Discussion
Here, we revealed the novel mechanism by which GS promote mitochondrial
energy metabolism in cardiomyocytes and neurons, aerobic respiration-
dominated cells. In this study, GS pretreatment increased mitochondrial
aerobic respiration, ATP production, and cellular NAD+level. Moreover, GS activated the SIRT1-PGC-1α pathway to promote
mitochondrial biosynthesis and ETC complex II-IV levels, which was
inhibited by NAM. In hypoxia- and OGD-induced cell injuries, GS also
increased ATP content and OXPHOS protein levels by promoting the
activation of the SIRT1-PGC-1α pathway. Finally, major ginsenoside
monomers in GS were identified for increasing HK-II, MPC1, and SIRT1
levels to promote mitochondrial energy promotion. These findings
confirmed the potential role of GS for energy metabolism, which
supported the clinical application of ginseng for the benefit of human
health and ischemic disorders (Fig. 9) .
As previously reported, ginsenosides have potential health benefits in
improving immunity and energy, and combating cardiovascular,
cerebrovascular, and metabolic diseases
(Ratan et al., 2020), which might be
associated with the enhancement of glucose energy metabolism. In
individual cell types, glycolysis and OXPHOS are utilized to maintain
energy metabolic needs. Several studies have demonstrated that neurons
depend on mitochondria, and endothelial cells rely on glycolysis,
whereas skeletal cells utilize both OXPHOS and glycolysis for energy
supply (Chang, Singh, Gross & Kioussi,
2019; De Bock et al., 2013;
Zeiger, Stankowski & McLaughlin, 2011).
For the first time, we chose a couple of types of cell lines such as
cardiomyocytes (H9c2, primary cardiac myocytes), neurons (differentiated
PC12, primary neurons), bone marrow-derived stem cells, endotheliocytes
(HUVECs), and stem cells to determine the effect of GS on energy
metabolism. The analysis of basal OCR demonstrated that GS preferably
increased oxygen consumption in cells, cardiomyocytes, and neurons,
which were highly dependent on mitochondria, not those cell lines
depdendent on glycolysis for energy production, such as endothelial
cells, or stem cells. Furthermore, ATP measurement and mitochondrial
stress assay confirm the preferable effect of GS on mitochondrial
respiration-dominant cardiomyocytes and neurons, similar to previous
findings that ginseng extract containing Rg1, Re, Rb1, and Rd can
promote the OCR and ATP production in healthy cardiomyocytes
(Huang et al., 2019).
Glucose 6-phosphate is an essential metabolite in the first step of
glucose metabolism, which is converted from glucose by hexokinases
(John, Weiss & Ribalet, 2011). During
the early stage of glycolysis, the content of glucose-6-phosphate was
increased by GS in both cell lines. However, other major metabolites
before pyruvate were obviously decreased in cardiomyocytes and not in
endothelial cells. It suggests that the rapid metabolic process from
fructose 6-phosphate to pyruvate was induced by GS in cardiomyocytes,
which may be related to GS-mediated high expression of key rate-limiting
enzymes, HK-II, PFKP, and PKM2. Meanwhile, pyruvate transport to
mitochondria medicated by MPC1/2 and its conversion into acetyl-CoA by
PDH were enhanced by GS in cardiomyocytes, similar to the functions of
Rb1 (Li et al., 2017) and Rg5
(Peng et al., 2019). PDH is a vital
regulatory enzyme for the connection of glycolysis to the aerobic TCA
cycle, which was activated by GS in cardiomyocytes, not in endothelial
cells. As previously reported from heart tissue proteome and urine
metabolome, total ginsenosides or ginseng extract can modulate the
levels of TCA cycle proteins, such as pyruvate dehydrogenase complex in
rat heart tissues (Wang, Zhou, Yi, Jiang
& Liu, 2012; Yang et al., 2016), which
was deeply confirmed by our study. In addition, multiple metabolites in
cardiomyocytes from succinate to citrate were increased by GS, but GS
only enhanced the metabolic process from succinate to malate in
endothelial cells. Compared with current reports, our findings clearly
explore the role of GS on energy metabolism that GS pretreatment
preferably promotes glucose metabolism into the TCA cycle for providing
energy in cardiomyocytes, and has a slight function in endothelial cells
through regulating different key enzymes in a series of steps of glucose
metabolism. During the OXPHOS, for the first time, we found that GS
mainly promoted the expressions of ETC complex proteins I-IV to increase
the transfer of electrons in the inner membrane of mitochondria, which
were not changed in endothelial cells. Currently, only one previous
report has shown that ginsenoside Re can rescue complex IV deficiency in
PINK1 null neuronal cells (Kim, Song,
Yoon, Shehzad, Kim & Son, 2012). However, we need to perform the13C-based metabolic flux analysis to identify the
accurate mapping of GS-mediated aerobic glucose oxidation in the future.
