Introduction
Aerobic cellular respiration refers to the process by which organisms convert nutrients into chemical energy, adenosine triphosphate (ATP), using oxygen, which is dominated in highly aerobic tissues, such as cardiac and neural systems (Hall, Klein-Flugge, Howarth & Attwell, 2012; Hickey et al., 2009; Neubauer, 2007). During glucose aerobic metabolism, glucose with 6-carbon is broken down into 3-carbon pyruvate by multiple key enzymes such as hexokinase-I/II (HK-I/II), phosphofructokinase-1 (PFK-1), and pyruvate kinase (PK) in the cytoplasm. Then pyruvate is transported into the mitochondria through the mitochondrial pyruvate carrier (MPC) (Bricker et al., 2012) and metabolized to synthesize acetyl-coenzyme A (CoA) by pyruvate dehydrogenase (PDH) (Sugden & Holness, 2003). After the metabolism of tricarboxylic acid (TCA) cycle, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) from acetyl-CoA can pass their electrons into the mitochondrial electron transport chain (ETC) to produce ATP molecules, with the help of oxygen (Fernie, Carrari & Sweetlove, 2004). During this process, respiratory chain protein complexes I-V embedded in the inner mitochondrial membrane is involved in ATP production (Wallace, Fan & Procaccio, 2010). Abnormal energy metabolism is a critical target during the progression of disease, including myocardial ischemia, heart failure (Fillmore & Lopaschuk, 2013), stoke (Narne, Pandey & Phanithi, 2017), or neurodegenerative diseases (Butterfield & Halliwell, 2019). During the exposure to hypoxia, cells display decreased carbon flux into the TCA cycle and diminished electron flux through ETC (Papandreou, Cairns, Fontana, Lim & Denko, 2006; Wheaton & Chandel, 2011). Moreover, oxygen-glucose deprivation (OGD) can cause mitochondrial damage and ATP depletion in cardiomyocytes and neurons (Almeida, Delgado-Esteban, Bolanos & Medina, 2002; Kalogeris, Baines, Krenz & Korthuis, 2012). Therefore, the enhancements of glucose oxidation and mitochondrial bioenergy have been potential strategies against various disorders, such as ischemic conditions.
Oxidized nicotinamide adenine dinucleotide (NAD+) is a well-known small molecule that drives energy metabolism through its electron transfer function (Hopp, Gruter & Hottiger, 2019). Increased NAD+ level promotes the activity of sirtuin 1 (SIRT1), a strong deacetylase of class III histone deacetylase, which heavily implicates in a number of physical and pathological processes, such as aging-related diseases (Imai & Guarente, 2014). SIRT1 deacetylates its substrate, peroxisome proliferator initiated receptor gamma and coactivator 1 alpha (PGC-1α), to regulate mitochondrial biosynthesis and energy metabolism (Aquilano, Baldelli, Pagliei & Ciriolo, 2013). PGC-1α can bind and co-activate different transcription factors, such as nuclear respiratory factor-1 (Nrf-1) and Nrf-2, in the promoter of mitochondrial transcription factor A (TFAM), which increases the transcription of key mitochondrial enzymes, such as cytochrome c oxidase subunits (COXII, COXIV) and ATP synthase β subunit, and drives mitochondrial DNA transcription and replication through the interaction of TFAM (Irrcher, Adhihetty, Sheehan, Joseph & Hood, 2003; Wu et al., 1999). Previous studies have shown that the activation of SIRT1-PGC-1α pathway plays a protective role in hypoxia or OGD-induced injuries (Ma et al., 2017; Zhou, Wang, Li, Yu & Zhao, 2017), which improves mitochondrial function to prevent and treat ischemic heart or brain disorders (Yang, Mukda & Chen, 2018; Zhao et al., 2019).
Ginsenosides are major active components of Panax ginseng C.A. Meyer , a medicinal herb as new-resource health food approved by the Ministry of Health, China. They are extensively used for diverse beneficial effects on myocardial infarction, stroke, and ischemia/reperfusion (I/R) injury (Aravinthan et al., 2015; Jia, Zhang, Huang & Leung, 2012; Liu, Anderson, Fernandez & Dore, 2019). Currently, about 250 ginsenosides have been isolated and identified, which are mainly divided into three groups, protopanaxadiol (PPD, such as Rb1, Rb2, Rc, Rd, Rg3), protopanaxatriol (PPT, such as Re, Rf, Rg1, Rh1), and oleanolic acid (OA, such as Ro), based on their chemical structures (Wu, Kwaku, Li, Yang, Ge & Xu, 2019). Proteomic analysis has shown that total ginsenosides (GS) can increase the expression of proteins in the TCA cycle to enhance cardiac energy metabolism in the ischemic rat (Wang, Zhou, Yi, Jiang & Liu, 2012). Meanwhile, ginsenoside Rb1, Rb3, Rg3, Rg1, Rg5, and compound K exert cardioprotective and neuroprotective effects by increasing energy metabolism (Chen et al., 2019b; Xu, Ma, Fan, Chen, Zhang & Tang, 2019; Yang et al., 2017), improving mitochondrial dynamics and quality (Dong et al., 2016; Sun et al., 2013), or inhibiting mitochondrial apoptosis (Huang et al., 2020; Li et al., 2018). Importantly, several ginsenosides, such as Rb1, Rc, F1, Rg3, Rh2, and their metabolites M1-M4 have been identified as SIRT1 activators (Ma, Zhou & Yang, 2015; Wang, Liang, Chen & Zhao, 2016; Yang, Ma, Zhou, Xu & Zhang, 2017), which play protective effects on a series of pathological injuries, such as oxidative stress, inflammation, senescence, hypoxia, or OGD (Cheng et al., 2019; Song et al., 2014; Wang et al., 2019). However, the molecular mechanism underlying the effects of GS and each ginsenoside monomer on mitochondrial aerobic metabolism and biosynthesis in cardiomyocytes and neurons remains unclear and needs to be further investigated.
In this study, we first investigated the effect of GS on mitochondrial oxygen consumption rate (OCR) in a series of cell lines, including aerobic respiration-dominated cardiomyocytes, neuronal cells, and skeletal myoblasts (Di Lisa, Canton, Menabo, Kaludercic & Bernardi, 2007; Zeiger, Stankowski & McLaughlin, 2011), as well as anaerobic respiration-dominated vascular endothelial, epithelial, and stem cells (De Bock et al., 2013; Tang et al., 2016). Then ATP production and mitochondrial respiration capacity (MRC) were examined in untreated and GS-treated cardiomyocytes and neurons. Targeted metabolomics and Western blot analysis were performed to observe the effect of GS on the contents of major metabolites and the levels of key multiple enzymes from glycolysis and TCA cycle. Furthermore, NAD+ level, SIRT1-PGC-1α pathway, and mitochondrial biosynthesis in the cell, Drosophila melanogaster, and mice models were further evaluated to explore the molecular mechanism of GS. In addition, we observed the effect of GS on SIRT1-mediated mitochondrial function in hypoxia- and OGD-induced cell models. Of note, the ginsenoside monomer in GS for promoting SIRT1 activation and mitochondrial energy production was preliminarily identified in cardiomyocytes. This is the first study to explore the molecular mechanism by which GS promote mitochondrial energy metabolism, which may provide new insights into the clinical application of ginseng for the prevention and treatment of cardiac and neurological disorders.