Dihydronicotinamide riboside is a potent NAD concentration enhancer in vitro and in vivo

Article abstract of DOI:10.1074/jbc.RA118.005772

Interest in pharmacological agents capable of increasing cellular NAD concentrations has stimulated investigations of nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). NR and NMN require large dosages for effect. Herein, we describe synthesis of dihydronicotinamide riboside (NRH) and the discovery that NRH is a potent NAD concentration-enhancing agent, which acts within as little as 1 h after administration to mammalian cells to increase NAD concentrations by 2.5–10-fold over control values. Comparisons with NR and NMN show that in every instance, NRH provides greater NAD increases at equivalent concentrations. NRH also provides substantial NAD increases in tissues when administered by intraperitoneal injection to C57BL/6J mice. NRH substantially increases NAD/NADH ratio in cultured cells and in liver and no induction of apoptotic markers or significant increases in lactate levels in cells. Cells treated with NRH are resistant to cell death caused by NAD-depleting genotoxins such as hydrogen peroxide and methylmethane sulfonate. Studies to identify its biochemical mechanism of action showed that it does not inhibit NAD consumption, suggesting that it acts as a biochemical precursor to NAD. Cell lysates possess an ATP-dependent kinase activity that efficiently converts NRH to the compound NMNH, but independent of Nrk1 or Nrk2. These studies identify a putative new metabolic pathway to NAD and a potent pharmacologic agent for NAD concentration enhancement in cells and tissues.

Full text of DOI:10.1074/jbc.RA118.005772

JBC Papers in Press. Published on April 4, 2019 as Manuscript RA118.005772
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA118.005772
NRH increases NAD+ concentrations in cells and tissues
Dihydronicotinamide riboside is a potent NAD+ concentration enhancer in vitro and in vivo
Yue Yang1, Farheen Sultana Mohammed1, Ning Zhang1 Anthony A. Sauve1
From the 1Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065
Running title: NRH increases NAD+ concentrations in cells and tissues
To whom correspondence should be addressed: Anthony A. Sauve 212-746-6224,
aas2004@med.cornell.edu
Keywords: NAD+, NRH, nicotinamide riboside, dihydronicotinamide riboside, NAD+ precursor
NAD+ metabolism is largely comprised of
ABSTRACT
biosynthetic, redox-active and “NAD+-consuming”
Interest in pharmacological agents capable
of increasing cellular NAD+ concentrations has
stimulated investigations of nicotinamide riboside
(NR) and nicotinamide mononucleotide (NMN).
NR and NMN require large dosages for effect.
pathways (1,2). In biosynthetic pathways NAD+ is
made from different NAD+ precursors. In redox
active pathways NAD+ is used to conduct hydride
equivalents between reductants and oxidants. In
NAD+-consuming pathways, NAD+ is consumed
via enzymes that transfer the ADPR moiety to
cellular nucleophiles (3,4). Reactions in each group
are plentiful in cells making NAD+ one of the most
versatile of cellular metabolites. NAD+ is required
in abundant amounts, existing at concentrations of
100-500 M in most mammalian cells and tissues
(See Table 1 for examples)(5). NAD+ metabolism
has become a topic of renewed interest in recent
years (6), notably focused upon the involvement of
ADPR transfer/sirtuin deacylation reactions in
mediating key cellular processes (7), such as DNA
repair (8), metabolic adjustment (8), apoptosis (5),
cell survival (6), stem cell fitness (6,9), and even
lifespan regulation (9-11).
Herein
we
describe
synthesis
of
dihydronicotinamide riboside (NRH) and the
discovery that NRH is a potent NAD+ concentration
enhancing agent, which acts within as little as 1 hr
after administration to mammalian cells to increase
NAD+ concentrations by 2.5-10 fold over control
values. Comparisons to NR and NMN show that in
every instance NRH provides greater NAD+
increases at equivalent concentrations. NRH also
provides substantial NAD+ increases in tissues
when administered by intraperitoneal injection
to C57BL/6J mice. NRH substantially increases
NAD+/NADH ratio in cultured cells and in liver,
and no induction of apoptotic markers or significant
increases in lactate levels in cells. Cells treated with
NRH are resistant to cell death caused by NAD+-
depleting genotoxins such as hydrogen peroxide
and methylmethane sulfonate. Studies to identify its
biochemical mechanism of action showed that it
does not inhibit NAD+ consumption suggesting it
acts as a biochemical precursor to NAD+. Cell
lysates possess an ATP-dependent kinase activity
which efficiently converts NRH to the compound
NMNH, but independent of Nrk1 or Nrk2. These
studies identify a putative new metabolic pathway
to NAD+, and a potent pharmacologic agent for
NAD+ concentration enhancement in cells and
tissues.
The substrate role of NAD+ for key
signaling enzymes has sparked investigations to
determine whether increased NAD+ concentrations
provide an impetus for cellular changes. Of interest
are genetic or pharmacologic interventions that can
significantly alter NAD+ concentrations, probing
for
downstream
effects
mediated
by
ADPribosyltransferases/sirtuins. This work has
provided evidence that increased NAD+
concentrations drive signaling programs leading to
key biochemical processes such as enhanced
mitochondrial biogenesis (12), protection against
fatty acid induced liver disease (13), improved
1

NRH increases NAD+ concentrations in cells and tissues
exercise performance (14), and reduced
neurodegeneration (15,16) in a variety of animal
models. In turn, these results have turned attention
to nicotinamide riboside (NR)(17-19) and
nicotinamide mono-nucleotide (NMN) (18,19) as
possible NAD+ precursors, with findings that these
agents are promising for raising cellular and tissue
NAD+ concentrations. Very recently a number of
clinical studies of these agents in humans have been
published (20-22), confirming their activities as
NAD+ concentration boosters in humans, and
setting the stage for further development as human
therapeutics (1,6).
compound was required, preferably by direct
chemical synthesis. We prepared the compound
from nicotinamide riboside triflate, via published
synthetic methodology developed by our laboratory
(17). NR(triflate) was reduced in aqueous solution
in 50 mM potassium phosphate pH 8.6 with 0.80
equivalents of sodium dithionite, followed by
immediate purification on C-18 resin to provide
purified NRH (Scheme 1, See Experimental
1
Procedures). The H NMR spectrum agreed with a
prior literature report for this compound (see
Supporting Information for 1H NMR and 13C NMR
spectra). Additional properties of NRH are
provided in the Supporting Information, including
its UV spectrum which is centered at 340 nm (See
Figure S1). The compound is strongly fluorescent,
(excitation wavelength = 340 nm), similar to
NADH. NRH has moderate chemical stability at pH
values below 7, with a half-life of decomposition of
3 hr at 37 °C at pH 6.0 (Figure S2). Consequently,
only freshly prepared NRH solutions were used in
all experiments.
There are limitations of the current set of
NAD+ concentration enhancers, including maximal
reported cellular effects for NAD+ enhancement of
270 percent (17), and requirements of high doses to
achieve beneficial effects in animal models of
disease with reported doses typically 250-1000
mg/kg in mice (14,23,24). These limitations
stimulated our laboratory to investigate other
chemical structures related to NR and NMN as
possible NAD+ precursors. Of interest to us was
dihydronicotinamide riboside (NRH) which has
never been investigated as a precursor in NAD+
metabolism, to our knowledge. Interestingly, an
enzymatic activity that preferentially uses NRH as
a substrate had been characterized previously. The
enzyme NQO2, a quinoneoxidoreductase, is known
to use NRH preferentially as the upstream
reductant, in a reaction which converts quinones to
dihydroquinones (25,26). However, the effects of
NRH in altering cellular NAD+ levels have
remained unexamined in the published literature. In
this manuscript we report properties of NRH as an
enhancer of cellular NAD+ concentrations. NRH is
effective in this respect in mammalian cells and in
mice tissues. NRH acts at equal or lower
concentrations than those observed for NR and
NMN and surpasses NR and NMN in biological
effect in vitro and in vivo. We identify a putative
biochemical mechanism of action wherein NRH
represents a starting point for a novel biosynthetic
pathway to NAD+
NAD+ Concentration Increases Caused by
Exposure to NRH To assess if NRH can be
absorbed by cells and change NAD+ concentrations
we exposed a number of mammalian cell lines,
including insulinogenic (INS1, MIN6), neuron-like
(F98, U87, LN229), muscle-like (C2C12), and
HEK293 cells with vehicle, 1 mM NR or 1 mM
NRH for time periods of 6 hr. Cellular NAD+
concentrations were determined in accord with our
published assay (27). Changes in NAD+
concentrations were 2.4-10 fold for cells exposed to
NRH, well above levels reported for any other
NAD+ precursor yet studied (Figure 1a). In each cell
line, NRH exceeded the NAD+ concentration
enhancement caused by NR by at least 2 fold, and
in all cases greatly exceeded the levels induced by
NMN (See Figure S3). These data establish that
NRH is effective in stimulating NAD+ increases
compound in cultured mammalian cells superior to
NR or NMN. To further confirm the NAD+
enhancement effect of NRH, we examined an
additional neuron-derived cell line (Neuro2a)
treated with NRH, which caused an approximately
10 fold increase in NAD+ concentrations compared
to control (Figure 1b), an enhancement for an NAD
precursor which is unprecedented to our
RESULTS
Preparation of NRH In order to better investigate
the properties of NRH (Scheme 1) as a potential
NAD+ precursor, a method of preparation of the
2