NAD+ can modulate glycolysis in the cytoplasm and the
TCA cycle/OXPHOS in the mitochondria, and activate sirtuins to maintain
energy homeostasis (Canto, Menzies &
Auwerx, 2015). In our study, NAD+ levels in the
cardiomyocytes, primary neurons, and fly brains were increased by GS
treatment, which can activate SIRT1 to match cellular energy
requirements (Canto & Auwerx, 2012).
SIRT1-mediated deacetylation leads to higher levels of PGC-1a to induce
the nuclear transcription of Nrf1/2 for mitochondrial biogenesis
(Canto & Auwerx, 2012). In different
models, we found that SIRT1 and its downstream pathway were obviously
activated by GS, which may be the molecular mechanism of GS on the
induction of mitochondrial mass. As we showed, GS-mediated mitochondrial
respiration, ATP production, and higher levels of ETC proteins were
abolished by NAM, which confirm that GS promote mitochondrial
biosynthesis and function through the activation of SIRT1 pathway. In
the stress conditions, such as hypoxia or OGD, we obtained similar
findings that GS can activate the SIRT1-PGC-1α pathway to increase the
levels of all five complexes and mitochondrial bioenergetics to maintain
energy homeostasis in cardiomyocytes, which may be one of the potential
mechanisms of GS against ischemic disorders. Many studies demonstrate
that ginsenoside Rg3 or a prescription containing ginseng could enhance
mitochondrial respiration in healthy and unhealthy cells
(Lin et al., 2015;
Sun et al., 2013). Our results together
with previous findings can verify the beneficial functions of
ginsenosides on mitochondrial energy metabolism without ROS injury.
For glucose oxidation, very few ginsenosides, such as compound K, Rg3,
or Rg5, can regulate HK-II expression to inhibit tumor growth or protect
cardiomyocyte ischemic injury (Chen et
al., 2019a; Yang et al., 2017). In our
study, we found that other ginsenosides Rg1, Re, Rf, and Rc can
upregulate HK-II level. For SIRT1 activation, ginsenoside monomers,
including Rb1, Rb2, Rc, and Rg3, have been identified as potential SIRT1
activators (Wang, Liang, Chen & Zhao,
2016; Yang, Ma, Zhou, Xu & Zhang,
2017). Ginsenoside Rb2, Rc, and F1 can promote SIRT1 activation to
increase ATP production and inhibit oxidative stress in cardiomyocytes
(Wang, Liang, Chen & Zhao, 2016).
Another study showed that ginsenosides Rg1, Rb1, Re, Rh2, Rg3, and Rg5
can protect ischemic brain injury, which is associated with STIRT1
activation and Toll-like receptor 4/myeloid differentiation factor 88
signaling pathways (Cheng et al., 2019).
In our study, we found that most ginsenosides can activate SIRT1 and
enhance ATP content, similar to previous findings. According to the
content percentage of each ginsenoside in GS, we found that Rh1, Rb3,
Rb2, or Rf at the concentration of lower than 0.2 μg/mL had a good
effect on SIRT1 activation and mitochondrial respiration. Moreover, the
analysis from published findings speculated that two glucopyranosyl
groups on the C-3 position of ginsenosides are critical for SIRT1
activity (Ma, Zhou & Yang, 2015;
Wang, Liang, Chen & Zhao, 2016;
Yang, Ma, Zhou, Xu & Zhang, 2017).
Therefore, the binding mechanism of ginsenosides on the SIRT1 needs to
be well studied to explore the sites of key amino acids in SIRT1
proteins using molecular docking, protein interaction, and mutation
techniques. More importantly, the synergistic effects of different
ginsenosides targeting potential molecules of glucose metabolism and
SIRT1 pathway could be investigated in the future.