A total of 30 male Sprague-Dawley (SD) rats (6–8 weeks old) weighing 200±20 g were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). Rats were housed at 24±1°C with 40–50% humidity in a clean environment with a 12 h light/dark cycle. All animals had free access to food and purified water. All procedures were approved by the Animal Ethics Committee of the First Affiliated Hospital of Harbin Medical University (Harbin, China). Rats were fed with a high glucose, high fat diet (60% common chow, 10% lard, 10% egg yolk powder and 20% sucrose) for 8 weeks. Rats then received a 40 mg/kg intraperitoneal injection of STZ (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) once. At 72 h following STZ injection, rats with fasting blood glucose ≥16.67 mM (based on tail vein samples) were selected as T2DM animals for use in the following experiments (19). The normal control group (NC, n=10) was administered the same volume of sodium citrate buffer instead of STZ. T2DM rats were randomly assigned to either a diabetic group (DM, n=10) or a diabetic RSV group (DR, n=10). Rats in the DR group were administered once with 30 mg/kg RSV intragastrically (Sigma-Aldrich; Merck KGaA). Rats in the NC and DM groups were administered intragastrically with the same amount of 0.5% carboxymethyl cellulose. At weeks 0, 4 and 8 following induction, fasting and 2 h postprandial blood glucose were measured using the glucose oxidase method on the Beckman Glucose Analyzer II (Beckman Coulter, Inc., Brea, CA, USA) and plasma INS levels were determined using an RIA Assay system (Linco Research, Inc., St. Charles, MO, USA) (20) according to the manufacturer's protocol. Following 8 weeks of different treatments, total RNA was isolated from rat pancreas tissues using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and proteins were extracted using radioimmunoprecipitation assay buffer (RIPA; Beyotime Institute of Biotechnology, Haimen, China) for western blot analysis.

The rat insulinoma cell line clone 1E (INS-1E; provided by Professor Pierre Maechler; CMU; University of Geneva, Geneva, Switzerland) was cultured at 37°C in a humidified atmosphere containing 5% CO₂ in RPMI 1640 medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA), 11 mM glucose, 10 mM HEPES, 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol (Sigma-Aldrich; Merck KGaA), 50 µg/ml penicillin and 100 µg/ml streptomycin. When density reached 80%, cells were pretreated with PA (0, 0.125, 0.25, 0.5, or 1 mM) for 24 h. INS-1E cells were treated with 0.5 mM PA for 0, 12, 24, 48 or 72 h.

INS-1E cells were seeded into 6-well plates at a density of 5×10⁵ cells/well and divided into 5 groups: A normal control (NC) group, a high fat (HF) group, a HF+RSV group, a HF+RSV+SIRT small interfering (si)RNA group (HF+RSV+SIRT siRNA) and a HF+RSV+negative control siRNA group (HF+RSV+NC siRNA). To transfect cells with SITR siRNA or NC siRNA, cells in the HF+RSV+SIRT siRNA and HF+RSV+NC siRNA groups were incubated in 1 ml RPMI 1640 with SIRT1 siRNA (5′-CACCCCAGCAACTCAGCATTCATCGAAATGAATGCTGAGTTGCTGG-3′) or NC siRNA (5′-TTCTCCGAACGTGTCACGT-3′), respectively, and Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) without FBS. The final concentrations of siRNA and Lipofectamine 2000 used were 2 and 4 µg/ml, respectively. Following 48 h incubation at 37°C, cells in the HF+RSV, HF+RSV+SIRT siRNA and HF+RSV+NC siRNA groups were treated with 0.5 mM PA+10 µM RSV. The HF group received the same amount of PA and NC cells were treated with the same amount of vehicle. Following 24 h incubation at 37°C, total RNA or proteins were extracted for further experiments.

