NAD+, an essential coenzyme in energy production, has recently risen to prominence as a signalling molecule central in mediating cellular metabolism and mitochondrial function. NAD+ dependent protein deacetylase sirtuin (SIRT) proteins regulate key metabolic transcription factors, including FOXOs and PGC-1α in muscle, in response to cellular energy demands and metabolic stress. Declining NAD+, metabolic and mitochondrial function are hallmark features of many patho-physiological processes such as ageing and type 2 diabetes. Thus, boosting NAD+ availability may have beneficial and therapeutic potential. NAD+ consumption (e.g. SIRTS) requires its re-synthesis through precursor salvage to maintain appropriate levels. Here we further define a skeletal muscle specific pathway to NAD+ and energy metabolism. Through mRNA and protein expression analysis of NAD+ biosynthesis genes we show that skeletal muscle relies on a limited set of salvage enzymes for NAD+ biosynthesis and replenishment. The most highly expressed and skeletal muscle specific of which is nicotinamide riboside kinase 2 (Nmrk2), which salvages the NAD+ precursor molecule nicotinamide riboside (NR) to produce NAD+. Nmrk2 demonstrates muscle fibre type specificity, being fivefold enriched in type II fibres compared to type I. Nmrk2 expression is induced during mouse myotube differentiation and embryonic muscle development in zebrafish. Primary derived mouse muscle cells supplemented with 0.5 mM NR for 24 h showed a significant 30% increase (P<0.001) in NAD+ levels with unchanged NADH, suggesting a shift in the redox ratio in favour of the oxidised form. Moreover, oxygen consumption rate was increased by 20% in NR treated myotubes when assessed using a Seahorse extracellular flux analyser, indicating that the NR is enhancing mitochondrial respiration as a result of boosting cellular NAD+ availability. Our data provides evidence that Nmrk2 is important for skeletal muscle NAD+ bioavailability and highlights the therapeutic potential for NR supplementation to target mitochondrial function in skeletal muscle.
Disclosure: This work was supported by the BBSRC David Phillips Fellowship BB/G23468/1 and the Medical Research Council PhD studentship (grant DHAA GAS 0028).