NRH increases NAD+ concentrations in cells and tissues
knowledge. To unequivocally confirm increased
NAD+ production, lysate obtained from Neuro2a
cells treated for 6 hr with NRH was analyzed using
HPLC to directly detect the increased NAD+
amount using an untreated control as a comparison
and employing an NAD+ standard to confirm
identity. As shown in Figure 1c, NRH substantially
increased the peak corresponding to NAD+ by
many-fold in the NRH treated lysate mixture, well
above the level for control. To validate our other
NAD+ measurements, we checked for possible
interference of NRH in the plate reader assay
typically employed (27), but minimal interference
from NRH was observed when the assay was
performed at relevant NRH concentrations (See
Supporting Information for detail). Thus, we can
conclude by two independent assays (HPLC and
plate reader NAD+ assay) that NAD+ concentrations
are substantially increased by NRH exposure to
cultured mammalian cells.
concentrations of NRH for a period of 6 hr. At the
lowest concentration of NRH tested, 100 M,
neuronal NAD+ concentrations were approximately
5 fold elevated over corresponding controls, and
saturation was reached at 500 M, where
concentrations reached 7.5 fold over untreated
controls. To further investigate if NRH can alter
sub-compartment NAD+ concentrations, we
measured mitochondrial NAD+ content which is
known to increase with some NAD+ concentration
enhancers, such as NR (14). This compartment had
increased NAD+ concentrations of at least 3.5 fold
in Neuro2a cells treated with NRH at 1 mM after 18
hr (Figure 2c).
Time courses for NRH action were also
performed. Thus, Neuro2a cells and HEK293 cells
were treated with NRH for different incubation
times then harvested and NAD+ contents assayed.
We determined that the NAD+ concentration
elevating effects were present as early as 1 hour in
HEK293 cells, with full effects evident at 6 hr
(Figure 2d). We saw a similar strong effect in
Neuro2a cells at 6 hr (Figure 2e). Robust NAD+
elevation caused by treatment of NRH remained
evident after 18 hr in HEK293 cells (Figure 2d), in
part due to persistence of the majority NRH in
media past a time of 18 hr (Figure S2).
A dose-response profile for NRH on NAD+
concentration was obtained in Neuro2a cells.
Concentrations of NRH in media were varied from
100 M to 2000 M for a period of 6 hours. NAD+
concentration increase reached greater than 10 fold
(Figure 2a). Maximal NAD+ increase was
determined by theoretical fit of the curve
[NAD+] = Bmax[NRH]/([NRH] + EC50) + B0
Lack of Toxicity and Rescue Effects in
Genotoxicity These large increases in NAD+
concentrations caused us to wonder about possible
toxicity to cells. We did not observe any appreciable
loss of cells in culture from exposure to 1 mM NRH
for 24 hr (Figure S4). For example, cell counts were
similar between controls and NRH treated cells for
Neuro2a although cell counts were slightly lower in
HEK293 cells after 24 hr. Trypan blue positive cells
were similar in both cases, not exceeding 3 percent,
and there were no significant differences in
apoptotic markers. Annexin V or Caspase 3/7 in NR
and NRH treated cells versus controls, indicating no
changes in apoptotic pathway activation (Figure
S4).
where Bmax is maximum biological effect
caused by NRH, EC50 is concentration to achieve
50 percent of Bmax and B0 is the untreated control
NAD+ concentration. The values obtained were
Bmax = 11200 pmol/106 cells and EC50 was found to
be 310 M. The value for B0 was 690 pmol/106
cells. According to the curve, the concentration of
NRH required to achieve 260 percent NAD+
concentration increase as compared to control was
determined to be 34 M, whereas 1 mM NR is
required to achieve the same increase in this cell
line (17), and NMN does not achieve doubling of
NAD+ concentration at 1 mM (Figure S3). These
data provide a relative potency increase of
approximately 30 fold versus NR for this cell line.
A dose-response profile was also determined for
primary neurons, obtained as previously described
(28). These cells were treated with increasing
NRH is a reductant and we considered that
it could potentially cause changes to NAD+/NADH
ratio. Thus, we measured the ratio after NRH
treatment. NAD+/NADH ratio is regulated in cells
and is typically highly oxidizing under most
3

NRH increases NAD+ concentrations in cells and tissues
physiological conditions (29). However, the effect
on this ratio upon increasing NAD+ concentrations
up to 10 fold from resting levels has not been
assessed, to our knowledge. Thus, we treated cells
for 6 hr with NRH and measured NAD+
which are insulin containing cells and sensitive to
HP. Cell survival went completely to zero in HP
treatment but was increased to nearly 50 percent in
HP + NRH (Figure 3b). We then compared NR
effects with NRH in Neuro2a cells at a lower
concentration
and
NADH
concentration
concentration of 250 µM, and found that NRH is
still protective, of not only NAD+ level, but in cell
survival, whereas, although NR treatment is also
effective, it does so much less well, in both
preservation of NAD+ content as well as cell
viability (Figure 3c).
independently (27) to determine absolute amounts
of NAD+ and absolute amounts of NADH. We
found that NAD+ concentrations were substantially
increased as expected, but NADH concentrations
were not increased proportionally (Figure 2e).
Thus, a consistent effect of NRH treatment is a net
increase in the NAD+/NADH ratio in 4 different
mammalian cell lines (Figure 2f). Interestingly,
Canto et al. reported (14) that NR-induced NAD+
increase in animal tissues caused an increased
NAD+/NADH ratio. As another way to identify
perturbation in NAD+/NADH ratio, we measured
lactate concentrations in cell culture. Both
intracellular lactate levels and extracellular lactate
were measured at times of 6 hr. We saw no
statistically significant increases in lactate in media,
and intracellular lactate levels were largely
unaffected, except in one instance, where
intracellular lactate was substantially depleted
within HEK293 cells (Figure S5). The preceding
results showed that mM concentrations of NRH are
well tolerated by cells, and that highly elevated
NAD+ levels are well tolerated and do not lead to
obvious toxic effects, at least over a 24 hr period.
Intrigued by the magnitude of NAD+
concentration enhancement we wondered if NRH
could improve cell survival under stressful
conditions that deplete NAD+. Thus, we examined
genotoxicity, which causes DNA damage and
activates polyADPribosylpolymerases and causes
NAD+ depletion (30). Strong genotoxicity can lead
cells to severely deplete NAD+, leading to cell death
(5). Thus, we treated Neuro2a cells with hydrogen
peroxide (HP) for a period of 6 hr and then counted
cells and measured NAD+ in samples either co-
treated with NRH (1 mM) or with vehicle. We
found that cell survival improved from 10 percent
(HP) to nearly 80 percent with HP + NRH treatment
(Figure 3a). In addition, NAD+ levels remained
highly elevated in HP + NRH treated cells, whereas
in HP cells NAD+ concentrations became
substantially depleted below control values (Figure
3). Similar results were obtained with INS1 cells,
To broaden these findings, we examined
the alkylative genotoxin methylmethane sulfonate
(MMS), which we have used in past studies (5), on
Neuro2a and HEK293 cells. Cell survival in
Neuro2a cells improved with NRH co-treatment
with MMS, with survival reaching near 100
percent, indicating full protection in MMS +NRH
treated samples. NAD+ concentrations plummeted
in this case, to below untreated control value, but
were still higher than MMS treatment alone (Figure
3d). In HEK293 cells, survival was improved
considerably from 20 percent (MMS) to nearly 80
percent (MMS+NRH; Figure 3e). Furthermore,
MMS + NRH treated cells had higher NAD+
concentrations versus MMS treatment alone. Thus,
challenge to cells with genotoxicity identifies
conditions where NRH substantially improves cell
survivability, concurrently protecting the cellular
NAD+ pool from depletion. Again, we compared
NR with NRH at 250 µM (Figure 3f), and found that
NR could not provide protection of cells at this
concentration, and only modestly improved NAD+
levels versus untreated controls, whereas NRH
provided statistically significant increases in cell
survival as well as increased NAD+ concentrations
versus corresponding MMS treated control.
The differences in NAD+ concentrations
observed at 6 hours in cells for the two genotoxins
appear to arise from differing lifetimes of the
genotoxins in cell culture. Peroxide is depleted
rapidly in cell culture (See standard curve and
depletions in Figure 3g and Figure 3h respectively).
Rapid depletion of peroxide allows rebound to the
NAD+ concentrations in NRH treated cells at 6
hours. On the other hand, MMS persists in cell
culture past 6 hours (See standard curve and
depletions in Figure 3i and Figure 3j respectively).
This stability explains persistent depletion of NAD+
4

