Skeletal muscle overexpression of nicotinamide phosphoribosyl transferase in mice coupled with voluntary exercise augments exercise endurance

Abstract

Objective: Nicotinamide phosphoribosyl transferase (NAMPT) is the rate-limiting enzyme in the salvage pathway that produces nicotinamide adenine dinucleotide (NAD⁺), an essential co-substrate regulating a myriad of signaling pathways. We produced a mouse that overexpressed NAMPT in skeletal muscle (NamptTg) and hypothesized that NamptTg mice would have increased oxidative capacity, endurance performance, and mitochondrial gene expression, and would be rescued from metabolic abnormalities that developed with high fat diet (HFD) feeding.

Methods: Insulin sensitivity (hyperinsulinemic-euglycemic clamp) was assessed in NamptTg and WT mice fed very high fat diet (VHFD, 60% by kcal) or chow diet (CD). The aerobic capacity (VO₂max) and endurance performance of NamptTg and WT mice before and after 7 weeks of voluntary exercise training (running wheel in home cage) or sedentary conditions (no running wheel) were measured. Skeletal muscle mitochondrial gene expression was also measured in exercised and sedentary mice and in mice fed HFD (45% by kcal) or low fat diet (LFD, 10% by kcal).

Results: NAMPT enzyme activity in skeletal muscle was 7-fold higher in NamptTg mice versus WT mice. There was a concomitant 1.6-fold elevation of skeletal muscle NAD⁺. NamptTg mice fed VHFD were partially protected against body weight gain, but not against insulin resistance. Notably, voluntary exercise training elicited a 3-fold higher exercise endurance in NamptTg versus WT mice. Mitochondrial gene expression was higher in NamptTg mice compared to WT mice, especially when fed HFD. Mitochondrial gene expression was higher in exercised NamptTg mice than in sedentary WT mice.

Conclusions: Our studies have unveiled a fascinating interaction between elevated NAMPT activity in skeletal muscle and voluntary exercise that was manifest as a striking improvement in exercise endurance.

Keywords: Exercise; High fat feeding; Insulin sensitivity; Mitochondrial gene expression; Nicotinamide adenine dinucleotide; Nicotinamide phosphoribosyl transferase.

Figure 1

NAMPT overexpression in skeletal muscle increased NAMPT mRNA and protein content in skeletal muscle. Quantitative reverse transcriptase-PCR (qRT-PCR) analyses of NAMPT mRNA expression in (A) mixed gastrocnemius, (B) mixed quadriceps, and (C) heart; and western blot (WB) analyses of NAMPT protein content in (D) mixed gastrocnemius, (E) mixed quadriceps, and (F) heart. WT LFD = Wild Type Low Fat Diet; NamptTg LFD = NamptTg Low Fat Diet; WT HFD = Wild Type High Fat Diet; NamptTg HFD = NamptTg High Fat Diet. Letters indicate significant differences between groups (p < 0.050). Data are mean ± SEM.

Figure 2

NAMPT overexpression in skeletal muscle increased NAMPT enzyme activity, NMN and NAD⁺in skeletal muscle. NAMPT enzyme activity in (A) mixed gastrocnemius, (B) mixed quadriceps, (C) heart, and (D) liver; and ultra-performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS) analyses of (E) nicotinamide mononucleotide (NMN), (F) nicotinamide adenine dinucleotide (NAD⁺), and (G) nicotinamide (NAM) in mixed quadriceps of chow fed WT and NamptTg mice. Letters indicate significant differences between groups (p < 0.050). Data are mean ± SEM.

Figure 3

NAMPT overexpression in skeletal muscle increased mitochondrial gene expression in skeletal muscle. Gene expression of genes related to (A) mitochondrial biogenesis, (B) oxidative phosphorylation, (C) oxidative stress modulation, and (D) substrate selection. WT LFD = Wild Type Low Fat Diet; NamptTg LFD = NamptTg Low Fat Diet; WT HFD = Wild Type High Fat Diet; NamptTg HFD = NamptTg High Fat Diet. Letters indicate significant differences between groups (p < 0.050). ∞ p < 0.050 all WT versus all NamptTg. Data are normalized to WT LFD, and are mean ± SEM.

Figure 4

NAMPT overexpression in skeletal muscle partially protected against body weight gain but not against insulin resistance, without behavioral changes in mice fed VHFD. (A) body weight over time, (B) fat mass percentage over time, (C) glucose infusion rate (GIR) during the hyperinsulinemic-euglycemic clamp after 16 weeks fed very high fat diet (VHFD) or chow diet (CD), (D) glucose rate of disposal (R_d) during the hyperinsulinemic-euglycemic clamp after 16 weeks fed very high fat diet (VHFD) or chow diet (CD, (E) oxygen consumption (VO₂) over 24 h, and (F) respiratory exchange ratio (RER) over 24 h after 16 weeks fed VHFD or CD. WT CD = Wild Type Chow Diet; NamptTg CD = NamptTg Chow Diet; WT VHFD = Wild Type Very High Fat Diet; NamptTg VHFD = NamptTg Very High Fat Diet. Letters indicate significant differences between groups (p < 0.050). γ p < 0.050 WT VHFD versus WT CD and NamptTg CD; $ p < 0.050 all VHFD versus all CD; ε p < 0.050 WT VHFD versus NamptTg VHFD; δ p < 0.050 WT CD versus WT VHFD and NamptTg VHFD; *p < 0.050 basal versus clamp and light versus dark within the same group. Data are mean ± SEM.

Figure 5

NAMPT overexpression in skeletal muscle coupled with voluntary exercise training increased exercise endurance capacity. (A) body weight over time, (B) fat mass percentage over time, (C) aerobic capacity (VO₂max) pre- and post-voluntary exercise training or sedentary conditions, (D) endurance capacity (running time) pre- and post-voluntary exercise training or sedentary conditions, (E) running distance and total running time per 24 h, (F) average running speed and maximum running speed per 24 h. WT sed = sedentary Wild Type; NamptTg sed = sedentary NamptTg; WT ex = exercised Wild Type; NamptTg ex = exercised NamptTg. $ p < 0.050 all ex versus all sed; ε p < 0.050 WT sed versus NamptTg sed; δ p < 0.050 WT sed versus WT ex and NamptTg ex; γ p < 0.050 NamptTg ex versus WT sed and NamptTg sed; η p < 0.050 WT ex versus WT sed; *p < 0.050 pre-versus post-within the same group. Data are mean ± SEM.

Figure 6

NAMPT overexpression in skeletal muscle and voluntary exercise training increased mitochondrial gene expression but not SIRT1 gene expression and complex III protein content. Gene expression of (A) SIRT1 and genes related to (B) mitochondrial biogenesis, (C) oxidative phosphorylation, (D) oxidative stress modulation, and (E) substrate selection in red quadriceps. (F) Complex III protein content in red quadriceps. WT sed = sedentary Wild Type; NamptTg sed = sedentary NamptTg; WT ex = exercised Wild Type; NamptTg ex = exercised NamptTg. Letters indicate significant differences between groups (p < 0.050). Data are normalized to WT sed, and are mean ± SEM.