Endocrine Abstracts

Nicotinamide adenine dinucleotide (NAD⁺) levels increase during metabolic stress, which acts as a consumed substrate by, amongst other proteins, the sirtuins, which adapt transcriptional programmes to increase energy availability and regulate insulin sensitivity. Thus, maintaining appropriate skeletal muscle NAD⁺ availability is critical for regulating systemic energy homeostasis. In order to gain better insight into ageing muscle NAD+ dynamics we used targeted LC/MS-based metabolomics and assessed NAD⁺ associated metabolome in young (3-months) and aged (30-months) mouse quadriceps (n=8). In aged muscle we identified significantly reduced NAD+(~20%). ADPribose was also significantly reduced (~30%). ADPribose is a product of NAD+ consumption and signalling molecule, implying reduced sirtuin mediated NAD+ turnover and impaired energy harvesting. Thus, low NAD+ and ADPribose levels corroborate the significantly reduced ATP levels (~40%). Following NAD+ consumption, nicotinamide is also released. Nicotinamide is recycled by the enzyme nicotinamide phosphoribosyltransferase (NAMPT) and critical to maintaining intracellular NAD+ levels. To model aged-related NAD⁺ decline, we exposed primary muscle myotubes (C57BL/6J quadriceps) to the NAMPT inhibitor FK866 (100 nM, 48–72 h) to deplete NAD⁺. After 48–72 h basal oxygen consumption was significantly reduced by 50% implying severely impaired mitochondrial function. Furthermore we also observed increased caspase 3 activity after 72 h indicative of cellular apoptosis initiation. 72 h FK866 treated myotubes were supplemented with the NAD⁺ precursor nicotinamide riboside (NR) for the final 24 h, which fully restored NAD⁺ levels, mitochondrial function and cell viability. Nicotinamide was unable to rescue these effects and supports the notion that muscle has a limited NAD+ salvage system which comprises NAMPT, and NR kinases (NMRK) 1/2 to maintain NAD⁺ availability from extracellular sources. Overall these data identify perturbed NAD⁺ dynamics in aged muscle and its potential impact on energy homeostasis that may underpin age-related metabolic decline. Enhancing NAD⁺ by NR supplementation may prove a useful nutraceutical approach to combat age-related muscle decline.