Nicotinamide mononucleotide increases muscle insulin sensitivity in prediabetic women

Abstract

In rodents, obesity and aging impair nicotinamide adenine dinucleotide (NAD+) biosynthesis, which contributes to metabolic dysfunction. Nicotinamide mononucleotide (NMN) availability is a rate-limiting factor in mammalian NAD+ biosynthesis. We conducted a 10-week, randomized, placebo-controlled, double-blind trial to evaluate the effect of NMN supplementation on metabolic function in postmenopausal women with prediabetes who were overweight or obese. Insulin-stimulated glucose disposal, assessed by using the hyperinsulinemic-euglycemic clamp, and skeletal muscle insulin signaling [phosphorylation of protein kinase AKT and mechanistic target of rapamycin (mTOR)] increased after NMN supplementation but did not change after placebo treatment. NMN supplementation up-regulated the expression of platelet-derived growth factor receptor β and other genes related to muscle remodeling. These results demonstrate that NMN increases muscle insulin sensitivity, insulin signaling, and remodeling in women with prediabetes who are overweight or obese (clinicaltrial.gov NCT03151239).

Conflict of interest statement

Competing interests: S.I. receives a part of patent-licensing fees from MetroBiotech (USA) and Teijin Limited (Japan) through Washington University. S.I. also serves as Invited Chief Scientist at the Institute for Biomedical Research and Innovation in Kobe, Japan, which does not involve competing interests. J.Y. is an inventor of a patent application related to NMN (#20180228824). S.K. receives research support from Janssen Pharmaceuticals and serves on a scientific advisory board for Merck Sharp & Dohme Corp. The other authors have nothing to disclose.

Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Figures

Figure 1.. NMN metabolites and NAD+ in plasma, PBMCs, and skeletal muscle.

A, Plasma N-methyl-2-pyridone-5-carboxamide (2PY) and N-methyl-4-pyridone-5-carboxamide (4PY) before (white bars) and after (gray bars) treatment. B, Basal peripheral blood mononuclear cell (PBMC) NAD+ content before (white bars) and after (gray bars) treatment. C, PBMC NAD+ content before (Time 0) and for 240 minutes after ingesting a placebo capsule (white bars) or NMN (250 mg) (black bars) at the end of 10 weeks of treatment with placebo or NMN. D, PBMC NAD+ area under the curve (AUC) for 240 minutes after ingesting a placebo capsule or 250 mg of NMN. E to I, skeletal muscle NAD+, nicotinamide, N-methyl-nicotinamide, 2PY, and 4PY contents before (white bars) and after placebo or NMN treatment (gray bars). Two-way mixed model analysis of variance (ANOVA) with time (before- versus after- treatment) and group (placebo versus NMN) as factors was used to compare the effect of treatment with NMN and placebo on basal PBMC and tissue NAD+. A significant time by group interaction is followed by Tukey’s post-hoc test to locate significant mean differences. A t-test for independent samples (two-tailed) was used to determine differences between the mean PBMC AUC values in the two groups. *Value significantly different from corresponding before treatment value, P <0.01. **Value significantly different from corresponding before treatment value, P <0.05. †Value significantly different from corresponding value in the placebo group, P <0.01. Circles represent individual participant values: skeletal muscle NAD+ content was measured in 11 placebo and 12 NMN participants; skeletal muscle NMN metabolites were measured in 11 placebo and 10 NMN participants; all other measurements are in 12 placebo and 13 NMN participants. Bars represent means ± SEM.

Figure 2.. Effect of NMN on skeletal muscle insulin sensitivity and signaling.

A, Muscle insulin sensitivity, assessed as glucose disposal rate during basal conditions (white bar) and insulin infusion (gray bar) of a hyperinsulinemic-euglycemic clamp procedure. A three-way mixed model ANOVA with time (before- vs after- treatment), condition (basal vs insulin infusion), and group (placebo vs NMN) as factors, was used to compare the effect of NMN (n=13) and placebo (n=12) treatment on insulin-stimulated glucose disposal rate. A significant three-way interaction (P =0.022) was followed by Tukey’s post-hoc test to locate significant mean differences. Significant differences in mean values are represented by a line above the bars with the corresponding P value. B, Western blot densitometric analyses of phosphorylated AKT serine-473 (pAKT Ser473), threonine-308 (pAKT Thr308) and mTOR serine-2448 (pmTOR Ser2448), and total AKT and mTOR relative to ACTIN in skeletal muscle during basal conditions and insulin infusion before and after 10 weeks of treatment with placebo (n=12) or NMN (n=12). Raw images for individual Western blot analysis are provided in the supplementary materials (Figure S3). One-way ANOVA was performed to determine differences in the quantity of phosphorylated AKTs among each combination of time and condition (during basal and insulin infusion before and after each treatment) within each group. Following a significant omnibus test, Tukey’s post-hoc test was used to locate significant mean differences. C, Western blot densitometric analyses of pAKT Ser473, pAKT Thr308, pmTOR Ser2448, and total AKT and mTOR relative to ACTIN in skeletal muscle during insulin infusion before (light gray bars) and after (dark gray bars) placebo (n=12) or NMN (n=12) treatment. Two-way mixed model ANOVA with time (before- vs. after- treatment) and group (placebo vs NMN) as factors was used to determine the significance of differences between variables. A significant two-way interaction was followed by Tukey’s post-hoc test to locate significant mean differences. Significant differences in mean values are represented by a line above the bars with the corresponding P value. *Value significantly different from the corresponding basal value, P <0.05. Circles represent individual participant values. Bars represent means ± SEM.

Figure 3.. Effects of NMN on skeletal muscle global transcriptome profile.

Skeletal muscle tissue obtained during basal conditions and insulin infusion before and after treatment in placebo (n=12) and NMN groups (n=11) were evaluated by using RNA-sequencing (RNA-seq). A, Volcano plots of RNA-seq data of skeletal muscle with log2-fold change (FC) (X-axis) and -log10-P value (Y-axis). The number of differentially expressed genes (DEGs, FDR<0.05) between before and after treatment during basal conditions and during insulin infusion are shown in boxes in each panel. Significantly up-regulated and down-regulated DEGs between before and after treatment in the placebo and NMN groups are shown as red and blue dots, respectively. B, The top 10 Gene Ontology (GO) terms ranked by fold-enrichment. C, Skeletal muscle gene expression of selective proteins involved in PDGF signaling and muscle remodeling during insulin infusion before and after NMN treatment in each participant. PDGFRβ, CD90, CD109, COL1A1, COL5A1, and COL6A1 were identified as DEGs (Table S2). Gene expression is presented as log2-transformed counts per million (CPM) reads. Lines represent values of individual participants before and after NMN treatment. A t-test for paired samples (two-tailed) was performed to determine the difference between before and after NMN treatment for each gene; corresponding P values are indicated above the black lines.