\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage[round]{natbib}
\let\cite\citep




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,greek,english]{babel}










\begin{document}

\title{The NAD+~precursor nicotinamide riboside enhances oxidative metabolism
and protects against high-fat diet induced obesity}



\author[1]{Elysium}%
\affil[1]{Elysium Health}%


\vspace{-1em}



  
  \date{May 07, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup








\sloppy


\textbf{Original Publication:}~Cant\selectlanguage{ngerman}ó C, Houtkooper RH, Pirinen E, et al.
The NAD+~precursor nicotinamide riboside enhances oxidative metabolism
and protects against high-fat diet induced obesity.~\emph{Cell
metabolism}. 2012;15(6):838-847. doi:10.1016/j.cmet.2012.04.022.

\section*{Summary}

{\label{675052}}

As NAD+ is a rate-limiting co-substrate 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, resulting in SIRT1 activation and protection
against metabolic 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
lifespan 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.

\section*{Introduction}

{\label{946499}}

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 flushing, resulting in poor patient compliance
(Bogan and Brenner, 2008). These side effects are mediated by the
binding of NA to 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 (Bieganowski and Brenner, 2004)
constituting a dietary source for NAD+ production. Once it enters the
cell, NR is metabolized into nicotinamide mononucleotide (NMN) by a
phosphorylation step catalyzed 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 an 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 an
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
situations 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 against 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. Validation of this concept was achieved recently, by
demonstrating that pharmacological and genetic approaches aimed to
reduce 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.

\section*{Results}

{\label{735581}}

\subsection*{NR increases intracellular and mitochondrial NAD+ content in
mammalian cells and
tissues}

{\label{860630}}

NR treatment dose-dependently increased intracellular NAD+ levels in
murine and human cell lines (Fig.1A), with maximal effects at
concentrations between 0.5 and 1 mM. In C2C12 myotubes, the Km for NR
uptake was 172.3±17.6 \selectlanguage{greek}μ\selectlanguage{english}M, with a Vmax of 204.2\selectlanguage{ngerman}±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
(Fig.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 (Fig.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 one week. All compounds increased NAD+
levels in liver, but only NR and NA significantly enhanced muscle NAD+
content. (Fig.1D). These results illustrate how NR administration is a
valid tool to boost NAD+ levels in mammalian cells and tissues without
activating GPR109A.

\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=1.00\columnwidth]{figures/emss-52498-f0001/emss-52498-f0001}
\caption{\selectlanguage{ngerman}{\textbf{Nicotinamide Riboside supplementation increases NAD+ content and
sirtuin activity in cultured mammalian cells}
{\label{705430}}
(A) C2C12 myotubes, Hepa1.6 and HEK293T cells were treated with
nicotinamide riboside (NR) for 24 hrs and acidic extracts were obtained
to measure total NAD+ intracellular content. (B) GPR109A-expressing
Chem-4 cells were loaded with 3 \selectlanguage{greek}μ\selectlanguage{english}M Fura-2 acetoxymethyl ester derivative
(Fura-2/AM) for 30 min at 37 °C. Then, cells were washed with Hank's
balanced salt solution and calcium flux in response to nicotinic acid
(NA; as positive control), NR and nicotinamide mononucleotide (NMN) at
the concentrations indicated was determined as indicated in methods. (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 hrs. 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 one 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 hrs.
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 one 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 hrs. 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 hrs. Then
protein homogenates were obtained to test global PARylation and PARP-1
levels. Throughout the figure, all values are presented as mean +/- SD.
* indicates statistical significant difference vs. respective vehicle
group at P \textless{} 0.05. Unless otherwise stated, the vehicle groups
are represented by white bars, and NR groups are represented by black
bars.
{\label{705430}}%
}\selectlanguage{english}}
\end{center}
\end{figure}\selectlanguage{ngerman}

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 (Fig.1E) and mouse liver (Fig.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 (Fig.1G).
Similarly, PARP activity and PARP-1 content were not affected by NR
(Fig.1H). Altogether, these results suggest that NR increases NAD+ by
direct NAD+ biosynthesis rather than by indirectly 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.

\subsection*{NR treatment enhances SIRT1 and SIRT3
activity}

{\label{579211}}

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
(Fig 2A). This deacetylation of FOXO1 by SIRT1 upon NR treatment
resulted in its transcriptional activation, leading to higher expression
of target genes, such as Gadd45, Catalase, Sod1 and Sod2 (Fig.S1)
(Calnan and Brunet, 2008). The lack of changes in SIRT1 protein levels
upon NR treatment (Fig.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 negative regulator of Ucp2 expression (Bordone et al., 2006), NR
decreased Ucp2 mRNA levels (Fig.2B). Importantly, knocking down Sirt1
prevented the action of NR on Ucp2 expression (Fig.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 (Fig.2B). This
suggested that NR leads to a higher Sod2 expression thought the
activation of SIRT1, which then deacetylates and activates FOXO1.
Importantly, the knock-down of SIRT1 did not compromise the ability of
NR to increase intracellular NAD+ content, indicating that NR uptake and
metabolism into NAD+ is not affected by SIRT1 deficiency (Fig.2C).

