Materials and reagents

Liver cell lines HepG2 and L02 were obtained from National Collection of Authenticated Cell Cultures (Shanghai, China). The glucose detection kit was obtained from RSbio (Shanghai, China). The glycogen stain kit was purchased from Jiancheng (Nanjing, China). Fluorescent probe of 2-NBDG was purchased from Dojindo (Shanghai, China). Recombinant human insulin was purchased from CoSin (Beijing, China). Pure C3G sample (No. SC8740) was purchased from Solarbio (Beijing, China). The primary antibodies used were PTP1B (Abcam, ab244207, 1:1000), anti-p-IRS-2 (Bioss, bs-0173R-1, 1: 250) and GAPDH (Abcam, ab8245, 1:10000). The secondary antibodies used were goat anti-rabbit (Sangon Biotech, D111018, 1:1000). The BCA kit, ECL kit, MTT kit and RIPA lysis buffer were purchased from Beyotime (Shanghai, China).

Extraction and identification of anthocyanins from red bayberry

Anthocyanins in the fresh red bayberry fruit were extracted and identified according to the previously reported methods by our team [15]. For extraction, fresh red bayberry fruits were subjected to ultrasonic extraction in a 4-fold volume of anhydrous ethanol for 90 min. After sonicating, the mixture underwent filtration through a fine-grade mesh to discard the solid debris, and then the filtrate was evaporated at 49 °C using a rotary evaporator to remove the ethanol solvent. Subsequently, phase separation was performed by adding an equal volume of ethyl acetate to the concentrated extract and mixing. This mixture was allowed to stand for 10 min, and this process was repeated until a clear organic phase was achieved, after which the aqueous phase was collected for further purification. The aqueous phase was then loaded onto a D-101 macroporous resin column pre-equilibrated, and eluted with a solution of 1% formic acid in 80% methanol at a flow rate of 2 mL/min. The anthocyanin-rich fractions were concentrated under reduced pressure, and lyophilized using a vacuum freeze dryer operating at specified conditions to yield the dry anthocyanin extracts. Finally, the extracts were stored at −80 °C in an ultra-low temperature freezer until further use.

For identification of anthocyanins in the extracts, an ultra-high performance liquid chromatography (UPLC) system (Thermo UltiMate 3000, Thermo Fisher Scientific) was used. Chromatographic separation was achieved using a binary solvent system consisting of 1% formic acid in water (solvent A) and a mixture of acetonitrile with 0.1% formic acid aqueous solution (1:1, v/v; solvent B). Elution was performed following a linear gradient program where the concentration of solvent B was varied as follows: 10% to 38% over the first 40 min, held at 38% until 60 min, then increased to 48% by 60 min and further to 100% by 70 min, maintained at 100% for 5 min before returning to the original 10% composition over the next 5 min. The column was then equilibrated under initial conditions for 5 more min, resulting in a total run time of 80 min. The flow rate was maintained at 0.3 mL/min and the detection of anthocyanins was carried out at a wavelength of 520 nm.

Cell culture and treatments

After reaching 80% confluence, HepG2 and L02 cells were incubated with 0.2 mM PA and 30 mM glucose for 24 h to induce IR [16], and cells treated with 5 mM glucose were used as the control (Con group). Then, IR hepatocytes were incubated with various concentrations (0, 40, or 80 μg/mL) of C3G for another 24 h.

Cell viability assay

After treating with different concentrations of C3G for 24 h, HepG2 and L02 cells were treated with 100 μL of MTT with the final concentration of 0.5 mg/mL per well, and incubated at 37 °C for 4 h. Then, 150 µL of dimethyl sulfoxide was added to per well, shaking the plates until the formazan was completely dissolved. Finally, the absorbance value at 490 nm was measured by a microplate spectrophotometer.

Glucose consumption and insulin sensitivity assays

HepG2 and L02 cells were seeded into a 96-well plate, the cell culture medium was collected after treatment, the glucose content of each well was measured with a commercial glucose detection kit. Next, the glucose consumption was calculated by the glucose content of the blank group minus that of the treated groups, and the actual glucose consumption was adjusted using cell viability measured by MTT analysis.

Insulin sensitivity index (%) = (glucose consumption in C3G group with insulin - glucose consumption in C3G group without insulin)/(glucose consumption in control group with insulin - glucose consumption in control group without insulin) × 100%.

Glucose uptake assay

HepG2 and L02 cells were seeded into a 24-well plate. Then, 0.1 mM 2-NBDG dye was added for 30 min incubation at 37 °C after treatment, cells were washed with PBS for three times. Finally, images were obtained by a fluorescence microscope and the fluorescence intensity was calculated using the ipwin 32 software.