NRH increases NAD+ concentrations in cells and tissues
concentrations at harvest, even in NRH treated
cells.
biosynthesis. Since NRH provides a substantially
higher accumulation effect than for other NAD+
precursors, it suggests possible involvement of
Possible Biochemical Mechanisms of NRH-
NAD+
multiple biosynthetic pathways converging to
NAD+. One putative pathway is nicotinamide
salvage. To investigate this pathway as a possible
player in NRH effects, we treated cells with FK866
(31), a known inhibitor of the enzyme nicotinamide
phosphoribosyltransferase (Nampt) which is
responsible for NAM recycling to nicotinamide
mononucleotide (NMN)(32). FK866 causes
depletion of NAD+ concentrations in both HEK293
(Figure 4b) and Neuro2a (Figure 4b) cells. Decline
in NAD+ is attributable to failure of released NAM
to be resynthesized into NAD+. Co-administration
of NRH with FK866 completely rescues the effect
of FK866 on cells. Moreover, FK866 has no effect
on either NRH induced NAD+ increases. Thus NRH
induced NAD+ concentration increases do not
apparently require nicotinamide recycling for the
Induced
Increases
to
In order to better understand the possible
mechanisms where NRH exposure to cells causes
NAD+ concentrations to accumulate, we sought to
determine if NAD+ turnover is inhibited by NRH
treatment. Therefore, we treated cells with
[carbonyl-14C]nicotinamide
(NAM)
which
becomes converted to [carbonyl-14C]NAD+ in cells
as shown by HPLC (0 time, Figure 4a). After
washing cells of residual radioactivity in media, we
exposed labeled cells to NRH or vehicle and
measured intracellular labeled NAD+ as a function
of time using HPLC and scintillation counting.
Extracellular radioactivity in the form of released
[carbonyl-14C]NAM was also determined at each
time point. Time dependent outcomes of the
experiment (NRH versus control) are expected as
follows: 1. No change in loss of label at each time
for NRH treated samples is indicative of no
inhibition of turnover by NRH. 2. Increased loss of
label at each time for NRH samples is indicative of
increased NAD+ turnover and no inhibition of
turnover by NRH and 3. Decreased label loss at
each time for NRH samples is indicative of
inhibition of NAD+ turnover by NRH. Radiolabeled
NAD+ acts as a reporter for the entire NAD+ pool,
as there is no expected isotope effect on turnover for
a carbonyl 14C-label. Exposure of radiolabeled
Neuro2a cells with 1 mM NRH resulted in rapid
loss of label from NAD+, with a half-life of less than
100 min (Figure 4a) whereas corresponding
controls not exposed to NRH lose label to NAD+
with an approximate half-life of 240 min (Figure
4a). Corresponding release of radioactivity to media
is correlated to label loss (Figure 4a). Thus,
exposure of cells to NRH accelerates radiolabel loss
from intracellular NAD+, which is consistent with
increased NAD+ reaction and degradation, i.e.
NAD+ consumption. This result provides evidence
that NRH does not inhibit NAD+ turnover. Thus,
inhibition of NAD+ consumption does not
obviously contribute to NRH-induced NAD+
concentration increase.
effect.
Similarly,
NR
increased
NAD+
concentrations and this effect was not very sensitive
to FK866 (Figure 4b), consistent with the
interpretation that NR utilizes a kinase dependent
pathway independent of Nampt to provide the
majority of its NAD+ biosynthesis. Thus, NRH
causes marked NAD+ increases even when Nampt
is inhibited, indicating a nicotinamide independent
and Nampt independent mechanism of action.
Next, we asked if it is possible that cells
possess an activity that can convert NRH to a
subsequent product, which could be on the
biosynthetic pathway to NAD+. We thus treated
Neuro2a cell lysates with ATP (2 mM) and NRH
(100 M) and monitored for formation of product.
We found that in the absence of ATP no reaction of
NRH was observed (data not shown), but in the
presence of ATP a new product was formed, of
identical retention time to the compound
dihydronicotinamide mono-nucleotide (Figure 4c
NMNH; prepared by degradation of NADH with
snake venom diesterase). To our knowledge, this
conversion of NRH to NMNH (Figure 4c, Neuro2a
lysate at 37° C: specific activity = 0.38 ± 0.05
pmol/min/µg protein. See Figure 5 for structure of
NMNH) has not previously been reported for cell
lysates and provides a putative first metabolic step
of NRH to NAD+.
Increased NAD+ consumption in the
presence of NAD+ accumulation in cells suggests
that NRH causes a marked increase in NAD+
5

NRH increases NAD+ concentrations in cells and tissues
One possible mechanism for the conversion
of NRH to NMNH could be action of known NR
dependent kinases, Nrk1 and/or Nrk2. To ascertain
whether NRH induction of NAD+ accumulation in
cells requires Nrk1, we obtained HAP1 wildtype
and Nrk1 KO cell lines (See Experimental
Procedures), and prepared protein lysates from
these cell lines. With these lysates, we measured
NR kinase activity by measurement of [14C-
carbonyl]NR conversion to [14C-carbonyl]NMN in
the presence of ATP. Conversion of NR to NMN in
wildtype HAP1 lysate was easily measureable, but
this activity was completely lost in corresponding
Nrk1 KO lysate (Figure 4d). However, both
wildtype and Nrk1 KO lysates retained NRH kinase
activity, as shown by formation of NMNH in the
elimination of Nrk1 or Nrk2 as possible kinases)
depicted in Figure 5 likely accounts for the majority
of biological action of NRH, and that Path B is not
required for NRH effects on NAD+ concentrations.
NRH in vivo Finally, in order to assess for
biological effects in mice, we treated C57BL/6J
mice by intraperitoneal injection with 1000 mg/kg
NRH, and assessed NAD+ contents in tissues at 4
hr. As can be observed in Figure 6, NAD+ contents
increased several fold in blood, brain, adipose, and
kidney, with a greater than 5 fold increase in liver
NAD+ concentrations at 4 hr (Figure 5a). Weakest
effects were observed in skeletal muscle, where
increases were above control, but not statistically
significant. Table 1 shows measured NAD+
contents and observed errors. As shown by the data,
NRH is an effective NAD+ concentration enhancer
in many animal tissues and organs upon single dose
administration. To assess for possible perturbation
of redox potential, we measured NADH content in
liver, which is the most profoundly affected organ
presence of ATP (Figure 4e).
The biological
activity of Nrk1 is known to regulate NR
conversion to NAD+ in some cell lines. In HAP1
this also appears to be the case. HAP1 WT cells
have statistically significant increase in NAD+
concentrations when treated with NR, increasing to
190 % of untreated control, whereas the
corresponding Nrk1 KO shows no effect for NR in
increasing NAD+ content (Figure 4f). Thus, Nrk1
is required for NR-induced NAD+ increase in this
cell line. On the other hand, WT and Nrk1 KO cell
lines are both able to respond to NRH treatment,
increasing 6.8 fold over untreated controls (Figure
4f). This result confirms that Nrk1 is not required
for NRH conversion to NAD+, consistent with
results showing Nrk1 is also not required for
conversion of NRH to NMNH (Figure 4e).
with NRH treatment.
We determined
a
NAD/NADH ratio 68 ± 10 in NRH treated liver.
In order to determine if NRH is more
effective than other NAD+ precursors in vivo, we
injected 250 mg/kg intraperitoneal doses of NRH or
NMN or NR or vehicle only and measured NAD+
contents in kidney and liver at 4 hr (Figure 5b). We
found that in kidney NR, NRH and NMN
statistically increased NAD+ contents above
control, with NRH providing the highest effect at
180 % with NR and NMN providing 159 and 156
% respectively. In liver, NRH increased NAD
concentration by 540 %, while NR and NMN
provided 132 % and 149 % respectively (Figure 5b).
Thus, NRH provides larger NAD+ increases than
NR and NMN at equivalent doses, and in liver
provides quite significant NAD+ increase at a low
dose, consistent with improved pharmacologic
potency versus other NAD+ precursors. These
findings, recapitulate observations made with cell
culture studies.
Finally,
we
investigated
whether
recombinant Nrk2 could phosphorylate NRH, and
found that despite changes in pH from pH =7-9,
recombinant Nrk2 could not measurably
phosphorylate NRH (rate <0.005 s-1) to the extent
observed by Neuro2a lysate (Figure 4g). On the
other hand, using [14C-carbonyl]NR as a substrate,
recombinant Nrk2 was clearly very active,
exhibiting an observed rate of 3.07 s-1 under the
same reaction conditions.
Collectively, these
experiments indicate that NRH is phosphorylated
by a kinase not previously recognized to participate
in NAD+ metabolism to convert to it to NMNH.
Moreover, NRH effects on NAD+ concentrations
appear to be independent of Nrk1 and Nrk2. These
results lead us to propose that Path A (with
DISCUSSION
The discovery of novel agents to increase
cellular NAD+ is of recent high interest in the
6