\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=1.00\columnwidth]{figures/emss-52498-f0002-(1)/emss-52498-f0002-(1)}
\caption{{\textbf{Nicotinamide Riboside supplementation increases sirtuin activity
in cultured mammalian cells}
{\label{856222}}
(A) HEK293T cells were transfected with a pool of either scramble siRNAs
or SIRT1 siRNAs. After 24 hrs, cells were treated with vehicle (PBS) or
NR at the concentrations indicated, and, after an additional 24 hrs,
total protein extracts were obtained. FOXO1 acetylation was tested after
FOXO1 immunoprecipitation (IP) from 500 \selectlanguage{greek}μ\selectlanguage{english}g 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 hrs, cells were treated with NR (0.5
mM; black bars) or vehicle (PBS; white bars) for additional 24 hrs. Then
total mRNA was extracted and the mRNA expression levels of the markers
indicated was evaluated by qRT-PCR. (C) HEK293T cells were transfected
with a pool of either scramble siRNAs, FOXO1 siRNAs or SIRT1 siRNAs.
After 24 hrs, cells were treated with NR (0.5 mM; black bars) or vehicle
(PBS; white bars) for additional 24 hrs. Then acidic extracts were
obtained to measure intracellular NAD+ levels. (D-E) HEK293T cells were
treated with NR (0.5 mM) or vehicle (PBS) for 24 hrs 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-G). SIRT3+/+ and SIRT3-/- mouse embryonic fibroblasts
(MEFs) were treated with NR (0.5 mM) or vehicle (PBS) for 24 hrs 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. *
indicates statistical significant difference vs. respective vehicle
group at P \textless{} 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 Fig.S1
{\label{856222}}%
}}
\end{center}
\end{figure}

In line with the increase in mitochondrial NAD+ levels (Fig.1E-F) and
the potential consequent activation of mitochondrial sirtuins, NR also
reduced the acetylation status of Ndufa9 and SOD2 (Fig.2D and 2E,
respectively), 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 SOD2
activity (Fig.2E). To ensure that NR-induced SOD2 deacetylation was
consequent to SIRT3 activation, we used mouse embryonary fibroblast
(MEFs) established from SIRT3 KO mice. The absence of SIRT3 was
reflected by the higher basal acetylation of SOD2 (Fig.2F). Importantly,
NR was unable to decrease the acetylation status of SOD2 in SIRT3-/-
MEFs (Fig.2F), despite that NAD+ levels increased to similar levels as
in SIRT3+/+ MEFs (Fig.2G). These results clearly indicate that NR
triggers SIRT3 activity, probably by increasing mitochondrial NAD+
levels, inducing the concomitant deacetylation of its mitochondrial
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).

\section*{NR supplementation enhances energy
expenditure}

{\label{986796}}

Given the promising role of sirtuins to protect against metabolic
disease, we next evaluated the effects of long-term NR administration in
vivo. We fed 10-week-old male C57Bl/6J mice with either chow (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 body weight
gain was significantly attenuated by NR (Fig.3A), due to reduced fat
mass (Fig.3B). This was visibly translated into a significant lower
weight of the epididymal depot in NR-fed mice (Fig.S2A). Importantly,
this was not due to redistribution of lipids to other tissues (Fig.S2A),
most notably to liver, which actually contained 40\% less triglycerides
(Fig.S2B).

\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=1.00\columnwidth]{figures/emss-52498-f0003/emss-52498-f0003}
\caption{{\textbf{NR supplementation prevents diet-induced obesity by enhancing
energy expenditure and reduces cholesterol levels}
{\label{834324}}
10-week-old C57Bl/6J mice were fed with either chow (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 during 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-G) Blood glucose and insulin levels were
measured in animals fed with their respective diets for 16 weeks after a
6 hr fast. (H-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 represent the vehicle group and black represent the
NR-supplemented mice. All values are presented as mean +/- SD. *
indicates statistical significant difference vs. respective vehicle
treated group. This figure is complemented by Fig.S2
{\label{834324}}%
}}
\end{center}
\end{figure}

The reduced body weight 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 (Fig.3C). Similarly, NR did not affect the
activity pattern of mice (Fig.3D), indicating 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 (Fig.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.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 conditions (Fig.3F).
However, fasting insulin levels were much lower in NR-supplemented mice
(Fig.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 (Fig.3H) in mice fed a
HFD, accompanied by a robust reduction in insulin secretion (Fig.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 tolerance 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 (Fig.3J). In agreement,
mice supplemented with NR required an almost 2-fold higher glucose
infusion rate to maintain euglycemia in hyperinsulinemic-euglycemic
clamps (Fig.3K). Together, these observations unequivocally demonstrate
that NR-fed mice are more insulin-sensitive. Furthermore, NR partially
prevented the increase in total (Fig.3K) and LDL cholesterol levels
(Fig.S2D) induced by HFD, even though HDL-cholesterol levels were
unaffected (Fig.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).

\subsection*{NR enhances the oxidative performance of skeletal muscle and
brown adipose
tissue}

{\label{537655}}

NR-fed mice had a clear tendency to display a better endurance
performance than vehicle fed mice (Fig.S3A). This tendency was
significantly accentuated upon HFD (Fig.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 temperature during cold exposure (Fig,S3B and Fig.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 performance, we
performed some histological analysis. Gastrocnemius muscles from NR mice
displayed a more intense SDH staining than 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 abundant cristae
(Fig.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.

