Male, 8-week-old db/db mice and male -+/db mice (n=28; weight, ~18 g) were purchased from Shanghai Xipu-bikai Experimental Animal Co., Ltd. The mice were housed at 22–25°C with 60±5%, 12-h light/dark cycles and sufficient water and were fed a commercial diet over one week of adaptation. The mice were then randomly divided into four groups. In the normal group (n=8), male -+/db mice were fed a commercial diet. Mice with fasting blood glucose >11.1 mmol/l were considered diabetic and selected in the T2DM groups. In the T2DM group (n=8), mice were fed a high-fat diet (T2DM model group). The mice in the acarbose-treated diabetic group (T2DM+acarbose group; n=8) were given 9 g/kg/day acarbose extract (cat. no. 1906208B; Zhongmei Huadong Pharmaceutical Co., Ltd.) by gavage for four weeks. The mice in the alkylresorcinol (AR; cat. no. 3158-56-3; Sigma-Aldrich; Merck KGaA)-treated T2DM group (T2DM+AR group; n=8) received 60 mg/kg/day AR extract by gavage for 4 weeks as a positive control (16). This study was performed in strict accordance with the NIH guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 85-23 Rev. 1985) and was approved by the Animal Care and Use Committee of Zhejiang Chinese Medical University (approval no. ZSLL-2019-102). Food intake, body weight, serum total cholesterol (TC) and triglyceride (TG) levels, glucagon, and fasting blood glucose (FBG) were measured. All mice were euthanized with 5% isoflurane. Mice were monitored until complete cessation of the heartbeat, sustained involuntary respiration for 2–3 min and absence of blinking or toe contraction reflex.

During the experimental period, food intake, body weight, and FBG were recorded weekly. Before measuring FBG, the mice were fasted for 24 h (with sufficient water), blood (1 ml; total 5 ml) was collected from the tail vein, and FBG was measured with a blood glucose meter (Jiangsu Yuyue Medical Equipment & Supply Co., Ltd.) weekly until the mice were sacrificed. Four weeks after treatment, glycosylated hemoglobin (HbA1c) was measured using a DCA2000 analyzer (Bayer AG). The levels of glucagon were measured by ELISA (cat. no. MOES01072; USCN Kit, Inc.).

The serum total cholesterol (TC) and triglyceride (TG) levels were measured by direct enzymatic methods using a CX-7 Biochemical Autoanalyser (Beckman Coulter, Inc.).

Intraperitoneal glucose tolerance tests (IPGTTs) and insulin tolerance tests (ITTs) were performed on different days. Four weeks after treatment, all mice were fasted overnight before analysis. Glucose was administered at a dose of 2 g/kg by intraperitoneal injection, and tail vein blood glucose levels were measured using a glucose meter (Yuyue301) at 0, 30, 60, and 120 min. The mice were injected with 1 U/kg humulin R (Eli Lilly) after fasting for 4 h for the IPGTT. The glucose levels were measured at 0, 30, 60, 90 and 120 min after injection.

The MSP was performed on a 7300 thermocycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers for Methylation PDX-1 and unmethylated PDX-1 were based on the genomic sequence (accession nos. NT_024524100 and NT_024524) of the PDX-1 gene. Primer sequences were as follows: i) Unmethylated PDX-1-forward, 5′-GCCTCGAGCGCAGACTGAGTCCAGAGTG-3′ and reverse, 5′-CGAAGCTTCGGTGATTTCTTCAGGGAAA-3′; ii) Methylation PDX-1-forward, 5′-GTTTTTTTCGGAGGGAGTC-3′ and reverse, 5′-TTTCCACGCGTAAACTTTAA-3′. Cellular DNA was extracted using the QIamp DNA mini kit (Qiagen GmbH) from mouse pancreatic samples according to the manufacturers instructions. Bisulphate modification of the DNA was performed using the EZ Methylation-Gold kit (Zymo Research, Corp.) according to the manufacturers protocol. DNA elements with methylated cytosins remained unmodified after bisulfite treatment, whereas unmethylated cytosins were completely converted into uracil nucleosides and further to thymine after PCR, thus giving different primer recognition sequences for MSP. PCR-mix (2X; 12.5 µl; Fermentas; Thermo Fisher Scientific, Inc.) was added to 2 µl bisulfonated/unmodified DNA sample followed by 0.8 µl forward and reverse primers (400 nM each) and 8.5 µl water.