NRH increases NAD+ concentrations in cells and tissues
biomedical research community with a number of
ongoing clinical studies and several recent
published ones involving the newly discovered
NAD+ enhancing agents NR and NMN (18, 19). In
the current work the previously uninvestigated
compound NRH was synthesized, and determined
to be a potent and effective enhancer of NAD+
concentrations in cultured cells as well as primary
neurons. The maximal concentration enhancements
were many fold over control values, and reached
unprecedented levels of up to 10 fold in Neuro2a
cells and over 7 fold in cultured primary neurons.
These effects raise the bar for a pharmacological
agent of this kind. NRH was found to exceed NR
and NMN effects by several to many fold in this
respect, suggesting a unique mechanism of action.
Moreover in terms of potency, NRH achieved a
260% NAD+ concentration versus control in
Neuro2a cells at 34 M, whereas NR required 1
mM for this same effect (17), indicating that NRH
is approximately 30 times more potent than NR
under these conditions. Similar doubling of NAD+
concentrations occurred at NRH concentrations
below 100 M in primary neurons.
perturbations to NAD+/NADH ratio. Cell counts
were slightly decreased in HEK293 cells in 24 hr
incubations as determined by cell counts, but there
was no evidence for activation of apoptosis.
Moreover, NAD+/NADH ratio was markedly
changed. NRH treatment after 6 hr caused marked
increases in NAD+ accumulation in cultured cells,
but less pronounced increases to NADH
concentrations. Consequently, the accumulation of
NAD+ shifts the NAD+/NADH ratio higher, and
suggests a strong homeostasis that keeps NADH
concentrations relatively lower. At the highest
dosage tested, NAD+/NADH ratio in liver was
increased to 68 ± 10 with NRH treatment. Prior
reports in the literature for mouse liver
NAD+/NADH range from 1-10 (33) and in rat liver
about 2-8 (29). However, in rat liver free
NAD+/NADH is in the range 10-700 depending on
whether cytoplasm or mitochondria is measured
(29). We speculate that the higher NAD+/NADH
ratio with NRH treatment likely is due to shifting
the ratio toward the protein-unbound NAD+/NADH
ratio which is typically much higher than the
protein-bound ratio (29). This effect could be a
NRH
has
NAD+
concentration
consequence of saturation of protein binding sites
for NAD+ and NADH in liver at higher NAD+
concentrations, causing the overall ratio to move
higher. The ramifications of consistent increase of
NAD+/NADH ratio in cells and tissues is an open
question, although arguably it poises cells to
become more oxidative and may increase metabolic
capacity.
enhancement effects requiring 1-6 hours to occur in
cell culture and its effects can remain sustained for
up to 18 hr. Effects reaching doubling of NAD+
concentrations occurred in as fast as 1 hr (Figure
2d). Time courses for the effects of NAD+
precursors on intracellular NAD+ concentrations
have remained largely unreported. Dose response
profiles of NRH in both Neuro2a cells and primary
neurons indicate EC50s (defined in Figure 2
legend) between 100-300 M indicating a large
dynamic concentration range for NRH action. Cell
culture studies in at least 8 cell lines showed NRH
was also able to substantially raise NAD+
concentrations, and this activity of NRH was also
observed in vivo. NRH injected by intraperitoneal
administration increased NAD+ concentrations
many fold in a variety of mouse tissues (Table 1).
The dose-response profile for NRH in mouse or any
other animal remains to be established and is an
objective of future research in the laboratory.
Increasing cellular NAD+ pools by up to 10
fold suggests that NRH could compensate NAD+
depletion caused by genotoxic stress. Cells treated
with either peroxide or the alkylative genotoxin
MMS were depleted of cellular NAD+ and
experienced cell death. In contrast, cells treated
with toxin and NRH maintained higher NAD+
concentrations and experienced increased cell
survival. These studies provide proof of concept for
NRH as a cellular NAD+ protection agent and cell
survival agent. Differences in cell responses to the
two treatments appear to arise from lifetimes of
MMS and HP in media in the presence of cells.
MMS is persistent in media, and cells have depleted
NAD+ even after 6 hr treatment. HP treated cells
experience rapid depletion of HP in media , leading
to recovery in NAD+ homeostasis at 6 hr.
A concern with pharmacologic use of
NAD+ concentration enhancers is the prospect that
they may cause an undesired cell toxicity or cause
7

NRH increases NAD+ concentrations in cells and tissues
Nevertheless, NRH is effective in protection of cell
NAD+ and cell viability in both genotoxic
treatments and is more effective than NR in
HEK293 in peroxide treatments (Figure 3c) and
Neuro2a cells in MMS treatments (Figure 3f).
To our knowledge, previous research had
transporter, or transporters and is internalized intact
as NRH (Figure 5). NRH is then be acted upon by a
kinase which converts NRH to a putative species
NMNH (Path A). We showed that cell lysates
possess an ATP dependent activity capable of
converting NRH to NMNH. The identity of this
kinase is not Nrk1 as shown by use of HAP1
wildtype and HAP1 Nrk1 KO cells. KO cells lack
the ability to phosphorylate NR, and to respond to
NR in NAD+ increase. However, HAP1 Nrk1 KO
cells respond fully to NRH and respond
equivalently to HAP1 wildtype cells. Moreover, we
showed that recombinant Nrk2 cannot convert NRH
to NMNH, although it can readily convert NR to
NMN. The independence of NRH effects from
Nrk1 activity implies that NRH is not acting as a
precursor to NR (Path B, Figure 5) since Nrk1
regulates NR effects in HAP1 cells but is not
required for NRH effects in this cell line. Moreover,
there is virtually no remaining NR kinase activity in
HAP1 Nrk1 KO cells as measured by activity
assays (Figure 4d), implying poor to non-existent
Nrk2 activity in this cell line. The current data
indicates to us that an unidentified kinase is
involved in the activity of NRH, acting most likely
to form NMNH (Path A Figure 5) as demonstrated
for HAP1 and Neuro2a lysates. The identification
of this kinase and characterization of its role in
NRH mediated NAD+ biosynthesis is an objective
of future work by our laboratory.
not investigated NRH as an NAD+ precursor, and
prior work had only uncovered a substrate role for
NRH in the action of NQO2 (34). The biogenic
origin of NRH in cells and tissues is currently
unknown, and its occurrence in mammalian cells is
also undocumented. One possible downstream
metabolic fate of NRH is conversion to NMNH, as
demonstrated in cell lysates in this report. Studies
to understand how NRH augments cellular NAD+
concentrations revealed it is probably not inhibiting
NAD+ consumption. This was investigated by
measurement of loss of [carbonyl-14C]NAD+ in
Neuro2a cells treated with NRH or with vehicle. In
fact, NRH treated cells had increased NAD+
turnover, and this is likely because increased NAD+
concentrations drive increased cellular demand for
NAD+. The specific origin of this increased demand
for NAD+ is unclear, but could be due to sub-
saturation of enzymes that consume NAD+ as a
substrate. This idea has been postulated to account
for the actions of NAD+ precursors as drivers of
cellular adaptation.
Consequently, the simplest explanation of
NRH effects on NAD+ concentration is that it acts
as a biosynthetic precursor to NAD+ via very
efficient biosynthetic pathways. Figure 5 presents
likely metabolic pathways emanating from
extracellular NRH and converging upon NAD+.
Although it is possible NRH is converted outside of
cells to another species, we consider it unlikely that
oxidation to NR or further breakdown to
nicotinamide is responsible for NRH action, since
neither NR nor nicotinamide alone provide the
NAD+ concentration enhancement effects observed
for NRH. Moreover, breakdown of NRH to
nicotinamide in either extracellular or intracellular
compartments is apparently not required for NRH
effects. This can be inferred from lack of effect of
FK866, a potent inhibitor of Nampt and
nicotinamide recycling. It had no effect on NAD+
concentration increases induced by NRH.
In summary, these findings reveal a
powerful naturally occurring pharmacologic agent
that can raise NAD+ levels in mammalian cells and
tissues, providing an exceptional new tool to
investigate how changes in NAD+ metabolism can
alter cellular physiology.
EXPERIMENTAL PROCEDURES
Synthesis of dihydronicotinamide riboside
“NRH”
(1-((2R,3S,4R,5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-1,4-
dihydropyridine-3-carboxamide). In a flame dried
flask under an Argon atmosphere, Nicotinamide
riboside (NR) (100mg, 0.24 mmol) was added to
10 ml of 50 m M potassium phosphate pH = 8.5 at
o
0 C. After 5 minutes, 0.8 eq of sodium dithionate
(Na2S2O4) was added and then run reaction at 0 oC
for 30 minutes. (Progress of the reaction was
We hypothesize that NRH encountering
cell membranes is internalized, via an unknown
8

