Glucose test kit

100 μL of the cell suspension (5 × 10⁴ /mL) was seeded to the 96-well cell culture plate and cultured at 37 °C and 5% CO₂. After the cells were cultured for 24 h, the old medium was aspirated and the wells were washed with PBS solution twice, then serum-free DMEM medium with insulin solution was added synchronously to the cells. The supernatant was aspirated after cultured for 36 h and the cells were poured with serum-free drug-containing or drug-free DMEM added. The experiment was divided into four groups: components treatment group (30-120 μg/mL), blank control group, Metformin (Met) group (30-120 μg/mL), and insulin group (10^-5 mmol/L). The glucose content was detected at 505 nm after 24 h of culture according to the glucose test kit (Beijing Applygen Technologies Inc., China). The glucose consumption rate was calculated with the following formula: ∆GC = (glucose concentration of blank wells - glucose concentration of cell cell-inoculated).

The IR of HepG2 cells was established based on the study of Zhang et al. [24 (link)] with modification. Briefly, cells were cultured and serum-starved for 2 h in 96-well plates, and the medium was replaced by serum-free DMEM supplemented with 10⁻⁵, 10⁻⁶, 10⁻⁷, and 10⁻⁸ mol/L insulin (Sigma) for 36 h. Subsequently, the medium was collected and the glucose content was measured using a glucose test kit (Applygen, China). In addition, glucose uptake was measured using the fluorescent glucose analog, 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose; Thermo Fisher Scientific). In this study, the glucose consumption and uptake of the 10⁻⁶ mol/L insulin-treated groups significantly decreased, indicating that the HepG2-IR model was established successfully (Figure 2S).