Perturbations of NAD⁺salvage systems impact mitochondrial function and energy homeostasis in mouse myoblasts and intact skeletal muscle

Abstract

Nicotinamide adenine dinucleotide (NAD) can be synthesized by nicotinamide phosphoribosyltransferase (NAMPT). We aimed to determine the role of NAMPT in maintaining NAD levels, mitochondrial function, and metabolic homeostasis in skeletal muscle cells. We generated stable Nampt knockdown (sh Nampt KD) C2C12 cells using a shRNA lentiviral approach. Moreover, we applied gene electrotransfer to express Cre recombinase in tibialis anterior muscle of floxed Nampt mice. In sh Nampt KD C2C12 myoblasts, Nampt and NAD levels were reduced by 70% and 50%, respectively, and maximal respiratory capacity was reduced by 25%. Moreover, anaerobic glycolytic flux increased by 55%, and 2-deoxyglucose uptake increased by 25% in sh Nampt KD cells. Treatment with the NAD precursor nicotinamide riboside restored NAD levels in sh Nampt cells and increased maximal respiratory capacity by 18% and 32% in control and sh Nampt KD cells, respectively. Expression of Cre recombinase in muscle of floxed Nampt mice reduced NAMPT and NAD levels by 38% and 43%, respectively. Glucose uptake increased by 40%, and mitochondrial complex IV respiration was compromised by 20%. Hypoxia-inducible factor (HIF)-1α-regulated genes and histone H3 lysine 9 (H3K9) acetylation, a known sirtuin 6 (SIRT6) target, were increased in shNampt KD cells. Thus, we propose that the shift toward glycolytic metabolism observed, at least in part, is mediated by the SIRT6/HIF1α axis. Our findings suggest that NAMPT plays a key role for maintaining NAD levels in skeletal muscle and that NAMPT deficiency compromises oxidative phosphorylation capacity and alters energy homeostasis in this tissue.

Aerobic and resistance exercise training reverses age‐dependent decline in NAD+salvage capacity in human skeletal muscle

“…; Agerholm et al. ), our findings highlight the importance of exercise training as an effective intervention to prevent aging‐associated declines in skeletal muscle function.…”

“…Moreover, in the older individuals, exercise training was able to completely restore skeletal muscle NAMPT to levels observed in young individuals. Given the role of NAMPT for maintaining the functional capacity (e.g., contractility and oxidative respiration) of skeletal muscle (Frederick et al 2016;Agerholm et al 2018), our findings highlight the importance of exercise training as an effective intervention to prevent aging-associated declines in skeletal muscle function. Lifelong (>10 years) football training, which consists of a combination of aerobic and resistance training, increases NAMPT, TFAM and PGC1a mRNA levels in skeletal muscle (Mancini et al 2017) highlighting the possible involvement of the NAD + salvage pathway for exercise training-induced improvements in mitochondrial function (Alfieri et al 2015).…”

“…NAMPT is a ubiquitously expressed protein, and whole-body knockout of Nampt in mice is embryonically lethal (Revollo et al 2007;Zhang et al 2017). Knockdown of Nampt in C2C12 cells reduces NAD + and decreases maximal respiratory capacity (Agerholm et al 2018). Muscle-specific Nampt knockout mice exhibit an 85% decrease in intramuscular NAD + levels, which subsequently causes muscle fiber degeneration and progressive loss of muscle strength and endurance capacity (Frederick et al 2016).…”

Nicotinamide riboside does not alter mitochondrial respiration, content or morphology in skeletal muscle from obese and insulin‐resistant men

“…; Agerholm et al . ). Interestingly, the capacity for NAD + biosynthesis was shown to be reduced in obese and old mice resulting in limited NAD + availability in a number of tissues, including skeletal muscle (Yoshino et al .…”

“…NAMPT is crucial for maintaining appropriate NAD + levels in skeletal muscle. In muscle-specific Nampt knockout (KO) mouse models, low NAD + levels have deleterious effects on muscle mitochondrial function that can be rescued by NR administration (Frederick et al 2016;Agerholm et al 2018). Interestingly, the capacity for NAD + biosynthesis was shown to be reduced in obese and old mice resulting in limited NAD + availability in a number of tissues, including skeletal muscle (Yoshino et al 2017).…”