\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=1.00\columnwidth]{figures/emss-52498-f0004/emss-52498-f0004}
\caption{{\textbf{NR enhances skeletal muscle and BAT oxidative function}
{\label{639240}}
10-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. (D) Electron microscopy of the BAT was
used to analyze mitochondrial content and morphology. The size and
cristae content of mitochondria was quantified as specified in methods.
Throughout the figure, all values are shown as mean +/- SD. * indicates
statistical significant difference vs. vehicle supplemented group at
P\textless{} 0.05. This figure is complemented by Fig.S3
{\label{639240}}%
}}
\end{center}
\end{figure}

\subsection*{Chronic NR feeding increases NAD+ in vivo in a
tissue-specific
manner}

{\label{132035}}

We next wondered how chronic NR feeding would affect NAD+ metabolism in
mice. Chronic NR supplementation increased NAD+ levels in both CD
(Fig.S4A) and HFD (Fig.5A) conditions in some tissues, including liver
and muscle, but not in others, such as brain or white adipose tissue
(WAT). Interestingly, NAD+ was also higher in the BAT of NR-fed mice,
but only on HFD (Fig.5B and Fig.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 (Fig.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 (Fig.S4C), in line with the better
ability of these tissues to respond to NR.

\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=1.00\columnwidth]{figures/emss-52498-f0005/emss-52498-f0005}
\caption{{\textbf{Chronic NR supplementation increases plasma and intracellular
NAD+ content in a tissue-specific manner}
{\label{270895}}
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 gastrocnemius muscle. (C) Quadriceps muscle protein homogenates were
obtained 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. * indicates statistical
significant difference vs. respective vehicle treated group.
{\label{270895}}%
}}
\end{center}
\end{figure}

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 in muscles from NR-fed mice
(Fig.5B), indicating that NR specifically increases NAD+, but not
necessarily other by-products 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 (Fig.1G-H),
PARP-1 levels and global PARylation were similar in muscle (Fig.5C) and
livers (Fig.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.5D) and protein
(Fig.5C, Fig.S4E and data not shown) levels were also similar in NR and
vehicle fed mice, suggesting 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 (Fig.5D). Altogether, these results reinforce the notion
that the higher NAD+ levels observed in tissues from NR-fed mice is
consequent to an increase in direct NAD+ synthesis from NR.

\subsection*{NR enhances sirtuin activity in
vivo}

{\label{169592}}

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 (Fig.6A and Fig.S5A). We also evaluated
PGC-1\selectlanguage{greek}α \selectlanguage{english}acetylation as a second readout of SIRT1 activity (Rodgers et
al., 2005). We were unable to detect PGC-1\selectlanguage{greek}α \selectlanguage{english}in total lysates from WAT or
brain (Fig.S5B), but in muscle, liver and BAT PGC-1\selectlanguage{greek}α \selectlanguage{english}was deacetylated
upon NR treatment (Fig.S5C). These observations highlight how NR can
only induce sirtuin activity in tissues where NAD+ accumulates. 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.

\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=1.00\columnwidth]{figures/emss-52498-f0006/emss-52498-f0006}
\caption{{\textbf{NR stimulates sirtuin activity in vivo and enhances
mitochondrial gene expression}
{\label{401860}}
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) Total protein extracts were obtained from
quadriceps muscle and brain indicated to evaluate the acetylation levels
of FOXO1 and SOD2 through immunoprecipitation assays, using 1 and 0.5 mg
of protein, respectively. (B) Total mRNA from quadriceps muscle and
brain was extracted to measure the abundance of the markers indicated by
RT-qPCR. (C) Mitochondrial DNA content was measured in DNA extracted
from quadriceps muscle and brain. The results are expressed a
mitochondrial copy number relative to genomic DNA (D) The abundance of
mitochondrial marker proteins in 20 \selectlanguage{greek}μ\selectlanguage{english}g of protein from total quadriceps
muscle and brain lysates. Throughout the figure, all values are shown as
mean +/- SD. * indicates statistical significant difference vs. vehicle
supplemented group at P\textless{} 0.05. This figure is complemented by
Fig.S4
{\label{401860}}%
}}
\end{center}
\end{figure}

In line with the changes in acetylation levels of PGC-1\selectlanguage{greek}α, \selectlanguage{english}a key
transcriptional regulator of mitochondrial biogenesis, we could observe
either an elevated expression or a strong tendency towards an increase
(P \textless{} 0.1) of nuclear genes encoding transcriptional regulators
of oxidative metabolism (Sirt1, PGC-1\selectlanguage{greek}α, \selectlanguage{english}mitochondrial transcription
factor A (Tfam)) and mitochondrial proteins (Mitofusin 2 (Mfn2),
Cytochrome C (Cyt C), Medium Chain Acyl-coA Dehydrogenase (MCAD),
Carnitine palmitoyltransferase-1b (CPT-1b), Citrate Synthase (CS) or ATP
synthase lipid binding protein (ATP5g1)) in quadriceps muscles from
NR-fed mice (Fig.6B). Conversely, in brain, where NAD+ and sirtuin
activity levels were not affected by NR feeding, the expression of these
genes was not altered (Fig.6B). Consistently also with enhanced
mitochondrial biogenesis, mitochondrial DNA content was increased in
muscle, but not in brain from NR-fed mice (Fig.6C). Finally,
mitochondrial protein content also confirmed that mitochondrial function
was only enhanced in tissues in which NAD+ content was increased (Fig.6D
and Fig.S5D). This way, while muscle, liver and BAT showed a prominent
increase in mitochondrial proteins (Complex V - ATP synthase subunit \selectlanguage{greek}α
\selectlanguage{english}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 (Fig.4C) would
contribute to explain the higher oxidative profile, energy expenditure
and protection against metabolic damage observed upon NR feeding.

