FIG. 6.

Effects of wortmannin and various mutant proteins on insulin-induced activation of PDE3B in 3T3-L1 adipocytes. (A) Effect of wortmannin on insulin activation of PDE3B. Cells were incubated in the absence or presence of 100 nM wortmannin for 30 min and then with or without 100 nM insulin for 15 min. PDE activity in solubilized membrane fractions was assayed in the absence or presence of cilostamide as described in Materials and Methods. Open columns show cilostamide-sensitive PDE activity, and closed columns show cilostamide-insensitive PDE activity. (B) Comparison of the activities of endogenous and recombinant PDE3B. Cells that had been infected (or not) with AxCAPDE3B-WT at an MOI of 30 PFU/cell were incubated in the absence or presence of insulin, after which solubilized membrane fractions were prepared as described in Materials and Methods and diluted 1:50 with solubilization buffer. The diluted samples were then assayed for PDE3B activity. (C to F) Effects of various mutant proteins on insulin activation of endogenous PDE3B. Cells that had been infected with AxCAAkt-AA (200 PFU/cell), AxCAAkt-K179D (200 PFU/cell), or AxCAMyr-Akt (30 PFU/cell) (C and D), or with AxCAλΔNKD (150 PFU/cell) or AxCAλΔPD (150 PFU/cell) (E and F), were incubated in the absence or presence of 100 nM insulin for 15 min, after which total cell lysates (C and E) or solubilized membrane fractions (D and F) were prepared and then subjected to immunoblot analysis (C and E) or assayed for cilostamide-sensitive PDE activity (D and F). Quantitative data are means ± standard errors from three independent experiments.