| NAD+ is one of the central cellular metabolites and shown to play important roles in DNA repair, transcription, apoptosis, metabolism, endocrine signaling, circadian rhythm and lifespan regulation through activities of NAD+ consuming enzymes, such as sirtuins and PARPs. Our goal was to investigate the NAD+ metabolism pathways and examine small molecule agents to enhance the production of NAD + in mammalian system.;Here, we have identified nicotinamide riboside (NR) as a NAD+ precursor that upregulates NAD+ levels in total cellular and mitochondrial pools in mammalian cells and various tissues in animal. We tested three different mammalian metabolic pathways of NR, Nrkl phosphorylation-depndent, Nrkl -independent, and Pnp phosphorolysis to determine the major path of NR conversion to NAD+. We report that NR is dependent on Nrkl phosphorylation for its conversion to NAD+ and is not affected by Pnp phosphorolysis in cells. Furthermore, we have synthesized NR ester and acid derivatives that showed the similar effect on NAD+ enhancement.;Based on NAD+ upregulation by NR treatment in cells, we examined the effects of NR under acute genotoxicity and hydrogen peroxide toxicity by testing cells treated with genotoxin MMS or H20 2. We report that NR treatment increases cell viability through enhancing NAD+ levels under toxicity. More importantly, increased NAD + levels by NR treatment in animals showed physiological consequences, such as increasing energy expenditure and weight gain prevention under high fat diet. We discovered that NR protective effects on cells and animals are sirtuin-dependent.;To date, there are no direct activators of sirtuins. In our study, we showed that NR is a direct activator of mitochondrial sirtuin, SIRT5. We characterized kinetic properties of SIRT5 as a deacetylase and desuccinylase. The deacetylase activity of SIRT5 has been upregulated by 2-fold with 1 mM NR. We report this NR activation is via decreasing Km of protein substrate and co-substrate NAD+. Finally, we confirmed the direct interaction and activation by SIRT5 mutagenesis and STD-NMR methodologies.;Collectively, these above studies offer insights into mammalian NAD + metabolism and mitochondrial sirtuin activities that are important for metabolism regulation. We also provide evidence to support NR as a direct activator of SIRT5. Our studies demonstrated that NR is a potential vitamin supplement that promotes protection against diseases that are implicated with NAD+ depletion. |
