GS significantly promote aerobic glucose metabolism in
cardiomyocytes
Increased OCR and ATP production are the manifestations of enhanced
energy metabolism; thus, we performed targeted metabolomic analysis of
glucose metabolites by LC-MS/MS in H9c2 and HUVECs untreated or treated
with GS for 48 h. A total of 15 different intermediates of glycolysis
and TCA cycle, such as pyruvate, lactate, citrate, and succinate were
identified in these two cell lines. In H9c2 cells, GS led to decreases
of seven metabolites and increases of eight metabolites, compared with
the control group (Fig. 2A) . The analysis of relative intensity
for each metabolite showed that GS decreased the levels of fructose
6-phosphate, fructose 1, 6-bisphosphate, 3-phosphoglycerate,
dihydroxyacetone phosphate, and phosphoenolpyruvate, and increased the
levels of glucose 6-phosphate and pyruvate in H9c2 cells, suggesting
that GS increased the conversion of glucose to pyruvate. Moreover, key
metabolites of TCA cycle such as citrate, succinate, fumarate, malate,
oxaloacetate, and glycolytic metabolite, lactate were clearly increased
by GS pretreatment (Fig. 2B) , suggesting that GS mainly
accelerated TCA metabolism in the cardiomyocytes. In HUVECs, only six
metabolites were upregulated by GS pretreatment, including
glucose-6-phosphate, fructose-6-phosphate, pyruvate, succinate, malate,
and fumarate. There were no obvious differences in the other nine
metabolites between the control and GS groups (Fig. 2C,
Supplementary Fig. 5A) . These data suggest that GS mainly increased the
conversion from glucose to fructose-6-phosphate and pyruvate, and
enhanced the metabolic process from succinate to malate in HUVECs.
Collectively, GS had more obvious effects on promoting glucose oxidation
in cardiomyocytes than endothelial cells.
Multiple key enzymes regulate glucose breakdown and TCA metabolism
reaction, which were examined by Western blot analysis in the control
and GS-treated H9c2 cells. GS pretreatment upregulated the levels to the
rate-limiting enzymes such as HK-II, PFKP, PKM2, and PDH, and did not
change GAPDH expression in H9c2 cells (Fig. 2D, Supplementary
Fig. 5B) . Mitochondrial pyruvate carriers, MPC1 and MPC2, which play a
potential role in the transport of pyruvate to mitochondria, were
increased by GS compared with the control group (Fig. 2D,
Supplementary Fig. 5B) . Furthermore, CS, the first and rate-limiting
enzyme of the TCA cycle; DLST, one of the core enzymes of
α-ketoglutarate dehydrogenase complex; and fumarase, a mitochondrial
metabolic enzyme, were significantly higher in GS-pretreated H9c2 cells
than the control group (Fig. 2D, Supplementary Fig. 5B) . Two
isocitrate dehydrogenase isoforms in cytoplasm (IDH1) and mitochondria
(IDH2) were not regulated by GS. In addition, ACO2, which is responsible
for catalyzing the isomerization of citrate to isocitrate and SDHA, were
decreased by GS pretreatment (Fig. 2D, Supplementary Fig. 5B) .
Importantly, the activity of PDH, as a rate-limiting metabolic step for
glucose oxidation was measured in untreated- and GS-pretreated H9c2 and
HUVECS. We found a significant induction in PDH activity in GS-treated
H9c2 cells and no change of PDH activity in GS-treated HUVECs compared
with control cells (Fig. 2E) . Taken together, our findings
suggest that GS pretreatment mainly accelerates glucose conversion to
pyruvate, and increases pyruvate transporting to promote TCA metabolism
in the cardiomyocytes. In future experiments, we will focus on the
effect and molecular mechanism of GS on cardiomyocytes and neurons
dominated by aerobic respiration.
GSincrease
mitochondrial biogenesis incardiomyocytes
and neurons
To investigate the role of GS in
mitochondrial biogenesis, we first
examined mitochondrial count in cardiomyocytes and neurons using the
Mitotracker probe. We found that pretreatment with GS induced a
significant increase in mitochondrial content in a dose-dependent manner
in H9c2 cells (Fig. 3A-3B) and primary neurons (Fig. 3A
and 3C) . Moreover, high-resolution micrographs of mitochondria were
obtained in control and GS-pretreated H9c2 cells using TEM. As shown inFig. 3D-3E , GS pretreatment at 5 μg/mL for 48 h increased the
number of mitochondria in the cardiomyocytes and had no effect on the
size of myocardial mitochondria, compared with control cells. To
determine if the observed changes in mitochondrial respiration and
biogenesis translate into altered mitochondrial function, we performed
immunoblotting with antibodies against five complexes of the oxidative
phosphorylation. Compared with the control group, GS induced increases
in complex I-IV levels in H9c2 cells and mainly increased the levels of
CII-SDHB and CI-NDUFB8 in PC12 cells (Fig. 3F-3G) . The
enhancement of mitochondrial function can increase ROS generation, which
damages the cells, so we examined ROS level in untreated- and GS-treated
H9c2 cells. Flow cytometry analysis showed that GS had no significant
effect on the production of intracellular and mitochondrial ROS in H9c2
cells (Supplementary Fig. 6A) . Additionally, mitochondrial
amount and complex protein level were further investigated in HUVECs. We
did not observe the effect of GS pretreatment on mitochondrial mass in
HUVECs (Supplementary Fig. 6B-6C) . Collectively, GS
preferentially promote the mitochondrial biosynthesis of cardiomyocytes
and neurons, and not that of endothelial cells.