Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (PS) and trypsin-EDTA were purchased from Gibco-BRL (Grand Island, NY, USA). The dichlorodihydrofluorescein-diacetate (H₂DCF-DA) apoptotic assay kit and nuclear extraction kit were obtained from Molecular Probes (Carlsbad, CA, USA). The rat/mouse insulin enzyme-linked immunosorbent assay kit was obtained from Linco Research, Inc. (St. Charles, MO, USA). Kuromanin chloride (C3G standard), Hoechst 33342 and the mitochondria isolation kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against c-Jun NH₂-terminal kinase (JNK; 1:1,000 dilution; rabbit polyclonal antibody; #9252), phosphorylated (p-)JNK (1:1,000 dilution; Thr183/Tyr185 mouse monoclonal antibody; #9255S), extracellular signal-related kinase (ERK; 1:1,000 dilution; rabbit monoclonal antibody; #4695), p-ERK (1:1,000 dilution; Thr202/Tyr204 rabbit polyclonal antibody; #9101S), p38 (1:1,000 dilution; rabbit polyclonal antibody; #9212), p-p38 (1:1,000 dilution; Thr180/Tyr182 rabbit monoclonal immunoglobulin (Ig)G antibody; #4631S), nuclear factor-κB(NF-κB) p65 (1:1,000 dilution; rabbit polyclonal antibody; #3034) and horseradish peroxidase (HRP)-linked anti-rabbit IgG (1:2,000 dilution; #7074) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibodies against β-actin (1:1,000 dilution; mouse monoclonal IgG₁; sc-47778), Bcl-2 (1:1,000 dilution; mouse monoclonal IgG₁; sc-7382), Bax (1:1,000 dilution; rabbit polyclonal IgG; sc-493), caspase-3 (1:1,000 dilution; mouse monoclonal IgG_2a; sc-7272) and HRP-linked goat anti-mouse IgG (1:2,000 dilution; sc-2005) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All other chemicals were obtained from Sigma-Aldrich.

Mulberry fruit was purchased from a local company (Sujuchon, Yecheon, Korea). For anthocyanin extraction, the mulberry fruits were placed in 70% ethanol for 24 h at room temperature (RT). Following centrifugation of the extract (600 × g, 20 min), the supernatants were filtered with Whatman filter paper (0.45 μm), concentrated using a vacuum rotary evaporator (Eyela, Tokyo, Japan), redissolved in triple distilled water and lyophilized using a freeze dryer (IlShin, Seoul, Korea). The powder was soaked in n-hexane for 24 h to remove the fats and oils. The anthocyanins in the powder were then extracted with acidified methanol (methanol and 1.0 N HCl; 85:15, v/v), centrifuged at 12,000 × g for 15 min to remove the precipitate and then filtered through a 0.45 μm filter. The purified anthocyanin extract was evaporated at 46°C to dryness and stored at 4°C. The anthocyanin powder was redissolved in the culture medium and filtered using a 0.22 μm filter prior to cell culture. The pure C3G standard and purified anthocyanin, isolated from mulberry fruits, were identified using high-performance liquid chromatography (HPLC) retention time and their purity was >99% (data not shown). Reversed-phase HPLC was performed using Waters 486 detector (Waters, Milford, MA, USA) under the following conditions: Column, μBondapakC18 (Waters; 3.9×300 mm); flow rate, 0.5 ml/min; injection volume, 10 μl; solvent, methanol/H₂O/formic acid (75:20:5); and column temperature, 46°C. The absorbance was recorded at 520 nm using a Thermo Spectronic Genesys ultraviolet-visible spectrophotometer (Spectronic Instruments; Thermo Fisher Scientific, Waltham, MA, USA).

The MIN6N pancreatic β-cells were derived from a mouse pancreatic islet. The cells were provided by Professor H. Y. Kwon (College of Medicine, Hallym University, Chuncheon, Korea). The MIN6N β-cells were cultured in DMEM (5.5 mM glucose) supplemented with 10% inactivated FBS and 1% PS and were maintained at 37°C in a humidified 5% CO₂ incubator. The cells were cultured to ~85% confluence and were harvested using 0.25% trypsin-EDTA. The cells were then subcultured in 6- or 12-well plates for 12 h until they reached confluence. The cells were then treated with various concentrations (0, 10, 20, 50, 70, 100 and 200 μg/ml) of C3G for 18 h and then cultured for an additional 18 h in DMEM containing either 25 mM glucose or 25 mM mannitol (mannitol with 5.5 mM glucose in culture medium). The cells were maintained in these culture conditions for all experiments.

