NAD+ Peptide (1000MG)

This 2016 study in Cell Metabolism (Mills et al., Washington University School of Medicine) gave nicotinamide mononucleotide (NMN), an NAD+ precursor, to wild-type mice in their drinking water for 12 months. Treatment ran from 5 to 17 months of age. The researchers compared treated mice with untreated controls on body weight, food intake, energy metabolism, activity, insulin sensitivity, plasma lipids, eye measures, bone density, blood counts, tissue gene expression and muscle mitochondrial function.

Important distinction: This study tested NMN, not NAD+. NMN is converted to NAD+ inside cells. Findings for NMN cannot be assumed to apply to NAD+ itself or to any other compound.

Scope: This was a study in male mice. It did not test NMN or NAD+ in people.

• Citation: Mills KF, Yoshida S, Stein LR, et al. Cell Metabolism. 2016;24(6):795–806.
• Compound tested: Nicotinamide mononucleotide (NMN)
• Animals: Male C57BL/6N mice on regular chow, treated from 5 to 17 months of age
• Groups: Control, 100 mg/kg/day NMN, and 300 mg/kg/day NMN in drinking water (about 9–15 mice per group for body weight)
• Short-term tests: Oral gavage with isotope-labeled NMN to trace its conversion to NAD+
• Funding: Sponsored research agreement with Oriental Yeast Co., which supplied the NMN and employed two co-authors

Earlier studies reported that NAD+ content falls with age in organs such as liver, muscle, fat and brain, and that short-term NMN or NR treatment changed outcomes in several mouse disease models. Most of that work was short-term. This study asked a different question: what happens with long-term administration in normal, healthy wild-type mice as they age?

The researchers first checked that NMN stayed 93–99% intact in drinking water for 7–10 days. In short-term tests, mice received NMN by oral gavage. Researchers tracked plasma NMN and liver NAD+, and they used isotope-labeled NMN to see whether labeled NAD+ appeared in liver and muscle.

For the long-term study, 5-month-old male mice received plain water or water containing NMN at 100 or 300 mg/kg/day for 12 months. These amounts describe the published mouse protocol only. Assessments included:

• Body weight, food and water intake, and body composition
• Oxygen consumption, energy expenditure and respiratory quotient by indirect calorimetry at 6 and 12 months
• Locomotor activity at 12–15 months of age
• Glucose and insulin tolerance tests, liver triglycerides and plasma lipids
• Blood counts, blood chemistry and urine for signs of toxicity
• Fundus imaging, electroretinography (ERG) and tear production
• Bone density
• Microarray gene expression in skeletal muscle, white fat and liver, and mitochondrial respiration in muscle

After oral gavage, plasma NMN rose within 2.5 minutes, and liver NAD+ rose between 15 and 30 minutes. Labeled NAD+ appeared in the liver within 10 minutes and in muscle by 30 minutes. The authors concluded that orally given NMN was quickly absorbed and used to make NAD+ in these tissues.

Over 12 months, NMN reduced age-associated body-weight gain in a dose-dependent way. Average body weight was about 4% (100 mg/kg/day) and 9% (300 mg/kg/day) lower than in controls. Treated mice ate and drank somewhat more than controls as they aged, so the lower weight was not explained by reduced food intake.

Oxygen consumption was higher in both NMN groups. Energy expenditure was higher over 24 hours in the 100 mg/kg/day group and during the light period in the 300 mg/kg/day group. A lower respiratory quotient suggested a shift toward fat as a fuel source. Between 6 and 12 months, oxygen consumption and energy expenditure declined in control mice but were largely maintained in NMN-treated mice.

Activity results differed by dose. The 100 mg/kg/day group showed more nighttime walking movement than controls. The 300 mg/kg/day group showed slightly less walking and less rearing.

After 12 months (age 17 months), NMN-treated mice showed better insulin tolerance than body-weight-matched controls. Liver triglycerides were lower in both NMN groups at 6 months and in the 300 mg/kg/day group at 12 months. Plasma free fatty acids rose over time in control and 100 mg/kg/day mice, but not in the 300 mg/kg/day group.

C57BL/6N mice carry the rd8 mutation, which causes light-colored spots on the retina with age. At 17 months, all five control mice examined had many such spots. Two of five mice in the 100 mg/kg/day group and four of five in the 300 mg/kg/day group showed marked reductions. On ERG, NMN-treated mice had higher scotopic a-wave amplitudes at some stimulus levels. NMN also increased tear production in a dose-dependent way.

The researchers also reported small, dose-dependent increases in bone density. Neutrophil counts were lower at both doses, and lymphocyte counts were higher at 300 mg/kg/day.

Between 6 and 12 months of treatment, control mice showed significant changes in 300 genes in skeletal muscle, 360 in white fat and 513 in liver. In NMN-treated mice, 76.3%, 73.1% and 41.7% of those genes, respectively, were not significantly altered. The top affected genes differed between tissues. The authors described the effect as tissue-specific.

In skeletal muscle, NMN-treated mice showed higher maximal mitochondrial respiration and a shifted ratio of mitochondrial-encoded to nuclear-encoded mitochondrial proteins.

Blood cell counts, blood chemistry and urine did not show signs of obvious toxicity at either dose. Survival over the treatment period did not differ significantly between groups. These observations apply to male C57BL/6N mice given NMN in drinking water under this protocol.

• Only male mice of one strain were studied, and several measures used small subgroups (for example, n = 4–5 per group for microarrays and calorimetry).
• Effects varied by dose, and the higher dose was not better on every measure (for example, physical activity).
• The study tested NMN. It did not test NAD+ or other NAD+-related compounds.
• The study was funded through a sponsored research agreement with the company that supplied the NMN.
• Results in mice do not establish effects, safety or dosing in people.

Mills et al. (2016) reported that 12 months of NMN in drinking water was associated with less age-associated weight gain, maintained energy expenditure, better insulin tolerance, and a range of eye, bone, blood-cell, gene-expression and muscle mitochondrial differences in aging male mice. They also reported no obvious signs of toxicity. The study shows that orally given NMN is rapidly used for NAD+ synthesis in mouse liver and muscle.

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