, NR is metabolized into nico-
`tinamide mononucleotide (NMN) by a phosphorylation step cata-
`lyzed by the nicotinamide riboside kinases (NRKs) (Bieganowski
`and Brenner, 2004). In contrast to NR, NMN has not yet been
`found in dietary constituents, and its presence in serum is
`a matter of debate (Hara et al., 2011; Revollo et al., 2007). This
`highlights how NR might be an important vehicular form of
`a NAD+ precursor whose levels could be modulated through
`nutrition.
`The sirtuins are a family of enzymes that use NAD+ as
`a cosubstrate to catalyze the deacetylation and/or mono-ADP-
`ribosylation of target proteins. One of their major particularities
`is that their Km for NAD+ is relatively high, making NAD+ a rate-
`limiting substrate for their reaction (Canto and Auwerx, 2012).
`Initial work by yeast biologists indicated that the activity of Sir2
`(the yeast SIRT1 homolog) as a NAD+-coupled enzyme could
`provide a link between metabolism and gene silencing (Imai
`et al., 2000a; Imai et al., 2000b). In this way, Sir2 was proposed
`to mediate metabolic transcriptional adaptations linked to situa-
`tions of nutrient scarcity, which are generally coupled to
`increased NAD+ levels (for review, see Houtkooper et al.,
`2010). During the last decade, an overwhelming body of
`evidence indicates that
`the activity of mammalian sirtuins,
`most notably SIRT1 and SIRT3, have the ability to enhance fat
`oxidation and prevent metabolic disease (Hirschey et al., 2011;
`Lagouge et al., 2006; Pfluger et al., 2008). Therefore, strategies
`aimed to increase intracellular NAD+ levels have gained interest
`in order to activate sirtuins and battle metabolic damage. Valida-
`tion of this concept was achieved recently, by demonstrating
`that pharmacological and genetic approaches aimed to reduce
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`Cell Metabolism
`NR Increases NAD+ and Prevents Metabolic Disorders
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`Figure 1. Nicotinamide Riboside Supplementation Increases NAD+ Content and Sirtuin Activity in Cultured Mammalian Cells
`(A) C2C12 myotubes, Hepa1.6, and HEK293T cells were treated with nicotinamide riboside (NR) for 24 hr, and acidic extracts were obtained to measure total
`NAD+ intracellular content.
`(B) GPR109A-expressing Chem-4 cells were loaded with 3 mM Fura-2 acetoxymethyl ester derivative (Fura-2/AM) for 30 min at 37
`C. Then cells were washed
`three times with Hank’s balanced salt solution. Finally, calcium fluxes in response to increasing concentrations of nicotinic acid (NA; as positive control), NR, or
`nicotinamide mononucleotide (NMN) were determined as indicated in the Experimental Procedures.
`(C) C2C12 myotubes, Hepa1.6, and HEK293T cells were treated with either PBS (as vehicle) or 0.5 mM of NR, NMN, or NA for 24 hr. Then total NAD+ intracellular
`content was determined as in (A).
`(D) C57Bl/6J mice were fed with chow containing vehicle (water) or either NR, NMN,or NA at 400 mg/kg/day (n = 8 mice per group). After 1 week, NAD+ content
`was determined in liver and quadriceps muscle.
`(E) HEK293T cells were treated with NR (0.5 mM, black bars) or vehicle (white bars) for 4 hr. Then cells were harvested, and mitochondria were isolated for NAD+
`measurement.
`(F) C57Bl/6J mice were fed with chow containing vehicle (water) or NR at 400 mg/kg/day (n = 8 mice per group). After 1 week, mitochondria were isolated from
`their livers to measure NAD+ content.
`(G) HEK293T cells were treated with either PBS (as vehicle) or 0.5 mM of NR for 24 hr. Then mRNA and protein was extracted to measure Nampt levels by
`RT-qPCR and western blot, respectively.
`(H) HEK293T cells were treated with either PBS (as vehicle) or 0.5 mM of NR for 24 hr. Then protein homogenates were obtained to test global PARylation and
`PARP-1 levels. Throughout the figure, all values are presented as mean ±SD. Asterisk indicates statistical significant difference versus respective vehicle group at
`p < 0.05. Unless otherwise stated, the vehicle groups are represented by white bars, and NR groups are represented by black bars.
