Response to Brenner: Growth Stage-dependent Regulation of Nicotinamide Riboside (NmR) Production

This is a response to a letter by Brenner (1) In this study (2), we aimed to identify factors regulating NmR metabolism. Multiple components of the phosphate signaling (PHO) pathways were identified in a cell-based genetic screen (Fig. 1). The interaction between the NAD+ and PHO pathways (Figs. 2–6) supports the importance of the PHO pathway in NAD+ metabolism. Our studies of the Fun26 transporter (Fig. 4) and Pho8 phosphatase (Fig. 3) suggest that NmR metabolism is regulated by various factors localized at different cellular compartments in response to metabolic changes. Although our method for extracting intracellular NmR may not be sensitive enough to reflect NmR levels in vivo, it is unlikely that it simply determines the degradation of NAD+/NMN to NmR after cell lysis. A similar method has been used to extract NAD+ without significant degradation (3). The discrepancies of Isn1 and Sdt1 deletions on intracellular NmR levels (2, 4) may also be due to growth stage (nutrient)-dependent regulation of NmR metabolic factors. Isn1 expression has been shown to decrease in glucose-depleted cells (5). Therefore, deleting Isn1 is anticipated to decrease NmR levels more significantly in early-log phase (glucose-abundant) cells (4) than in late-log phase (glucose-depleted) cells (2). Our studies employed late-log phase cells because we observed a growth stage-dependent increase of NmR level, which reached maximum at ∼24 h. Finally, the intracellular NAD+ level declines over time in vivo (6). If our methods were simply detecting NAD+/NMN degradation after cell lysis, we would have observed a decrease (rather than an increase) of NmR at late growth stage.