Figure 2.

PI-3 kinase inhibitors block insulin-stimulated, but not basal, ACRP30 secretion. Eight 10-cm plates of 3T3-L1 adipocytes were serum-starved and then treated or not with wortmannin, LY294002, or rapamycin at the indicated concentrations for 45 min. As in Fig. 1, total cellular protein was labeled with a 15-min pulse of ³⁵S-labeled cysteine and methionine, followed by inhibition of further protein synthesis with cycloheximide. During the chase period, cells were maintained in the presence or absence of 160 nM insulin and the continued presence or absence of inhibitors, and the medium was collected and replaced at 30-min intervals. ACRP30 was quantitatively immunoprecipitated from the media collected at each time point. The immunoprecipitates were separated by SDS-PAGE and the amount of ACRP30 present at each data point was quantified using a PhosphorImager. Cumulative ACRP30 is plotted for cells chased in the absence (open squares) or presence (filled diamonds) of insulin. The total amount of protein secreted by the untreated cells in the presence of insulin is taken as 100%; all other data are plotted relative to this amount.