FIG. 1.

Differential effects of Akt inhibition at low and high concentrations of isoproterenol. (A) Floxed Akt2 fibroblasts stably expressing PPARγ were infected with either Adeno-GFP or Adeno-Cre and then differentiated into adipocytes. These cells were serum starved and treated with 1 nM insulin and analyzed by immunoblotting using Licor Odyssey for the excision of Akt2 and the loss of phospho-Akt Ser473 signal (left panel; representative of two experiments). The quantification of immunoblot analysis of phospho-Akt Ser473 of Akt2 ^lox/lox adipocytes infected with Ad-GFP or Ad-Cre is shown (middle panel). A glycerol release assay (right panel) was performed with increasing doses of isoproterenol in the presence and absence of 1 nM insulin for 2 h. Data are expressed as means ± standard deviations (SD) from two experiments performed in duplicate. (B) RNA inteference-mediated reduction in Akt2 does not affect insulin inhibition of glycerol release. 3T3-L1 preadipocytes were infected with an shRNA lentiviral construct that targets Akt2 and sorted for the low and high expression of GFP. Adipocytes were treated with the indicated concentrations of insulin and subjected to the immunoblot analysis of Akt2 and phospho-Akt Thr308, confirming the efficiency of knockdown (left). Highly expressing cells were differentiated into adipocytes, and a glycerol release assay was performed using 2 nM isoproterenol with increasing doses of insulin (upper right). Data are expressed as means ± SD from two experiments performed in duplicate. A glucose uptake assay also was performed on differentiated control, highly, and lowly expressing cells (bottom right).