Total RNA was isolated from INS-1E cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. The quality of RNA was determined by agarose electrophoresis and the concentration was measured using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA) at 260 nm and 280 nm. RT was performed using 1 µg total RNA using a reverse transcriptase cDNA synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. qPCR was performed using SYBR-Green qPCR SuperMix (Invitrogen; Thermo Fisher Scientific, Inc.) on a real-time RT-PCR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The thermocycling conditions were as follows: 94°C for 5 min followed by 35 cycles of 94°C for 30 sec, 56°C for 30 sec and 72°C for 30 sec, with a final elongation step at 72°C for 5 min. All amplification reactions were performed in triplicate and the relative expression of target genes were normalized against β-actin using the 2^−∆∆Cq method (21). The primers for RT-qPCR are presented in Table I.

Proteins were extracted from rat pancreatic tissues or INS-1E cells using RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China) and centrifuged at 15,000 × g for 30 min at 4°C. The concentration of proteins was determined using a bicinchoninic acid kit (Pierce; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Equal amounts of protein (25 µg/lane) were subjected to 12.5% SDS-PAGE. Proteins were subsequently electrotransferred to a polyvinylidene fluoride membrane and blocked with 5% non-fat milk in Tris-buffered saline with 0.05% Tween-20 (TBST) for 1 h at room temperature. The membrane was incubated with mouse anti-SIRT1 (sc-15404), anti-FOXO3a (sc-20680), anti-PGC-1α (sc-13067) and anti-β-actin (sc-1616) antibodies (all 1:1,000 dilution; all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. Following three washes with TBST, the membrane was incubated with horseradish peroxidase-conjugated rabbit anti-mouse secondary antibody (1:2,000; sc-203; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Membrane bands were developed using an enhanced chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.). The relative densities of target protein bands were normalized against β-actin using Quantity One software v4.4 (Bio-Rad Laboratories, Inc.).

Total heart blood was harvested from rats following 8 weeks of different treatments and centrifuged at 3,500 × g for 15 min at 4°C to separate the serum. Serum SOD (cat. no. SES134Hu) and MDA (cat. no. CEA597Ge) levels were detected using a commercially available sandwich ELISA kit (Cloud-Clone Corp., Katy, TX, USA) according to the manufacturer's protocol. Absorbance was measured at 450 nm on Bio-Rad 680 Microplate reader (Bio-Rad Laboratories, Inc.). The quantity of SOD and MDA in the serum was estimated using a constructed calibration curve.

All statistical analyses were performed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). All results are presented as the mean ± standard error of the mean. Significant differences in mean values were evaluated using one-way analysis of variance with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To confirm that the diabetic rat model was successfully established, blood glucose levels were measured following STZ injection. No significant differences in blood glucose levels were observed in the NC group at weeks 0, 4 or 8 (Table II). Compared with the NC group, fasting glucose levels in the DM group were significantly higher at week 8 (P<0.05; Table II). However, the administration of RSV significantly attenuated the STZ-induced increase in fasting glucose (P<0.05; Table II). The 2 h postprandial blood glucose levels were subsequently assessed. At weeks 4 and 8 post-induction, postprandial blood glucose was significantly higher in the DM group compared with the NC group (P<0.01; Table II); however, in the DR group, this increase was significantly attenuated (P<0.01; Table II).

The fasting and 2 h postprandial levels of INS were also assessed. Compared with the NC group, fasting and 2 h postprandial blood INS levels were significantly elevated in the DM group at weeks 4 and 8 (P<0.05; Table III). However, INS levels in the DR group were significantly lower at week 8 compared with the DM group (P<0.05; Table III). Taken together, these results suggest that STZ injection successfully induced DM in rats and that RSV administration modulated glucose and INS levels in rats with STZ-induced DM.

To investigate the mechanism by which RSV functions in DM, levels of PGC-1α, SIRT1 and FOXO3a mRNA and protein were assessed using RT-qPCR and western blotting, respectively. Compared with the NC group, levels of PGC-1α, SIRT1 and FOXO3a mRNA and protein were significantly suppressed in the DM group (P<0.01; Fig. 1A and B). However, pretreatment with RSV significantly attenuated these decreases (P<0.05; Fig. 1A and B). These data suggest that RSV administration upregulates PGC-1α, SIRT1 and FOXO3a expression in mice with STZ-induced DM.

