NMN is an effective anti-aging intervention that could be translated to humans

NAD + availability decreases with age and in certain disease conditions. Nicotinamide mononucleotide (NMN), a key NAD + intermediate, has been shown to enhance NAD + biosynthesis and ameliorate various pathologies in mouse disease models. In this study, we conducted a 12-month-long NMN administration to regular chow-fed wild-type C57BL/6N mice during their normal aging. Orally administered NMN was quickly utilized to synthesize NAD + in tissues. Remarkably, NMN effectively mitigates age-associated physiological decline in mice. Without any obvious toxicity or deleterious effects, NMN suppressed age-associated body weight gain, enhanced energy metabolism, promoted physical activity, improved insulin sensitivity and plasma lipid profile, and ameliorated eye function and other pathophysiologies. Consistent with these phenotypes, NMN prevented age-associated gene expression changes in key metabolic organs and enhanced mitochondrial oxidative metabolism and mitonuclear protein imbalance in skeletal muscle. These effects of NMN highlight the preventive and therapeutic potential of NAD + intermediates as effective anti-aging interventions in humans.

To examine whether long-term administration of NMN shows preventive effects on age-associated pathophysiological changes, we treated regular chow-fed wild-type mice for 12 months with two different doses of NMN in their drinking water. We assessed a variety of functional traits, as well as long-term safety and toxicity, and found that NMN is remarkably capable of ameliorating age-associated physiological decline in mice. Our findings from this long-term administration study provide a proof of concept to develop NMN as an effective anti-aging compound that prevents age-associated physiological decline, hoping to translate the results to humans.

Interestingly, it has been demonstrated that enhancing NADbiosynthesis extends lifespan in yeast, worms, and flies (). In rodents and humans, a number of studies have reported that NADcontent declines with age in multiple organs, such as pancreas, adipose tissue, skeletal muscle, liver, skin, and brain (). Thus, enhancing NADbiosynthesis with NMN or NR is expected to provide significant preventive effects on various pathophysiological changes in the natural process of aging. To address this critical question, long-term administration studies need to be performed under normal conditions in wild-type mice.

In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences.

Nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), key NADintermediates in mammals, could be such candidates (). NMN is synthesized from nicotinamide (Nic), an amide form of vitamin B, and 5′-phosphoribosyl-pyrophosphate (PRPP) by nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in this particular NADbiosynthetic pathway (). NR is phosphorylated to NMN by nicotinamide riboside kinases (NRKs) (). Once NMN is synthesized, it is converted to NADby three NMN adenylyltransferases, NMNAT1-3. The short-term administration of either NMN or NR has been reported to have remarkable therapeutic effects on metabolic complications and other disease conditions. For example, we have shown that NMN ameliorates impairments in glucose-stimulated insulin secretion in aged wild-type mice and some genetic mouse models (). NMN treatment also significantly improves both insulin action and secretion in diet- and age-induced type 2 diabetic or obese mouse models (). Furthermore, NMN protects the heart from ischemia/reperfusion injury by preventing NADdecrease induced by ischemia (), maintains the neural stem/progenitor cell population, and restores skeletal muscle mitochondrial function and arterial function in aged mice (), ameliorates mitochondrial function, neural death, and cognitive function in Alzheimer’s disease rodent models (). NR is also able to ameliorate mitochondrial dysfunction in obese mouse models () and various mitochondrial disease models (), attenuate cognitive deterioration in Alzheimer’s disease model mice (), prevent DNA damage and hepatocellular carcinoma formation (), improve noise-induced hearing loss (), and maintain muscle stem cell function (). Collectively, these findings strongly suggest that enhancing NADbiosynthesis by administering NMN or NR is an efficient therapeutic intervention against many disease conditions ().

NAD(+) metabolism and the control of energy homeostasis: a balancing act between mitochondria and the nucleus.

Historically unprecedented worldwide trends in population aging are predicted to become an incessant burden on governmental healthcare finances (). To make the process of aging healthy and prevent expensive age-associated health problems, efforts to develop effective, affordable, anti-aging interventions have recently been intensified, leading to some promising compounds, such as metformin, rapamycin, and SIRT1 activators (). Whereas these compounds were originally developed as pharmaceutical drugs, some endogenous compounds might also have the potential to achieve healthy and productive lives even at a very old age ().

Finally, we found that NMN was indeed contained in some daily natural food sources ( Table 1 ). For example, vegetables such as edamame (immature soybeans), broccoli, cucumber, and cabbage contained 0.25–1.88 mg of NMN per 100 g. Fruits such as avocado and tomato also contained 0.26–1.60 mg/100 g. Raw beef meat and shrimp contained relatively low levels of NMN (0.06–0.42 mg/100 g). Given that human red blood cells contain ∼50 mg of NMN as a total (unpublished data), a physiologically relevant amount of NMN might be absorbed from various daily food sources to our body and help sustain NADbiosynthesis and many physiological functions throughout the body.

