Fig. 7. Butein selectively inhibits Akt1 and Akt1 is necessary for effects of butein in adipocytes.

a C3H10T1/2 adipocytes were treated with 10, 20, 40 μM of Pan Akt inhibitor (Akt1/2 i) for 6 h and expression levels of Ucp1 and Prdm4 mRNA were determined by real-time PCR. b C3H10T1/2 adipocytes were treated with Akt1/2 i (10, 20, or 40 μM) for 6 h and expression levels of Prdm4 and Ucp1 protein were measured by western blotting. c C3H10T1/2 adipocytes were treated with 20 μM of Pan Akt inhibitor (Akt1/2 i) for 6 h and consumption rates (OCR) was measured in approximately 8 min intervals using XF24 Extracellular Flux Analyzer. Data represent means ± s.d. (n = 3). d C3H10T1/2 adipocytes were treated with butein, and Akt1 and Akt2 phosphorylation levels were determined by western blot analysis. e Akt1 and Akt2 phosphorylation levels in epididymal adipose tissue (eWAT) of HFD-fed C57BL/6J mice treated with butein or control for 3 weeks (n = 4 per group) were determined by western blot analysis. f Mouse embryonic fibroblast isolated from wild-type (WT) mice or Akt1 knockout (KO) mice were treated with DMSO (control) or butein for 12 h and expression of Prdm4 and thermogenic genes were determined. g Expression of Akt1 in WT and KO MEF was verified by western blot analysis. h Model of adipocyte browning by butein. Butein inhibit the PI3Kα–Akt1 pathway in adipocytes, leading to upregulation of Prdm4 followed by expression of thermogenic genes. Data represent mean ± s.e.m. and statistically significant differences were determined by Student’s t-test. *P < 0.05; **P < 0.005