The viability of the treated cells was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 500 μg/ml MTT solution was added to each well and incubated for 2.5 h at 37°C. The formazan crystals in each well were dissolved in isopropyl alcohol and the absorbance of each well was measured at 595 nm using an ELISA microplate reader (Bio-Rad model 550; Bio-Rad Laboratories Inc., Hercules, USA).

Following treatment of the cells, 5 μM H₂DCF-DA in phosphate-buffered saline (PBS, pH 7.38; Gibco-BRL) was added and the fluorescence was measured at excitation and emission wavelengths of 485 nm and 535 nm, respectively, using a microplate spectrofluorometer (Molecular Devices Corp., Sunnyvale, CA, USA). The production of intracellular ROS was determined by image analysis of the cells, which were seeded into coverslip-loaded 6-well plates. Subsequently, H₂DCF-DA solution (500 μl per well) was added to each well of the plate, which was incubated for 2 h at 37°C. Images of the stained cells were acquired using a fluorescence microscope (Nikon Eclipse TE 300; Nikon, Tokyo, Japan).

The cells were treated with Hoechst 33342, a dye used to detect DNA condensation and/or fragmentation, followed by incubation at RT for 15 min. Images of the stained cells were acquired using a fluorescence microscope to examine the degree of DNA fragmentation.

The apoptotic cells were examined using a fluorescein isothiocyanate (FITC)-labeled Annexin V/propidium iodide (PI) apoptosis detection kit (Moleclular Probes, Carlsbad, CA, USA) according to the manufacturer’s instructions. The treated cells were harvested and washed with PBS and then centrifuged at 600 × g for 5 min to collect the cell pellet. Subsequently, the cells were resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2; pH 7.4) and stained with FITC-labeled Annexin V/PI at RT for 15 min in light-protected conditions. Flow cytometric analysis was performed using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA, USA) within 1 h of the supravital staining. The apoptotic cell rate was calculated as the sum of cells in the early and late phases of apoptosis divided by the total number of events.

The treated cells were washed in 1X PBS and lysed in lysis buffer [10 mM Tris-HCl (pH 7.5), 10 mM NaH₂PO₄/NaHPO₄ (pH 7.5), 130 mM NaCl, 1% Triton X-100, 10 mM NaPPi, 1 mM phenylmethylsulphonyl fluoride, 2 μg/ml pepstatin A] for 40 min on ice. The lysates were centrifuged at 12,000 × g for 30 min at 4°C. The supernatant was then collected and the protein content of the supernatant was measured using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc.) prior to analysis. The total or fractionated protein samples were loaded and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc.). The membranes were inhibited with 5% non-fat powdered milk in 1X Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 1 h and were incubated with primary antibodies at 4°C overnight. Finally, the membranes were treated with HRP-linked secondary antibodies for 1 h at RT. The membranes were washed with TBS-T following the binding reaction with each antibody. The detection of each protein was performed using an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA).

The treated MIN6N β-cells were lysed in 500 μl lysis buffer consisting of 10 mM Tris-HCl (pH 7.5), 10 mM NaH₂PO₄/NaHPO₄ (pH 7.5), 130 mM NaCl, 1% Triton X-100 and 10 mM NaPPi. The activity of caspase-3 in the lysates was evaluated using a caspase assay kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions.

The culture medium was collected from the treated cells and the level of insulin released in the medium was measured using a rat/mouse insulin enzyme-linked immunosorbent (ELISA) assay kit (Linco Research Inc., St. Charles, MO, USA) according to the manufacturer’s instructions.

All the measurements were obtained from at least three independent experiments and the values are expressed as the mean ± standard error. Statistical analysis was performed using Student’s t-test to evaluate significant differences and analysis of variance and Duncan’s multiple range tests (SAS, version 9.1; SAS-Institute, Cary, NC, USA) for comparing multiple groups when appropriate. P<0.05 was considered to indicate a statistically significant difference.