NMN was extracted from each food and measured by HPLC. Several different sources were examined for some foods, showing ranges of values.

In addition to metabolic functions, we assessed other pathophysiological changes in control and NMN-administered mice. The C57BL/6N mouse strain has been reported to carry the rd8 mutation, a single nucleotide deletion in the Crb1 gene (). This particular mutation causes the age-dependent accumulation of subretinal microglia and macrophages, which corresponds to the development of light-colored spots in the fundus of the eye (). Thus, we conducted fundus biomicroscopy for control and NMN-administered mice. Interestingly, whereas all five control C57BL/6N mice at 17 months of age showed many light-colored spots in their fundus, two and four out of five mice at 100 and 300 mg/kg/day doses, respectively, showed dramatic reduction in these spots, implicating that age-associated pathological changes caused by the rd8 mutation may be suppressed by NMN in C57BL/6N mice ( Figure 6 A). In order to assess the effects of long-term NMN administration on retinal response, we conducted electroretinography (ERG). In the ERG analysis, there was a significant interaction between stimulus and group (p = 0.009 from the two-way repeated-measures ANOVA) for the scotopic a wave, and NMN-administered mice at both doses showed significantly higher amplitudes at 0 and 5 db (p = 0.035 and 0.022 for the 300 mg/kg group at 0 and 5 db, respectively; p = 0.009 for the 100 mg/kg group at 5 db from the Dunnett’s T3 test in the one-way repeated-measures ANOVA within groups), suggesting that NMN is able to prevent the decline in rod cell function in aged C57BL/6N mice ( Figure 6 B). Furthermore, improvements for the scotopic b and phototic b waves, which represent Müller/bipolar cell function and cone cell function, respectively, were observed through entire ranges of stimulus in both 100 and 300 mg/kg/day groups ( Figures 6 C and 6D), although statistically significant interactions between stimulus and group were not achieved. Because the function of the lacrimal gland decreases with age in humans and rodents (), we also assessed tear production in control and NMN-administered mice with a modified Schirmer’s test. Remarkably, NMN increased tear production in a dose-dependent manner at 12 months of NMN administration ( Figure 6 E). The tear production observed in the 300 mg/kg/day group was comparable to the maximal tear production throughout the mouse lifespan. In addition to these effects of NMN on eye function, we detected small but significant increases in bone density in NMN-administered mice in a dose-dependent manner ( Figure 6 F). NMN administration at both doses also significantly decreased the number of neutrophils, whereas it increases the number of lymphocytes at the dose of 300 mg/kg/day ( Figures S2 D–S2E). These additional findings provide support for our notion that NMN brings significant anti-aging effects on a variety of age-associated pathophysiological changes.

(F) Bone mineral density (BMD) was evaluated by dual-energy X-ray absorptiometry (DEXA) and analyzed by one-way ANOVA (n = 4–5 per group). All values are presented as mean ± SEM. Asterisks indicate statistical significances compared to controls ( ∗ p < 0.05; ∗∗ p < 0.01).

(B–D) Scotopic a (B), scotopic b (C), and photopic b (D) waves were measured by electroretinography (ERG) and analyzed using two-way RANOVA with Dunnett’s T3 post hoc test (n = 10–24 per group).

(A) Representative fundus biomicroscopy photos from control, 100, and 300 mg/kg/day NMN-administered mice (n = 5 per group). Light-colored spots due to the rd8 mutation carried by C57BL/6N mice were seen in all five control mice. Two and four out of five mice at 100 and 300 mg/kg/day, respectively, showed dramatic reductions in these spots.

Eye function and bone density were analyzed after 12 months of NMN administration (at 17 months of age). Results from control and 100 and 300 mg/kg/day NMN-administered mice are shown in blue, green, and red, respectively.

The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes.

Given that skeletal muscle exhibited the most profound preventive effects of NMN, we conducted high-resolution respirometry on permeabilized skeletal muscles from control and 300 mg/kg/day NMN-treated mice. Skeletal muscle from NMN-treated mice showed significant enhancement of the maximum respiration rate induced by addition of a mitochondrial uncoupler FCCP and also a trend of increased mitochondrial oxidative metabolism stimulated by pyruvate, ADP, and succinate ( Figure 5 D), suggesting that NMN stimulates mitochondrial oxidative metabolism in skeletal muscle. Interestingly, NMN administration also increased the ratios of a mtDNA-encoded mitochondrial protein MTCO1 (cytochrome c oxidase subunit 1) and nuclear DNA-encoded mitochondrial proteins ATP5A (ATP synthase subunit 5α) or SDHB (succinate dehydrogenase complex subunit B) in mitochondrial extracts of skeletal muscle ( Figure 5 E), suggesting that NMN induces two critically correlated mitochondrial alterations, mitonuclear protein imbalance and mitochondrial oxidative metabolism, in skeletal muscle (). Taken together, these findings suggest that NMN mediates its anti-aging effect, at least in part, by preventing age-associated gene expression changes in a tissue-specific manner and also enhancing mitonuclear protein imbalance and mitochondrial oxidative metabolism in skeletal muscle.