`
`
`
`the activity of major NAD+-consuming activities in the cell, such
`as PARP-1 (Bai et al., 2011b) and CD38 (Barbosa et al., 2007),
`prompted an increase in NAD+ bioavailability and enhanced
`SIRT1 activity, ultimately leading to effective protection against
`metabolic disease. In this work we aimed to test whether similar
`effects could be achieved through dietary supplementation with
`a natural NAD+ precursor, such as NR.
`
`RESULTS
`
`NR Increases Intracellular and Mitochondrial NAD+
`Content in Mammalian Cells and Tissues
`NR treatment dose-dependently increased intracellular NAD+
`levels in murine and human cell lines (Figure 1A), with maximal
`
`effects at concentrations between 0.5 and 1 mM. In C2C12 my-
`otubes, the Km for NR uptake was 172.3 ± 17.6 mM, with a Vmax of
`204.2 ± 20.5 pmol/mg of protein/min. Unlike NA, both NR and
`another well-described NAD+ precursor, NMN (Revollo et al.,
`2007), did not activate GPR109A (Figure 1B), hence constituting
`valuable candidates to increase NAD+ levels without activating
`GPR109A. Strikingly, the ability of NR to increase intracellular
`NAD+ in mammalian cells was, at least, similar to that of these
`other precursors (Figure 1C). We next evaluated the efficacy of
`NR, NMN, and NA to increase NAD+ in vivo by supplementing
`mouse chow with NR, NMN, or NA at 400 mg/kg/day for
`1 week. All compounds increased NAD+ levels in liver, but only
`NR and NA significantly enhanced muscle NAD+ content. (Fig-
`ure 1D). These results illustrate how NR administration is a valid
`
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`Cell Metabolism
`NR Increases NAD+ and Prevents Metabolic Disorders
`
`tool to boost NAD+ levels in mammalian cells and tissues without
`activating GPR109A.
`Given the existence of different cellular NAD+ pools and the
`relevance of mitochondrial NAD+ content for mitochondrial and
`cellular function (Yang et al., 2007a), we also analyzed whether
`NR treatment would affect mitochondrial NAD+ levels.
`In
`contrast to what has been observed with other strategies aimed
`to increase NAD+ bioavailability, such as PARP inhibition (Bai
`et al., 2011a), we found that mitochondrial NAD+ levels were
`enhanced in cultured cells (Figure 1E) and mouse liver (Figure 1F)
`after NR supplementation.
`To further solidify our data, we also wondered whether the
`enhanced NAD+ levels upon NR treatment could derive from
`alterations in the NAD+ salvage pathway or PARP activity.
`However, we could not see any change in Nampt mRNA or
`protein content in response to NR treatment (Figure 1G). Simi-
`larly, PARP activity and PARP-1 content were not affected by
`NR (Figure 1H). Altogether,
`these results suggest
`that NR
`increases NAD+ by direct NAD+ biosynthesis rather than by indi-
`rectly affecting the major NAD+ salvage (Nampt) or consumption
`(PARPs) pathways. Importantly, this increase in NAD+ was not
`linked to changes in cellular glycolytic rates or ATP levels (data
`not shown), which would be expected if NAD+/NADH ratios
`had been altered to the point of compromising basic cellular
`functions.
`
`NR Treatment Enhances SIRT1 and SIRT3 Activity
`The ability of NR to increase intracellular NAD+ levels both in vivo
`and in vitro prompted us to test whether it could activate sirtuin
`enzymes. Confirming this hypothesis, NR dose-dependently
`decreased the acetylation of FOXO1 (Brunet et al., 2004) in
`a SIRT1-dependent manner (Figure 2A). This deacetylation of
`FOXO1 by SIRT1 upon NR treatment resulted in its transcrip-
`tional activation, leading to higher expression of target genes,
`such as Gadd45, Catalase, Sod1, and Sod2 (see Figure S1 on-
`line) (Calnan and Brunet, 2008). The lack of changes in SIRT1
`protein levels upon NR treatment (Figure 2A) suggests that NR
`increases SIRT1 activity by enhancing NAD+ bioavailability.