\section*{Discussion}

{\label{807813}}

While increased NAD+ levels in response to NR supplementation were
already reported in yeast (Belenky et al., 2007; Bieganowski 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+
availability (Houtkooper et al., 2010).

The fact that the activity of both SIRT1 and SIRT3 are positively
regulated by NR both in vitro and in vivo favours the hypothesis that
the increase in NAD+ promoted by NR affects, at least, the mitochondrial
and the nuclear compartments. This is a key difference compared to other
strategies that boost intracellular NAD+ levels. For example, Parp-1
knock-out mice show a two-fold increase in intracellular NAD+, yet SIRT3
activity is not affected (Bai et al., 2011b). Given that PARP-1 is a
nuclear enzyme, it is tempting to hypothesize that, in the Parp-1
knockout mice, the increase in NAD+ is mainly confined to the nucleus,
while the NR-induced increase in NAD+ levels, despite being more
discrete in amplitude, reaches different subcellular compartments.

The inability of NR to activate GPR109A illustrates how NR provides an
alternative way to increase NAD+ and ameliorate metabolic homeostasis in
the absence of the undesirable effects seen with NA. Since NR does not
activate GPR109A, we speculate that NR reduces cholesterol levels
through the activation of SIRT1 in liver, which influences the activity
of a number of transcription factors and cofactors linked to cholesterol
homeostasis (Kemper et al., 2009; Li et al., 2007; Shin et al., 2003;
Walker et al., 2010). Interestingly, the mechanism of action of NA on
VLDL/LDL and hepatic metabolism is not clear, as GPR109A is not
expressed in the liver (Tunaru et al., 2003). Our data raise the
possibility that NA, like NR, achieves such effects by elevating NAD+
levels, activating SIRT1 and its downstream targets. However, future
experiments will be required to verify this hypothesis. The activation
of SIRT1 and SIRT3, impacting on targets such as PGC-1\selectlanguage{greek}α, \selectlanguage{english}FOXOs and SOD2,
also provides a likely explanation for the mitochondrial fitness and
metabolic flexibility of NR-supplemented mice. In this sense, it is
remarkable that while SIRT1 transgenic mice have some shared phenotypes
with NR-treatment mice, such as protection against metabolic damage,
there are also a number of discrepancies. For example, SIRT1 transgenics
show similar body weight gain upon HFD to wild-type littermates (Pfluger
et al., 2008). The differences between NR supplemented mice and SIRT1
trangenics can be explained by numerous reasons. First, NR affects
different sirtuin activities; second, sirtuin expression does not
necessarily correspond with sirtuin activity; third, NR actions are
tissue-specific. Importantly, we should also consider that NR might
trigger other actions unrelated to sirtuin activity that might also
contribute to the beneficial effects of NR (Fig.7).

\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=1.00\columnwidth]{figures/emss-52498-f0007/emss-52498-f0007}
\caption{{\textbf{Schematic representation of the different actions of NR in
metabolic homeostasis}
{\label{387606}}
The scheme summarizes the hypothesis by which NR supplementation would
increase NAD+ content in key metabolic tissues, leading to SIRT1 and
SIRT3 activation and the deacetylation and modulation of the activity of
key metabolic regulators. This model does not rule out the participation
of additional mechanisms of action for NR to achieve its beneficial
effects. Abbreviations can be found in the text and enzymes are
indicated in italics.
{\label{387606}}%
}}
\end{center}
\end{figure}

In conclusion, our work shows that NR is a powerful supplement to boost
NAD+ levels, activate sirtuin signalling and improve mitochondrial
function, suggesting that this vitamin could be used to prevent and
treat the mitochondrial decline that is a hallmark of many diseases
associated with aging. Very recent work, showing that intraperitoneal
administration of NMN could improve the metabolic damage induced by high
fat feeding (Yoshino et al., 2011) further supports this concept. To
date, however, only NR has been identified as a naturally occurring
component of the human diet (Bieganowski and Brenner, 2004).
Furthermore, NR protects against metabolic dysfunction at lower
concentrations than those reported for NMN and we proved that it is
effective after oral administration when mixed with food, which is in
contrast to NMN which is injected intraperitoneally (Yoshino et al.,
2011). So, how might NR and NMN biology intertwine and impact on NAD+
synthesis in mammalian physiology? Recent observations suggest that, at
least for some cell types, NMN needs to be converted into NR through
dephosphorylation performed by extracellular 5'-nucleotidases in order
to penetrate into the cell (Nikiforov et al., 2011). Given that the
presence of NMN in plasma is debated (Hara et al., 2011), the most
likely scenario is that NR, rather than NMN, is the entity directly
taken up by the cell, to then become metabolized in the cytosolic
compartment to NMN. The lack of NRK1 and NRK2 in the mitochondrial
compartment (Nikiforov et al., 2011) supports that NMN might act as the
precursor entering the mitochondria or other compartments for further
metabolism into NAD+ by the various NMNAT enzymes (see Fig.7 for a
scheme).

Our data, combined with the evidence that other NAD+ precursors can
improve age-related insulin resistance (Yoshino et al., 2011) and that
NR increases yeast replicative lifespan (Belenky et al., 2007),
therefore warrant future investigations to see if boosting NAD+ levels
by NR supplementation might also improve the health- and lifespan of
humans.