Next, we conducted principal component analysis (PCA) on the entire gene sets to further examine the global effects of NMN administration on age-associated transcriptional changes ( Figure 5 C). The first principal component (PC1) explained 20%–23% of the variance within the datasets and appeared to separate the expression profiles of young mice (blue and green) from those of old mice (red and orange) in skeletal muscle, WAT, and liver. Interestingly, compared to aged control mice (red), NMN-administered mice (orange) exhibited a shift toward young mice (blue and green) along the PC1 axis, particularly in skeletal muscle, supporting our notion that NMN administration prevents age-associated transcriptional changes.

Assessments of metabolic parameters in NMN-administered mice revealed significant effects of NMN to mitigate age-associated physiological decline. To further evaluate these anti-aging effects of NMN on molecular events, we conducted microarray analyses and compared global gene expression profiles in key metabolic organs including skeletal muscle, WAT, and the liver between 6- and 12-month time points in each group of control and 300 mg/kg/day NMN-administered mice. Between 6 and 12 months of NMN treatment (11 and 17 months of age), 300, 360, and 513 genes were significantly changed in skeletal muscle, WAT, and the liver, respectively, in the control cohorts. Most of these genes were downregulated between these time points ( Figure 5 A). Remarkably, 76.3%, 73.1%, and 41.7% of the genes changed in skeletal muscle, WAT, and the liver of control mice, respectively, were not significantly altered in NMN-administered mice ( Figure 5 A), suggesting that NMN is able to prevent age-associated transcriptional changes in these three peripheral tissues. Nonetheless, among these three key metabolic tissues, no common genes were observed in top 20 genes based on the sum of the absolute values of Z ratios between two comparisons (Z difference) ( Figure S5 A), implying that the effect of NMN on transcription is tissue-specific. We also conducted parametric analysis of gene set enrichment (PAGE) and found that 55.5%, 54.4%, and 32.2% of biological pathways that show significant changes in skeletal muscle, WAT, and the liver of control mice, respectively, were not significantly altered in NMN-administered mice ( Figure 5 B). NMN affected diverse sets of biological pathways in these tissues, and no obvious common pathways were observed ( Figure S4 B). However, it should be noted that several WAT biological pathways related to immune function and inflammation were significantly upregulated in aged control mice, whereas these age-induced alterations were blunted in NMN-administered mice (e.g., CYTOKINE_ACTIVITY, LEUKOCYTE_ACTIVATION, IMMUNE_RESPONSE; Figure S5 B), suggesting that NMN administration ameliorated age-associated increase in adipose tissue inflammation, a hallmark of obesity and insulin resistance ().

(E) Protein levels of nuclear DNA-encoded and mtDNA-encoded mitochondrial proteins (ATP5A, UQCRC2, SDHB, and VDAC1 versus MTCO1, respectively) were measured in mitochondrial extracts from control and 300 mg/kg/day NMN-treated skeletal muscles at 6 months (left panel; n = 4). The protein ratios of MTCO1 to ATP5A or SDHB were calculated by quantitating signal intensities of each band (right panel). All values are presented as mean ± SEM.

(D) High-resolution respirometry was performed for permeabilized skeletal muscles from control (n = 7) and 300 mg/kg/day NMN-treated (NMN300; n = 9) mice at 6 months. Mitochondrial oxidative respiration was measured by addition of pyruvate, ADP, succinate, a mitochondrial uncoupler FCCP, and a complex I inhibitor rotenone.

(C) Principal component analysis (PCA) was performed on the entire gene sets. The x and y axes indicate the first and the second principal components (PC1 and PC2), respectively. All values are presented as mean ± SEM (n = 4).

(A and B) Microarray analysis was conducted to compare gene expression profiles of skeletal muscle, white adipose tissue (WAT), and the liver in control and 300 mg/kg/day NMN-administered mice at 6 and 12 months after NMN administration. Throughout the figure, upregulated genes are shown in red and downregulated genes are shown in blue (n = 4 per group). Heatmaps in (A) reveal changes in individual gene expression. Genes that were significantly changed in control mice were selected by the rank product method (false discovery ratios [FDR] < 0.05) and ordered based on Z ratios of control mice. NMN administration inhibits age-induced changes in gene expression of skeletal muscle (76.3% of 300 genes), WAT (73.1% of 360 genes), and liver (41.7% of 513 genes). Heatmaps in (B) are shown from the parametric analysis of gene-set enrichment (PAGE). Biological pathways that were significantly changed in control mice were selected by PAGE (p < 0.05) and ordered based on Z scores of control mice. NMN administration inhibits these age-induced changes in pathways of skeletal muscle (55.5% of 299 pathways), WAT (54.4% of 226 pathways), and liver (32.2% of 174 pathways).