`The higher SIRT1 activity in NR-treated cells was supported by
`mRNA expression analysis. Consistent with SIRT1 being a nega-
`tive regulator of Ucp2 expression (Bordone et al., 2006), NR
`decreased Ucp2 mRNA levels (Figure 2B). Importantly, knocking
`down Sirt1 prevented the action of NR on Ucp2 expression (Fig-
`ure 2B). Similarly, the higher expression of a FOXO1 target gene,
`Sod2, upon NR treatment was also prevented by the knockdown
`of either Foxo1 or Sirt1 (Figure 2B). This suggested that NR leads
`to a higher Sod2 expression through the activation of SIRT1,
`which then deacetylates and activates FOXO1. Importantly, the
`knockdown of Sirt1 did not compromise the ability of NR to
`increase intracellular NAD+ content, indicating that NR uptake
`and metabolism into NAD+ are not affected by SIRT1 deficiency
`(Figure 2C).
`In line with the increase in mitochondrial NAD+ levels (Figures
`1E and 1F) and the potential consequent activation of mitochon-
`drial sirtuins, NR also reduced the acetylation status of Ndufa9
`immunoprecipitates and SOD2 (Figures 2D and 2E, respec-
`tively), both targets for SIRT3 (Ahn et al., 2008; Qiu et al.,
`2010). SOD2 deacetylation has been linked to a higher intrinsic
`activity. In line with these observations, NR treatment enhanced
`
`840 Cell Metabolism 15, 838–847, June 6, 2012 ª2012 Elsevier Inc.
`
`SOD2 activity (Figure 2E). To ensure that NR-induced SOD2 de-
`acetylation was consequent to SIRT3 activation, we used mouse
`embryonic fibroblasts (MEFs) established from SIRT3 KO mice.
`The absence of SIRT3 was reflected by the higher basal acetyla-
`tion of SOD2 (Figure 2F).
`Importantly, NR was unable to
`/
`MEFs (Fig-
`decrease the acetylation status of SOD2 in SIRT3
`ure 2F), even though NAD+ levels increased to similar levels as in
`SIRT3+/+ MEFs (Figure 2G). These results clearly indicate that NR
`triggers SIRT3 activity, probably by increasing mitochondrial
`NAD+ levels, inducing the concomitant deacetylation of its mito-
`chondrial targets. Strikingly, not all sirtuins were affected by NR,
`as the acetylation of tubulin, a target of the cytoplasmic SIRT2
`(North et al., 2003), was not altered (data not shown).
`
`NR Supplementation Enhances Energy Expenditure
`Given the promising role of sirtuins to protect against metabolic
`disease, we next evaluated the effects of long-term NR adminis-
`tration in vivo. We fed 10-week-old male C57Bl/6J mice with
`either chow diet (CD) or high-fat diet (HFD), supplemented or
`not with NR at 400 mg/kg/day. While NR had no effect on the
`body weight (BW) on CD, HFD-induced BW gain was signifi-
`cantly attenuated by NR (Figure 3A), due to reduced fat mass
`(Figure 3B). This was visibly translated into a significant lower
`weight of the epididymal depot in NR-fed mice (Figure S2A).
`Importantly, this was not due to redistribution of lipids to other
`tissues (Figure S2A), most notably to liver, which actually con-
`tained 40% less triglycerides (Figure S2B).
`The reduced BW gain of NR-fed mice upon HFD was not due
`to reduced food intake, as NR-fed mice actually had a tendency
`to eat more, especially on HFD conditions (Figure 3C). Similarly,
`NR did not affect the activity pattern of mice (Figure 3D), indi-
`cating that the lower BW on HFD was not consequent to different
`physical activity. Rather, the phenotype was due to enhanced
`energy expenditure (EE). Mice on CD had a marked tendency
`to display higher O2 consumption rates when fed with NR, and
`this tendency became clearly significant under HFD conditions
`(Figure 3E). Of note, NR-fed mice became more flexible in their
`use of energy substrates, as reflected in the higher amplitude
`of the changes in RER between feeding and fasting periods (Fig-
`ure S2C) in CD conditions. Altogether, these results indicate that
`NR lowers HFD-induced BW gain by enhancing EE.
`From a metabolic perspective, NR- and vehicle-fed mice had
`similar fasting blood glucose levels in either CD or HFD condi-
`tions (Figure 3F). However, fasting insulin levels were much lower
`in NR-supplemented mice (Figure 3G). This lower insulin/glucose
`ratio is indicative of insulin sensitization after NR administration.