\section*{Experimental Procedures}

{\label{675059}}

\subsection*{Materials}

{\label{412333}}

All chemicals and reagents were purchased from Sigma-Aldrich unless
stated otherwise. Nicotinamide Riboside was custom synthesized as
previously described (Yang et al., 2007b) and was kindly provided by
Amazentis and Merck Research Laboratories.

\subsection*{Animal experiments}

{\label{146917}}

8 weeks-old male C57Bl/6J mice were purchased from Charles River and
powder chow (D12450B) and high fat (D12492) diets were from Research
Diets Inc (New Brunswick, NJ, USA). 80 ml of water per kg of powder CD
were used to make food pellets. 40 ml of water per kg of powder HFD were
used to make food pellets. For NR, NMN and NA supplemented diets, the
appropriate amount of these compounds was added to the water used to
create the pellets, taking into account the differences in the daily
intake of each diet. Mice were housed separately, had ad libitum access
to water and food and were kept under a 12h dark-light cycle. Mice were
fed with homemade pellets from 10 weeks of age. To make the pellets, the
powder food was mixed with water (vehicle) or with NR. All animal
experiments were carried according to national Swiss and EU ethical
guidelines and approved by the local animal experimentation committee of
the Canton de Vaud under license \#2279.

\subsection*{Animal phenotyping}

{\label{853230}}

Mice were weighed and the food consumption was measured each week on the
same day. Most clinical tests were carried out according to standard
operational procedures (SOPs) established and validated within the
Eumorphia program (Champy et al., 2008). Body composition was determined
by Echo-MRI (Echo Medical Systems, Houston, TX, USA) and oxygen
consumption (VO2), respiratory exchange ratios (RER), food intake and
activity levels were monitored by indirect calorimetry using the
comprehensive laboratory animal monitoring system (Columbus Instruments,
Columbus, OH, USA). Treadmill, cold tests and
hyperinsulinemic-euglycemic clamps were performed as described (Lagouge
et al., 2006). Glucose and insulin tolerance was analyzed by measuring
blood glucose and insulin following intraperitoneal injection of 2 g/kg
glucose or 0.25 U insulin/kg, respectively, after an overnight fast.
Plasma insulin was determined in heparinized plasma samples using
specific ELISA kits (Mercodia). All animals were sacrificed at 13:00,
after a 6 hr fast, using CO2 inhalation. Blood samples were collected in
heparinized tubes and plasma was isolated after centrifugation. All
plasma parameters were measured using a Cobas c111 (Roche Diagnostics).
Tissues were collected upon sacrifice and flash-frozen in liquid
nitrogen.

\subsection*{Histology and microscopy}

{\label{286459}}

Succinate dehydrogenase staining and transmission electron microscopy
(TEM) was performed as described (Bai et al., 2011b). Mitochondrial size
and cristae content was analyzed as previously described (St-Pierre et
al., 2003).

\subsection*{Cell culture and
transfection}

{\label{414142}}

Murine C2C12 myoblasts were grown and differentiated into myotubes as
described (Canto et al., 2009). Murine Hepa1.6 and human HEK293T cells
were cultured in DMEM (4,5 g/l glucose, 10\% FCS). Human FOXO1 and SIRT1
siRNAs were obtained from Dharmacon (Thermo Scientific) and used
following the instructions from the manufacturer. SIRT3 MEFs were
established according to standard techniques (Picard et al., 2002) from
conditional SIRT3-/- mice, whose generation will be described separately
(Fernandez-Marcos et al., submitted). Deletion of the SIRT3 gene was
induced via infection with adenovirus encoding for the Cre recombinase.

\subsection*{Gene expression and mitochondrial DNA
abundance}

{\label{203211}}

RNA was extracted and quantified by qRT-PCR as described (Lagouge et
al., 2006). The qPCR primers used have been previously described.
Mitochondrial DNA abundance (mtDNA) was quantified as described (Lagouge
et al., 2006) after isolating total DNA using a standard phenol
extraction.

\subsection*{Immunoprecipitation, SDS-PAGE, Western
blotting}

{\label{803879}}

Cells were lysed in lysis buffer (50 mM Tris, 150 mM KCl, EDTA 1 mM,
NP40 1\%, nicotinamide 5mM, Na-butyrate 1 mM, protease inhibitors
pH7,4). Immunoprecipitation from cultured cells or tissue samples was
performed exactly as described (Canto et al., 2009). For western
blotting, proteins were separated by SDS-PAGE and transferred onto
nitrocellulose membranes. Anti-Acetyl-Lysine antibodies were from Cell
Signalling; FOXO1 and SOD2 antibodies were from Santa Cruz
Biotechnology; SIRT1 and PGC-1\selectlanguage{greek}α \selectlanguage{english}antibodies were from Millipore; Tubulin
and Acetyl-tubulin antibodies were from Sigma Inc. Antibodies for
mitochondrial markers were purchased from Mitosciences. Antibody
detection reactions were developed by enhanced chemiluminescence
(Amersham, Little Chalfont, UK).

\subsection*{NAD+, NADH and NAM
determination}

{\label{221814}}

NAD+ levels were determined using a commercial kit (Enzychrom, BioAssays
Systems, CA) and tissue samples were also measured by HPLC (Ramsey et
al., 2009). Other NAD metabolites were determined as previously
described (Yang and Sauve, 2006).