High glucose conditions increase the osmolarity in cells, therefore, the MIN6N β-cells were also cultured with 25 mM mannitol as an osmotic control agent to distinguish between the effects of glucose and osmotic pressure. High glucose significantly decreased the viability of the cells to 72.9% of the control, whereas no significant decrease in viability was observed following culture with mannitol. The pretreatment of the cells with C3G at a concentration of 70 μg/ml restored the cell viability to 83.8% of the control (Fig. 1A). The intracellular ROS levels in the high glucose-treated cells were determined using the ROS-sensitive fluorescent probe, H₂DCF-DA. High glucose markedly increased the levels of intracellular ROS; however, no effects on the intracellular ROS levels were observed following culture with mannitol. Compared with the cells under high glucose conditions, the cells pretreated with 70 μg/ml C3G exhibited a marked decrease in the high glucose-dependent increase in intracellular ROS levels and green fluorescence intensity (Fig. 1B and C).

To evaluate whether the growth-inhibitory effect of high glucose was associated with apoptosis, double-staining was performed using FITC-labeled Annexin V/PI. High glucose caused apoptosis in 33.9% of the cells, which was significantly higher compared with mannitol. However, pretreatment with 70 μg/ml C3G significantly inhibited the high glucose-induced apoptotic cell death (24.85%; Fig. 2A). The fragmentation of DNA upon apoptosis induced by high glucose was confirmed by staining the chromatin of the MIN6N β-cells using Hoechst 33342. Only the high glucose-treated cells exhibited DNA fragmentation, however, these chromatin changes were decreased following pretreatment with 70 μg/ml C3G (Fig. 2B).

The present study performed western blot analysis to determine the effects of C3G on the mitogen-activated protein kinase (MAPK) signaling pathway in the MIN6N β-cells. The cells were cultured under high glucose conditions, with or without C3G, followed by examination of the phosphorylation levels of ERK, JNK and p38. Treatment with C3G (50 and 70 μg/ml) led to dose-dependent inhibition of the high glucose-dependent phosphorylation of all the MAPK proteins (Fig. 3A). Apoptosis is initiated via two pathways, the intrinsic pathway is characterized by the release of cytochrome c and the activation of Bcl-2 family proteins, whereas the extrinsic pathway involves activation of apoptosis inducing factor (AIF), caspase-8 and caspase-10 (11). A previous study demonstrated that the phosphorylation of MAPK proteins is involved in the regulation of the mitochondrial permeability-mediated activation of apoptotic proteins, including Bcl-2 family proteins and cytochrome c (12). In the present study, the release of cytochrome c and expression of Bcl-2 family proteins, including Bcl-2 and Bax were confirmed (Fig. 3B). The protein expression of Bcl-2 decreased, whilst that of Bax increased in the high glucose-treated cells. The expression of the Bcl-2 family proteins in the group pretreated with 70 μg/ml C3G was regulated in a manner similar to the control group. In addition, the present study investigated whether high glucose induced the release of cytochrome c from the mitochondria into the cytosol. Western blot analysis of the cytosolic fraction revealed a significant release of cytochrome c from mitochondria in the cells cultured with high glucose. However, C3G (50 and 70 μg/ml) treatment inhibited the release of cytochrome c (Fig. 3C). To investigate whether high glucose induced the extrinsic apoptotic pathway, the expression of AIF and caspase-8 was examined in the MIN6N β-cells treated with high glucose. However, no significant promotion of AIF or caspase-8 activation was observed in high glucose-induced glucotoxicity (data not shown).

NF-κB is involved in oxidative stress-induced cell death in different cell types (12), therefore, the present study examined the translocation of NF-κB from the cytosol into the nucleus. High glucose induced the nuclear translocation of NF-κB p65, however pretreatment with C3G (50 and 70 μg/ml) inhibited the high glucose-induced nuclear translocation of NF-κB p65 (Fig. 4A). Caspase-3 is important in the execution of apoptosis, therefore, the present study examined the effect of C3G on the high glucose-induced activation of caspase-3. The cleaved form of caspase-3 was detected in the high glucose-treated group. However, compared with the high glucose-treated group, the group pretreated with C3G (50 and 70 μg/ml) exhibited a decrease in the level of cleaved caspase-3 (Fig. 4B). To further investigate the effects of C3G on caspase-3 activity, the activity of caspase-3 was analyzed using a caspase-3 assay kit. High glucose treatment significantly increased the activity of caspase-3, however, C3G (70 μg/ml) significantly decreased the caspase-3 activity (Fig. 4C). The antidiabetic efficacy of C3G was determined by measuring insulin release in the MIN6N β-cells using a rat/mouse insulin ELISA kit. Treatment with C3G led to an increase in insulin secretion compared with the high glucose-treated control (74.8, vs. 84.2%; Fig. 4D).