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. 2011 Dec 5;287(3):1861–1873. doi: 10.1074/jbc.M111.305789

FIGURE 2.

FIGURE 2.

Insulin signaling mediates feeding induction of CYP7A1 in wild type C57BL6J mice. A, feeding effects on total and phosphorylated AKT, GSK3β p70 S6K, ERK1/2, and AMP-activated protein kinase in mouse liver were determined by immunoblot; samples were pooled from four mice. B, mice were fasted from 9 a.m. and given a single dose of the ERK inhibitor U0126 (5 mg/kg) or the PI3K inhibitor, wortmannin (0.8 mg/kg) by intravenous injection at 11 p.m. One h later, mice were either fasted or refed chow for an additional 3 h. Liver mRNA expression was determined by real-time PCR. C, mice were injected (intravenously) with adenovirus (2−9 pfu/mouse) expressing GFP (left) or a dominant negative form of AKT (Ad-DN-AKT) (right). Seven days later, mice were fasted for 15 h or refed chow for an additional 3 h, and liver CYP7A1 mRNA was measured. D, mice fasted for 15 h were refed chow or given a single dose of glucose (8 g/kg, oral gavage), medium chain triglycerides (MCT) (4 mg/kg, oral gavage), or insulin (0.8 unit/kg, intraperitoneal injection). Liver CYP7A1 mRNA levels were determined 3 h later. E, ChIP assay of FoxO1 binding to the cyp7a1 gene promoter in mouse livers. Liver nuclei were isolated from fasted and refed (3 h) mice. Pooled samples from four mice were used. ChIP primers detecting the proximal cyp7a1 promoter region (−1k) were used. Real-time PCR was done in triplicate, and mean values were plotted. F, effect of adenovirus-mediated transduction of constitutively active nuclear form of FoxO1 on CYP7A1 mRNA expression levels in mouse liver. Mice were injected (intravenously) with adenovirus (2−9 pfu/mouse) expressing either GFP as control or expressing a constitutively active nuclear form of FoxO1 (Ad-FoxO1). Seven days later, liver CYP7A1 mRNA was measured in overnight fasted mice. G, liver CYP7A1 mRNA was determined in fasted and refed (3 h) wild type and FXR knock-out (KO) mice. Results are expressed as mean ± S.E. (error bars) (n = 4). *, statistical significance versus fasted mice; **, statistical significance versus fasted + U0126 or fasted FXR knock-out mice (p < 0.05).