Figure 2. Silencing ADRB3 in human brown/beige adipocytes lowers cAMP and affects β-AR–stimulated lipolysis.

(A and B) Basal, forskolin-stimulated (Fsk 10 μM) and isoproterenol-stimulated (Iso 1 μM), cAMP concentration (A) and glycerol release (B) in siRNA-Ctrl and siRNA-ADRB3 adipocytes. (C) RNA levels of lipolytic genes PNPLA2, LIPE, and ABHD5 in siRNA-Ctrl and siRNA-ADRB3 adipocytes. (D) Protein expression of pHSL563, pHSL660, HSL, ATGL, CGI-58, perilipin, and actin in cell lysates following Fsk treatment. (E–G) Quantification of HSL (E), ATGL (F), and CGI-58 (G) from Western blotting analysis. (H) Glycerol released into the incubation media following dose-dependent treatment with mirabegron (1-10-100-1000-10,000 nM) in siRNA-Ctrl– and siRNA-ADRB3–transfected adipocytes. (I) Glycerol released by siRNA-Ctrl and siRNA-ADRB3 adipocytes treated with 10 μM of relatively selective human β₁ agonist dobutamine, β₂ agonist terbutaline, and β₃ agonist mirabegron. Fsk (10 μM) and Iso (1 μM) were used as positive controls of agonist-induced lipolysis. Data are represented as mean ± SEM. Two-tailed unpaired Student’s t test and a 2-way ANOVA were used for statistical analysis. Gene expression data are normalized to siRNA-Ctrl adipocytes and expressed on a log₁₀ scale. For cAMP and lipolysis data, *when comparing basal with stimulated in siRNA-Ctrl, ^$when comparing basal with stimulated in siRNA-ADRB3 adipocytes, ^#when comparing same doses between siRNA-Ctrl and siRNA-ADRB3 adipocytes. *^,$,#P < 0.05; **^,$$,##P < 0.01; ***^,$$$P < 0.001; ****^,$$$$,####P < 0.0001.