Given the existence of different cellular NAD+ pools and the relevance of mitochondrial NAD+ content for mitochondrial and cellular function (Yang et al., 2007a), we also analyzed whether NR treatment would affect mitochondrial NAD+ levels. In contrast to what has been observed with other strategies aimed to increase NAD+ bioavailability, such as PARP inhibition (Bai et al., 2011a), we found that mitochondrial NAD+ levels were enhanced in cultured cells (Fig.1E) and mouse liver (Fig.1F) after NR supplementation.
To further solidify our data, we also wondered whether the enhanced NAD+ levels upon NR treatment could derive from alterations in the NAD+ salvage pathway or PARP activity. However, we could not see any change in Nampt mRNA or protein content in response to NR treatment (Fig.1G). Similarly, PARP activity and PARP-1 content were not affected by NR (Fig.1H). Altogether, these results suggest that NR increases NAD+ by direct NAD+ biosynthesis rather than by indirectly affecting the major NAD+ salvage (Nampt) or consumption (PARPs) pathways. Importantly, this increase in NAD+ was not linked to changes in cellular glycolytic rates or ATP levels (data not shown), which would be expected if NAD+/NADH ratios had been altered to the point of compromising basic cellular functions.

NR treatment enhances SIRT1 and SIRT3 activity

The ability of NR to increase intracellular NAD+ levels both in vivo and in vitro prompted us to test whether it could activate sirtuin enzymes. Confirming this hypothesis, NR dose-dependently decreased the acetylation of FOXO1 (Brunet et al., 2004) in a SIRT1-dependent manner (Fig 2A). This deacetylation of FOXO1 by SIRT1 upon NR treatment resulted in its transcriptional activation, leading to higher expression of target genes, such as Gadd45, Catalase, Sod1 and Sod2 (Fig.S1) (Calnan and Brunet, 2008). The lack of changes in SIRT1 protein levels upon NR treatment (Fig.2A) suggests that NR increases SIRT1 activity by enhancing NAD+ bioavailability. The higher SIRT1 activity in NR-treated cells was supported by mRNA expression analysis. Consistent with SIRT1 being a negative regulator of Ucp2 expression (Bordone et al., 2006), NR decreased Ucp2 mRNA levels (Fig.2B). Importantly, knocking down Sirt1 prevented the action of NR on Ucp2 expression (Fig.2B). Similarly, the higher expression of a FOXO1 target gene, Sod2, upon NR treatment was also prevented by the knockdown of either Foxo1 or Sirt1 (Fig.2B). This suggested that NR leads to a higher Sod2 expression thought the activation of SIRT1, which then deacetylates and activates FOXO1. Importantly, the knock-down of SIRT1 did not compromise the ability of NR to increase intracellular NAD+ content, indicating that NR uptake and metabolism into NAD+ is not affected by SIRT1 deficiency (Fig.2C).