We also compared plasma concentrations of cholesterol, triglycerides, and free fatty acids (FFAs) among three groups. Plasma concentrations of cholesterol and triglycerides showed similar changes over time. For plasma FFA levels, the interaction between time and group was statistically significant (p = 0.007 from the two-way repeated-measures ANOVA), and the 300 mg/kg/day group did not show any statistically significant increases over time, whereas the control and the 100 mg/kg/day groups showed significant increases over time (p < 0.01 from tests of within-subjects effects in the one-way repeated-measures ANOVA). Indeed, plasma FFA levels tended to be lower in the 100 and 300 mg/kg groups compared to those in the control group at 9 and 12 months of NMN treatment, although the differences at each time point did not reach statistical significance ( Figure 4 D). This tendency appears to be consistent with lower respiratory quotient ( Figure 3 C), lower intrahepatic triglyceride levels ( Figure 4 C), and greater insulin sensitivity ( Figure 4 A) in NMN-administered mice, compared to the body-weight matched control mice.

We assessed the effect of NMN on glucose and lipid metabolism by conducting intraperitoneal glucose and insulin tolerance tests (IPGTTs and ITTs, respectively) and measuring plasma lipid panels at 3, 6, 9, and 12 months of treatment. To eliminate the possible confounding effects induced by body weight differences between control and NMN-administered mice ( Figure 2 A), we evaluated these metabolic parameters in body weight-matched groups of control and NMN-administered mice. We did not observe any significant differences in glucose tolerance among the control and the 100 and 300 mg/kg/day NMN-administered groups through all time points ( Figure S4 A). No difference was observed in plasma insulin levels during IPGTTs among three groups ( Figure S4 B). Fasted insulin levels increased over time in all three groups. However, the 300 mg/kg/day NMN-administered group tended to show lower fasted insulin levels after 3–9 months of treatment ( Figure S4 C). Interestingly, fed insulin levels showed a clearer tendency of lower insulin levels in NMN-administered groups compared to control groups ( Figure S4 D), whereas fed blood glucose levels were not different among three groups ( Figure S4 E). Consistent with these insulin results, after 12 months of NMN administration (mice were 17 months old), NMN-administered mice showed significantly improved insulin sensitivity compared to the body weight-matched control group ( Figure 4 A). There was a statistically significant interaction between time and group (p = 0.023 from the Greenhouse-Geisser test in two-way repeated-measures ANOVA), and the linear dose-dependent effects were statistically significant or close to significance at the 30 min and 45 min time points, respectively (p = 0.026 and p = 0.061 in the one-way repeated-measures ANOVA with unweighted linear term). This improved insulin sensitivity in the 100 and 300 mg/kg groups became more evident when plotting percent glucose changes relative to the glucose levels at 0 min time point ( Figure 4 B), although the interaction between time and group did not reach statistical significance in this assessment (p = 0.091 from the Greenhouse-Geisser test in two-way repeated-measures ANOVA). Similar improvement of insulin sensitivity was still observed even when all mice were included ( Figure S4 F). Consistent with this improved insulin sensitivity, intrahepatic triglyceride levels, a surrogate of insulin resistance, were lower in both NMN-administered groups at 6 months after and in the 300 mg/kg/day group at 12 months after NMN administration ( Figure 4 C). These results suggest that long-term NMN administration can ameliorate age-associated decline in insulin sensitivity, independent of its effect on body weight.

All values are presented as mean ± SEM. Asterisks indicate statistical significances compared to controls using one-way ANOVA ( ∗ p < 0.05; ∗∗ p < 0.01).

(D) Plasma concentrations of cholesterol, triglycerides, and free fatty acids (FFA) were measured at 3, 6, 9, and 12 months of NMN treatment, after an overnight fast (n = 10–15 per group).

(C) Intrahepatic triglyceride levels were measured after an overnight fast from animals in each group at 6 and 12 months after NMN administration (n = 4–5 per group).

(B) Relative blood glucose levels were calculated for body weight-matched mice (n = 10–15 per group). Glucose levels at each time point are normalized to that at 0 min. AUCs are also shown at right.

(A) Blood glucose levels were measured at the indicated times in insulin tolerance tests for body weight-matched mice after a 4 hr fast (n = 10–15 per group). Areas under the curves (AUCs) are also shown at right.

