Fig. 5.

Cell proliferation induced by insulin is reduced and pERK1/2 levels are increased when lipid rafts are disrupted. A: bromodeoxyuridine (BrdU) uptake in HepG2 cells before and after insulin (300, 600, and 1,200 nM, 14 h) stimulation. Ten percent of serum was used as additional positive control for cell proliferation [0% serum = 100%, 10% serum = 176 ± 12%, insulin (300 nM) = 135 ± 8%, insulin (600 nM) = 146 ± 8%, insulin (1,200 nM) = 157 ± 8%; n = at least 3 individual experiments in triplicate]. B: BrdU uptake in control and metyl-β-cyclodextrin (MβCD)-treated (45min) HepG2 cells before and after insulin (300, 600, and 1,200 nM, 14 h) stimulation. Ten percent serum and (2-hydroxypropyl)-γ-cyclodextrin (HYCD) was used as additional positive and negative controls, respectively, for cell proliferation [0% serum = 100%, 10% serum = 176 ± 12%, MβCD + 10% serum = 110 ± 11%, insulin (300 nM) = 135 ± 8%, MβCD + insulin (300 nM) = 94.2 ± 4.1%, insulin (600 nM) = 146 ± 8%, MβCD + insulin (600 nM) = 85 ± 6%, insulin (1,200 nM) = 157 ± 8%, MβCD + insulin (1,200 nM) = 84 ± 8%; n = 3 experiments in triplicate]. C and D: C and D: HepG2 cells (C) and rat hepatocytes (D) (control, insulin stimulated, MβCD treated) were stimulated with insulin and pERK1/2 levels were evaluated by immunoblot. MβCD + insulin samples shown in C and D were run on a separate gel. Densitometric analysis of the C and D, respectively, shows an increase on pERK1/2 levels in cells treated with MβCD [HepG2: control = 0.29 ± 0.03 arbitrary units (a.u.), insulin (300 nM) = 6.61 ± 1.6 a.u., MβCD + insulin = 30.7 ± 0.5 a.u.; hepatocytes: control = 9.3 ± 1.5 a.u., insulin (300 nM) = 31.19 ± 0.8 a.u, MβCD + insulin = 66.33 ± 9.4 a.u.; n = 3 independent experiments]. pERK and ERK blots shown in C and D are from the same experiments for each sample analyzed. The values indicate the means ± SD. *P < 0.01, difference between groups was statistically significant; ns, not significant. Data were analyzed by one-way ANOVA followed by Bonferroni posttests.