According to the method of Kato et al (26), 3-DG was synthesized from glucose. A hot solution of 6 g D-glucose, 3.3 g p-toluidine, 6.6 ml acetic acid and 135 ml ethanol was stirred vigorously and heated in an oil bath at 90°C for 30 min. Then, 9.9 g benzoylhydrazine was added, followed by refluxing for 6–7 h. The reaction solution was incubated at 4°C overnight, then the resulting precipitate (3DG-bisbenzoylhydrazone) was collected by filtration through a Buchner funnel (Aladdin Industrial Corporation, Shanghai, China), and washed successively with 75 ml methanol and 75 ml diethyl ether 3 times. The product was then dried at room temperature. A solution of the 3DG-bisbenzoylhydrazone product (3 g), 90 ml ethanol, 1.5 ml acetic acid, 50 ml water and 1.6 ml freshly distilled benzaldehyde (at 40–50 mmHg, 120–130°C) was refluxed at 90°C for 4 h. The reaction mixture was incubated overnight at room temperature and then the filtrate was collected through a Buchner funnel and then concentrated to 70 ml using an RE-52 rotary evaporator (Shanghai Yarong Biochemical Instrument Factory, Shanghai, China) and washed 6 times with 30 ml diethyl ether, then decolorized with 2 g activated carbon. The concentrate was evaporated down to 3 ml and 10 ml of 95% ethanol was added. The solution was then charged on Amberlite IR120B (H⁺ form) and Amberlite IR4B (OH⁻ form) ion-exchange resin columns. The final solution was evaporated to a thick syrup, then the 3DG was purified further using a flash column (silicagel 60; chloroform/methanol/water ratio, 8.0:2.0:0.1). All chemical reagents used were purchased from J&K Chemical, Ltd. (Shanghai, China) without further purification. The product was identified using infrared, ¹H-nuclear magnetic resonance (NMR), ¹³C-NMR and mass spectroscopy (27).

The HepG2 hepatocellular carcinoma cells were provided by the School of Biology and Basic Medical Sciences, Soochow University (Suzhou, China), and were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% (v/v) fetal bovine serum (Zhejiang Tianhang Biological Technology Co., Ltd., Huzhou, China) and antibiotic solution (100 U/ml penicillin and 100 µg/ml streptomycin; Beyotime Institute of Biotechnology, Shanghai, China) at 37°C in a humidified atmosphere containing 5% CO₂. The cells were grown to 70–80% confluence and were seeded into 96/48-well plates at a density of 5×10⁴ cells/well. The cells were then either pre-treated with 3DG (10, 80 or 300 ng/ml) in serum-free medium for 24 h, or remained untreated, with or without subsequent exposure to insulin (2.0 or 6.6 IU/ml), for the experimental groups.

Cell viability was measured using a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay (Wuxi Zhanwang Chemical Co., Ltd., Wuxi, China). Briefly, 5×10⁴ cells/well were seeded in 96-well microtiter plates for 24 h at 37°C; the cells were then pre-incubated with or without 3DG at final concentrations of 10, 50, 80, 300, 500, 800 and 1,000 ng/ml in serum-free medium supplemented with high (H-DMEM; 25 mmol/l) or low (L-DMEM; 5.6 mmol/l) glucose (Gibco; Thermo Fisher Scientific, Inc.) for 24 h at 37°C. The medium was subsequently removed, and 200 µl of MTT was added to a final concentration of 0.5 mg/ml. After 4 h, 150 µl dimethyl sulfoxide solution (Wuxi Zhanwang Chemical Reagent Co., Ltd., Wuxi, China) was added to solubilise the MTT formazan. The plates were placed on a mechanical shaker (Shanghai Centrifuge Institute Co., Ltd., Shanghai, China) for 10 min at room temperature, and the optical density (OD) was read at 490 nm using an enzyme-linked immunometric meter SpectraMax M2 (Molecular Devices, LLC, Sunnyvale, CA, USA), as previously described (28).