\subsection*{GPR109A -- calcium mobilization
assay}

{\label{834276}}

Ready-to-Assay GRP109A Nicotinic Acid Receptor Cells were used to
measure calcium mobilization as specified by the manufacturer
(Millipore). Calcium flux was determined using excitation at 340 and 380
nm in a fluorescence spectrophotometer (VictorX4, Perkin Elmer) in a 180
seconds time course, adding the ligand at 60 seconds. Internal
validation was made using 0,1\% Triton X-100 for total fluorophore
release and 10 mM EGTA to chelate free calcium. Similarly, GPR109A
specificity was internally validated using control cells devoid of
GRP109A overexpression.

\subsection*{Liver triglyceride
measurement}

{\label{340754}}

Liver triglycerides were measured from 20 mg of liver tissue using a
variation of the Folch method, as described (Bai et al., 2011a).

\subsection*{Statistics}

{\label{786362}}

For the statistical analysis of the animal studies, all data was
verified for normal distribution. To assess significance we performed
Student's t-test for independent samples. Values are expressed as mean \selectlanguage{ngerman}±
SD unless otherwise specified.

\section*{Highlights}

{\label{720075}}

\begin{itemize}
\tightlist
\item
  NR efficiently increases NAD+ levels in mammalian cells and tissues
\item
  NR supplementation increases SIRT1 and SIRT3 activities.
\item
  NR largely prevents the detrimental metabolic effects of high-fat
  feeding.
\item
  NR enhances mitochondrial function and endurance performance
\end{itemize}

\section*{Acknowledgements}

{\label{306063}}

This work was supported by grants of the École Polytechnique Fédérale de
Lausanne, Swiss National Science Foundation, the European Research
Council Ideas programme (Sirtuins; ERC-2008-AdG231-118) and the Velux
foundation. RHH has been supported by a Rubicon fellowship of the
Netherlands Organization for Scientific Research and by an AMC postdoc
fellowship. EP is funded by the Academy of Finland. JA is the Nestle
chair in energy metabolism. AAS received grants to support this research
from the Ellison Medical Foundation New Scholar Award 2007 and contract
C023832 from NY State Spinal Cord Injury Board. The authors thank Robert
Myers, Peter Meinke and Thomas Vogt at Merck Research Laboratories,
Rahway and Charles Thomas at Amazentis, Lausanne, for the kind gift of
NR, all the members of the Auwerx lab for inspiring discussions and
Graham Knott of the BioEM facility at EPFL for EM imaging.

