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.