It has been reported that oxidaωtive stress due to the excessive production of reactive oxygen species (ROS) and mitochondrial dysfunction are major factors in the development of INS resistance in T2DM (22). ELISA assays were used to analyze the activity of SOD and MDA to evaluate oxidative stress. SOD activity was significantly suppressed in the DM group compared with the NC group (P<0.01; Fig. 1C). However, RSV treatment significantly increased SOD activity in the DR group compared with the DM group (P<0.05; Fig. 1C). By contrast, MDA activity was significantly upregulated in the DM group compared with the NC group (P<0.01; Fig. 1D). In the DR group, pretreatment with RSV significantly attenuated the DM-induced increase in MDA activity (P<0.05; Fig. 1D). These data suggest that RSV regulates the activity of SOD and MDA in rats with STZ-induced DM.

It has been demonstrated that PA serves a role in the development of obesity and T2DM (17). Varying concentrations of PA were used to treat INS-1E cells and the results indicated that PA significantly suppressed SIRT1 mRNA in a dose-dependent manner (P<0.01; Fig. 2A). The association between SIRT1 expression and PA induction time was subsequently investigated and the results indicate that PA treatment also significantly reduced SIRT1 mRNA expression in a time-dependent manner (P<0.01; Fig. 2B).

To further investigate the role of RSV in vitro, levels of PGC-1α, SIRT1 and FOXO3a in INS-1E cells were evaluated. PA significantly suppressed the levels of PGC-1α, SIRT1 and FOXO3a mRNA and protein compared with the NC group (P<0.01; Fig. 3A-C); however, administration of RSV significantly attenuated this effect (P<0.01; Fig. 3A-C). Furthermore, transfection with SIRT1 siRNA in conjunction with PA significantly reduced the expression of PGC-1α, SIRT1 and FOXO3a compared with the HF+RSV+NC siRNA group (P<0.01; Fig. 3A-C). These data suggest that RSV mitigates the HF-induced inhibition of PGC-1α and FOXO3a expression via SIRT1.

To examine the mechanism by which SIRT1 functions, the expression of a cascade of genes was investigated, including mitochondrial biogenesis-associated NRF and mtTFA, lipid metabolism-associated CPT-1, ACC and LCAD, and β-cells-associated PDX-1, MAFA and INS. PA treatment significantly suppressed mRNA levels of SIRT1, NRF, mtTFA, CPT-1, LCAD, PDX-1, MAFA and INS mRNA, whereas it significantly upregulated the expression of ACC mRNA compared with the NC group (P<0.01; Fig. 4). Treatment with RSV significantly attenuated these PA-induced alterations in gene expression (P<0.01; Fig. 4). However, SIRT1 knockdown significantly attenuated the PA-induced increases in SIRT1, NRF, mtTFA, CPT-1, LCAD, PDX-1, MAFA and INS expression as well as the PA-induced decrease in ACC expression compared with the HF+RSV+NC siRNA group (P<0.01; Fig. 4). These data suggest that RSV modulates the expression of NRF, mtTFA, CPT-1, LCAD, PDX-1, MAFA and INS via SIRT1 in PA-induced INS-1E cells.

The expression of SOD and MDA mRNA in INS-1E cells following SIRT1 knockdown was assessed to determine the effect of SIRT1 on oxidative stress. Compared with the NC group, PA significantly suppressed SOD mRNA expression (P<0.01; Fig. 5A) and RSV treatment significantly reversed this effect (P<0.01; Fig. 5A). However, SIRT1 knockdown significantly decreased SOD expression compared with the HF+RSV+NC siRNA group (P<0.01; Fig. 5A). The administration of PA significantly increased MDA expression compared with the NC group (P<0.01; Fig. 5B) and RSV significantly attenuated this upregulation (P<0.01; Fig. 5B). SIRT1 knockdown significantly upregulated MDA mRNA expression compared with the HF+RSV+NC siRNA group (P<0.01; Fig. 5B). These data indicate that RSV regulates SOD and MDA levels via SIRT1 in PA-treated INS-1E cells.