Shengqiang Han,Long You,Yeye Hu,Shuai Wei,Tingwu Liu,Jae Youl Cho,Weicheng Hu
Ginsenoside F2 enhances glucose metabolism by modulating insulin signal transduction in human hepatocarcinoma cells
Ginsenoside F2,PI3K/AKT signaling pathway,Glucose metabolism
Background Ginsenoside F2 (GF2), a minor component of Panax ginseng, has been reported to possess a wide variety of pharmacological activities. However, its effects on glucose metabolism have not yet been reported. Here, we investigated the underlying signaling pathways involved in its effects on hepatic glucose. Methods HepG2 cells were used to establish insulin-resistant (IR) model and treated with GF2. Cell viability and glucose uptake-related genes were also examined by real-time PCR and immunoblots. Results Cell viability assays showed that GF2 up to 50 μM did not affect normal and IR-HepG2 cell viability. GF2 reduced oxidative stress by inhibiting phosphorylation of the mitogen-activated protein kinases (MAPK) signaling components such as c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 MAPK, and reducing the nuclear translocation of NF-κB. Furthermore, GF2 activated PI3K/AKT signaling, upregulated the levels of glucose transporter 2 (GLUT-2) and GLUT-4 in IR-HepG2 cells, and promoted glucose absorption. At the same time, GF2 reduced phosphoenolpyruvate carboxykinase and glucose-6-phosphatase expression as well as inhibiting gluconeogenesis. Conclusion Overall, GF2 improved glucose metabolism disorders by reducing cellular oxidative stress in IR-HepG2 cells via MAPK signaling, participating in the PI3K/AKT/GSK-3β signaling pathway, promoting glycogen synthesis, and inhibiting gluconeogenesis.
Ginsenoside F2 enhances glucose metabolism by modulating insulin signal transduction in human hepatocarcinoma cells

Ginsenoside F2 (GF2), a minor component of Panax ginseng, has been reported to possess a wide variety of pharmacological activities. However, its effects on glucose metabolism have not yet been reported. Here, we investigated the underlying signaling pathways involved in its effects on hepatic glucose.

HepG2 cells were used to establish insulin-resistant (IR) model and treated with GF2. Cell viability and glucose uptake-related genes were also examined by real-time PCR and immunoblots.

Cell viability assays showed that GF2 up to 50 μM did not affect normal and IR-HepG2 cell viability. GF2 reduced oxidative stress by inhibiting phosphorylation of the mitogen-activated protein kinases (MAPK) signaling components such as c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 MAPK, and reducing the nuclear translocation of NF-κB. Furthermore, GF2 activated PI3K/AKT signaling, upregulated the levels of glucose transporter 2 (GLUT-2) and GLUT-4 in IR-HepG2 cells, and promoted glucose absorption. At the same time, GF2 reduced phosphoenolpyruvate carboxykinase and glucose-6-phosphatase expression as well as inhibiting gluconeogenesis.

Overall, GF2 improved glucose metabolism disorders by reducing cellular oxidative stress in IR-HepG2 cells via MAPK signaling, participating in the PI3K/AKT/GSK-3β signaling pathway, promoting glycogen synthesis, and inhibiting gluconeogenesis.