Glycogen synthesis and glycogen staining assays

To determine the glycogen synthesis, different concentrations of glucose solutions were prepared, and a glucose standard curve was established with glucose concentration as the horizontal axis and absorbance value as the vertical axis. Hepatocytes were collected into a centrifuge tube and centrifuged for 5 min at 4 °C and 4000 × g, the supernatant was discarded and dried naturally. The cell mass was obtained by subtracting the mass of centrifuge tube from the total mass. 0.5 mL of 30% KOH solution was added to each sample and shaken well before heating for 30 min using a boiling-water bath. Each sample was cooled sufficiently before adding 1.5 mL of ethanol and allowing it to stand for 30 min. The sample was then centrifuged at 12,000 × g for 15 min at 4 °C, and the supernatant was discarded. Immediately, the precipitate was collected, dissolved in 0.5 mL dd-H2O, and then mixed with 1 mL of 0.2% anthrone solution. Heated in a water bath at 100 °C for 20 min, and the optical density (OD) value was measured at 620 nm. The glucose concentration of each sample was calculated using the glucose standard curve formula and divided by the corresponding cell mass to obtain the glycogen content per unit mass of cells.

For the glycogen staining assay, after treatment with different concentrations of C3G, the medium was removed and washed three times with PBS. A glycogen staining kit was used to measure the glucose content of cells, and images were obtained with a light microscope.

RNA isolation, reverse transcription, and qRT-PCR analysis

An RNA miniprep kit was used to isolate total RNA from cells, and the concentration and purity were determined by a nucleic acid and protein detector (Nanodrop, Thermo). Then reverse transcription was performed with an RT kit to obtain the corresponding cDNA. The qRT-PCR was performed with cDNA (1:20, v:v) on an Applied Biosystems Step One RT-PCR system by using the TB Green kit (Takara, Japan). Table 1 shows the primer sequences of target genes.

Table 1 Primer sequences for the qRT-PCR assay.Western blotting analysis

Proteins were extracted from treated cells by using a cell lysis buffer kit, and the total protein concentration was measured using a BCA kit. PTP1B, p-IRS-2 and GAPDH from the total protein were separated by SDS-PAGE (12% gel). The targeted proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane, and incubated with blocking solution containing 10% skim milk (w/v) for 1 h at room temperature, followed by overnight incubation with different primary antibodies at 4 °C. Subsequently, PVDF membranes were washed three times with TBST, and transferred to horseradish peroxidase-labeled goat anti-rabbit or anti-mouse secondary antibodies at room temperature for 1 h. Finally, PVDF membranes were washed with TBST three times and exposed to the luminescence reagent ECL. Images were captured with an image digital imaging system (General Electric Company, USA).

Animals and experimental design

Four-week-old wild-type C57BL/6J male mice and db/db male mice (C57BL/6 J genetic background), were obtained from SLAC Laboratory Animal Co. Ltd. All mice were housed in a specific pathogen-free (SPF) barrier facility. The criteria for housing conditions were controlled temperature (23 °C ± 3 °C), 12 h light/dark cycle, standard chow feeding (Table S1) and free access to water.

The animals were randomized divided into three groups based on the gavage solution, including: (a) negative control group, C57BL/6J mice with water (n = 6); (b) positive control group, db/db mice with water (n = 6); (c) intervention group, db/db mice with C3G (n = 6). Professional technicians were employed to conduct the daily gavage with C3G aqueous solution (150 mg/kg/day) or equivalent volume of vehicle (pure water) for 6 weeks by using a soft, flexible and properly sized gavage needle. All procedures were strictly followed to prevent esophageal irritation or puncture, ensuring they were performed slowly and gently. Meanwhile, health monitoring of daily visual observations, behavioral evaluation, and weight measurements were included. A glucose monitor (Sinocare, China) was used to measure the fasting blood glucose of all mice.

All our experiments involving laboratory animals were conducted in strict adherence to the principles set forth by the National Institutes of Health guidelines on the care and use of laboratory animals. Additionally, the Animal Ethics Committee of Zhejiang Chinese Medical University granted approval for these procedures, dedicated by the authorization number 20201103-08.

OGTT and HOMA-IR analysis

For oral glucose tolerance test (OGTT), mice were fasted for 12 h before experiment and glucose was administered orally (1.5 g/kg body weight) at 0 min. Blood glucose was measured at 0, 30, 60, 90, 120 and 150 min with a blood glucose monitor from a tail prick.

For homeostasis model assessment-insulin resistance (HOMA-IR) analysis, whole blood from the mice was centrifuged for 15 min at 4 °C and 4000 rpm to separate serum and plasma. The serum was collected and stored in a −80 °C freezer for later use. The levels of GLP-1 and insulin in mouse serum were determined using an ELISA kit according to the manufacturer’s instructions. HOMA-IR = serum glucose (mmol/L) × serum insulin (mU)/22.5.

Statistical analyses

Fluorescent images and intensities of protein bands were counted by applying Image-Pro Plus-6.0 software and Image J software. SPSS 16.0 software was used to perform statistical analysis of data. Data were expressed as means ± standard deviation (SD). Statistical analyses for significance were performed using one-way ANOVA, Duncan’s multiple range test, and t-test, results were considered statistically significant if p < 0.05.