Metabolic tests were performed after 12 months of their respective NMN doses. Results from control, 100, and 300 mg/kg/day NMN-administered mice are shown in blue, green, and red, respectively.

We also evaluated the general locomotor activity in control and NMN-administered mice at 12–15 months of age. Ambulations (whole-body movements) and rearing (vertical activity) were measured ( Figures 3 F and 3G). Compared to control mice, mice administered with 100 mg/kg/day NMN showed significantly higher hourly ambulations during the dark period, whereas mice administered with 300 mg/kg/day NMN showed slightly lower ambulations ( Figure 3 F). In rearing activity, there was no significant difference between control and 100 mg/kg/day groups ( Figure 3 G). However, the 300 mg/kg/day group exhibited decreases in rearing activity throughout the dark period ( Figure 3 G). Thus, whereas NMN administration can stimulate energy metabolism and general locomotor activity in aged mice, a lower dose appears to be optimal for these particular parameters.

Because NMN-administered mice showed higher food intake but lower body weight compared to control mice during the process of aging, we measured oxygen consumption, energy expenditure, and respiratory quotient at the 6- and 12-month time points for control, 100, and 300 mg/kg/day NMN-administered mice ( Figures 3 A–3E ). Oxygen consumption significantly increased in both 100 and 300 mg/kg/day groups during both light and dark periods ( Figure 3 A). Energy expenditure also showed significant increases through 24 hr in the 100 mg/kg/day group and during the light period in the 300 mg/kg/day group ( Figure 3 B). Respiratory quotient significantly decreased in both groups during both light and dark periods ( Figure 3 C), suggesting that NMN-administered mice switched their main energy source from glucose to fatty acids. Body temperature did not significantly change, although NMN-administered mice occasionally showed a tendency of higher body temperatures ( Figure S3 B). Interestingly, whereas oxygen consumption and energy expenditure significantly decreased, particularly during the dark period, from 6 months to 12 months in control mice, mice treated with NMN for 12 months were able to maintain both oxygen consumption and energy expenditure close to those of control mice at 6 months after NMN administration ( Figures 3 D and 3E). Taken together, these results strongly suggest that NMN has significant preventive effects against age-associated impairment in energy metabolism in regular chow-fed wild-type mice.

(F and G) Ambulations (F) and rearings (G) were measured for control (blue) and 100 (green) and 300 (red) mg/kg/day NMN-administered mice at 12–15 months of age. Hourly counts from 3 p.m. to 8 a.m. (left) and total counts during the dark time (6 p.m.–5 a.m., right) were presented. Red and green asterisks indicate statistically significant differences between 100 (green) or 300 (red) mg/kg/day NMN-administered and control mice by Wilcoxon matched-pairs singled-ranks test with Bonferroni adjusted p values ( ∗ p < 0.017, ∗∗ p < 0.003; n = 9–10 per group). Black bars (A–E) and gray shaded areas (F and G) represent the dark period.

(D and E) VO 2 (D) and EE (E) were compared between 6 and 12 months after NMN administration (6-month controls, black circles; 12-month controls, black triangles; 12-month 100 mg/kg/day NMN-administered mice, green triangles; 12-month 300 mg/kg/day NMN-administered mice, red triangles). Black, green, and red asterisks indicate statistically significant differences between controls at 6 and 12 months after NMN administration, between 100 mg/kg/day and control groups at 12 months, and between 300 mg/kg/day and control groups at 12 months of NMN treatment, respectively. Wilcoxon matched-pairs singled-ranks test with Bonferroni adjusted p values was used ( ∗ p < 0.01, ∗∗ p < 0.002; n = 5 per group).

(A–C) Oxygen consumption (VO 2 ) (A), energy expenditure (EE) (B), and respiratory quotient (RQ) (C) were measured after 12 months of NMN administration using indirect calorimetry (n = 5 per group). Red and green asterisks indicate statistically significant differences between 100 (green) or 300 (red) mg/kg/day NMN-administered and control mice by Wilcoxon matched-pairs singled-ranks test with Bonferroni adjusted p values ( ∗ p < 0.017, ∗∗ p < 0.003).

