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

Nicotinamide adenine dinucleotide (NAD) can be synthesized by nicotinamide phosphonbosyltransferase (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 (shNampt KD) C2C12 cells using a shRNA lentivirai approach. Moreover, we applied gene electrotransfer to express Cre rccombinasc in tibialis anterior muscle of floxed Nampt mice. In shNampt KD C2CJ 2 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 shNampt KD cells. Treatment with the NAD (+) precursor nicotinamide riboside restored NAD (+) levels in shNampt cells and increased maximal respiratory capacity by 18% and 32% in control and shNampt 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 inereased by 40%, and mitochondrial complex IV respiration was compromised by 20%. Hypoxiainducible factor (HIF)-l alpha-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/ HIT l alpha 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.