NRH increases NAD+ concentrations in cells and tissues
monitored by HPLC: 70% of starting material was
consumed after 30 minutes). The crude product was
purified by C-18 column using water as eluent to
obtain light yellow solid (2). Yield 70%.
Cell numbers were counted using hemocytometer
and trypan blue. The harvested cells were pelleted
at 3000 × g for 3 min. After removing the remaining
media, cells were lysed with 7% perchloric acid to
preserve NAD+, then neutralized with 2 M NaOH
and 500 mM K2HPO4. The cellular NAD+ levels
were measured as previously published (27).
Cell culture HEK293, Neuro2a, F98, U87, LN229
and C2C12 cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented
with 10% fetal bovine serum, 100 U/ml penicillin
and 100 µg/ml streptomycin. MIN6 cells were
cultured in DMEM with 15% fetal bovine serum,
100 U/ml penicillin and 100 µg/ml streptomycin.
INS1 cells were maintained in Roswell Park
Memorial Institute (RPMI)-1640 with 11.1 mmol/l
D-glucose supplemented with 10% fetal bovine
serum, 100 U/ml penicillin and 100 µg/ml
streptomycin, 10 mmol/l HEPES, 2 mmol/l L-
glutamine, 1 mmol/l sodium pyruvate, and 50
µmol/l 2-mercaptoethanol. Primary neurons were
harvested from the brains of neonatal rats and
plated for overnight. Human nicotinamide riboside
kinase (Nrk1) knockout HAP1 cell line and the
corresponding wildtype HAP1 cells were
purchased from Horizon Discovery, UK. HAP1
cells were cultured in Iscove's Modified Dulbecco's
Medium (IMDM) with 10% fetal bovine serum,
100 U/ml penicillin and 100 µg/ml streptomycin.
Cells were maintained in a humidified incubator
supplied with 5% CO2/95% air at 37°C.
NADH measurement To measure intracellular
NADH levels, cell pellets were harvested after
NRH treatment as previously described and lysed
in 0.1 M NaOH/1 mM EDTA then incubated at
80°C for 10 min to avoid NAD+ contamination. The
samples are then neutralized with 0.5 M HCl and
500 mM KH2PO4. The NADH levels were
measured as published (27).
Mitochondrial isolation
To measure the
individual NAD+ concentration in mitochondria,
Neuro2a cells were seeded in a 10 cm2 petri dish,
then treated with 1 mM NRH for overnight. The
cells were harvested with trypsin and pelleted by
spinning at 3000x g for 5 min. The mitochondrial
fractions were isolated using Mitochondrial
Isolation Kit for Mammalian Cells (Thermo
Scientific) according to manufacturer’s manual.
Protein concentrations were later measured using
Bradford assay for normalization. NAD+
concentrations were measured as reported (27).
Identification of NRH and NAD+ on HPLC
Samples containing NRH or NAD+ were injected
into EC 250/4.6 Nucleosil 100-5 C18 column on a
Hitachi Elite Lachrom HPLC system equipped with
Diode Array Detector L-2450. The C18 column
was eluted with 20 mM ammonium acetate at 1
mL/min for 25 min, then with 20 mM ammonium
acetate and 20% methanol for 20 min. NRH was
characterized by its peak at 340 nm, while NAD+
was characterized by its peak at 260 nm.
Hydrogen peroxide stability test Hydrogen
peroxide (HP) in growth media was measured by a
plate reader assay (Sigma Aldrich Fluorimetric
Hydrogen Peroxide Assay Kit MAK165). 100 M
HP was incubated with either HEK293 or Neuro2a
cells, both at 80-90% confluency. Media was
aliquoted at 0, 10, 20, 30, 40 and 60 min and
assayed by dilution into 96 well plates according to
manufacturers instructions. Curve fit of the points
is to the equation [Peroxide]= A1 + A2*exp(-kt)
where k is the observed rate constant, A1 is residual
concentration of peroxide at infinite time and A2 is
the starting peroxide concentration. A standard
curve was generated with known peroxide
concentrations to achieve quantitation for the
biological samples.
Cellular NAD+ measurement For NAD+
measurements, cells were seeded in 6-well plates
until they reached approximately 90% confluency.
Cells were treated with the desired concentrations
of NRH or NR or NMN from concentrated stocks
dissolved in water. Cells were harvested with
trypsin digestion after treatment time. Unless
indicated, the NRH treatment duration was 6 hr.
9

NRH increases NAD+ concentrations in cells and tissues
controls with no lysate or no ATP were also
incubated. The reactions were then ceased by
incubating at 60 C for 2 min and then spun by
Methyl methanesulfonate stability test
Growth medium containing 1 mM methylmethane
sulfonate (MMS) was incubated with 80%
confluent HEK293 cells or Neuro2a cells in 6 well
plates. At 0, 2 and 4 hr the concentration of MMS
in media was determined by mixing 50 L media
with 5 L 1 M nicotinamide. Samples were heated
at 80 °C for 10 min. The reaction was subsequently
diluted with 200 L water, then injected onto a C-
18 column on HPLC and eluted with 20 mM
ammonium acetate followed by 20 mM ammonium
acetate with 20% MeOH. N-methyl-nicotinamide
(260 nm detection) eluted before unreacted
nicotinamide and the peak area of N-
methylnicotinamide was quantified. A standard
curve with a range of MMS concentrations was
obtained similarly in order to quantitate samples.
o
microcentrifuge at maximum speed for 10 min at 4
oC to remove precipitated protein. Supernatant was
injected onto a C-18 column on an HPLC system
and eluted with 20 mM ammonium acetate. The
conversion of NRH to NMNH was characterized by
integration of peaks with detection wavelength of
340 nm. Authentic NMNH was prepared by
digestion of NADH with snake venom diesterase.
Authentic NMNH was used as a chromatographic
standard.
Synthesis of 14C-labeled NR To test NR
phosphorylation activity of enzymes, [carbonyl-
14C]NR was prepared from [carbonyl-14C]NAM
(Moravek Biochemicals). First, 5 µCi radioactive
NAM was added to 1 mM unlabeled NAM, 600 µM
NAD+ and 200 µM acetylated peptide JB12
(Rockefeller University Proteomics Resource
Center) in the presence of 1 M recombinant SirT1
protein in 100 L of 20 mM KH2PO4 pH = 7.
Reaction was incubated at 37°C for 1 hr and then
quenched with 5 % TFA. The [carbonyl-14C]NAD+
product was purified by injection onto a C-18
column on HPLC eluted with 0.1% TFA at 1
mL/min for 25 min, then with 0.1% TFA and 20%
MeOH for 20 min. The fraction containing
NAD+ turnover To measure the NAD+ turnover
rate in presence or absence of NRH, Neuro2a cells
were seeded into 6-well plate until ~90% confluent,
and treated with 700,000 CPM of 14C-NAM
(Movarek Biochemicals) per well overnight. Cells
were then washed with PBS twice and incubated
with or without 1 mM NRH. Both media and cells
were harvested at 0, 1.5, 3, 5 and 7 hr. Radioactivity
of media was directly counted by scintillation
counter (Beckman Coulter). Cells were trypinsized
and pelleted, then extracted with 7% perchloric acid
and injected onto HPLC. The NAD+ peak was
collected using the above mentioned elution system
and counted for radioactivity.
[carbonyl-14C]NAD+
was dried by vacuum.
[carbonyl-14C]NAD+ dissolved in 100 L of 20 mM
KH2PO4 pH = 7 and treated with snake venom
diesterase and alkaline phosphatase at 37°C for 2
hr. The reaction was quenched by addition of 5 %
TFA. The resulting [carbonyl-14C]NR was purified
by injection onto a C-18 column on HPLC and
collected and dried, similar to the procedure
described above. The concentration of [carbonyl-
14C]NR was determined by peak area and compared
to an authentic weighed NR standard. Specific
activity was determined by scintillation counting of
a known amount of [carbonyl-14C]NR.
FK866 effect on NAD+ levels To understand
effects of nicotinamide recycling on NRH action,
Neuro2a and HEK293 cells were treated with
vehicle or 20 nM FK866, an NAMPT inhibitor that
blocks the synthesis of NMN from nicotinamide.
Additional added components included vehicle, 1
mM NR or 1 mM NRH. Cells were harvested after
6 hr and NAD+ measured as previously described
(27).
Identification of NMNH on HPLC To measure
NRH kinase activity, 100 M NRH, 2 mM ATP and
5 mM Mg2+ were combined withNeuro2a protein
lysate (122 g protein) all prepared in 100 L RIPA
buffer (Amresco, Cat No. 653-100ml). Reactions
were incubated for 30 min at 37 oC. Corresponding
HAP1 cell lysate activity test Human Nrk1
sequence was disrupted in HAP1 cell line by 1 bp
insertion in exon 5 of transcript NM_017881 by
Horizon Discovery, UK and the mutation was
validated by sequencing result by the company.
10