Diabetes, one of the fastest growing chronic diseases [1], leads to the occurrence of metabolic syndrome, characterized by retinopathy, nephropathy, dyslipidemia, and cardiovascular disease [2], resulting in almost 5 million deaths each year. A total of around 425 million cases of diabetes were reported by the International Diabetes Federation worldwide in 2017; this number is expected to increase to 640 million by 2045 [3]. Diabetes is divided into type 1 diabetes and type 2 diabetes (T2DM); whereas T2DM accounts for 90% of cases [4]. The role of insulin in glucose homeostasis is specific to the muscle, liver, and hypothalamus, which may underlie the pathogenesis of T2DM, and is linked to disorders of glucose and lipid metabolism [5]. Meanwhile, hyperglycemia can cause oxidative stress and an accumulation of oxidized proteins, which intensifies IR and contributes to the development of T2DM [6]. As a major target organ of insulin action, liver plays an important role in maintaining systemic glucose homeostasis. With the help of insulin or glucagon action, hepatocytes regulate the uptake and use of glucose and the production of hepatic glucose. Insulin receptor substrate 1 (IRS-1)/ PI3K/ AKT signaling is the main pathway of insulin signal transduction [7]. In the presence of insulin, IRS-1 is tyrosine phosphorylated, which activates PI3K kinase to bind phosphoinositide-dependent kinase-1 (PDK1); activated PDK1 directly phosphorylates AKT. Activated AKT phosphorylates downstream effectors at serine/threonine residues to regulate sugar metabolism and gluconeogenesis [8]. When IR occurs in the liver, the insulin signaling pathway is damaged, cell glucose metabolism is disordered, gluconeogenesis is enhanced, liver glucose output is increased, and symptoms of hyperglycemia occur [9]. Studies have reported that a high glucose level can induce oxidative stress in the liver accompanied by the release of mitochondrial cytochrome C, and high levels of reactive oxygen species (ROS) activate mitogen-activated protein kinases (MAPKs) and PI3K/AKT signaling, further aggravating the development of IR or T2DM [10]. Oxidative stress regulates glucose metabolism of hepatocytes via IR signaling. Therefore, inhibition of oxidative stress has been considered an effective strategy to improve IR and prevent the progression of T2DM. Ginsenosides are bioactive components of Panax ginseng (P. ginseng) with multiple pharmacological functions. Among them, 20(S)-ginsenoside F2 (GF2) can be obtained in small amounts from natural P. ginseng leaves or in large quantities through biotransformation of the ginsenosides Rb1, Rb2, Rb3, Rc, and Rd, the contents of which are high in the roots and leaves of P. ginseng (Fig. 1A) [11]. It has recently been reported that ginsenoside Rb1, Rb2, Rb3, Rc and Rd improved glucose uptake via different mechanisms [[12], [13], [14], [15]]. Protopanaxadiol type ginsenosides Rb1, Rb2, Rb3, Rc and Rd share the same chemical structure with at least one glucoside residue attached to C-3 and C-20 of the aglycone, respectively, which might be associated with glucose metabolism [16]. Our previous studies showed that GF2 can prevent obesity by directly binding peroxisome proliferator-activated receptor gamma (PPARγ) and promoting mitochondrial biosynthesis via activation of the AMP-activated protein kinase (AMPK) pathway [17]. However, the mechanism through which GF2 inhibits diabetic complications and promotes glucose uptake is unclear. The aim of this study is to investigate the effects of GF2 on IR using HepG2 cells and elucidate the underlying mechanism.

GF2 (purity,≥99%) was purchased from Chengdu PhytoElite Biotech (Chengdu, China). The MEM medium was obtained from Hyclone (Logan, UT, USA). Antibiotics (100 × penicillin/streptomycin) and Trypsin-EDTA were purchased from Gibco (California, USA). Fetal bovine serum (FBS) was purchased from Corning (New Zealand). 2-NBDG and TRIzol reagent were from Invitrogen (Carlsbad, CA, USA). cDNA synthesis kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). FastStart Universal SYBR Green Master was provided from Roche (Mannheim, Germany). Malondialdehyde (MDA) and superoxide dismutase (SOD) assay kits were acquired form Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). The kits for reactive oxygen species assay, glycogen PAS stain and glycogen content assay were purchased from Solarbio (Beijing, China). Cell lysis buffer and eECL kit were purchased from CWBio (Jiangsu, China). The primary antibodies against p-JNK (#4671), JNK (#9258), p-ERK1/2 (#8544), ERK1/2 (#4348), p-p38 MAPK (#4511), p38MAPK (#9212), NF-κB p65 (#8242), p-AKT (#4060), AKT (#9272), p-PDK1 (#3438), PDK1 (#3062), p-GSK-3β (#5558), GSK3β (#12456), Anti-β-actin pAb-HRP-DirecT (#PM053-7) was from Medical & Biological Laboratories CO., LTD (Nagoya, Japan). Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 647) (ab150075) was from Abcam (Cambridge, MA, USA).

Human HepG2 hepatocytes were provided by China Center for Type Culture Collection and cultured in MEM medium with 10% FBS and 1% antibiotics at 37 °C in a humidified atmosphere with 5% CO2. The IR-HepG2 cell model was established as described previously [10,18]. Briefly, HepG2 cells were seeded into 96-well plate at 1 × 104 cells/well. After cell attachment, the medium was removed, cells were cultured in serum-free medium with different concentrations of high glucose (35, 45, 55, or 65 mM) for 24 h. Afterwards, cellular glucose uptake was monitored with 2-NBDG [19] and fluorescence intensity was measured by a microplate reader (Tecan infinite 200, Switzerland).

Normal cultured cells and IR model cells were treated with various concentrations of GF2 for 24 h and the cell viability evaluated by MTT assay as described previously [20].