The glucose uptake was measured using 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose (2-NBDG; Cayman Chemical Co., Ann Arbor, MI, USA), comparable to the study by Engelbrecht et al (29). Following treatment of the cells (5×10⁷ cells/well) with or without different concentrations of 3DG in L-DMEM for 24 h, the cells were washed with Krebs-ringer bicarbonate (KRb) buffer (4A Biotech Co. Ltd., Beijing, China) comprising 129 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.0 mM CaCl₂, 1.2 mM MgSO₄, 5.0 mM NaHCO₃ and 10.0 mM HEPES. In the insulin-treated group, the cells were then exposed to 3.60 IU/ml insulin, and 100 µl serum-free KRb buffer (supplemented with 160 µM 2-NBDG) was added to the medium and incubated for 30 min at 37°C. The cells were washed twice with KRb buffer and the radioactivity incorporated into the cells was measured using a fluorescence microplate reader (excitation/emission, 488/520; Model 680, Bio-Rad Laboratories, Inc., Hercules, CA, USA). Subsequently, the medium was replaced with L-DMEM supplemented with 200 µl MTT, and continued to culture. After 4 h incubation at 37°C, the OD at 490 nm was measured using a microplate reader. The error was corrected using an MTT assay.

The glycogen levels in the HepG2 cells were determined using a glycogen assay kit in the presence or absence of 3.6 IU/ml insulin. Briefly, following 3DG treatment of the cells for 24 h, the medium containing L-DMEM was removed and the cells were washed twice with ice-cold phosphate-buffered saline (PBS; Beyotime Institute of Biotechnology) and incubated with serum-free L-DMEM and insulin for 24 h. Subsequently, glycogen in the cells was extracted using 66% (v/v) ethanol and centrifuged for 10 min at 8,000 × g at 4°C to remove the supernatant. The precipitates of each sample were mixed with 0.5 ml water, and the medium was subsequently added into 1 ml 0.2% (w/v) anthrone reagents (Rsbio, Shanghai, China). The tubes were boiled for 30 min. Absorbance at 620 nm was measured using an enzyme-linked immunometric meter. A standard glycogen curve (50, 25, 12.5, 6.25, 3.12 and 1.60 mg/ml) was calculated using the above method.

The HepG2 cells were rendered insulin resistant by treatment with 3DG, as described above. The medium containing L-DMEM and 3DG was removed from the cells, and the cells were then washed twice with ice-cold PBS and solubilised in IP lysin buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 2 mM SDS, 25 mM β-glycerophoaphate, 1 mM EDTA, 1 mM Na₃VO₄ and 0.5 µg/ml leupeptin (Beyotime Institute of Biotechnology). Following centrifugation at 12,000 × g at 4°C for 20 min, the supernatants were collected and used for Western blot analysis. The total protein concentrations were determined using a bicinchoninic acid protein (BCA) assay kit (cat. no. P0012; Beyotime Institute of Biotechnology) comprised of BCA kit A (cat. no P0012-1), BCA kit B (cat. no. P0012-2) and standard proteins (cat. no. P0012-3). The proteins (80–120 µg) were loaded onto a 12% SDS-polyacrylamide gel (Thermo Fisher Scientific, Inc.), then subjected to electrophoresis and transferred onto polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany). The membranes were blocked for 1 h in Tris-buffered saline with 1% Tween (TBST; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) containing 5% dry milk. The membranes were washed with PBS containing 0.05% Tween-20 three times, and incubated at 4°C overnight with the following antibodies (dilution, 1:1,000): Rabbit polyclonal anti-glucose transporter 2 (GLUT-2; cat. no. ab54460; Abcam, Cambridge, UK); and rabbit monoclonal anti-glycogen synthase kinase-3 (GSK-3)a/β (cat. no. 5676), rabbit polyclonal anti-p-insulin receptor substrate 1 (IRS-1; cat. no. 3070), rabbit polyclonal anti-phosphoinositide 3-kinase (PI3K)-p85 (cat. no. 4292), rabbit polyclonal anti-PI3K-p110 (cat. no. 4255) and rabbit polyclonal anti-AKT (cat. no. 9272), all purchased from Cell Signaling Technology, Inc., Danvers MA, USA. Following washing 4 times for 5 min each in TBST, the membranes were incubated with goat anti-rabbit secondary antibody [1:1,000; cat. no. GAR0072; Multi Sciences (Lianke) Biotech Co., Ltd., Hangzhou, China] for 2 h and visualized using an ECL detection kit (Beijing Solarbio Science & Technology Co., Ltd.). Quantification of protein bands was performed using Image-J software (version 1.42).

