Figure 6.

SIRT1 transgenic adipocytes display an exhacerbated response to β3-adrenergic stimulation. Brown pre-adipocytes from wild-type (WT) and SIRT1 transgenic (Tg) mice were isolated and immortalized. (A) The morphology of immortalized brown adipocytes was evaluated before (day 0) and after (day 6) differentiation. (B) Total triglyceride content in differentiated brown adipocytes was evaluated. (C) Total mRNA levels were extracted from pre-adipocytes and differentiated adipocytes and used for qPCR analysis. (D) Total protein extracts were used to evaluate diverse differentiation markers in differentiated and undifferentiated adipocytes. (E) Ucp1 expression was measured in total mRNA extracts of differentiated brown adipocytes. (F) Differentiated WT and Tg adipocytes were stimulated with 1 μM of norepinephrine (NE) or 1 μM of CL316,243 (CL) during 5 h at 37 °C. Then, total proteins were extracted and used for western blot analysis. (G) WT and Tg brown adipocytes were treated with CL in a dose–response fashion for 5 h at 37 °C and then total mRNA was extracted to measure Ucp1 expression. (H) WT and Tg brown adipocytes were treated with 1 μM CL and incubated at 37 °C. Then, total mRNA was extracted at the times indicated to measure Ucp1 expression. All values are presented as mean ± SEM of at least n = 4 independent experiments, each of them run in triplicate. * indicates statistical significant difference between WT (white bars and circles) and Tg mice (black bars and circles) at P < 0.05.