NRH increases NAD+ concentrations in cells and tissues
Protein lysates from wildtype and human Nrk1
knockout HAP1 cells were extracted with RIPA
buffer and quantified by Bradford assay. To test the
lysate activity against NR, equal protein masses of
wildtype and Nrk1 knockout HAP1 lysates were
incubated with 100 µM NR containing 8,000 CPM
[carbonyl-14C]NR, 2 mM ATP, 5 mM Mg2+, and
150 mM KH2PO4 at pH 7. After incubation at 37°C
for 30 min, the reactions were quenched by adding
10% volume of 20% trifluoroacetic acid and
incubated on ice for 5 min. [carbonyl-14C]NMN and
[carbonyl-14C]NR were collected after injection
onto a C-18 column on HPLC eluted with 20 mM
ammonium acetate and 20% MeOH. Fractions
containing NMN and NR were counted separately
by scintillation counter. To test the lysate activity
against NRH, equal mass of cell lysates were
incubated with 2 mM NRH, 2 mM ATP, 5 mM
Mg2+, and 150 mM KH2PO4 at pH 9 at 37°C for 30
min. The protein in reaction was removed by
passing the reaction mixture through a 10K Amicon
Ultra centrifugal filter (Millipore). NMNH was
identified at 340 nm using HPLC system mentioned
above.
ATP including 8,000 CPM [carbonyl-14C]NR in the
presence of 150 mM KH2PO4 at pH7, 37°C for 1 hr.
NMN and NR were separated by HPLC and
counted by scintillation counter for 14C activity. For
activity with NRH, assays were performed as
above, except that pH was varied from 7-9. No NR
was added, and 1 mM NRH was used, and reactions
were monitored by HPLC as described previously.
NRH effects on NAD+ in vivo Eight
male C57BL/6J mice at 8 weeks old were
purchased from Charles River and were housed in
a polycarbonate cage under a 12-hour-light/-dark
cycle with free access to food and water. Then the
mice were randomly assigned to three groups and
received an intraperitoneal injection of either 1000
mg/kg NRH dissolved in PBS, or only PBS in
control group. After 4 hours, these mice were
subjected to cardiac puncture for blood collection
and sacrificed. Blood was collected into tubes
containing EDTA (BD Vacutainer) and centrifuged
for 20 min at 5000 × g at 4°C and then plasma was
collected and stored at −80°C. Liver, kidney, brain,
muscle, and epididymal adipose tissue were
harvested, immediately frozen in liquid nitrogen,
then stored at -80ºC until NAD+ analyses. To
understand the effect of NRH in comparison with
NMN and NR at a lower dosage, a similar
experiment was done with intraperitoneal injection
Human Nrk2 protein cloning and activity test
Human Nrk2 sequence (NM_017881.2) was cloned
into a pET28a plasmid and sequenced. The plasmid
was transfected into BL21-Codon Plus cells (Cat
#230280 Agilent Technologies)
for protein
of one of the following 250 mg/kg NRH, 250 mg/kg
NR or 250 mg/kg NMN or PBS and the mice were
sacrificed after 4 hr for tissue collection. All
procedures were approved by the Institutional
Animal Care and Use Committee of Weill Cornell
Medicine. For NAD+ analysis, ~100 mg frozen
tissue was pulverized in liquid nitrogen and
homogenized in 7% perchloric acid by sonication,
then the solution was neutralized and subjected to
NAD+ measurement as described above. For
NADH measurement, ~100 mg frozen tissue was
pulverized in liquid nitrogen and homogenized in
water by sonication, then heated at 80 ºC for 2 min.
The extractions were then centrifuged and injected
onto HPLC. NADH peak was quantified at 340 nm
and the concentration determined by an NADH
standard.
expression. Induction of cultures at OD600= 0.5 at
37 °C was followed by 4 hours continued growth,
followed by centrifugation to pellet. Pellets were
resuspended in 75 mM KH2PO4 pH = 7 (5 volumes)
and lysozyme and DNase added to break cells and
digest DNA. After 3 freeze-thaw cycles, lysates
were clarified by centrifugation and added to Ni-
agarose. His-tagged Nrk2 protein was eluted with
250 mM of imidazole, 75 mM KH2PO4, 200 mM
NaCl pH 7.0. Bradford assay and silver staining
were used to quantify the protein concentration and
to visualize the purity of protein. Protein was
combined with 15% glycerol, 1 mM DTT and
frozen in aliquots at -80 °C until use. To determine
protein activity, 100 nM Nrk2 protein was
incubated with 500 µM NR, 5 mM Mg2+, 2 mM
11

NRH increases NAD+ concentrations in cells and tissues
Acknowledgments: This work was funded in part by NIH grant GM R01-106072 (AAS) and from NY
Spinal Cord Research Board Contract C32098GG (AAS).
Conflict of interest: The authors Anthony A Sauve, Yue Yang and Farheen Sultana Mohammed have filed
a patent on aspects of this work in conjunction with Cornell University. Anthony A Sauve has intellectual
property related to NR and derivatives of NR. Chromadex Inc of Irvine California has a license on
intellectual property related to production and uses of NR. Anthony A Sauve is a consultant and a co-
founder of Metro MidAtlantic Biotech LLC and Metro International Biotech LLC.
12

NRH increases NAD+ concentrations in cells and tissues
REFERENCES
1.
2.
3.
Yang, Y., and Sauve, A. A. (2016) NAD+ metabolism: Bioenergetics, signaling and
manipulation for therapy. Biochimica et biophysica acta 1864, 1787-1800
Belenky, P., Bogan, K. L., and Brenner, C. (2007) NAD+ metabolism in health and disease.
Trends in biochemical sciences 32, 12-19
Canto, C., Menzies, K. J., and Auwerx, J. (2015) NAD(+) Metabolism and the Control of
Energy Homeostasis: A Balancing Act between Mitochondria and the Nucleus. Cell
metabolism 22, 31-53
4.
5.
Hassa, P. O., Haenni, S. S., Elser, M., and Hottiger, M. O. (2006) Nuclear ADP-
ribosylation reactions in mammalian cells: where are we today and where are we going?
Microbiology and molecular biology reviews : MMBR 70, 789-829
Yang, H., Yang, T., Baur, J. A., Perez, E., Matsui, T., Carmona, J. J., Lamming, D. W.,
Souza-Pinto, N. C., Bohr, V. A., Rosenzweig, A., de Cabo, R., Sauve, A. A., and Sinclair,
D. A. (2007) Nutrient-sensitive mitochondrial NAD+ levels dictate cell survival. Cell 130,
1095-1107
6.
7.
8.
Katsyuba, E., and Auwerx, J. (2017) Modulating NAD(+) metabolism, from bench to
bedside. The EMBO journal 36, 2670-2683
Sauve, A. A. (2008) NAD+ and vitamin B3: from metabolism to therapies. The Journal of
pharmacology and experimental therapeutics 324, 883-893
Scheibye-Knudsen, M., Mitchell, S. J., Fang, E. F., Iyama, T., Ward, T., Wang, J., Dunn,
C. A., Singh, N., Veith, S., Hasan-Olive, M. M., Mangerich, A., Wilson, M. A., Mattson,
M. P., Bergersen, L. H., Cogger, V. C., Warren, A., Le Couteur, D. G., Moaddel, R.,
Wilson, D. M., 3rd, Croteau, D. L., de Cabo, R., and Bohr, V. A. (2014) A high-fat diet
and NAD(+) activate Sirt1 to rescue premature aging in cockayne syndrome. Cell
metabolism 20, 840-855
9.
Zhu, X. H., Lu, M., Lee, B. Y., Ugurbil, K., and Chen, W. (2015) In vivo NAD assay
reveals the intracellular NAD contents and redox state in healthy human brain and their
age dependences. Proceedings of the National Academy of Sciences of the United States of
America 112, 2876-2881
10.
11.
Verdin, E. (2015) NAD(+) in aging, metabolism, and neurodegeneration. Science 350,
1208-1213
Mouchiroud, L., Houtkooper, R. H., and Auwerx, J. (2013) NAD(+) metabolism: a
therapeutic target for age-related metabolic disease. Critical reviews in biochemistry and
molecular biology 48, 397-408
12.
Ryu, D., Zhang, H., Ropelle, E. R., Sorrentino, V., Mazala, D. A., Mouchiroud, L.,
Marshall, P. L., Campbell, M. D., Ali, A. S., Knowels, G. M., Bellemin, S., Iyer, S. R.,
Wang, X., Gariani, K., Sauve, A. A., Canto, C., Conley, K. E., Walter, L., Lovering, R. M.,
Chin, E. R., Jasmin, B. J., Marcinek, D. J., Menzies, K. J., and Auwerx, J. (2016) NAD+
repletion improves muscle function in muscular dystrophy and counters global
PARylation. Science translational medicine 8, 361ra139
13.
Gariani, K., Menzies, K. J., Ryu, D., Wegner, C. J., Wang, X., Ropelle, E. R., Moullan, N.,
Zhang, H., Perino, A., Lemos, V., Kim, B., Park, Y. K., Piersigilli, A., Pham, T. X., Yang,
13

