Figure 4.

Effect of cholesterol-binding drugs on clathrin-coated pit mediated endocytosis and insulin-stimulated glucose transport. (A) Tf internalization was measured in 3T3-L1 adipocytes grown on 6-well plates. Tf internalization in control cells (dark symbols) and cells treated with 50 μg/ml nystatin (open symbols) was measured by incubating the 3T3-L1 adipocytes at 37°C for different times with iodinated Tf. Tf internalization values are means ± SEM of counts per minute (cpm) (n = 4) corrected for the nonspecific values (obtained for each time point by including a 200-fold excess of unlabeled Tf) from a representative experiment. (B) Insulin binding (open bars) and internalization (dark bars) was measured in 3T3-L1 adipocytes grown on 6-well plates. Cells were pretreated for 15 min in the absence or in the presence of 50 μg/ml nystatin, and insulin binding was determined by incubating the cells with iodinated insulin [1 nM, 0.5 μCi per well (1 Ci = 37 GBq)] for 4 h at 4°C. Insulin internalization was measured by incubating adipocytes with iodinated insulin (1 nM, 0.5 μCi per well) at 37°C for 30 min and by washing at low pH at 4°C. Data are means ± SEM (n = 4) of cpm corrected for the nonspecific values (obtained by including 1000-fold excess of unlabeled insulin) from a representative experiment. (C) Adipocytes were incubated in the absence or the presence of 5 μg/ml filipin or 50 μg/ml nystatin for 15 min. Thereafter, cells were incubated for 30 min in the absence (open bars) or presence of 100 nM insulin (filled bars) and 2-deoxyglucose uptake was measured for 5 min. Results are means ± SEM from a representative experiment run in triplicate. (D) Adipocytes were incubated for 15 min with (open symbols) or without (dark symbols) 50 μg/ml nystatin. Subsequently, 100 nM insulin was added for various times and 2-deoxyglucose uptake was measured for 1 min. Data are means ± SEM from a representative experiment run in triplicate.