We also tested whether the increase in NAD+ would be concomitant to changes in other NAD+ metabolites. Strikingly, NADH and nicotinamide (NAM) levels were largely diminished in muscles from NR-fed mice (Fig.5B), indicating that NR specifically increases NAD+, but not necessarily other by-products of NAD+ metabolism. We analyzed in vivo whether the activity of major NAD+ degrading enzymes or the levels of Nampt could also contribute to the increase in NAD+ after chronic NR supplementation. As previously observed in HEK293T cells (Fig.1G-H), PARP-1 levels and global PARylation were similar in muscle (Fig.5C) and livers (Fig.S4D) from NR- and vehicle-fed mice, indicating that the enhanced NAD+ content cannot be explained by differential NAD+ consumption through PARP activity. Nampt mRNA (Fig.5D) and protein (Fig.5C, Fig.S4E and data not shown) levels were also similar in NR and vehicle fed mice, suggesting that NAD+ salvage pathways do not explain the differences in NAD+ levels. We furthermore could not detect differences in mRNA expression of the different NMN adenylyltransferase (NMNAT) enzymes (Fig.5D). Altogether, these results reinforce the notion that the higher NAD+ levels observed in tissues from NR-fed mice is consequent to an increase in direct NAD+ synthesis from NR.
NR enhances sirtuin activity in vivo
Higher NAD+ levels were also accompanied by higher sirtuin activity in vivo. A prominent deacetylation of SIRT1 and SIRT3 targets (FOXO1 (Brunet et al., 2004) and SOD2 (Qiu et al., 2010), respectively) was observed in the skeletal muscle, liver and BAT, where NAD+ content was induced by NR, but not in brain and WAT, where NAD+ levels were unaffected by NR supplementation (Fig.6A and Fig.S5A). We also evaluated PGC-1α acetylation as a second readout of SIRT1 activity (Rodgers et al., 2005). We were unable to detect PGC-1α in total lysates from WAT or brain (Fig.S5B), but in muscle, liver and BAT PGC-1α was deacetylated upon NR treatment (Fig.S5C). These observations highlight how NR can only induce sirtuin activity in tissues where NAD+ accumulates. Like in cultured cells, we could not detect changes in the acetylation status of the SIRT2 target tubulin (data not shown), suggesting either that increasing NAD+ might not affect the activity of all sirtuins equally, that the increase is only compartment-specific or that additional regulatory elements, like class I and II HDACs, also contribute to tubulin acetylation status.