GS increase NAD+-dependent SIRT1 activation in cardiomyocytes and neurons
NAD+ level and its dependent deacetylase, SIRT1, play key roles in regulating mitochondrial function (Katsyuba et al., 2018). Based on the findings above, we determined if GS could alter the intracellular level of NAD+ and regulate SIRT1 activation in cardiomyocytes and neurons. Consistent with our hypothesis, the level of NAD+ luminescence was obviously higher in GS-treated H9c2 and primary neurons than in the control group (Fig. 4A) . Moreover, the targeted metabolomic analysis showed that NAD+ level was significantly increased by GS pretreatment in H9c2 cells (Fig. 4B) . As reported, NAD+-dependent SIRT1 can deacetylate and activate PGC-1α to stimulate mitochondrial biogenesis (Aquilano, Baldelli, Pagliei & Ciriolo, 2013). As shown in Fig. 4C , qPCR analysis showed that GS incubation for 8 h increased SIRT1 and PGC-1α mRNA levels in H9c2 and primary neurons. Western blot analysis demonstrated that GS pretreatment for 48 h led to increases in SIRT1 and its targets PGC-1α, Nrf1, and Nrf2 in H9c2 cells (Fig. 4D) . Furthermore, similar findings in cardiomyocytes were observed in GS-treated PC12 cells (Fig. 4E) . In addition, the wound-healing ability in H9c2 cells and neurons was increased by GS pretreatment, suggesting that GS promoted cell migration (Supplementary Fig. 7A-7C) .
Intriguingly, GS were administered to the flies and mice to further validate the function of GS in NAD+-dependent SIRT1 activation. Consistent with the in vitro results, GS administration for 7 days led to a significant increase in the intensity of NAD+ luminescence and the ratio of NAD+/NADH in the drosophila brain compared with the control group (Fig. 5A) . The increased SIRT1 in response to NAD+ was confirmed in the brain of drosophila treated with GS (Fig. 5B) . Meanwhile, the number of activities and climbing distance of 30 flies fed with GS were greatly increased, compared to the control group with common food (Fig. 5C) . In addition, IHC staining showed that SIRT1 expression was significantly increased in the heart and brain tissues of mice after 21 days of GS administration (Fig. 5D) . Together, these in vitro and in vivo results indicate that GS obviously increase NAD+-dependent SIRT1 activation in cardiomyocytes and neurons.