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.