\section*{References}

{\label{565520}}\par\null

\begin{itemize}
\tightlist
\item
  Ahn BH, Kim HS, Song S, Lee IH, Liu J, Vassilopoulos A, Deng CX,
  Finkel T. A role for the mitochondrial deacetylase Sirt3 in regulating
  energy homeostasis.~Proc Natl Acad Sci U S
  A.~2008;105:14447--14452.~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2567183/}{PMC
  free
  article}{]}~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/18794531}{PubMed}{]}
\item
  Bai P, Canto C, Brunyanszki A, Huber A, Szanto M, Cen Y, Yamamoto H,
  Houten SM, Kiss B, Oudart H, et al. PARP-2 regulates SIRT1 expression
  and whole-body energy expenditure.~Cell
  Metab.~2011a;13:450--460.~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3108571/}{PMC
  free
  article}{]}~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/21459329}{PubMed}{]}
\item
  Bai P, Canto C, Oudart H, Brunyanszki A, Cen Y, Thomas C, Yamamoto H,
  Huber A, Kiss B, Houtkooper RH, et al. PARP-1 inhibition increases
  mitochondrial metabolism through SIRT1 activation.~Cell
  Metab.~2011b;13:461--468.~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3086520/}{PMC
  free
  article}{]}~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/21459330}{PubMed}{]}
\item
  Barbosa MT, Soares SM, Novak CM, Sinclair D, Levine JA, Aksoy P, Chini
  EN. The enzyme CD38 (a NAD glycohydrolase, EC 3.2.2.5) is necessary
  for the development of diet-induced obesity.~FASEB
  J.~2007;21:3629--3639.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/17585054}{PubMed}{]}
\item
  Belenky P, Racette FG, Bogan KL, McClure JM, Smith JS, Brenner C.
  Nicotinamide riboside promotes Sir2 silencing and extends lifespan via
  Nrk and Urh1/Pnp1/Meu1 pathways to
  NAD+~Cell.~2007;129:473--484.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/17482543}{PubMed}{]}
\item
  Benyo Z, Gille A, Kero J, Csiky M, Suchankova MC, Nusing RM, Moers A,
  Pfeffer K, Offermanns S. GPR109A (PUMA-G/HM74A) mediates nicotinic
  acid-induced flushing.~J Clin
  Invest.~2005;115:3634--3640.~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1297235/}{PMC
  free
  article}{]}~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/16322797}{PubMed}{]}
\item
  Bieganowski P, Brenner C. Discoveries of nicotinamide riboside as a
  nutrient and conserved NRK genes establish a Preiss-Handler
  independent route to NAD+ in fungi and
  humans.~Cell.~2004;117:495--502.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/15137942}{PubMed}{]}
\item
  Bogan KL, Brenner C. Nicotinic acid, nicotinamide, and nicotinamide
  riboside: a molecular evaluation of NAD+ precursor vitamins in human
  nutrition.~Annu Rev
  Nutr.~2008;28:115--130.{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/18429699}{PubMed}{]}
\item
  Bordone L, Motta MC, Picard F, Robinson A, Jhala US, Apfeld J,
  McDonagh T, Lemieux M, McBurney M, Szilvasi A, et al. Sirt1 regulates
  insulin secretion by repressing UCP2 in pancreatic beta cells.~PLoS
  Biol.~2006;4:e31.~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1318478/}{PMC
  free
  article}{]}~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/16366736}{PubMed}{]}
\item
  Brunet A, Sweeney LB, Sturgill JF, Chua KF, Greer PL, Lin Y, Tran H,
  Ross SE, Mostoslavsky R, Cohen HY, et al. Stress-dependent regulation
  of FOXO transcription factors by the SIRT1
  deacetylase.~Science.~2004;303:2011--2015.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/14976264}{PubMed}{]}
\item
  Calnan DR, Brunet A. The FoxO
  code.~Oncogene.~2008;27:2276--2288.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/18391970}{PubMed}{]}
\item
  Canto C, Auwerx J. Targeting sirtuin 1 to improve metabolism: all you
  need is NAD+?~Pharmacol
  Rev.~2012;64:166--187.~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3616312/}{PMC
  free
  article}{]}~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/22106091}{PubMed}{]}
\item
  Canto C, Gerhart-Hines Z, Feige JN, Lagouge M, Noriega L, Milne JC,
  Elliott PJ, Puigserver P, Auwerx J. AMPK regulates energy expenditure
  by modulating NAD+ metabolism and SIRT1
  activity.~Nature.~2009;458:1056--1060.~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3616311/}{PMC
  free
  article}{]}~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/19262508}{PubMed}{]}
\item
  Champy MF, Selloum M, Zeitler V, Caradec C, Jung B, Rousseau S,
  Pouilly L, Sorg T, Auwerx J. Genetic background determines metabolic
  phenotypes in the mouse.~Mamm
  Genome.~2008;19:318--331.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/18392653}{PubMed}{]}
\item
  Hara N, Yamada K, Shibata T, Osago H, Tsuchiya M. Nicotinamide
  phosphoribosyltransferase/visfatin does not catalyze nicotinamide
  mononucleotide formation in blood plasma.~PLoS
  One.~2011;6:e22781.~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3149623/}{PMC
  free
  article}{]}~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/21826208}{PubMed}{]}
\item
  Hirschey MD, Shimazu T, Jing E, Grueter CA, Collins AM, Aouizerat B,
  Stancakova A, Goetzman E, Lam MM, Schwer B, et al. SIRT3 Deficiency
  and Mitochondrial Protein Hyperacetylation Accelerate the Development
  of the Metabolic Syndrome.~Mol
  Cell.~2011~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3563434/}{PMC
  free
  article}{]}{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/21856199}{PubMed}{]}
\item
  Houtkooper RH, Canto C, Wanders RJ, Auwerx J. The secret life of NAD+:
  an old metabolite controlling new metabolic signaling pathways.~Endocr
  Rev.~2010;31:194--223.~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2852209/}{PMC
  free
  article}{]}{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/20007326}{PubMed}{]}
\item
  Imai S, Armstrong CM, Kaeberlein M, Guarente L. Transcriptional
  silencing and longevity protein Sir2 is an NAD-dependent histone
  deacetylase.~Nature.~2000a;403:795--800.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/10693811}{PubMed}{]}
\item
  Imai S, Johnson FB, Marciniak RA, McVey M, Park PU, Guarente L. Sir2:
  an NAD-dependent histone deacetylase that connects chromatin
  silencing, metabolism, and aging.~Cold Spring Harb Symp Quant
  Biol.~2000b;65:297--302.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/12760043}{PubMed}{]}
\item
  Karpe F, Frayn KN. The nicotinic acid receptor--a new mechanism for an
  old
  drug.~Lancet.~2004;363:1892--1894.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/15183629}{PubMed}{]}
\item
  Kemper JK, Xiao Z, Ponugoti B, Miao J, Fang S, Kanamaluru D, Tsang S,
  Wu SY, Chiang CM, Veenstra TD. FXR acetylation is normally dynamically
  regulated by p300 and SIRT1 but constitutively elevated in metabolic
