EGCG was purchased from Shanghai SolarBio Bioscience and Technology Co., Ltd. (Shanghai, China). To determine the dose-response association of EGCG on glycogen synthesis and lipogenesis, a series of molar concentrations (0.01, 0.1, 1.0 and 10 µM) of EGCG were prepared as previously reported (15).

The human hepatocyte cell line HepG2 (ref. no. ATCCHB8065; American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing normal glucose (5 mM), supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin (Thermo Fisher Scientific, Inc.), in an incubator at 37°C and 5% CO₂. HepG2 cells were cultured in complete media (CM) with 10% FBS until 70% confluence was reached. At 24 h prior to all experimental procedures, 5 or 30 mM D-glucose (Thermo Fisher Scientific, Inc.), termed low glucose (LG) and high glucose (HG) respectively, were added to cells.

Cells (4×10³) were seeded on 24-well plates for all assays. When the cells reached 90% confluence, the CM was discarded and starvation medium (SM) containing 0.5% FBS was added. Following incubation at 37°C with SM for 6 h, 100 nM insulin (Eli Lilly Australia Pty, Ltd., Melrose Park, NSW, Australia) was added to each well, followed by the addition of 0.01–10 µM EGCG to the appropriate wells in duplicate. Plates were then maintained for 24 h in 5% CO₂ at 37°C. This treatment procedure was used for the purposes of all experiments in the present study.

A single male C57BL/6J mouse (weight, 20–25 g; age, 12 weeks) was obtained from the Peking University Health Science Center (Beijing, China). It was maintained in a constructed shelter at 20–26°C, 30–60% humidity, 12:12 light/dark cycle, and provided with adequate supplies of food and fresh water. All experiments were approved by the Institutional Animal Care and Use Committee at the Fourth Military Medical University (Xi'an, China; no. 20150302). The liver was removed and primary hepatocytes were isolated using a two-step collagenase perfusion method (0.2 mg/ml type IV collagenase in Hanks' balanced salt solution; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) as described previously (15). The hepatocytes were collected by centrifugation at 100 × g for 8 min at room temperature. The cells were immediately re-suspended in pre-warmed William's E medium (Merck Millipore) supplemented with 10% FBS, 20 ng/ml dexamethasone (Merck Millipore), an insulin (5 mg/l)-transferrin (5 mg/l)-sodium selenite (5 g/l) solution (Merck Millipore), and 10 g/ml gentamicin (Invitrogen; Thermo Fisher Scientific, Inc.). The hepatocytes were then plated in collagen-coated 25 cm² flasks at 1×10⁶ cells/flask.

Following pretreatment with 5 and 30 mM D-glucose, glycogen levels were measured in the HepG2 cells and primary hepatocytes at 37°C for 3 h in the presence of 100 nmol/l insulin (US Biological, Salem, MA, USA), using a Glycogen Assay kit (BioVision, Inc., Milpitas, CA, USA), according to the manufacturer's protocol. Briefly, for hydrolysis, the Enzyme Mixture was added and the plates were incubated for 30 min at 37°C; then the Glycogen Fluorometric Detector solution, which contains 10-acetyl-3,7-dihydroxyphenoxazine, was added and the fluorescence signal was monitored with an excitation wavelength of 530–540 nm and an emission wavelength of 585–595 nm.

Cellular proteins were extracted using radioimmunoprecipitation assay buffer [50 mM Tris/HCl, pH 7.4; 150 mM NaCl; 1% (v/v) nonidet P-40; 0.1% (w/v) SDS; Shanghai SolarBio Bioscience and Technology, Co., Ltd.] containing 1% (v/v) phenylmethanesulfonyl fluoride (Shanghai SolarBio Bioscience and Technology, Co., Ltd.), 0.3% (v/v) protease inhibitor (Merck Millipore) and 0.1% (v/v) phosphorylated proteinase inhibitor (Merck Millipore). Lysates were centrifuged at 1,000 × g and 4°C for 15 min, and the supernatant was collected to obtain total protein. A Bicinchoninic Acid Protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to determine the protein concentration. Equal amounts of protein (15 µg) were loaded onto a SDS-PAGE gel [10% (v/v) polyacrylamide] and transferred onto a polyvinylidene difluoride membrane. Nonspecific binding was blocked using 8% (w/v) milk in TBST (5% Tween-20) for 2 h at room temperature. The membranes were then incubated with primary antibodies against phosphorylated (p)-insulin receptor substrate 1 (IRS1, Ser307; ab1194; 1:250), IRS1 (ab52167; 1:500), p-glycogen synthase kinase (p-GSK3β, Ser9; ab131097; 1:500), GSK3β (ab170191; 1:1,000), p-protein kinase B (p-AKT; ab8933; 1:500), AKT (ab64148; 1:1,000) (Abcam, Shanghai, China) and β-actin (sc130300; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. Following several washes with TBST, the membranes were incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit (ab97051) or goat anti-mouse immunoglobulin G (ab6789; Abcam) at a 1:5,000 dilution for 2 h at room temperature and then washed with phosphate-buffered saline (PBS). The target proteins were visualized using enhanced chemiluminescence (Merck Millipore) according to the manufacturer's recommendations, quantified using densitometry analysis and normalized against β-actin. Data are expressed as the fold-change compared to the β-actin.

To further determine whether the EGCG-associated enhancement of insulin signaling was due to scavenging reactive oxygen species (ROSs), the antioxidant N-acetyl-cysteine (NAC; 10 mM; Beyotime Institute of Biotechnology, Jiangsu, China) was added either with or without EGCG, and the cells were incubated for 1 h. Then the whole proteins were extracted and western blotting was performed as aforementioned.

