Fig 2: 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 PI3Ka–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

Fig 3: aP2-Prdm4 Tg mice improve glucose homeostasis and induce thermogenic program.a Glucose tolerance test and b insulin tolerance test in control NonTg (n = 7) and aP2-Prdm4 Tg group (n = 7). HFD-fed mice were fasted for 6 h before intraperitoneal injection of glucose (1 g/kg) or insulin (0.5 U/kg) for GTT and ITT experiments, respectively. Tail blood samples were collected at different time points to measure blood glucose levels. c Heat maps of relative expression levels of brown or beige-selective, white-selective, and Ucp1-independent genes in iWAT of aP2-Prdm4 Tg and NonTg mice. d Expression analysis of selected brown genes (Ucp1, Cidea, and Prdm16) and white adipocyte-selective gene (Retn) in iWAT of NonTg and aP2-Prdm4 Tg mice. Gene expression was determined by real-time PCR. Data were normalized to 36b4 and expressed as mean ± s.e.m. statistically significant differences in gene expression data were determined by Student’s t-test and two-way ANOVA was used to determine significance in GTT and ITT. *P < 0.05

Fig 4: aP2-Prdm4 Tg male mice increase energy expenditure in HFD feeding.a, b Energy expenditure was evaluated by oxygen consumption and carbon dioxide production. a O2 consumption and b CO2 production rates of littermate control NonTg and Prdm4 Tg male mice were measured by indirect calorimetry using CLAMS after 6 weeks on HFD (n = 7 per group). Bar graph (right panel) represents the average of O2 consumption or CO2 production in each group. c Rectal temperature was measured in NonTg and aP2-Prdm4 Tg male mice during cold exposure at 4 °C. d Corrected food intake in NonTg and aP2-Prdm4 Tg male mice. e Ambulatory activities, and f Respiratory exchange ratio (RER) of NonTg and aP2-Prdm4 Tg HFD-fed mice (n = 7 per group). Data represent mean ± s.e.m. and statistically significant differences between control NonTg and aP2-Prdm4 Tg mice were determined by Student’s t-test. Statistically significant differences were determined by two-way ANOVA for the rectal temperature. *P < 0.05; **P < 0.005

Fig 5: Prdm4 expression in adipose tissue (aP2-Prdm4) protect from obesity and fat expansion in HFD feeding.a A schematic transgenic construct used to generate adipose tissue-specific Prdm4 transgenic mice. b Representative Prdm4 and actin expression in iWAT, eWAT, and BAT from male littermate of non-transgenic (NonTg) and two lines of aP2-Prdm4 transgenic (Tg#1 and Tg#2) mice. c Body weight gains of Prdm4 Tg and NonTg male mice. Male NonTg or Prdm4 Tg mice were fed with a low-fat diet (LFD, 10% fat) or high-fat diet (HFD, 60% fat) for 8 weeks. (HFD: n = 7 (NonTg), n = 7 (Tg); LFD: n = 6 (NonTg), n = 7 (Tg)). d Differences in epididymal fat (eWAT) and inguinal fat (iWAT) weight gains in NonTg and Prdm4 Tg male mice. e Adipocyte size of epididymal fat (eWAT) from Prdm4 Tg and NonTg HFD-fed male mice. f Body composition of mice after 8 weeks of HFD-fed NonTg and Prdm4 Tg male mice. Data represent mean ± s.e.m. and statistically significant differences between control NonTg and Prdm4 Tg mice were determined by Student’s t-test. *P < 0.05; **P < 0.005