Skeletal muscle NAMPT is induced by exercise in humans

Abstract

In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is responsible for the first and rate-limiting step in the conversion of nicotinamide to nicotinamide adenine dinucleotide (NAD+). NAD+ is an obligate cosubstrate for mammalian sirtuin-1 (SIRT1), a deacetylase that activates peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), which in turn can activate mitochondrial biogenesis. Given that mitochondrial biogenesis is activated by exercise, we hypothesized that exercise would increase NAMPT expression, as a potential mechanism leading to increased mitochondrial content in muscle. A cross-sectional analysis of human subjects showed that athletes had about a twofold higher skeletal muscle NAMPT protein expression compared with sedentary obese, nonobese, and type 2 diabetic subjects (P < 0.05). NAMPT protein correlated with mitochondrial content as estimated by complex III protein content (R(2) = 0.28, P < 0.01), MRS-measured maximal ATP synthesis (R(2) = 0.37, P = 0.002), and Vo(2max) (R(2) = 0.63, P < 0.0001). In an exercise intervention study, NAMPT protein increased by 127% in sedentary nonobese subjects after 3 wk of exercise training (P < 0.01). Treatment of primary human myotubes with forskolin, a cAMP signaling pathway activator, resulted in an approximately 2.5-fold increase in NAMPT protein expression, whereas treatment with ionomycin had no effect. Activation of AMPK via AICAR resulted in an approximately 3.4-fold increase in NAMPT mRNA (P < 0.05) as well as modest increases in NAMPT protein (P < 0.05) and mitochondrial content (P < 0.05). These results demonstrate that exercise increases skeletal muscle NAMPT expression and that NAMPT correlates with mitochondrial content. Further studies are necessary to elucidate the pathways regulating NAMPT as well as its downstream effects.

Trial registration: ClinicalTrials.gov NCT00401791 NCT00402012.

Figures

Fig. 1.

Skeletal muscle nicotinamide phosphoribosyltransferase (NAMPT) protein expression and mitochondrial content in metabolically distinct subjects. FH, nonobese sedentary subjects with or without family history of T2DM; AU, arbitrary units. A: NAMPT protein in skeletal muscle (means ± SD, n = 5, one-way ANOVA with Tukey's post hoc test, *P < 0.05, **P < 0.01 vs. Athletes). B: complex III protein in skeletal muscle (means ± SD, n = 5, one-way ANOVA with Tukey's post hoc test, **P < 0.01 vs. Athletes). C: mitochondrial DNA in skeletal muscle (means ± SD, n = 2–5, one-way ANOVA with Tukey's post hoc test, *P < 0.05, **P < 0.01 vs. Athletes).

Fig. 2.

Relationship between skeletal muscle NAMPT protein and markers of mitochondrial content and function. A: Spearman correlation between NAMPT and complex III protein expression in skeletal muscle (n = 25, R2 = 0.28, P < 0.01). B: Pearson correlation between skeletal muscle NAMPT protein expression and ATPmax (n = 24, R2 = 0.37, P = 0.002). C: Pearson correlation between skeletal muscle NAMPT protein expression and V̇o2max (n = 18, R2 = 0.63, P < 0.0001). D: Spearman correlation between skeletal muscle NAMPT protein and %body fat mass (n = 24, R2 = 0.36, P = 0.002). E: Spearman correlation between skeletal muscle NAMPT protein and glucose disposal rate (n = 20, R2 = 0.20, P < 0.05).

Fig. 3.

Skeletal muscle NAMPT and PPARγ coactivator-1α (PGC-1α) expression pre- and postexercise training. A: skeletal muscle NAMPT mRNA (means ± SD, n = 13, paired Student's t-test, ***P < 0.001). B: skeletal muscle NAMPT protein (means ± SD, n = 10, paired Student's t-test, **P < 0.01). C: skeletal muscle PGC-1α mRNA (means ± SD, n = 13, paired Student's t-test, ***P < 0.001). D: Spearman correlation between skeletal muscle NAMPT and PGC-1α mRNA (pretraining n = 20, posttraining n = 13, R2 = 0.27, P = 0.002).

Fig. 4.

A: Spearman correlation between baseline level of skeletal muscle NAMPT mRNA and %change in NAMPT mRNA with exercise training (n = 13, R2 = 0.31, P < 0.05). B: Spearman correlation between baseline level of skeletal muscle PGC-1α mRNA and %change in PGC-1α mRNA with exercise training (n = 13, R2 = 0.62, P = 0.002).

Fig. 5.

NAMPT protein expression and mitochondrial content in forskolin- and/or ionomycin-treated primary human myotubes. DAPI staining of nuclei and OXPHOS staining of mitochondria and overlay in control (untreated; A) and forskolin- and ionomycin-treated primary human myotubes (B). C: quantification of mitochondrial volume by OXPHOS fluorescence (means ± SD, n = 4, paired Student's t-test, P < 0.05) in control and forskolin- and ionomycin-treated cells. D: complex III protein in control and forskolin- and/or ionomycin-treated primary human myotubes (means ± SD, n = 5, one-way ANOVA with Dunnett post hoc test, *P < 0.05, **P < 0.01). E: NAMPT protein in control and forskolin- and/or ionomycin-treated primary human myotubes [means ± SD, n = 5, one-way ANOVA with Dunnett's post hoc test, not significant (NS)]. F: %increase in NAMPT protein vs. control cells (means ± SD, n = 5, one-way ANOVA with Tukey's post hoc test, **P < 0.01).

Fig. 6.

Effect of AMPK activation on NAMPT expression and mitochondrial content. A: phospho-AMPKα (Thr172)-to-AMPKα ratio in control and forskolin- and/or ionomycin-treated primary human myotubes (means ± SD, n = 4, one-way ANOVA with Dunnett's post hoc test, *P < 0.05). B: NAMPT mRNA in control and AICAR-treated primary human myotubes (means ± SD, n = 3, paired Student's t-test, *P < 0.05). C: NAMPT protein in control and AICAR-treated primary human myotubes (means ± SD, n = 4, paired Student's t-test, *P < 0.05). D: complex III protein in control and AICAR-treated primary human myotubes (means ± SD, n = 4, paired Student's t-test, *P < 0.05).