Fig. 4

Effect of cholesterol depletion on redistribution of signaling proteins, insulin-mimetic signaling, and glucose transport activation by various stimuli. Isolated rat adipocytes were incubated in the absence or presence of increasing concentrations of mβCD (50 min, 30°C) prior to subsequent treatment with 3 µM PIG41, 30 µM glimepiride, or 300 µM CBDP. Purified plasma membranes were analyzed for cholesterol (set at 100% = 0.27 ± 0.07 nmol/µg protein in the absence of mβCD). hcDIGs were prepared and used for immunoprecipitation of caveolin-1 in nondissociating buffer. The immunoprecipitates were photoaffinity labeled for Gce1 (dissociation of caveolin-Gce1 interaction) and immunoblotted for pp59^Lyn (dissociation of caveolin-pp59^Lyn interaction). From total cell lysates, pp59^Lyn and IRS-1 were immunoprecipitated and then immunoblotted for phosphotyrosine (pp59^Lyn and IRS-1 tyrosine phosphorylation). Glucose transport was determined in addition. Each value represents the mean ± SD from three independent adipocyte incubations with determinations in quadruplicate each (absence of mβCD set at 100% each).