`This speculation was further supported by glucose tolerance
`tests. NR promoted a slight, albeit not significant, improvement
`in glucose tolerance (Figure 3H) in mice fed a HFD, accompanied
`by a robust reduction in insulin secretion (Figure 3I). Therefore,
`NR-fed mice on HFD display a better glucose disposal with lower
`insulin levels. In order to conclusively establish whether NR
`fed mice were more insulin sensitive, we performed insulin toler-
`ance tests and hyperinsulinemic-euglycemic clamps on CD and
`CD-NR mice. We chose not to perform this analysis on the HFD
`groups in order to avoid the possible influence of differential BW.
`Glucose disposal upon insulin delivery was largely enhanced in
`NR-fed mice (Figure 3J). In agreement, mice supplemented
`with NR required an almost 2-fold higher glucose infusion rate
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`Cell Metabolism
`NR Increases NAD+ and Prevents Metabolic Disorders
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`Vehicle
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`Figure 2. Nicotinamide Riboside Supplementation Increases Sirtuin Activity in Cultured Mammalian Cells
`(A) HEK293T cells were transfected with a pool of either scramble siRNAs or SIRT1 siRNAs. After 24 hr, cells were treated with vehicle (PBS) or NR at the
`concentrations indicated, and, after an additional 24 hr, total protein extracts were obtained. FOXO1 acetylation was tested after FOXO1 immunoprecipitation (IP)
`from 500 mg of protein, while tubulin and SIRT1 levels were evaluated in the supernatant of the IP.
`(B) HEK293T cells were transfected with a pool of either scramble siRNAs, FOXO1 siRNAs, or SIRT1 siRNAs. After 24 hr, cells were treated with NR (0.5 mM; black
`bars) or vehicle (PBS; white bars) for an additional 24 hr. Then total mRNA was extracted and the mRNA expression levels of the markers indicated were evaluated
`by qRT-PCR.
`(C) HEK293T cells were transfected with a pool of either scramble siRNAs, FOXO1 siRNAs, or SIRT1 siRNAs. After 24 hr, cells were treated with NR (0.5 mM; black
`bars) or vehicle (PBS; white bars) for an additional 24 hr. Then acidic extracts were obtained to measure intracellular NAD+ levels.
`(D and E) HEK293T cells were treated with NR (0.5 mM) or vehicle (PBS) for 24 hr and total protein extracts were obtained to measure (D) Ndufa9 or (E) SOD2
`acetylation after IP. The extracts were also used to measure SOD2 activity (E, bottom panel).
`/
`(F and G) SIRT3+/+ and SIRT3
`mouse embryonic fibroblasts (MEFs) were treated with NR (0.5 mM) or vehicle (PBS) for 24 hr, and either (F) total extracts to test
`SOD2 acetylation were obtained or (G) acidic extracts were used to measure intracellular NAD+ content. Throughout the figure, all values are presented as
`mean ±SD. Asterisk indicates statistical significant difference versus respective vehicle group at p < 0.05. Unless otherwise stated, the vehicle groups are
`represented by white bars, and NR groups are represented by black bars. This figure is complemented by Figure S1.
`
`to maintain euglycemia in hyperinsulinemic-euglycemic clamps
`(Figure 3K). Together, these observations unequivocally demon-
`strate that NR-fed mice are more insulin sensitive. Furthermore,
`NR partially prevented the increase in total (Figure 3K) and LDL
`cholesterol levels (Figure S2D) induced by HFD, even though
`HDL-cholesterol
`levels were unaffected (Figure S2E). The
`amelioration of cholesterol profiles is fully in line with previous
`observations from the use of other NAD+ precursors, such as
`NA (Houtkooper et al., 2010).
`
`NR Enhances the Oxidative Performance of Skeletal
`Muscle and Brown Adipose Tissue
`NR-fed mice had a clear tendency to display a better endurance
`performance than vehicle-fed mice (Figure S3A). This tendency
`
`was significantly accentuated upon HFD (Figure 4A), suggesting
`an enhanced muscle oxidative performance. Similarly, NR-fed
`mice, both on CD and HFD, showed enhanced thermogenic
`capacity, as manifested in the ability to maintain body tempera-
`ture during cold exposure (Figure S3B and Figure 4B). The latter
`observation hints toward an improvement in brown adipose
`tissue (BAT) oxidative performance. To gain further insight into
`the ability of BAT and muscle to enhance their oxidative perfor-
`mance, we performed some histological analysis. Gastrocne-
`mius muscles from NR mice displayed a more intense SDH
`staining than gastrocnemius muscles from their vehicle-fed
`littermates, indicating a higher oxidative profile (data not shown).
