FIG. 5.
PI 3-kinase inhibitors increased insulin-induced Grb-2-associated IRS-2 and the mitogenic response in IRS-1-deficient brown adipocytes. (A) Quiescent IRS-1+/+ (+/+) and IRS-1−/− (−/−) brown adipocytes were pretreated for 45 min with either 40 nM wortmannin (W) or 5 μM LY294002 (Ly) and then stimulated with 10 nM insulin (ins; also designated i) for a further 5 min. Cells were lysed and immunoprecipitated (IP) with the anti-Grb-2 antibody and analyzed by Western blotting (WB) with anti-IRS-2 or anti-Grb-2 antibodies. Results from a representative experiment are shown. The autoradiograms corresponding to three independent experiments, which used two different clones of each cell type, were quantitated by scanning densitometry. Results are expressed as arbitrary units of Grb-2-associated IRS-2 and are means ± standard errors. (B) Cells were preincubated with 40 nM wortmannin (W) or 5 μM LY294002 (Ly) as described for panel A. Then cells were further stimulated for 5 min with various doses of insulin and were lysed. Equal amounts of protein (30 to 50 μg) were submitted to Western blot analysis with anti-phospho p42-p44 MAPK and anti-MAPK antibodies. Results from a representative experiment of three experiments are shown. (C) Quiescent wild-type and IRS-1−/− brown adipocytes were cultured for 24 h with 100 nM insulin in both the absence and presence of either 40 nM wortmannin or 5 μM LY294002. [3H]thymidine incorporation into acid-insoluble material was determined during the last 4 h of culture. Results are expressed as disintegrations per minute (dpm) per dish and are means ± standard errors from six independent experiments, which used three different clones of each cell type.