Fig. 8.

Reduction of insulin signaling by knocking down the insulin receptor prevents development of insulin resistance induced by the prolonged exposure to insulin. The cognate siRNA against the insulin receptor or a scramble siRNA was introduced into hep1c1c7 cells by transient transfection with Lipofectamine 2000 for 36 h, as described in materials and methods. A: Western blot of insulin receptor. Levels of insulin receptor proteins were evaluated by immunoblotting with the specific anti-insulin receptor antibody and normalized to the expression level of β-actin. B: cells after knocking down of insulin receptor for 36 h were treated with 10 nM insulin for 24 h (chronic insulin treatment). After an extensive washing with warm PBS, the cells were exposed to 1 nM insulin for 5 min (acute insulin treatment). Activation of Akt was determined by immunoblottings with specific antibodies as indicated. C: the GSH/GSSG ratio was evaluated in the cells in the presence or absence of 10 nM insulin for 24 h. Results represent means ± SE of 3 independent experiments, each in triplicate. INSR, insulin receptor. *P < 0.05, **P < 0.01 vs. vehicle.