`Electron microscopy revealed that mitochondria in BAT of
`NR-fed mice, despite not being significantly larger, had more
`
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`Cell Metabolism
`NR Increases NAD+ and Prevents Metabolic Disorders
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`Figure 3. NR Supplementation Prevents Diet-Induced Obesity by Enhancing Energy Expenditure and Reduces Cholesterol Levels
`Ten-week-old C57Bl/6J mice were fed with either chow diet (CD) or high-fat diet (HFD) mixed with either water (as vehicle) or NR (400 mg/kg/day) (n = 10 mice
`per group).
`(A) Body weight evolution was monitored for 12 weeks.
`(B) Body composition was evaluated after 8 weeks of diet through Echo-MRI.
`(C–E) Food intake, activity, and VO2 were evaluated using indirect calorimetry.
`(F and G) Blood glucose and insulin levels were measured in animals fed with their respective diets for 16 weeks after a 6 hr fast.
`(H and I) After 10 weeks on their respective diets (CD, squares; HFD, circles), an intraperitoneal glucose tolerance test was performed in mice that were fasted
`overnight. At the indicated times, blood samples were obtained to evaluate either (H) glucose or (I) insulin levels. Areas under the curve are shown at the top right
`of the respective panels.
`(J) Insulin tolerance tests were performed on either CD or CD-NR mice (4 weeks of treatment). At the indicated times, blood samples were obtained to evaluate
`blood glucose levels. The area above the curve is shown at the top right of the panel.
`(K) Hyperinsulinemic-euglycemic clamps were performed on either CD or CD-NR mice (4 weeks of treatment). Glucose infusion rates (GIR) were calculated after
`the test.
`(L) Serum levels of total cholesterol were measured in animals fed with their respective diets for 16 weeks, after a 6 hr fast. Throughout the figure, white represents
`the vehicle group, and black represents the NR-supplemented mice. All values are presented as mean ±SD. Asterisk indicates statistical significant difference
`versus respective vehicle-treated group. This figure is complemented by Figure S2.
`
`abundant cristae (Figure 4C), which has been linked to
`increased respiratory capacity (Mannella, 2006). Altogether,
`the above results suggest that NR-supplemented mice display
`a higher oxidative capacity due to enhanced mitochondrial
`function.
`
`Chronic NR Feeding Increases NAD+ In Vivo in a Tissue-
`Specific Manner
`We next wondered how chronic NR feeding would affect NAD+
`metabolism in mice. Chronic NR supplementation increased
`NAD+ levels in both CD (Figure S4A) and HFD (Figure 5A) condi-
`tions in some tissues, including liver and muscle, but not in
`others, such as brain or white adipose tissue (WAT). Interest-
`
`842 Cell Metabolism 15, 838–847, June 6, 2012 ª2012 Elsevier Inc.
`
`ingly, NAD+ was also higher in the BAT of NR-fed mice, but
`only on HFD (Figure 5B and Figure S4A). These differences could
`be due to the differential expression of NRKs in tissues. NRKs
`initiate NR metabolism into NAD+ (Houtkooper et al., 2010).
`There are two mammalian NRKs: NRK1 and NRK2 (Bieganowski
`and Brenner, 2004). While we found Nrk1 expressed ubiquitously
`(Figure S4B), Nrk2 was mainly present in cardiac and skeletal
`muscle tissues, as previously described (Li et al., 1999), but
`also detectable in BAT and liver (Figure S4C), in line with the
`better ability of these tissues to respond to NR.
`We also tested whether the increase in NAD+ would be
`concomitant to changes in other NAD+ metabolites. Strikingly,
`NADH and nicotinamide (NAM) levels were largely diminished
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`Cell Metabolism
`NR Increases NAD+ and Prevents Metabolic Disorders
`
`that NAD+ salvage pathways do not explain the differences in
`NAD+ levels. We furthermore could not detect differences in
`mRNA expression of the different NMN adenylyltransferase
`(NMNAT) enzymes (Figure 5D). Altogether, these results rein-
`force the notion that the higher NAD+ levels observed in tissues
`from NR-fed mice are consequent to an increase in direct NAD+
`synthesis from NR.