We next assessed a variety of physiological, biochemical, and molecular parameters in control and NMN-administered mice. We found that NMN administration significantly and dose-dependently suppressed age-associated body weight gain ( Figures 2 A and 2B ). There was a statistically highly significant interaction between time and group (p < 0.001 from the two-way repeated-measures ANOVA). Additionally, the linear dose-dependent effects were statistically significant at all time points through 4–12 months (p < 0.05 from the one-way repeated-measures ANOVA with the unweighted linear term) ( Figure 2 A). The average numbers of percent body weight reduction normalized to control mice were 4% and 9% in 100 and 300 mg/kg/day groups, respectively. This suppressive effect of NMN on age-associated body weight gain became more evident by plotting body weight gain in each group ( Figure 2 B). Again, the interaction between time and group was statistically highly significant (p < 0.001 from the two-way repeated-measures ANOVA), and the linear dose-dependent effects were significant at all points through 2–12 months (p < 0.01 from the one-way repeated-measures ANOVA with the unweighted linear term). At 12 months, the 300 mg/kg/day group tended to have a decreased fat mass and an increased lean mass compared to controls ( Figure S1 F). NMN-administered and control mice did not show any recognizable difference in body length ( Figure S2 A). Interestingly, when mice became older, NMN-administered mice were able to maintain higher levels of food and water consumption in a dose-dependent manner compared to control mice ( Figures 2 C and 2D). These results confirm that the effect of NMN on body weight was not due to a growth defect or loss of appetite. Furthermore, analyses of blood cell counts ( Figures S2 B–S2E), blood chemistry panels ( Figures S2 F–S2W), and urine ( Figure S2 X) did not detect any sign of obvious toxicity from NMN at either dose. No statistical difference was detected by the log-rank test in survival of mice over the entire intervention period between control, 100 and 300 mg/kg/day NMN-administered mouse cohorts. No obvious differences were observed for the causes of death, which included urinary tract obstruction, thrombosis, and myocardial infarction, between control and NMN-administered mice ( Figure S3 A). These results suggest that NMN administration can significantly suppress age-associated body weight gain in a dose-dependent manner in regular chow-fed mice, without showing any serious side effects during the entire 12-month intervention period.

(C and D) Food (C) and water intake (D) were measured throughout the 12-month intervention period. Symbol (‡) indicates statistically significant differences within control groups (p < 0.05); 6- and 11-month (dark gray and black bars, respectively) versus 1-month (white bar) in food intake, and 6- and 11-month (dark gray and black bars, respectively) versus 2-month (white bar) in water intake. Asterisks indicate statistically significant differences compared to controls at indicated time points ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 by ANOVA with Tukey post hoc test; n = 9–15 per group). All values are presented as mean ± SEM.

(A and B) Body weight (A) and body weight gain (B) were plotted for each group (n = 9–15 per group). Red arrows indicate the starting point for both the statistically significant differences between control and 300 mg/kg/day NMN groups (p < 0.001 from two-way repeated-measures ANOVA), and statistically significant dose-dependent effects (p < 0.05 [A] or p < 0.01 [B] from one-way repeated-measures ANOVA with the unweighted linear term).

Body weight and food and water intake were monitored throughout the 12-month intervention period in control (blue) and 100 (green) and 300 (red) mg/kg/day NMN-administered mice.

NMN-Administered Mice Exhibit Suppression of Age-Associated Weight Gain while Maintaining Food and Water Intake during the Process of Aging

Figure 2 NMN-Administered Mice Exhibit Suppression of Age-Associated Weight Gain while Maintaining Food and Water Intake during the Process of Aging

To determine the effects of long-term NMN administration on age-associated pathophysiologies, we performed a 12-month-long NMN administration study using regular chow-fed wild-type C57BL/6N mice ( Figures 1 D and S1 E). We tested two doses of NMN, 100 and 300 mg/kg/day, from 5 months to 17 months of age. During this period, we carefully monitored the tolerance of mice to NMN by measuring water intake in control and NMN-administered mice and found that NMN administration was well tolerated over 12 months (see below). This long-term oral administration regimen could cause very small increases in the steady-state levels of plasma or tissue NMN and NADwhen mice take a sip of NMN-containing water. However, it is technically very difficult to detect such small fluctuations of plasma or tissue NMN and NADlevels. Nonetheless, we observed a tendency of dose-dependent NADincrease over time in the liver and BAT, but not in other tissues including skeletal muscle and WAT ( Figure 1 E).

