Fig. 2. Establishment of an insulin-resistant cell model. (A) Effects of different concentrations (0, 1 × 10−9, 1 × 10−8, 1 × 10−7, 1 × 10−6, 1 × 10−5 mol L−1) of insulin on glucose uptake in HepG2 cells. Black, red and blue lines represented insulin treatment for 12, 24, & 36 h respectively, and the results were expressed as percentage of mean fluorescence intensity relative to control cells. (B) Cell viability analysis of cells treated with different concentrations (0, 1 × 10−9, 1 × 10−8, 1 × 10−7, 1 × 10−6 and 1 × 10−5 mol L−1) of insulin for 24 h, by CCK-8 method, and the results were expressed as percentage viability relative to the control group. (C) Glycogen content estimation in cells after treatment with 1 × 10−6 mol L−1 insulin for 24 h. The results were expressed as percent of control. (D) Flow cytometry-based assessment of intracellular ROS levels in cells treated with 1 × 10−6 mol L−1 insulin for 24 h, using DCFH-DA fluorescent reagent. The results were expressed as mean fluorescence intensity. (E) Analysis of the 1 × 10−6 mol L−1 insulin treatment effects after 24 h on IRS-1 and AMPK phosphorylation status in HepG2 cells. (F) Analysis of insulin-resistant HepG2 cells stability, as measured by glucose uptake after treatment of cells first with 1 × 10−6 mol L−1 insulin for 24 h, and later further culturing them in medium without insulin for another 24, 48 and 72 h. Represented values are means ± SD from three separate experiments. *p < 0.05 vs. control cells. IR, insulin resistance.