`
`*
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`*
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`20
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`15
`
`10
`
`5
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`0
`
`AOC (a.u.)
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`*
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`NR Enhances Sirtuin Activity In Vivo
`Higher NAD+ levels were also accompanied by higher sirtuin
`activity in vivo. A prominent deacetylation of SIRT1 and
`SIRT3 targets (FOXO1, Brunet et al., 2004; and SOD2 Qiu
`et al., 2010, respectively) was observed in the skeletal muscle,
`liver, and BAT, where NAD+ content was induced by NR, but
`not in brain and WAT, where NAD+ levels were unaffected by
`NR supplementation (Figure 6A and Figure S5A). We also eval-
`uated PGC-1a acetylation as a second readout of SIRT1
`activity (Rodgers et al., 2005). We were unable to detect
`PGC-1a in total
`lysates from WAT or brain (Figure S5B), but
`liver, and BAT, PGC-1a was deacetylated upon
`in muscle,
`NR treatment (Figure S5C). These observations highlight how
`NR can only induce sirtuin activity in tissues where NAD+ accu-
`mulates. Like in cultured cells, we could not detect changes in
`the acetylation status of the SIRT2 target tubulin (data not
`shown), suggesting either that increasing NAD+ might not affect
`the activity of all sirtuins equally, that the increase is only
`compartment specific, or that additional regulatory elements,
`like class I and II HDACs, also contribute to tubulin acetylation
`status.
`In line with the changes in acetylation levels of PGC-1a, a
`key transcriptional regulator of mitochondrial biogenesis, we
`could observe either an elevated expression or a strong
`tendency toward an increase (p < 0.1) of nuclear genes encoding
`transcriptional
`regulators of oxidative metabolism (Sirt1,
`Ppargc1a, mitochondrial transcription factor A [Tfam]) and mito-
`chondrial proteins (Mitofusin 2 [Mfn2], cytochrome c [Cycs],
`medium-chain acyl-coA dehydrogenase [Acadm], carnitine
`palmitoyltransferase-1b [Cpt1b], citrate synthase [Cs], or ATP
`synthase lipid binding protein [Atp5g1]) in quadriceps muscles
`from NR-fed mice (Figure 6B). Conversely,
`in brain, where
`NAD+ and sirtuin activity levels were not affected by NR feeding,
`the expression of these genes was not altered (Figure 6B).
`Consistently also with enhanced mitochondrial biogenesis,
`mitochondrial DNA content was increased in muscle, but not in
`brain from NR-fed mice (Figure 6C). Finally, mitochondrial
`protein content also confirmed that mitochondrial function was
`only enhanced in tissues in which NAD+ content was increased
`(Figure 6D and Figure S5D). This way, while muscle, liver, and
`BAT showed a prominent increase in mitochondrial proteins
`(complex V—ATP synthase subunit a and porin), such change
`was not observed in brain or WAT. Altogether, these results
`suggest that NR feeding increases mitochondrial biogenesis in
`a tissue-specific manner, consistent with the tissue-specific
`nature of the increases in NAD+ and sirtuin activity observed in
`NR-fed mice. The higher number of mitochondria, together
`with the different morphological mitochondrial profiles found in
`NR-fed mice (Figure 4C), would contribute to explain the higher
`oxidative profile, EE, and protection against metabolic damage
`observed upon NR feeding.
`
`Cell Metabolism 15, 838–847, June 6, 2012 ª2012 Elsevier Inc. 843
`
`0
`
`1
`
`2
`
`3
`
`4
`
`5
`
`Time at 4ºC (hours)
`
`*
`
`2
`
`1,5
`
`1
`
`0,5
`
`0
`
`7
`
`6
`
`5
`
`4
`
`3
`
`2
`
`1
`
`0
`
`(relative to vehicle group)
`
`(relative to vehicle group)
`
`Mitochondrial size
`
`Cristae content
`
`38
`
`37
`
`36
`
`35
`
`34
`
`33
`
`B
`
`Body temperature ºC
`
`*
`
`A
`
`1000
`
`800
`
`600
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`400
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`200
`
`0
`
`Distance run (m)
`
`C
`
`Veh
`
`NR
`
`Figure 4. NR Enhances Skeletal Muscle and BAT Oxidative Function
`Ten-week-old C57Bl/6J mice were fed a high-fat diet (HFD) mixed with either
`water (as vehicle; white bars and circles) or NR (400 mg/kg/day; black bars and
`circles) (n = 10 mice per group).