In our previous study, we showed that a bolus intraperitoneal administration of NMN (500 mg/kg body weight) increased tissue NMN and NADlevels within 15 min in the liver, pancreas, and white adipose tissue (WAT) in regular chow-fed wild-type mice (). To make long-term NMN administration possible, we decided to test lower doses, which could potentially be translatable to humans, and add it to drinking water. We confirmed that 93%–99% of NMN was maintained intact in drinking water at room temperature for 7–10 days ( Figure S1 A). We next administered NMN at a dose of 300 mg/kg body weight to mice by oral gavage and measured plasma NMN and hepatic NADlevels over 30 min. Plasma NMN levels exhibited a steep increase at 2.5 min, further increases from 5 to 10 min, and then went back to original levels at 15 min ( Figure 1 A), implicating very fast absorption in the gut. Consistent with this notion, hepatic NADlevels showed a steady increase from 15 to 30 min ( Figure 1 A). We also measured tissue NADlevels 60 min after oral gavage of NMN. Although differences did not reach statistical significance at this particular dose, relatively small increases in NADlevels were observed in the liver, skeletal muscle, and cortex of the brain, but not in WAT or brown adipose tissue (BAT) ( Figures 1 B and S1 B). To further confirm whether orally administered NMN is utilized to synthesize NADin tissues, we used doubly-labeled isotopic NMN (C13-D-NMN; Figure S1 C) and traced these labels in NADin the liver and soleus muscle by mass spectrometry. Interestingly, in the liver, after administering C13-D-NMN by oral gavage, we clearly detected doubly labeled NAD(C13-D-NAD) at 10 min, and the level of C13-D-NADfurther increased at 30 min ( Figures 1 C and S1 D). In the soleus muscle, we detected C13-D-NADat 30 min, but not at 10 min ( Figures 1 C and S1 D). These results suggest that orally administered NMN is quickly absorbed, efficiently transported into blood circulation, and immediately converted to NADin major metabolic tissues.

(E) NAD + was measured in the liver, skeletal muscle, white adipose tissue (WAT), and brown adipose tissue (BAT) of control (blue) and 100 (green) and 300 (red) mg/kg/day NMN-administered mice 6 months after NMN administration (at 11 months of age) (n = 4–5 per group). All values are presented as mean ± SEM.

(D) A scheme showing the long-term NMN administration and various analyses. NMN (100 or 300 mg/kg/day) was dissolved into the drinking water and administered ad libitum to C57BL/6N male mice for 12 months, starting at 5 months of age. Experiments were performed at intervals to document the changes over time, as indicated.

(C) Doubly-labeled isotopic NAD + (C13-D-NAD + ) were measured in the liver and soleus muscle by mass spectrometry at 10 and 30 min time points after orally administering doubly-labeled isotopic NMN (C13-D-NMN) (n = 3 at 10 min and n = 1 at 30 min). The areas under the curves of C13-D-NAD + were calculated by subtracting the background values of PBS controls.

(B) NAD + levels 1 hr after oral gavage (300 mg/kg) in the liver, skeletal muscle, and cortex of control (blue) and NMN-administered (red) mice (n = 10 mice per group).

(A) Plasma NMN (red circles) and liver NAD + (blue squares) levels were measured by HPLC after oral gavage (300 mg/kg) (n = 5–13 per time point). ∗∗ p < 0.01 compared to 0 min.

Three- to four-month-old C57BL/6N mice were given nicotinamide mononucleotide (NMN) either by oral gavage (300 mg/kg) or ad libitum in the drinking water (100 or 300 mg/kg/day).

Discussion

+ decline or NMN uptake in each tissue or organ might determine an optimal dose of NMN to restore each physiological function. Given that 100 mg/kg/day of NMN was able to mitigate most age-associated physiological declines in mice, an equivalent surface area dose for humans would be ∼8 mg/kg/day ( Freireich et al., 1966 Freireich E.J.

Gehan E.A.

Rall D.P.

Schmidt L.H.

Skipper H.E. Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey, and man. It should be noted that NMN administration did not generate any obvious toxicity, serious side effects, or increased mortality rate throughout the 12-month-long intervention period, suggesting the long-term safety of NMN. Nonetheless, an optimal dose of NMN to maximize its efficacy appears to differ depending on physiological functions. For example, whereas the effects of NMN on body weight gain, insulin sensitivity, tear production, and bone mineral density were dose-dependent, 100 mg/kg/day of NMN improved oxygen consumption, energy expenditure, and physical activity better than 300 mg/kg/day. For rod and cone photoreceptor function, both doses had similar effects. Indeed, we found that expression of Ox2r and Prdm13, two downstream genes in the SIRT1-mediated signaling pathway in the hypothalamus, exhibited significant decreases in the hypothalami of 300 mg/kg/day NMN-treated mice (our preliminary finding), which could partly explain some of the observed differences in the effects of NMN, particularly those on physical activity, between two tested doses. Additionally, the extent of age-dependent NADdecline or NMN uptake in each tissue or organ might determine an optimal dose of NMN to restore each physiological function. Given that 100 mg/kg/day of NMN was able to mitigate most age-associated physiological declines in mice, an equivalent surface area dose for humans would be ∼8 mg/kg/day (), providing hope to translate our findings to humans.

Houtkooper et al., 2013 Houtkooper R.H.

Mouchiroud L.

Ryu D.

Moullan N.

Katsyuba E.

Knott G.

Williams R.W.

Auwerx J. Mitonuclear protein imbalance as a conserved longevity mechanism. Mouchiroud et al., 2013 Mouchiroud L.