The results of the experiments are expressed as the mean ± standard deviation. SPSS software (version 14.0; SPSS, Inc., Chicago, IL, USA) was used for statistical analysis The statistical significance of differences were analyzed using Student's t-test. P≤0.05 was considered to indicate a statistically significant difference. In the following, n describes the number of repetitions of independent experiments.

The HepG2 cells were exposed to different concentrations of 3DG supplemented with H-DMEM or L-DMEM for 24 h to examine the effects of 3DG on cell viability. Different concentrations of 3DG were assessed, ranging between 10 and 1,000 ng/ml, and cell viability was measured using an MTT assay. As shown in Fig. 1, compared with the untreated control, 3DG did not alter HepG2 cell viability at concentrations between 10 and 300 ng/ml, whereas 3DG exhibited a degree of inhibitory activity against the growth of the HepG2 cells at concentrations of 500 or 1,000 ng/ml.

To determine whether 3DG leads to hepatic insulin resistance, the uptake of 2-NBDG and glycogen content in the HepG2 cells were measured following treatment with non-cytotoxic concentrations (10–300 ng/ml) of 3DG for 24 h. Compared with the untreated control cells, 2-NBDG uptake was significantly increased following insulin stimulation (112.61±8.42, vs. 198.85±15.65; P<0.001). The uptake of 2-NBDG remained unchanged in the 3DG-only treated cells, however, the cells co-incubated with 80 ng/ml (162.93±20.49; P<0.05) or 300 ng/ml (151.44±11.79; P<0.01) 3DG and insulin showed significant dose-dependent decreases in 2-NBDG uptake, compared with the cells exposed to insulin only (198.85±15.65; Fig. 2A). As shown in Fig. 2B, exposure to insulin significantly increased glycogen content, compared with the control (29.743±3.712, vs. 10.151±1.102 µmol/mg, respectively). The cells treated with different concentrations of 3DG showed significant dose-dependent decreases in glycogen content, at concentrations of 80 ng/ml (22.060±1.821; P<0.05) and 300 ng/ml (16.568±1.200 µmol/mg; P<0.01), compared with the cells exposed to insulin alone (29.743±3.712 µmol/mg) However, the glycogen content remained unchanged in the 3DG-only treated cells, compared with the normal group. The effect of 3DG treatment became significant from a concentration of 80 ng/ml. Lower concentrations of 3DG did not significantly induce decreased glucose uptake or glycogen content.

To further elucidate the action of 3DG, the present study evaluated the expression profile of proteins, which are important for glucose uptake and glycogen content in HepG2 cells. In the present study, treatment of the HepG2 cells with insulin alone induced a significant increase in the expression levels of GLUT2 and p-GSK-3a/β, compared with the control. Although the expression levels of GLUT2 and p-GSK-3a/β were not altered by non-cytotoxic concentrations of 3DG in the 3DG-only treated celss, significant decreases in the protein expression levels of GLUT2 and p-GSK-3a/β were observed following exposure of the cells to 3DG at concentrations of 80 and 300 ng/ml for 24 h with insulin stimulation (Fig. 3).

Glucose uptake and glycogen synthesis in the liver are regulated via the activation of IRS-1, PI3K and AKT (30). Exogenous 3DG treatment had no significant effect on the expression levels of IR-β and IRS-1, however, treatment of the cells with 300 ng/ml 3DG reduced the insulin-stimulated tyrosine phosphorylation of IRS-1 (Fig. 4A). To investigate the consequences of the reduced tyrosine phosphorylation of IRS-1, the expression levels of the downstream target proteins, PI3K-p85, PI3K-p110, AKT and p-AKT, were determined. As shown in Fig. 4A, 3DG treatment induced significant decreases in the protein expression levels of the PI3K p85 and p110 subunits. Furthermore, 3DG led to a dose-dependent decrease in the phosphorylation of AKT, which was not accompanied by changes in the level of AKT (Fig. 4B).