Roswell Park Memorial Institute medium (RPMI)-1640 medium, antibiotics, and trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco (Grand Island, NY, USA). Other reagents for cell culturing were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). INS-1 cells from rat insulinoma were grown in RPMI-1640 medium containing 11.1 mM pyruvate, 10 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 50 µM 2-mercaptoethanol, 100 U/mL penicillin, and 100 g/mL streptomycin in 5% CO₂-95% air at 37℃. The RPMI-1640 medium used for all of the experiments contained the supplements noted above. All studies were performed using the INS-1 cells between passages 21 and 29. For the screening of the stimulating effect of OS in insulin mRNA expression, we treated various OS extracts for 12 hours. We found that the hexane extract of OS stimulated insulin mRNA expression. Glucotoxicity was induced by treatment with high glucose (HG; 30 mM) levels for 3 days and was confirmed by observing impaired glucose-stimulated insulin secretion (GSIS) in the INS-1 cells. The INS-1 cells cultured at HG or at the normoglycemic condition were treated with the hexane OS extract (200 µM) for 12 hours and then harvested to measure insulin mRNA expression, insulin secretory capacity, and reactive oxygen species (ROS) levels. The signaling proteins (PI3K and Akt) were analyzed after 1 hour of treatment with the hexane OS extract at 0, 50, 100, or 200 µM concentrations in the INS-1 cells cultured at a normal glucose condition.

Dried OS leaves imported from Indonesia were kindly donated by Dongbang FTL Co. (Seoul, Korea). A voucher specimen (OS201103) has been deposited at the Yeungnam University College of Pharmacy (Gyeongsan, Korea). Dried OS powder (78.6 g) was subjected to extraction with 70% ethanol by reflux for 12 hours as previously described by Choi et al. [20] The ethanol solution was then completely evaporated. The ethanol extract residue (8.8 g) was suspended in H₂O (1.5 L), and the H₂O layer was partitioned with n-hexane, ethylacetate, and n-butanol (each 1.5 L×3). The resulting four extracts were completely evaporated to recover the n-hexane (445 mg), H₂O (2,409 mg), n-butanol (934.3 mg), and ethylacetate (1,175 mg) fractions.

The INS-1 cells were incubated in Krebs-Ringer buffer (KRB; 118 mmol/L NaCl, 4.7 mmol/L KCl, 2.5 mmol/L CaCl₂, 1.18 mmol/L KH₂PO₄, 1.18 mmol/L MgSO₄, 25 mmol/L NaHCO₃, 10 mmol/L HEPES; and 0.1% bovine serum albumin (BSA, pH 7.4) that contained either normal (5.6 mM) or stimulatory (16.7 mM) concentrations of glucose for 2 hours. The incubation period was terminated by adding 1 mL of an ethanol:hydrochloric acid (95:5, v:v) solution, and the KRB was collected. Insulin levels in the buffer samples were measured with an enzyme-linked immunosorbent assay (rat insulin-specific ELISA kit; Mercodia, Uppsala, Sweden) according to the manufacturer's protocol.

Total RNA was obtained from the INS-1 cells using Trizol reagent (Bio Science Technology, Daejeon, Korea). cDNA was synthesized using 1 µg of total RNA with oligo-(dT) primers (Bioneer, Daejeon, Korea) and Prime RT Premix (GeNet Bio, Seoul, Korea). Real time-polymerase chain reaction was performed with a LightCycler (Roche, Mannheim, Germany) as previously described [20]. The following primers were used: 5'-ACC CAA GTC CCG TCG TGA AGT-3' (forward) and 5'-CCA GTT GGT AGA GGG AGC AGA TG-3' (reverse) for insulin, 5'-GGC TTA ACC TAA ACG CCA CA-3' (forward) and 5'-GGG ACC GTC CAA GTT TGT AA-3' (reverse) for PDX-1, and 5'-TAC TGC CCT GGC TCC TAG CA-3' (forward) and 5'-TGG ACA GTG AGG CCA GGA TAG-3' (reverse) for β-actin.

The intracellular peroxide levels were measured by flow cytometry using an oxidation-sensitive fluorescein-labeled dye, carboxylated dichlorodi-hydrofluorescein diacetate (carboxy-H2DCFDA; Molecular Probes, Carlsbad, CA, USA). The non-fluorescent dye is converted into a fluorescent form through an oxidative process by intracellular ROS. The INS-1 cells were labeled with 100 M carboxy-H2DCFDA for 1 hour at 37℃. After loading with the dye, the cells were washed twice with phosphate buffered saline (PBS) and further incubated for 2 hours. The INS-1 cells were then harvested, washed twice with PBS, and re-suspended in trypsin-EDTA (0.25% trypsin and 2 mM Na₄-EDTA) for 5 minutes at 37℃. To disperse the cells in a single suspension, the INS-1 cells were gently passed 20 times in and out of a 1-mL pipette tip. The cells were then washed twice with ice-cold PBS and analyzed with a 488-nm argon laser EPICS XL-MCL flow cytometer with EXPO 32-ADC software (Beckman Coulter, Fullerton, CA, USA). The ROS concentrations were measured based on fluorescence intensity.

Proteins (30 µg) in an INS-1 cell lysate were separated in 10% polyacylamide gels and were transferred onto nitrocellulose membranes. The levels of phosphorylated PI3K, p-Akt, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were measured by incubating the blots with primary antibodies specific for these factors (1:1,000 dilution). Antibodies for p-PI3K and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and the anti-p-Akt antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). The secondary horseradish peroxidase-conjugated anti-rabbit antibody (1:2,000 dilution) was used to detect primary antibody binding. The blots were developed with an enhanced chemiluminescence reagent (Amersham Biosciences, Little Chalfont, UK), and band intensities were quantified using Multi Gauge software (Fujifilm, Tokyo, Japan).

All of the results are expressed as the mean±standard error. One-way analysis of variance was used for comparisons of dose-dependent changes. Post hoc comparisons were performed using Duncan's test. Statistical comparisons of insulin mRNA expression and peroxide level between the control and OS-treated groups were evaluated by Student t-test. Values of P<0.05 were considered statistically significant.

Crude OS extract elevated insulin mRNA expression in the INS-1 cells under both normal and HG conditions. Furthermore, the hexane and ethylacetate fractions elevated insulin mRNA expression in the INS-1 cells under normal and HG conditions. In contrast, the water and butanol OS fractions failed to affect insulin mRNA expression (Fig. 1). The hexane OS extract elevated insulin and PDX-1 mRNA levels in a dose-dependent manner in the INS-1 cells (Fig. 2).

Three days of HG completely suppressed GSIS, indicating that glucotoxicity had been induced in the INS-1 cells. The hexane OS extract (200 µM) protected the INS-1 cells from glucotoxicity. Moreover, treatment with the hexane extract slightly elevated insulin secretion in both the basal and glucose-stimulated states (Fig. 3).

To investigate possible mechanisms underlying the induction of insulin expression by the hexane OS extract, we evaluated signaling molecules that influence insulin expression. The hexane OS extract increased p-PI3K levels in a dose-dependent manner. Akt phosphorylation was also increased by treatment with 100 and 200 µmoL of the OS extract (Fig. 4).

To determine whether the ability of the OS extract to protect β-cells is mediated by changes in oxidative stress, we measured the cellular peroxide levels. HG elevated the peroxide concentrations in the INS-1 cells. Treatment with the OS extract did not affect the levels in the cells under either normal or hyperglycemic conditions (Fig. 5).