Figure 9

A-769662 facilitated thermogenesis in differentiated iWAT-SVF cells through AMPK signaling pathway. (A–D) iWAT-SVF cells were induced to differentiation toward brown-like adipocytes and were treated with the indicated compounds on day 7. (A) Relative mRNA levels of thermogenic genes in differentiated iWAT-SVF cells treated with the indicated compounds for 6 h were analyzed by quantitative RT-PCR. n = 4. (B) Western blot analysis of UCP1, PGC-1α, p-AMPKα (T172), AMPKα, p-ACC (S79), and ACC expression levels in differentiated iWAT-SVF cells treated with indicated compounds on day 7 for 24 h. (C) Relative protein expression levels of UCP1 and PGC-1α and the relative phosphorylation levels of AMPKα and ACC were determined by densitometric quantification of the immunoblots shown in (B). n = 3. (D) Basal and uncoupled oxygen consumption rate (OCR) of differentiated iWAT-SVF cells treated with NE (10 μM) for 3 h or with DMSO or A-769662 at different concentrations on day 7 for 12 h. n = 4. (E–G) iWAT-SVF cells were isolated from iWAT of 5-week-old AMPK α1/α2-floxed mice and induced to differentiate toward beige adipocytes. Cells were infected with NC and Cre lentivirus on day 6 to knockdown AMPKα expression and were treated with the indicated compounds on day 8. (E) Relative mRNA levels of the indicated genes in differentiated iWAT-SVF cells treated with the indicated compounds for 6 h on day 8 were analyzed by quantitative RT-PCR. n = 4. (F) Western blot analysis of UCP1, PGC-1α, p-AMPKα (T172), AMPKα, p-ACC (S79), and ACC expression levels in differentiated iWAT-SVF cells treated with indicated compounds on day 8 for 24 h. (G) Relative protein expression levels of UCP1 and PGC-1α and the relative phosphorylation levels of AMPKα and ACC were determined by densitometric quantification of the immunoblots shown in (F). n = 3. Data are presented as the means ± SEM. One-way ANOVA. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with the indicated DMSO group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared with the indicated NC group.