Briefly, IR-HepG2 cells were treated with different concentrations of GF2 (12.5, 25, or 50 μM) for 12 h. Afterwards, the medium was removed, and the fluorescence intensity was described as described above.

IR-HepG2 cells were treated with different concentration of GF2 for 12 h. The medium was then replaced with serum-free medium containing 10 μM DCFH-DA probes and incubated at 37°C for 30 min. The fluorescence intensity was observed and photographed under the IX71 inverted microscope (Olympus, Tokyo, Japan) and analyzed with ImageJ (National Institutes of Health, Bethesda, MD, USA).

Followed the treatment, total protein was isolated using RIPA lysis buffer. The MDA content and SOD activity was determined manufacturer instructions.

Following the treatment, glycogen staining was carried out according to the manufacturer's protocol. Briefly, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature. After being reacted with oxidant for 20 min, cells were then washed with PBS and stained with Schiff Reagent for 20 min. Cells were finally washed 3 times with distilled water and visualized the IX71 inverted microscope. Following the treatment, total protein was isolated using RIPA lysis buffer. The glycogen content was determined manufacturer instructions.

IR-HepG2 cells were treated with different concentration of GF2 for 12 h. Cells were then washed by PBS and lysed directly in Trizol for RNA isolation. After cDNA synthesis, the reaction was carried out using the CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). The results were normalized to endogenous GAPDH by 2−ΔΔCT method. The sequences of primers for this study were listed in Table 1.

IR-HepG2 cells were treated with GF2 for different points. The nuclear proteins were prepared using a nuclear and cytoplasmic extraction kit (CWbio, Taizhou, China). The total proteins were prepared using a RIPA lysis buffer (50mM Tris pH7.4, 150mM NaCl, 1% NP-40, 0.25% sodium deoxycholate) with Roche complete protease inhibitor and phosphatase inhibitor cocktails (Mannheim, Germany). The concentration of total protein or nuclear protein was determined using BCA protein assay. The protein was separated by Tris-glycine SDS gels and transferred onto PVDF membranes. Western blot analysis was carried out as described previously [20].

IR-HepG2 cells were treated with 50 μM GF2 for 12 h. Cells were then immobilized by 4% polyformaldehyde for 15 min. After washing with PBS, cells were permeabilized in 0.5% Triton X-100 for 10 min. The cells were then blocked with 3% BSA for 1h and incubated with primary antibody for 2 h at a room temperature. After being rinsed three times with PBS, cells were incubated with Alexa Fluor 647 Donkey Anti-Rabbit IgG (1:400) in the dark for 1 h. Immunofluorescent images were obtained using a confocal laser scanning microscope.

The results have been represented as the mean ± standard deviation (SD). Variances among two groups were analyzed by Student's t-test. All statistical analyses were completed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). A p-value less than 0.05 indicated significant differences.

IR is one of the main causes of T2DM. Despite years of research, the mechanism of IR remains unclear. The known causes of IR include endoplasmic reticulum stress, inflammation, and ROS [21,22]. T2DM has also been linked to increased oxidative stress caused by a variety of conditions [23]. Natural products, such as corylin and trilobatin, have been shown to alleviate IR. Chen et al found that corylin, derived from Psoralea corylifolia L., not only dramatically reduced body weight and fat accumulation but also improved insulin sensitivity [24]. In addition, trilobatin showed potential to ameliorate IR in T2DM mice [25]. Trilobatin could also inhibit the effects of a high-fat diet and IR via IRS/AKT/GLUT-4 signaling. An imbalance of active oxygen in the body is the primary driver of oxidative stress; this plays a causal role in IR and contributes to the development of T2DM [26]. Studies have shown that high glucose results in cellular oxidative stress [27]. For instance, T2DM caused by high glucose accumulation disrupted the functions of mitochondria, which enhanced ROS production via ERK1/2-PI3K/Akt-IRF3 signaling [28]. High levels of glucose can induce oxidative stress and IR in HepG2 cells [29,30]. In this study, we treated HepG2 cells with 35-65 mM glucose for 24 h and found that glucose uptake was inhibited; this inhibitory effect was significant and increased as the concentration of glucose increased (Fig. 1B). At 55 and 65 mM glucose, cellular 2-NBDG uptake decreased by 45.10% and 47.27%, respectively. MTT assay results showed that high glucose (65 mM) reduced cell viability (Fig. 1C). We therefore used 55 mM glucose to establish a model of IR for subsequent experiments. We previously showed that GF2 could inhibit adipogenesis in vitro and could be used to treat obesity in vivo; these effects were AMPK signaling-mediated [17]. Qi et al reported that zinc was able to relieve obesity and blood glucose levels, demonstrating a relationship between obesity and glucose metabolism [31]. We thus investigated whether GF2 had similar effects on glucose metabolism in T2DM. To exclude a cytotoxic effect of GF2, cell viability was examined by MTT assay. GF2 at 12.5-50 μM had no negative effect on HepG2 cell viability (Fig. 1D). However, cell viability was significantly reduced at 100 μM GF2. Similarly, 12.5-50 μM GF2 and 1 mM metformin did not have cytotoxic effects on high glucose (55 mM)-induced IR-HepG2 cells (Fig. 1E). Thus, 55 mM glucose was determined to be the most suitable dose for establishing an IR model in HepG2 cells. Since HepG2 cells were used to mimic normal hepatic cells, we did not want GF2 to have cytotoxic effects in the IR model. Thus, 12.5-50 μM GF2 was used to evaluate the effects of GF2 on glucose metabolism.