Houtkooper R.H.

Moullan N.

Katsyuba E.

Ryu D.

Cantó C.

Mottis A.

Jo Y.S.

Viswanathan M.

Schoonjans K.

et al. The NAD(+)/sirtuin pathway modulates longevity through activation of mitochondrial UPR and FOXO signaling. + biosynthesis and mitochondrial oxidative metabolism in human skeletal muscle ( van de Weijer et al., 2015 van de Weijer T.

Phielix E.

Bilet L.

Williams E.G.

Ropelle E.R.

Bierwagen A.

Livingstone R.

Nowotny P.

Sparks L.M.

Paglialunga S.

et al. Evidence for a direct effect of the NAD+ precursor acipimox on muscle mitochondrial function in humans. Satoh et al., 2013 Satoh A.

Brace C.S.

Rensing N.

Cliften P.

Wozniak D.F.

Herzog E.D.

Yamada K.A.

Imai S. Sirt1 extends life span and delays aging in mice through the regulation of Nk2 homeobox 1 in the DMH and LH. Yoon et al., 2015 Yoon M.J.

Yoshida M.

Johnson S.

Takikawa A.

Usui I.

Tobe K.

Nakagawa T.

Yoshino J.

Imai S. SIRT1-mediated eNAMPT secretion from adipose tissue regulates hypothalamic NAD+ and function in mice. Gomes et al., 2013 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.

et al. Declining NAD(+) induces a pseudohypoxic state disrupting nuclear-mitochondrial communication during aging. Yoshino et al., 2011 Yoshino J.

Mills K.F.

Yoon M.J.

Imai S. Nicotinamide mononucleotide, a key NAD(+) intermediate, treats the pathophysiology of diet- and age-induced diabetes in mice. Detailed molecular mechanisms responsible for the pleiotropic effects of NMN need to be investigated further. Nonetheless, our analyses of global gene expression profiles in skeletal muscle, WAT, and the liver revealed that NMN administration was able to prevent age-associated transcriptional changes in a tissue-specific manner. In particular, skeletal muscle exhibited the most profound preventive effects of NMN on age-associated transcriptional changes. Consistent with this finding, mitochondrial oxidative metabolism was significantly enhanced in NMN-treated skeletal muscle. Additionally, NMN-treated skeletal muscle showed enhanced mitonuclear protein imbalance (), which has recently been reported to correlate to enhanced NADbiosynthesis and mitochondrial oxidative metabolism in human skeletal muscle (). Thus, it is likely that skeletal muscle is one of the most sensitive target tissues for the anti-aging effects of NMN. One would also suspect that some of the anti-aging effects of NMN administration might be modified by the function of SIRT1 in the brain, particularly in the hypothalamus (). Other nuclear and mitochondrial sirtuins, such as SIRT6 and SIRT3-5, might mediate these pleiotropic effects of NMN in a tissue-dependent manner. It is also possible that target organs and mediators causing the effects of long-term NMN administration might not be the same as those causing the effects of acute NMN administration (). Therefore, further careful investigation will be necessary to identify major target organs and effector molecules for each effect of NMN administration at different time points.

+ in tissues. These in vivo pharmacokinetic data imply that there may be an effective transporter that directly uptakes NMN into cells and tissues. Similarly, it has been suggested that NR is directly transported into cells through nucleoside transporters ( Nikiforov et al., 2011 Nikiforov A.

Doelle C.

Niere M.

Ziegler M. Pathways and subcellular compartmentation of NAD biosynthesis in human cells: from entry of extracellular precursors to mitochondrial NAD generation. + precursors and thereby developing effective interventions for age-associated physiological decline by using NMN, NR, or their combination. Our present study clearly shows that NMN is quickly absorbed from the gut into blood circulation within 2–3 min and also cleared from blood circulation into tissues within 15 min. The isotopic tracing experiment with C13-D-NMN also confirmed this fast absorption of NMN and its immediate conversion to NADin tissues. These in vivo pharmacokinetic data imply that there may be an effective transporter that directly uptakes NMN into cells and tissues. Similarly, it has been suggested that NR is directly transported into cells through nucleoside transporters (). Taken together, studying in vivo pharmacokinetics and identifying precise mechanisms responsible of NMN and NR uptake will provide critical insight into understanding tissue preferences of these two distinct NADprecursors and thereby developing effective interventions for age-associated physiological decline by using NMN, NR, or their combination.

In conclusion, our long-term NMN administration study provides compelling support to an effective anti-aging intervention using NMN, a key NAD+ intermediate. Given that NMN is contained in a variety of food sources such as vegetables, fruits, and meat, it will be of great interest to translate our study from mice to humans and examine whether this endogenous compound, NMN, is also an effective intervention that prevents age-associated physiological decline in humans.