FIGURE 7.

PKA and not EPAC activation mimics A2bAR agonism by BAY 60-6583. SVCs were isolated from the subcutaneous adipose tissue of WT mice, treated with DMSO (Vehicle), 1 μm BAY 60-6583 (BAY), 2 μm FSK, 500 μm 8-bromo-cAMP (activates EPAC and PKA, 8-Br), 200 μm 8-CPT-2′-O-Me-cAMP (activates EPAC, O-Me), or 200 μm 6-MB cAMP (activates PKA, 6-MB) on day 0 and day 3 of induction and induced to differentiate as described under “Experimental Procedures.” A, cAMP levels were assessed as described under “Experimental Procedures” (n = 4). B–D, mRNA expression of aP2 (B) and PPARγ (C) after 6 days of differentiation and of KLF4 (D) after 6 h of differentiation was determined using the ΔΔCT method and normalized to 18 S rRNA values (n = 3). E, SVCs were treated with 1 μm BAY 60-6583 and 2 μm FSK. Fluorescence at 340 and 380 nm was assessed throughout the treatments, and the 340:380 nm ratio was calculated using ImageJ software. Data are mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with vehicle treatment at the same time point. Analyses were performed by two-way ANOVA with Bonferroni multiple comparisons post hoc test (A), by one-way ANOVA with Bonferroni multiple comparisons post hoc test (B and C), or by Student's t test (D).