NRH increases NAD+ concentrations in cells and tissues
Y., Ku, C. S., Koo, S. I., Fomitchova, A., Canto, C., Schoonjans, K., Sauve, A. A., Lee, J.
Y., and Auwerx, J. (2016) Eliciting the mitochondrial unfolded protein response by
nicotinamide adenine dinucleotide repletion reverses fatty liver disease in mice.
Hepatology 63, 1190-1204
14.
Canto, C., Houtkooper, R. H., Pirinen, E., Youn, D. Y., Oosterveer, M. H., Cen, Y.,
Fernandez-Marcos, P. J., Yamamoto, H., Andreux, P. A., Cettour-Rose, P., Gademann, K.,
Rinsch, C., Schoonjans, K., Sauve, A. A., and Auwerx, J. (2012) The NAD(+) precursor
nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-
induced obesity. Cell metabolism 15, 838-847
15.
16.
Langley, B., and Sauve, A. (2013) Sirtuin deacetylases as therapeutic targets in the nervous
system. Neurotherapeutics : the journal of the American Society for Experimental
NeuroTherapeutics 10, 605-620
Qin, W., Yang, T., Ho, L., Zhao, Z., Wang, J., Chen, L., Zhao, W., Thiyagarajan, M.,
MacGrogan, D., Rodgers, J. T., Puigserver, P., Sadoshima, J., Deng, H., Pedrini, S., Gandy,
S., Sauve, A. A., and Pasinetti, G. M. (2006) Neuronal SIRT1 activation as a novel
mechanism underlying the prevention of Alzheimer disease amyloid neuropathology by
calorie restriction. The Journal of biological chemistry 281, 21745-21754
17.
Yang, T., Chan, N. Y., and Sauve, A. A. (2007) Syntheses of nicotinamide riboside and
derivatives: effective agents for increasing nicotinamide adenine dinucleotide
concentrations in mammalian cells. Journal of medicinal chemistry 50, 6458-6461
Yoshino, J., Baur, J. A., and Imai, S. I. (2018) NAD(+) Intermediates: The Biology and
Therapeutic Potential of NMN and NR. Cell metabolism 27, 513-528
Rajman, L., Chwalek, K., and Sinclair, D. A. (2018) Therapeutic Potential of NAD-
Boosting Molecules: The In Vivo Evidence. Cell metabolism 27, 529-547
Martens, C. R., Denman, B. A., Mazzo, M. R., Armstrong, M. L., Reisdorph, N., McQueen,
M. B., Chonchol, M., and Seals, D. R. (2018) Chronic nicotinamide riboside
supplementation is well-tolerated and elevates NAD(+) in healthy middle-aged and older
adults. Nature communications 9, 1286
18.
19.
20.
21.
Dellinger, R. W., Santos, S. R., Morris, M., Evans, M., Alminana, D., Guarente, L., and
Marcotulli, E. (2017) Repeat dose NRPT (nicotinamide riboside and pterostilbene)
increases NAD(+) levels in humans safely and sustainably: a randomized, double-blind,
placebo-controlled study. NPJ aging and mechanisms of disease 3, 17
22.
23.
Trammell, S. A., Schmidt, M. S., Weidemann, B. J., Redpath, P., Jaksch, F., Dellinger, R.
W., Li, Z., Abel, E. D., Migaud, M. E., and Brenner, C. (2016) Nicotinamide riboside is
uniquely and orally bioavailable in mice and humans. Nature communications 7, 12948
Gomes, A. P., Price, N. L., Ling, A. J., Moslehi, J. J., Montgomery, M. K., Rajman, L.,
White, J. P., Teodoro, J. S., Wrann, C. D., Hubbard, B. P., Mercken, E. M., Palmeira, C.
M., de Cabo, R., Rolo, A. P., Turner, N., Bell, E. L., and Sinclair, D. A. (2013) Declining
NAD(+) induces a pseudohypoxic state disrupting nuclear-mitochondrial communication
during aging. Cell 155, 1624-1638
24.
Gong, B., Pan, Y., Vempati, P., Zhao, W., Knable, L., Ho, L., Wang, J., Sastre, M., Ono,
K., Sauve, A. A., and Pasinetti, G. M. (2013) Nicotinamide riboside restores cognition
through an upregulation of proliferator-activated receptor-gamma coactivator 1alpha
14

NRH increases NAD+ concentrations in cells and tissues
regulated beta-secretase 1 degradation and mitochondrial gene expression in Alzheimer's
mouse models. Neurobiology of aging 34, 1581-1588
25.
26.
Riches, Z., Liu, Y., Berman, J. M., Walia, G., and Collier, A. C. (2017) The ontogeny and
population variability of human hepatic dihydronicotinamide riboside:quinone
oxidoreductase (NQO2). Journal of biochemical and molecular toxicology
Megarity, C. F., Gill, J. R., Caraher, M. C., Stratford, I. J., Nolan, K. A., and Timson, D. J.
(2014) The two common polymorphic forms of human NRH-quinone oxidoreductase 2
(NQO2) have different biochemical properties. FEBS letters 588, 1666-1672
Li, W., and Sauve, A. A. (2015) NAD(+) content and its role in mitochondria. Methods in
molecular biology 1241, 39-48
Brochier, C., Jones, J. I., Willis, D. E., and Langley, B. (2015) Poly(ADP-ribose)
polymerase 1 is a novel target to promote axonal regeneration. Proceedings of the National
Academy of Sciences of the United States of America 112, 15220-15225
Williamson, D. H., Lund, P., and Krebs, H. A. (1967) The redox state of free nicotinamide-
adenine dinucleotide in the cytoplasm and mitochondria of rat liver. The Biochemical
journal 103, 514-527
27.
28.
29.
30.
31.
Yang, T., and Sauve, A. A. (2006) NAD metabolism and sirtuins: metabolic regulation of
protein deacetylation in stress and toxicity. The AAPS journal 8, E632-643
Hasmann, M., and Schemainda, I. (2003) FK866, a highly specific noncompetitive
inhibitor of nicotinamide phosphoribosyltransferase, represents a novel mechanism for
induction of tumor cell apoptosis. Cancer research 63, 7436-7442
32.
33.
Wang, T., Zhang, X., Bheda, P., Revollo, J. R., Imai, S., and Wolberger, C. (2006)
Structure of Nampt/PBEF/visfatin, a mammalian NAD+ biosynthetic enzyme. Nature
structural & molecular biology 13, 661-662
Hayashida, S., Arimoto, A., Kuramoto, Y., Kozako, T., Honda, S., Shimeno, H., and Soeda,
S. (2010) Fasting promotes the expression of SIRT1, an NAD+ -dependent protein
deacetylase, via activation of PPARalpha in mice. Molecular and cellular biochemistry
339, 285-292
34.
Wu, K., Knox, R., Sun, X. Z., Joseph, P., Jaiswal, A. K., Zhang, D., Deng, P. S., and Chen,
S. (1997) Catalytic properties of NAD(P)H:quinone oxidoreductase-2 (NQO2), a
dihydronicotinamide riboside dependent oxidoreductase. Archives of biochemistry and
biophysics 347, 221-228
15