  disease states.~Cell
  Metab.~2009;10:392--404.~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785075/}{PMC
  free
  article}{]}{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/19883617}{PubMed}{]}
\item
  Lagouge M, Argmann C, Gerhart-Hines Z, Meziane H, Lerin C, Daussin F,
  Messadeq N, Milne J, Lambert P, Elliott P, et al. Resveratrol improves
  mitochondrial function and protects against metabolic disease by
  activating SIRT1 and
  PGC-1alpha.~Cell.~2006;127:1109--1122.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/17112576}{PubMed}{]}
\item
  Li J, Mayne R, Wu C. A novel muscle-specific beta 1 integrin binding
  protein (MIBP) that modulates myogenic differentiation.~J Cell
  Biol.~1999;147:1391--1398.~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2174245/}{PMC
  free
  article}{]}~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/10613898}{PubMed}{]}
\item
  Li X, Zhang S, Blander G, Tse JG, Krieger M, Guarente L. SIRT1
  deacetylates and positively regulates the nuclear receptor LXR.~Mol
  Cell.~2007;28:91--106.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/17936707}{PubMed}{]}
\item
  Mannella CA. Structure and dynamics of the mitochondrial inner
  membrane cristae.~Biochim Biophys
  Acta.~2006;1763:542--548.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/16730811}{PubMed}{]}
\item
  Nikiforov A, Dolle C, Niere M, Ziegler M. Pathways and subcellular
  compartmentation of NAD biosynthesis in human cells: from entry of
  extracellular precursors to mitochondrial NAD generation.~J Biol
  Chem.~2011;286:21767--21778.~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3122232/}{PMC
  free
  article}{]}~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/21504897}{PubMed}{]}
\item
  North BJ, Marshall BL, Borra MT, Denu JM, Verdin E. The human Sir2
  ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase.~Mol
  Cell.~2003;11:437--444.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/12620231}{PubMed}{]}
\item
  Pfluger PT, Herranz D, Velasco-Miguel S, Serrano M, Tschop MH. Sirt1
  protects against high-fat diet-induced metabolic damage.~Proc Natl
  Acad Sci U S
  A.~2008;105:9793--9798.~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2474520/}{PMC
  free
  article}{]}{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/18599449}{PubMed}{]}
\item
  Picard F, Gehin M, Annicotte J, Rocchi S, Champy MF, O'Malley BW,
  Chambon P, Auwerx J. SRC-1 and TIF2 control energy balance between
  white and brown adipose
  tissues.~Cell.~2002;111:931--941.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/12507421}{PubMed}{]}
\item
  Qiu X, Brown K, Hirschey MD, Verdin E, Chen D. Calorie restriction
  reduces oxidative stress by SIRT3-mediated SOD2 activation.~Cell
  Metab.~2010;12:662--667.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/21109198}{PubMed}{]}
\item
  Ramsey KM, Yoshino J, Brace CS, Abrassart D, Kobayashi Y, Marcheva B,
  Hong HK, Chong JL, Buhr ED, Lee C, et al. Circadian clock feedback
  cycle through NAMPT-mediated NAD+
  biosynthesis.~Science.~2009;324:651--654.~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738420/}{PMC
  free
  article}{]}~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/19299583}{PubMed}{]}
\item
  Revollo JR, Korner A, Mills KF, Satoh A, Wang T, Garten A, Dasgupta B,
  Sasaki Y, Wolberger C, Townsend RR, et al. Nampt/PBEF/Visfatin
  regulates insulin secretion in beta cells as a systemic NAD
  biosynthetic enzyme.~Cell
  Metab.~2007;6:363--375.~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2098698/}{PMC
  free
  article}{]}~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/17983582}{PubMed}{]}
\item
  Rodgers JT, Lerin C, Haas W, Gygi SP, Spiegelman BM, Puigserver P.
  Nutrient control of glucose homeostasis through a complex of
  PGC-1alpha and
  SIRT1.~Nature.~2005;434:113--118.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/15744310}{PubMed}{]}
\item
  Shin DJ, Campos JA, Gil G, Osborne TF. PGC-1alpha activates CYP7A1 and
  bile acid biosynthesis.~J Biol
  Chem.~2003;278:50047--50052.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/14522988}{PubMed}{]}
\item
  St-Pierre J, Lin J, Krauss S, Tarr PT, Yang R, Newgard CB, Spiegelman
  BM. Bioenergetic analysis of peroxisome proliferator-activated
  receptor gamma coactivators 1alpha and 1beta (PGC-1alpha and
  PGC-1beta) in muscle cells.~J Biol
  Chem.~2003;278:26597--26603.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/12734177}{PubMed}{]}
\item
  Tunaru S, Kero J, Schaub A, Wufka C, Blaukat A, Pfeffer K, Offermanns
  S. PUMA-G and HM74 are receptors for nicotinic acid and mediate its
  anti-lipolytic effect.~Nat
  Med.~2003;9:352--355.{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/12563315}{PubMed}{]}
\item
  Walker AK, Yang F, Jiang K, Ji JY, Watts JL, Purushotham A, Boss O,
  Hirsch ML, Ribich S, Smith JJ, et al. Conserved role of SIRT1
  orthologs in fasting-dependent inhibition of the lipid/cholesterol
  regulator SREBP.~Genes
  Dev.~2010;24:1403--1417.~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2895199/}{PMC
  free
  article}{]}~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/20595232}{PubMed}{]}
\item
  Yang H, Yang T, Baur JA, Perez E, Matsui T, Carmona JJ, Lamming DW,
  Souza-Pinto NC, Bohr VA, Rosenzweig A, et al. Nutrient-sensitive
  mitochondrial NAD+ levels dictate cell
  survival.~Cell.~2007a;130:1095--1107.~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3366687/}{PMC
  free
  article}{]}~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/17889652}{PubMed}{]}
\item
  Yang T, Chan NY, Sauve AA. Syntheses of nicotinamide riboside and
  derivatives: effective agents for increasing nicotinamide adenine
  dinucleotide concentrations in mammalian cells.~J Med
  Chem.~2007b;50:6458--6461.~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/18052316}{PubMed}{]}
\item
  Yang T, Sauve AA. NAD metabolism and sirtuins: metabolic regulation of
  protein deacetylation in stress and toxicity.~AAPS
  J.~2006;8:E632--643.~{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2751359/}{PMC
  free
  article}{]}~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/17233528}{PubMed}{]}
\item
  Yoshino J, Mills KF, Yoon MJ, Imai S. Nicotinamide mononucleotide, a
  key NAD(+) intermediate, treats the pathophysiology of diet- and
  age-induced diabetes in mice.~Cell
  Metab.~2011;14:528--536.{[}\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3204926/}{PMC
  free
  article}{]}~{[}\href{https://www.ncbi.nlm.nih.gov/pubmed/21982712}{PubMed}{]}
\end{itemize}

\selectlanguage{english}
\FloatBarrier
\end{document}