The liver is a primary site of glucose metabolism; it is also the primary organ for energy metabolism [32]. When postprandial blood glucose increased, the liver absorbs glucose and synthesizes and stores glycogen and lipids. During conditions of starvation, when levels of blood glucose become lower, the liver breaks down glycogen or produces glucose through gluconeogenesis to maintain the blood glucose balance [33]. When IR occurs, liver glucose metabolism is disordered, glucose absorption and use are reduced, gluconeogenesis is enhanced, and liver glucose output is increased. Studies have shown that the hypoglycemic effects of ginsenosides include improvement of IR, glucose tolerance, and glucose homeostasis; antioxidation; and a reduction in gluconeogenesis [12,16]. We previously showed that fluorescent intensity of 2-NBDG was decreased with increasing glucose (Fig. 1B). Whereas GF2 treatment counteracted these effects in IR-HepG2 cells and reversed glucose uptake (Fig. 2A). This result is consistent with the effects of Rg3, Rb2, and Re to promote glucose absorption [14,34]. In liver, glucose uptake is mediated by GLUT-2 and GLUT-4 [35,36]. Therefore, we assessed mRNA expression of GLUT-2 and GLUT-4 by conducting qRT-PCR. GF2 promoted mRNA expressions of GLUT-2 and GLUT-4 in IR-HepG2 cells in a dose-dependent manner (Fig. 2B and C). Overall, GF2 reversed the decrease in glucose uptake that was a result of the high glucose concentration; in fact, glucose uptake was accelerated. GF2 also unregulated mRNA expression of GLUTs in both insulin-dependent and -independent manners.

ROS can cause damage or death of cells and organs by directly impairing many cellular components, including proteins and nucleic acids [37]. The organelle of ROS production is the mitochondrion, which plays an important role in homeostasis. If glucose levels remain high, i.e., conditions of hyperglycemia, mitochondria become fragmented, resulting in dysregulation of the electron transport chain and increased ROS production [38]. Thus, many studies have tried to establish a model to mimic the effects of hyperglycemia to identify ways to control and recover glucose homeostasis. Since HepG2 cells have similar functions as normal hepatic cells in terms of lipid and glucose metabolism, this cell line was used to generate a model of IR [39,40]. HepG2 cells were incubated in high glucose to induce hyperglycemia, resulting in an overproduction of ROS. We then treated these cells with various concentrations of GF2 to assess scavenging. DCFH-DA staining was applied to detect ROS in IR-HepG2 cells (Fig. 3A). As the dose increased, ROS levels were significantly reduced by GF2. This result was confirmed and quantified using mean fluorescence intensity (Fig. 3B). Another two biomarkers of oxidative stress, MDA, and SOD were also assessed to evaluate the ROS scavenging effect of GF2. MDA, which is produced by polyunsaturated fatty acids, is commonly used to assess oxidative stress. The hyperglycemic environment caused an increased production of ROS, which resulted in increased levels of MDA [41]. Interestingly, GF2 treatment suppressed the production of MDA in a dose-dependent manner (Fig. 3C). SOD, however, showed potent antioxidant activity; this activity was inhibited at high concentrations of glucose. GF2 treatment remarkably relieved impairment of the hyperglycemic effect (Fig. 3D). Taken together, the overproduction of ROS induced by high levels of glucose was alleviated by GF2, indicating GF2 may have antioxidative properties that help relieve the complications of hyperglycemia.

