Fig. 8.

Prolonged exposure to insulin decreases mitochondrial membrane fluidity in a cholesterol synthesis-dependent manner. A and B, Hepa1c1c7cells were treated with either the vehicle solution or insulin (5 nm) in the presence or absence of simvastatin (10 nm) for 16 h as indicated (n = 3). Mitochondria were then isolated from these cells and labeled with either TMA-DPH or DPH. Fluorescence density was quantified at 366 nm (emission = 440 nm) using polarizing filters in excitation and emission planes and normalized to mitochondrial protein level. C and D, Mitochondria (50 mg proteins) isolated from Hepa1c1c7 cells were incubated cholesterol-BSA complex for 5 min at 4 C (n = 3), washed three times to eliminate the free cholesterol, and labeled with TMA-DPH or DPH. Fluorescence density was quantified at 366 nm (emission = 440 nm) using polarizing filters in excitation and emission planes and normalized to mitochondrial protein level. Results represent mean ± se of three independent experiments. **, P < 0.01 vs. control; ##, P < 0.01 vs. insulin alone.