Nicotinamide mononucleotide, a key NAD(+) intermediate, treats the pathophysiology of diet- and age-induced diabetes in mice

Abstract

Type 2 diabetes (T2D) has become epidemic in our modern lifestyle, likely due to calorie-rich diets overwhelming our adaptive metabolic pathways. One such pathway is mediated by nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD(+) biosynthesis, and the NAD(+)-dependent protein deacetylase SIRT1. Here, we show that NAMPT-mediated NAD(+) biosynthesis is severely compromised in metabolic organs by high-fat diet (HFD). Strikingly, nicotinamide mononucleotide (NMN), a product of the NAMPT reaction and a key NAD(+) intermediate, ameliorates glucose intolerance by restoring NAD(+) levels in HFD-induced T2D mice. NMN also enhances hepatic insulin sensitivity and restores gene expression related to oxidative stress, inflammatory response, and circadian rhythm, partly through SIRT1 activation. Furthermore, NAD(+) and NAMPT levels show significant decreases in multiple organs during aging, and NMN improves glucose intolerance and lipid profiles in age-induced T2D mice. These findings provide critical insights into a potential nutriceutical intervention against diet- and age-induced T2D.

Figure 1. NMN ameliorates defects in NAMPT-mediated NAD⁺ biosynthesis in HFD-induced diabetic mice

(A) NAMPT protein levels in the liver, WAT, and skeletal muscle. Female mice were fed a RC or a HFD for 6–8 months. NAMPT levels were normalized to ACTIN (liver) or TUBULIN (WAT and skeletal muscle) (n=4 to 5 mice per group). (B) Tissue NAD⁺ levels in the liver, WAT, and skeletal muscle from RC, HFD, and NMN-treated HFD mice (n=5 to 13 mice per group). NMN (500mg/kg body weight/day) was given intraperitoneally to HFD-fed female mice for 7 consecutive days. (C) Changes in NMN and NAD⁺ levels in the liver after administering a single dose of NMN to B6 mice (n=3 to 5 mice for each time point). (D and E) Intracellular NAD⁺ levels in mouse primary hepatocytes. Cells were treated with NMN at the indicated concentrations (D), or with enzyme inhibitors [500 nM FK866 or 100 μM gallotannin (GTN)] in the presence or absence of 100 μM NMN (E), for 4 hrs (n=3 per group). Data were analyzed by Student’s unpaired t test (A) and one-way ANOVA with the Fisher’s PLSD post-hoc test (B, D, E). All values are presented as mean ± SEM. *P < 0.05; **P <0.01; ***P < 0.001.

Figure 2. NMN administration improves impaired glucose tolerance in HFD-induced diabetic mice

(A and D) Glucose tolerance in HFD female (A) and male mice (D) before and after NMN treatment (n=10 for females, and n=6 for males). IPGTTs were conducted with the same individuals before (closed circles) and after (open circles) NMN. NMN (500mg/kg body weight/day) was administered to female and male mice for 7 and 10 consecutive days, respectively. The areas under each glucose tolerance curve are presented next to the glucose tolerance curves. (B and E) Plasma insulin levels in female (B) and male (E) mice during IPGTTs before and after NMN treatment (n=10 for females, and n=6 for males). (C and F) Insulin tolerance in HFD female (C) and male (F) mice before and after NMN treatment (n=10 for females, and n=6 for males). ITTs were performed before (closed circles) and after (open circles) NMN. ITTs were conducted several days before or after IPGTTs. The areas under each insulin tolerance curve are presented next to the insulin tolerance curves. Data were analyzed by Student’s paired t test. All values are presented as mean ± SEM. *P < 0.05; **P <0.01; ***P < 0.001.

Figure 3. NMN ameliorates hepatic insulin resistance and restores gene expression related to oxidative stress, inflammatory response, and circadian rhythm

(A) The phosphorylation status of AKT in HFD and NMN-treated HFD female livers (n=3 mice per group). Signal levels of phosphorylated AKT were normalized to total AKT protein levels. (B) Biological pathways that were altered by HFD and reversed by NMN in female livers. Parametric analysis of gene-set enrichment (PAGE) was performed to identify pathways that were significantly up-regulated (red) or down-regulated (blue) by either HFD or NMN using our microarray data (n=4 mice for each condition). Twenty top pathways are listed following the sum of the absolute values of Z scores between two comparisons. (C) Quantitative RT-PCR results for representative genes related to oxidative stress, inflammatory response, circadian rhythm, and metabolism (n=4 to 5 mice per group). Gsta2, glutathione S-transferase alpha 2; Il1b, interleukin 1 beta; Pdk4, pyruvate dehydrogenase kinase isozyme 4; Dbp, D site of albumin promoter (albumin D-box) binding protein; Dec1, deleted in esophageal cancer 1. (D) Acetylation status of NF-κB p65 in RC, HFD, and NMN-treated HFD livers. Two independent sets of mice were used for this analysis, and numbers below each panel represent normalized ratios of acetylated to total p65 levels. (E–G) The effects of TNF-α on NAMPT-mediated NAD⁺ biosynthesis and gene expression in mouse primary hepatocytes. Cells were treated with 50 ng/ml TNF-α for 72 hrs and given indicated reagents for 6 hrs prior to harvesting for measurements. (E) NAMPT protein levels were normalized to ACTIN (n=3 per group). (F) TNF-α-treated cells were given 100 μM NMN prior to NAD⁺ measurements (n=3–6 per group). (G) TNF-α-treated cells were cultured with NMN or NMN plus 40 μM EX527 and examined for Gsta2 expression (n=6–9 per group). Data were analyzed by Student’s unpaired t test (A, E). Differences in Ct values or NAD⁺ levels were analyzed with one-way ANOVA with the Fisher’s PLSD post-hoc test (C, F, G). All values are presented as mean ± SEM. *P < 0.05; **P <0.01; ***P < 0.001.

Figure 4. NMN improves glucose and lipid homeostasis in age-induced T2D

(A) NAD⁺ levels in metabolic tissues between young and old mice. Pancreas, liver, WAT, and skeletal muscle were collected from young (n=5–11) and old (n=5–15) mice at 3–6 and 25–31 months of age, respectively. (B) Glucose tolerance in aged, naturally occurring diabetic male mice before (closed circles) and after (open circles) a single dose of NMN (500 mg/kg body weight) (n=11). The areas under each glucose tolerance curve are presented next to the glucose tolerance curves. (C) Plasma insulin levels were measured during IPGTTs (n=5). (D and E) Glucose tolerance (D) and lipid levels (E) in aged HFD female mice before (closed circles) and after (open circles) NMN (n=5). Fasted plasma samples were collected from the same mice and subjected to the measurements of cholesterol (Chol), triglycerides (TG), and non-esterified free fatty acids (FFA). Data were analyzed by Student’s unpaired t test (A), paired t test (B–D), and one-way ANOVA with the Fisher’s PLSD post-hoc test (E). All values are presented as mean ± SEM. *P < 0.05; **P <0.01; ***P < 0.001.