Insulin is secreted by the pancreas and is induced by high levels of glucose in the blood. Insulin keeps blood sugar in balance by facilitating cellular uptake of glucose from the bloodstream [42]. These cells transport glucose from the blood and store it in the liver as glycogen [40]. However, if insulin levels are not sufficient, blood glucose levels will continue to rise, resulting in IR [39,43]. In the long term, this condition results in T2DM. Under conditions of hyperglycemia, hepatic IR occurs in addition to a decrease in hepatic glycogen synthesis [44]. This result was consistent with our periodic acid-Schiff staining data (Fig. 4A). Glycogen synthesis recovered following treatment with GF2. Moreover, 50 μM GF2 treatments resulted in higher hepatic glycogen levels than the control. This result was confirmed by glycogen quantitation (Fig. 4B), indicating that GF2 improves glycogen synthesis in hyperglycemia and has a potent effect against IR. Next, we assessed protein levels of glycogen synthase kinase (GSK)-3β and its phosphorylation. GSK-3β is an enzyme involved in glycogen synthesis in response to insulin [45]. When HepG2 cells were incubated in a high concentration of glucose, phosphorylation of GSK-3β was suppressed; phosphorylation was dramatically enhanced following treatment with GF2 (Fig. 4E). As the dose of GF2 increased, phosphorylation of GSK-3β also increased gradually, eventually exceeding the level observed in conditions of normoglycemia at 50 μM. This result is consistent with the glycogen synthesis results (Fig. 4B), suggesting that GF2 not only recovered glycogen synthesis inhibited by hyperglycemia but also directly activated its synthesis. Gluconeogenesis, a metabolic pathway that results in the formation of glucose and primarily occurs in the liver, also need to be considered in hyperglycemia. mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), which are involved in gluconeogenesis, were assessed by qRT-PCR. Normally, gluconeogenesis is important in balancing blood sugar in the body; it must also be considered in T2DM. Some studies have reported that insulin can effectively block gluconeogenesis [[46], [47], [48]], while T2DM is characterized by IR. PEPCK regulates the rate of gluconeogenesis and insulin targets its transcription. G6Pase adjusts levels of blood glucose as well as the last step of gluconeogenesis [47]. Our results showed that IR-HepG2 cells significantly increased levels of PEPCK and G6Pase mRNA while GF2 treatment downregulated transcription of these enzymes in a dose-dependent manner (Fig. 4C and D). Thus, GF2 can prevent gluconeogenesis in IR-HepG2 cells by decreasing mRNA levels of PEPCK and G6Pase, suggesting a novel way to relieve glucose formation in insulin-resistant conditions. Some researchers have reported crosstalk of GSK-3β with PI3K/AKT signaling [49,50]. Kuai er al. Also reported that Sang-Tong-Jian, a mixture medicine derived from mulberry leaf, can improve T2DM [51]. Treatment with this medicine not only ameliorated glycometabolism but also relieved IR by upregulating PI3K/AKT signaling. As a result, we investigated whether GF2 could also adjust glycometabolism by activating PI3K/AKT signaling. High level of glucose dramatically suppressed phosphorylation of PDK1, and AKT, whereas GF2 treatment increased p-PDK1, and p-AKT levels compared to the control (Fig. 4E).

Studies have reported that a high level of glucose can induce phosphorylation of MAPKs pathway components [[52], [53], [54]]. Previous studies demonstrated that ROS can also activate MAPKs signaling. As we illustrated, high levels of glucose markedly increased ROS production (Fig. 3A); this was decreased by GF2 in a dose-dependent manner. As shown in Fig. 5A, GF2 significantly repressed the phosphorylation of JNK, ERK and p38 in IR-HepG2 cells. GF2 at 50 μM also significantly suppressed translocation of p65 to nucleus (Fig. 5B). Immunofluorescence showed that a high level of glucose increased expression of NF-κB p65; treatment with 50 μM GF2 reversed the translocation of p65 (Fig. 5C). Taken together, GF2 can downregulate both MAPK and NF-κB signaling induced by hyperglycemia. In the present study, we discussed the hypoglycemic effect of GF2 in human insulin-resistant hepatic cells (Fig. 6). A high concentration of glucose resulted in IR in HepG2 cells, which may mimic T2DM in the human body. GF2 enhanced glycogen synthesis and inhibited gluconeogenesis. However, there is further work to be done, including investigation of different signaling components and their specific functions. Future studies will elucidate the signaling pathways that regulate glycogen synthesis, gluconeogenesis, and oxidative stress. Overall, GF2 has potential as a nutraceutical to treat insulin resistant T2DM.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.