The PCR reaction was run using the Taq GC Buffer amplification system (Takara Biotechnology Co., Ltd.) and the following thermocycling conditions: 94°C for 2 min; followed by 35 cycles at 94°C for 10 sec, 60–70°C for 20 sec and 72°C for 20 sec; and terminated at 72°C for 4 min followed by immediate cooling to 4°C.

After staining with 6X loading dye solution (Fermantas; Thermo Fisher Scientific, Inc.), the PCR products were analyzed on 2% agarose gels and visualized under UV illumination using a LabWorks gel imaging system (version 4.6; UVP, LLC).

Nuclear extracts were isolated from pancreatic tissues using NE-PER™ Extraction Reagent (Thermo Fisher Scientific, Inc.), according to the manufacturers protocol. Frozen pancreatic tissues were placed on ice and pulverized in a pre-cooled mortar with liquid nitrogen. Tissues were lysed in 500 µl RIPA buffer (Beyotime Institute of Biotechnology) supplemented with phosphatase and protease inhibitor cocktail (Thermo Fisher Scientific, Inc.) and then centrifuged at 10,000 × g for 20 min at 4°C. The soluble extracts were collected for western blotting. The determination of protein concentrations was performed using a bicinchoninic acid assay (Thermo Fisher Scientific, Inc.).

Soluble lysates (40 µg) and nuclear extracts (20 µg) were separated by SDS-PAGE on 8% gels and transferred to PVDF membranes (EMD Millipore). The membranes were blocked with 5% nonfat milk in TBS-T (10 mM Tris, pH 7.6, 150 mM NaCl, and 0.05% Tween-20) for 2 h at room temperature and probed with primary antibodies against PDX-1 (42 kDa; rabbit; 1:1,000; cat. no. 5679; Cell Signaling Technology), β-actin (45 kDa; rabbit; 1:1,000; cat. no. ab8227; Abcam) and PCNA (36 kDa; rabbit; 1:1,000; cat. no. 13110; Cell Signaling Technology) overnight at 4°C. A peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:2,000; cat. no. sc-516102; Santa Cruz Biotechnology, Inc.) was then added for 1.5 h at room temperature. The bands were visualized using an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology) and quantified using ImageJ software (version 6.0; National Institutes of Health).

After fixation by perfusion with 4% paraformaldehyde for 5 min at room temperature, the mouse pancreas was removed. After sectioning into 5-µm, dewaxing and microwave antigen recovery, the slides were briefly incubated with 0.2% Triton X-100 and 10% BSA (cat. no. A3858; Sigma-Aldrich; Merck KGaA), then kept overnight at 4°C with guinea pig anti-insulin (cat. no. ab7842; 1:1,000; Abcam) and mouse anti-BrdU (cat. no. ab8152; 1:1,000; Abcam) antibodies followed by Alexa Fluor 568 goat anti-guinea pig (cat. no. AP108B; 1:1,000; Sigma-Aldrich; Merck KGaA) and FITC goat anti-mouse (cat. no. ab6785; 1:1,000; Abcam) secondary antibodies. Nuclei were counterstained with DAPI (cat. no. ab104139; Abcam). All BrdU+ cells and insulin+ cells (representing total β-cells) from 50 pancreatic islets per mouse were counted using a confocal microscope (Zeiss AG). The percentage of β-cell proliferation was determined by dividing the number of BrdU+ β-cells by the total number of β-cells.

