In line with the increase in mitochondrial NAD+ levels (Fig.1E-F) and the potential consequent activation of mitochondrial sirtuins, NR also reduced the acetylation status of Ndufa9 and SOD2 (Fig.2D and 2E, respectively), both targets for SIRT3 (Ahn et al., 2008; Qiu et al., 2010). SOD2 deacetylation has been linked to a higher intrinsic activity. In line with these observations, NR treatment enhanced SOD2 activity (Fig.2E). To ensure that NR-induced SOD2 deacetylation was consequent to SIRT3 activation, we used mouse embryonary fibroblast (MEFs) established from SIRT3 KO mice. The absence of SIRT3 was reflected by the higher basal acetylation of SOD2 (Fig.2F). Importantly, NR was unable to decrease the acetylation status of SOD2 in SIRT3−/− MEFs (Fig.2F), despite that NAD+ levels increased to similar levels as in SIRT3+/+ MEFs (Fig.2G). These results clearly indicate that NR triggers SIRT3 activity, probably by increasing mitochondrial NAD+ levels, inducing the concomitant deacetylation of its mitochondrial targets. Strikingly, not all sirtuins were affected by NR, as the acetylation of tubulin, a target of the cytoplasmic SIRT2 (North et al., 2003), was not altered (data not shown).
NR supplementation enhances energy expenditure
Given the promising role of sirtuins to protect against metabolic disease, we next evaluated the effects of long-term NR administration in vivo. We fed 10-week-old male C57Bl/6J mice with either chow (CD) or high-fat diet (HFD), supplemented or not with NR at 400 mg/kg/day. While NR had no effect on the body weight (BW) on CD, HFD-induced body weight gain was significantly attenuated by NR (Fig.3A), due to reduced fat mass (Fig.3B). This was visibly translated into a significant lower weight of the epididymal depot in NR-fed mice (Fig.S2A). Importantly, this was not due to redistribution of lipids to other tissues (Fig.S2A), most notably to liver, which actually contained 40% less triglycerides (Fig.S2B).