NRH increases NAD+ concentrations in cells and tissues
ABBREVIATIONS AND FOOTNOTES
NAD+, nicotinamide adenine dinucleotide; NR, nicotinamide riboside; NRH,
dihydronicotinamide riboside; NMNH, dihydronicotinamide mononucleotide .
16

NRH increases NAD+ concentrations in cells and tissues
Table 1. Tissue NAD+ values in Ctrl and NRH treated C57/B6 mice
NAD+ concentration (pmol/mg weight)
Tissue
Ctrl
NRH
Blood
23.9 ± 2.2
834.9 ± 130.7
590.8 ± 83.9
51.0 ± 13.5
274.3 ± 11.6
15.2 ± 5.9
57.0 ± 4.6
Liver
4488.1 ± 142.0
1833.1 ± 148.9
112.9 ± 17.3
338.3 ± 91.9
41.3 ± 2.7
Kidney
Brain
Muscle
Adipose
Data are presented as mean ± SEM.
17

NRH increases NAD+ concentrations in cells and tissues
FIGURE AND SCHEME LEGENDS
Scheme 1. Synthesis of dihydronicotinamide riboside (NRH) from nicotinamide riboside
triflate.
Figure 1. NAD+ increases in different mammalian cell lines. (a) F98, U87, LN229,
HEK293, INS1, MIN6 and C2C12 cells were treated with either 1 mM NRH or 1 mM NR for 6
hr. Their cellular NAD+ changes are expressed as fold over control. Data are expressed as mean ±
SEM, n=6. (b) NRH induces NAD+ increase in Neuro2a cells. The cellular NAD+ content of
Neuro2a cells treated with either 1 mM NRH or 1 mM NR for 6 hr. Data are expressed as mean ±
SEM, n=4. **** indicates p<0.0001. (c) NAD+ peaks in HPLC chromatograms of control Neuro2a
cell lysates or cell lysates treated with 1 mM NRH or 1 mM NR. The absorbance is shown at 260
nm. Upper right panel shows representative spectrum of NAD+ peak.
Figure 2. Effects of NRH in different scenarios (a) Dose-response of NRH in Neuro2a
cells. Neuro2a cells were incubated with 100, 200, 500, 1000 and 2000 µM for 6 hour. Fit is
described in text. (b) Dose-response of NRH in primary neurons extracted from neonatal rat brains.
Primary neuron cells were incubated with 100, 250, 500, 750 and 1000 µM NRH for 6 hour. (c)
Mitochondrial NAD+ contents in Neuro2a cells treated with 1 mM NRH for 18 hr. Data are
expressed as mean ± SEM, n=4. (d) NRH increased NAD+ levels in a time dependent manner in
HEK293 and Neuro2a cells. HEK293 cells were incubated with 1 mM NRH for 1, 3, 6 and 18 hr.
Neuro2a cells were incubated with 1 mM NRH for 1 and 6 hr. Data are expressed as mean ± SEM,
n=4. (e) NAD+ and NADH values were measured side by side in Neuro2a, HEK293, INS1 and
C2C12 cells treated with 1 mM NRH for 6 hr. (f) shows NAD+/NADH ratios. Data are shown as
mean ± SEM, n=4. *, p<0.05; **, p<0.01, ***, p<0.001, ****, p<0.0001.
Figure 3. NRH protects cells from genotoxicity. (a) Neuro2a cells and (b) INS1 cells were
exposed to 500 µM hydrogen peroxide (HP) with or without the co-incubation of 1 mM NRH for
6 hr. (c) Neuro2a were treated with 500 µM HP in the presence either 250 µM NR or NRH for 6
hr. d) Neuro2a cells and (e) HEK293 cells were treated with 400 µM methylmethane sulfonate
(MMS) with or without the co-incubation of 1 mM NRH for 6 hr. (f) HEK293 cells were treated
with 400 µM MMS, with or without 250 µM NR or NRH. Their survival rate and cellular NAD+
content are expressed as mean ± SEM, n=4. Different letter indicates significant difference
18

NRH increases NAD+ concentrations in cells and tissues
(p<0.05) between groups. (g) Standard curve constructed with different concentrations of
hydrogen peroxide (HP). (h) stability of 100 M HP in media with cultured cells. Solid curve is
for HEK293 cells, and dashed curve is for Neuro2a cells. Fit provided k values of 0.094 min-1 for
Neuro2a cells and 0.160 min-1 for HEK293 cells. Maximal rate under the assay conditions was
determined to be 9.4 M min-1 at 100 M for Neuro2a cells, and 16 mM min-1 for HEK293 cells
corresponding to half-life of 7.25 min for Neuro2a cells and 4.33 min for HEK293 cells. (i) HPLC
assay to quantify MMS concentration and resulting standard curve. (j) The amount of MMS left
in media quantified with initial 1 mM MMS combined with HEK293 and Neuro2a cells at 0, 2 and
4 hr.
Figure 4. NRH conversion to NAD+ via a kinase mechanism. a) NAD+ turnover in Neuro2a
cells treated with or without NRH. Neuro2a cells were incubated with [carbonyl-14C]nicotinamide
overnight then treated with or without 1 mM NRH. Radioactivity was counted in both media and
NAD+ purified and counted at 0, 90, 180, 300 and 420 min after the treatment. b) FK866 did not
inhibit the NAD+ raising effect of NRH. HEK293 and Neuro2a cells were treated with the Nampt
inhibitor FK866 or vehicle with or without 1 mM NR or NRH for 6 hr. Data are expressed as mean
± SEM, n=4. Different letter indicates significant difference (p<0.05) between groups. c) NRH is
converted to NMNH by cell lysate. After incubation with Neuro2a protein lysate for 30 min at 37
oC, a new peak, detected at 340 nm wavelength on HPLC chromatography, is identified as NMNH.
Conditions are described in Experimental Procedures. Inset shows UV spectrum for this product.
d) Protein lysate from wildtype HAP1 cells can convert [carbonyl-14C]NR into [carbonyl-
14C]NMN, but the activity has been abolished in lysate from human Nrk1 knockout HAP1 cells.
e) When using NRH as substrate, both lysates from wildtype and human Nrk1 knockout HAP1
cells generate NMNH. f) Wildtype and human Nrk1 knockout HAP1 cells were treated with either
1 mM NR or NRH for 6 hr and measured for NAD+ content. Data are expressed as mean ± SEM,
n=4. Different letter indicates significant difference (p<0.05) between groups. g) Neuro2a protein
lysate produced NMNH from NRH in the presence of ATP. When using recombinant human Nrk2
protein in the reaction carried out in pH 7, 8 and 9 buffers, no significant NMNH peak can be
19

NRH increases NAD+ concentrations in cells and tissues
observed at 340 nm. The activity of recombinant human Nrk2 was validated using [carbonyl-
14C]NR as substrate, and compared to NRH activity quantified in the inset.
Figure 5. Illustration of putative pathways from extracellular NRH to NAD+. NRH may be
internalized via an unknown transporter as intact molecule, then is either directly phosphorylated
(Path A) or oxidized to NR (Path B). Phosphorylation of NRH generates putative species NMNH
(Path A), a putative intermediate, which would then be oxidized to NMN or further metabolized
by nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), Nmnat2 or Nmnat 3 to produce
NADH, then converge on NAD+.
Figure 6. NRH increased tissue NAD+ content in vivo. (a) After intraperitoneal injection
of 1000 mg/kg NRH in male C57BL/6J mice for 4 hours, NAD+ content in blood, liver, kidney,
brain and epididymal adipose tissue were increased compared to control group. Data are shown as
mean ± SEM, n=4, *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 when compared with
control. (b) Male C57BL/6J mice were intraperitoneal injected with one of 250 mg/kg NRH, 250
mg/kg NMN, 250 mg/kg NR or vehicle and then sacrificed at 4 hours. Liver and kidney NAD+
content were compared across groups. Data are presented as mean ± SEM, n=4. Different letter
indicates significant difference (p<0.05) between groups.
20

NRH increases NAD+ concentrations in cells and tissues
21

NRH increases NAD+ concentrations in cells and tissues
22

NRH increases NAD+ concentrations in cells and tissues
23

NRH increases NAD+ concentrations in cells and tissues
24

NRH increases NAD+ concentrations in cells and tissues
25

NRH increases NAD+ concentrations in cells and tissues
26

NRH increases NAD+ concentrations in cells and tissues
27

Dihydronicotinamide riboside is a potent NAD+ concentration enhancer in vitro and
in vivo
Yue Yang, Farheen Sultana Mohammed, Ning Zhang and Anthony A. Sauve
J. Biol. Chem. published online April 4, 2019
Access the most updated version of this article at doi: 10.1074/jbc.RA118.005772
Alerts:
• When this article is cited
• When a correction for this article is posted
Click here to choose from all of JBC's e-mail alerts