`(A) An endurance exercise test was performed using a treadmill in mice fed
`with either HFD or HFD-NR for 12 weeks.
`(B) A cold test was performed in mice fed with either HFD or HFD-NR for
`9 weeks. The area over the curve (AOC) is shown on the top right of the graph.
`(C) Electron microscopy of the BAT was used to analyze mitochondrial content
`and morphology. The size and cristae content of mitochondria were quantified
`as specified in the Experimental Procedures. Throughout the figure, all values
`are shown as mean ±SD. Asterisk indicates statistical significant difference
`versus vehicle-supplemented group at p < 0.05. This figure is complemented
`by Figure S3.
`
`in muscles from NR-fed mice (Figure 5B), indicating that NR
`specifically increases NAD+, but not necessarily other byprod-
`ucts of NAD+ metabolism. We analyzed in vivo whether the
`activity of major NAD+-degrading enzymes or the levels of
`Nampt could also contribute to the increase in NAD+ after
`chronic NR supplementation. As previously observed in
`HEK293T cells (Figures 1G and 1H), PARP-1 levels and global
`PARylation were similar in muscle (Figure 5C) and livers (Fig-
`ure S4D) from NR- and vehicle-fed mice, indicating that the
`enhanced NAD+ content cannot be explained by differential
`NAD+ consumption through PARP activity. Nampt mRNA (Fig-
`ure 5D) and protein (Figure 5C, Figure S4E, and data not shown)
`levels were also similar in NR- and vehicle-fed mice, suggesting
`
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`

`Cell Metabolism
`NR Increases NAD+ and Prevents Metabolic Disorders
`
`Figure 5. Chronic NR Supplementation Increases
`Plasma and Intracellular NAD+ Content
`in
`a Tissue-Specific Manner
`Tissues from C57Bl/6J mice were collected after 16 weeks
`of HFD supplemented with either water (as vehicle; white
`bars) or NR (400 mg/kg/day; black bars).
`(A) NAD+ levels were measured in acidic extracts obtained
`from different tissues.
`(B) NADH and NAM levels were measured in gastrocne-
`mius muscle.
`(C) Quadriceps muscle protein homogenates were ob-
`tained to test global PARylation, PARP-1, and Nampt
`protein levels.
`(D) Total mRNA was isolated from quadriceps muscles,
`and the mRNA levels of the markers indicated were
`measured by RT-qPCR. Throughout the figure, all values
`are expressed as mean ±SD. Asterisk indicates statistical
`significant difference versus respective vehicle-treated
`group.
`
`*
`
`*
`
`0,3
`
`0,2
`
`0,1
`
`0
`
`(mmol / kg tissue)
`
`B
`
`*
`
`*
`
`*
`
`Brain
`
`Muscle
`
`Liver
`
`BAT
`
`WAT
`
`NADH
`
`NAM
`
`Nampt NMNAT-1 NMNAT-2 NMNAT-3
`
`D
`
`2
`
`1,5
`
`1
`
`0,5
`
`0
`
`(relative to Vehicle)
`
` mRNA levels
`
`0,5
`
`0,4
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`0,3
`
`0,2
`
`0,1
`
`0
`
`(mmol / kg tissue)
`
`A
`
` content
`
`+
`
`NAD
`
`C
`
`PAR
`
`PARP-1
`
`Nampt
`
` Vehicle NR
`
`DISCUSSION
`
`While increased NAD+ levels in response to NR supplementation
`were already reported in yeast (Belenky et al., 2007; Bieganow-
`ski and Brenner, 2004) and cultured human cells (Yang et al.,
`2007b), we extend these observations to a wide range of
`mammalian cell lines and demonstrate that NR supplementation
`also enhances NAD+ bioavailability in mammalian tissues. Also,
`our work provides evidence that the increase in NAD+ after NR
`administration stimulates the activity of mammalian sirtuins.
`This further supports the role of sirtuins as a family of proteins
`whose basal activity can be largely modulated by NAD+ avail-
`ability (Houtkooper et al., 2010).
`The fact that the activity of both SIRT1 and SIRT3 is positively
`regulated by NR both in vitro and in vivo favors the hypothesis
`that the increase in NAD+ promote