HepG2 cells cultured on 6-well chamber slides (1×10⁶) were washed with PBS three times for 5 min/wash, and the slides were incubated with the ROS fluorescent probe dihydroethidium (DHE; Vigorous Biotechnology Beijing Co., Ltd., Beijing, China) in serum-free DMEM/F-12 medium (Thermo Fisher Scientific, Inc.) for 30 min at 37°C in the dark. The slides were then fixed in 4% paraformaldehyde for 30 min at room temperature. The slides were washed with PBS and mounted. Immunofluorescence images were captured by fluorescence microscopy.

Intracellular ROS generation was monitored by flow cytometry using the peroxide-sensitive fluorescent probe, 2′,7′-dichlorofluorescin diacetate (DCFH-DA; Molecular Probes; Thermo Fisher Scientific, Inc.) as described previously (16). DCFH-DA is converted by intracellular esterases to DCFH, which is oxidized to the highly fluorescent DCF in the presence of an oxidant.

HepG2 cells (50–60% confluent) were pre-incubated with LG (5 mM D-glucose) or HG (30 mM D-glucose) for 24 h at 37°C, then treated with 5 µM EGCG. Finally, cells were washed twice with 1X PBS and diluted to 2–3×10⁶ cells/ml in 10 mmol/l DCHF-DA dyes. Cells were then incubated at room temperature for 30 min in the dark. The cells were subsequently analyzed by flow cytometry (Becton Dickinson; BD Biosciences, Franklin Lakes, NJ, USA).

All experiments were repeated at least three times. Statistical analyses were performed using SPSS software, version 22.0 (IBM SPSS, Armonk, NY, USA). The data are expressed as the mean ± standard deviation and were analyzed using one-way analysis of variance followed by the Tukey-Kramer multiple comparison test. P<0.05 was considered to indicate a statistically significant difference.

To determine the effect of HG on the AKT/GSK signaling pathway, HepG2 cells and mouse primary hepatocytes were treated with 30 or 5 mM glucose for 24 h. HG treatment significantly decreased the protein expression levels of phosphorylated AKT and GSK when compared to that of the LG treatment in HepG2 cells (p-AKT, P<0.05; p-GSK, P<0.05) and mouse primary hepatocytes (p-AKT, P<0.01; p-GSK, P<0.01; Fig. 1). The effect of EGCG on the levels of phosphorylated AKT and GSK in HG-treated HepG2 cells and primary hepatocytes was then analyzed. Pretreatment with EGCG significantly restored the levels of p-AKT and p-GSK in HepG2 cells (p-AKT, P<0.05; p-GSK, P<0.05) and primary hepatocytes (p-AKT, P<0.05; p-GSK, P<0.05) when compared to cells exposed to HG alone (Fig. 1). These results indicated that HG impaired insulin signaling in HepG2 cells and mouse primary hepatocytes, and EGCG improved HG-induced insulin resistance in the two cell types.

To determine the effect of EGCG on glycogen synthesis, the glycogen content in HepG2 cells and hepatocytes pretreated with HG (30 mM) was determined. EGCG was added to the cell cultures at concentrations of 0.01–10 µM. Following stimulation of cells with 100 nM insulin, glycogen synthesis decreased by ~50% in HepG2 cells (P<0.01) and primary hepatocytes (P<0.01) exposed to HG when compared to those treated with 100 nM insulin plus LG, indicating that HG treatment may lead to insulin resistance (Fig. 2). By contrast, when cells were pre-treated with EGCG (0.01–10 µM), glycogen synthesis was gradually restored in HepG2 cells and primary hepatocytes (Fig. 2). Treatment of cells with 10 µM EGCG resulted in a two-fold increase in glycogen levels in HepG2 (P<0.01) and primary hepatocytes (P<0.01) when compared with HG-only treated cells (Fig. 2). These results indicated that glycogen synthesis was improved by EGCG in a dose-dependent manner.

To determine whether HG treatment induced ROS production in cultured HepG2 cells, ROS levels were measured using DHE staining. Following 24 h incubation with LG and HG, 10 µM EGCG was added, and ROS production was determined using immunofluorescence and flow cytometry (Fig. 3A and B, respectively). The microimages in Fig. 3A demonstrate that ROS content was markedly reduced in HG + EGCG-treated cells compared with HG-only treated cells. As shown in Fig. 3B, HG treatment enhanced ROS production by ~1-fold relative to LG-treated cells (P<0.01).

Owing to the vital role the insulin-signaling pathway serves in glycogen synthesis (17), the authors investigated whether EGCG affects the insulin-signaling pathway in hepatocytes treated with EGCG. As shown in Fig. 4, a significant increase in p-JNK expression was observed in response to HG-treatment of HepG2 cells (P<0.01). In addition to increased JNK phosphorylation, phosphorylation of the residue Ser307 in IRS-1 was significantly enhanced by HG treatment (P<0.01; Fig. 4). HG-induced activation of JNK may have been responsible for the impaired phosphorylation of AKT and GSK (Figs. 1A and 4). However, the alterations in JNK, IRS-1, AKT and GSK expression that were induced by HG were reversed by EGCG treatment (Figs. 1 and 4). When HepG2 cells were pretreated with NAC and exposed to HG, JNK activation and phosphorylation of the Ser307 residue of IRS-1 were significantly reduced in NAC-treated cells compared with HG-only treated cells (p-JNK, P<0.01; p-IRS-1, P<0.01; Fig. 4). These results suggested that EGCG ameliorates HG-induced insulin resistance in signaling hepatocytes by altering the ROS-induced JNK/IRS1/AKT/GSK signaling pathway.