Pancreatic tissues were fixed in 10% neutral-buffered formalin for 5 min at room temperature and embedded in paraffin. After a xylene wash to remove the paraffin and a rehydration step with serial dilutions of alcohol, the 5-µm thick sections were incubated in 0.3% H₂O₂ for 15 min to block endogenous peroxidases and heated at 98°C for 15 min for antigen retrieval. Samples were blocked with 5% BSA for 1 h at room temperature. The sections were then incubated overnight at 4°C with primary antibodies against inducible Neurogenin 3 (NGN3; cat. no. sc-374442; mouse; 1:60; Santa Cruz Biotechnology, Inc.), Insulin (mouse; cat. no. BM4310; 1:50; Wuhan Boster Biological Technology, Ltd.), PDX-1 (cat. no. 5679; rabbit; 1:50; Cell Signaling Technology, Inc.) and Forkhead box O1 (FoXO1; cat. no. 2880; rabbit, 1:100; Cell Signaling Technology). After a series of washes, peroxidase-conjugated goat anti-mouse IgG/goat anti-rabbit IgG secondary antibodies (cat. nos. sc-516102 and sc-2357; 1:100; Santa Cruz Biotechnology, Inc.) were applied. The sections were stained with diaminobenzidine solution (OriGene Technologies, Inc.) washed, dehydrated, permeabilized, mounted and examined under bright-field microscopy. Images were examined using an LSM 710 laser scanning confocal microscope (Carl Zeiss AG).

Data are presented as the mean ± SEM of at least three independent experiments. Statistical analysis was performed using SPSS version 13.0 (SPSS, Inc.). One-way ANOVA followed by Bonferroni correction was used for multi-group comparisons. A t-test was performed to identify significant differences between two groups. P<0.05 were considered to indicate a statistically significant difference.

T2DM mice exhibited a significant increase and body weight, compared with normal mice (Fig. 1A and B). Furthermore, T2DM mice demonstrated a significant increase in FBG compared with normal mice, and the increased FBG levels were maintained throughout the entire experiment (Fig. 1C). These results are considered common signs of diabetes. Importantly, in all diabetic mice mice, glucagon, HbA1c, TC, TG and LDL-C levels were all significantly increased, compared with the normal group (Fig. 1D-H), while HDL-C levels remained unchanged (Fig. 1I), indicating that hyperlipidemia symptoms were also successfully established in the diabetes group. Moreover, acarbose significantly decreased glucagon, HbA1c, TC, TG and LDL-C levels in serum, compared with untreated T2DM mice.

Blood glucose levels were measured at 30, 60, 90 and 120 min. In the normal group, the initial and final blood glucose levels were similar. By contrast, blood glucose levels remained high in T2DM mice at the 120-min timepoint, compared with the initial timepoint (Fig. 2A). Interestingly, blood glucose significantly decreased in the acarbose- or AR-treated groups, compared, with T2DM mice, suggesting that acarbose could reduce glucose rapidly and stably. Similarly, in the ITT, the acarbose-treated mice displayed reduced blood glucose levels at 30, 60, 90 and 120 min compared to the T2DM model mice (Fig. 2B).

HE staining of pancreatic tissue demonstrated that high-fat diet administration elicited severe injury to the pancreas, including decreased islet cells and diminished pancreatic islet diameter. Additionally, the structure of the pancreatic islets was disordered, vacuoles appeared, and nuclei were swollen in T2DM mice. These effects were reversed by acarbose (Fig. 3).

Active PDX-1 induces the efficient production of insulin. The strong nuclear expression of PDX-1 was shown in the islet β-cells of NC pancreatic tissues, while PDX-1 staining was weak in the DM group and was restored by acarbose (Fig. 4A). Conversely, strong nuclear expression of FoxO1 was observed in the pancreatic tissues of T2DM model mice, which was reversed by acarbose (Fig. 4A). The Ngn3 level in the pancreas was obviously increased in the T2DM model mice and was obviously decreased by acarbose (Fig. 4A). Moreover, strong insulin staining was observed in normal mice but weak staining was observed in T2DM model mice. However, acarbose increased insulin levels (Fig. 4A). the level of GLP-1 was determined, and the results showed that GLP-1 level was decreased in T2DM group compared to normal group, which partly reversed by acarbose and alkylresorcinol (Fig. 4B). Similarly, the number of insulin-positive/BrdU-positive β-cells increased dramatically in the T2DM + acarbose group compared with the T2DM group (Fig. 4C).

Hypermethylation of the PDX-1 promoter was not observed in normal mice but was significantly increased in T2DM model mice. Additionally, PDX-1 hypermethylation was inhibited by acarbose and alkylresorcinol (Fig. 5A-C). Moreover, a significant redistribution of PDX-1 from the nucleus to the cytoplasm was observed in pancreatic tissues of T2DM model mice compared with that in normal mice. In contrast, acarbose prevented the nuclear export of PDX-1 in T2DM model mice (Fig. 5D).