ACEA induces <i>FGF21</i> gene expression.

<p>(A–C) HepG2 cells, rat primary hepatocytes (RPH), and AML12 cells were treated with ACEA (10 μM) for the indicated time periods. (D) Wild-type or CB1^-/- mouse primary hepatocytes (MPH) were treated with ACEA (10 μM) for 3 h. (E) Mice were treated with ACEA (10 mg/kg) for the indicated number of days. Livers were harvested for mRNA analysis. (A–E) <i>FGF21</i> and <i>ERRγ</i> mRNA levels were measured by quantitative qPCR analysis and normalized to <i>actin</i> mRNA levels. All data are the means ± standard errors of at least three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 by one-way ANOVA.</p

GSK5182 inhibits ACEA-mediated induction of <i>FGF21</i> gene expression.

<p>(A) AML12 cells were transfected with mFGF21-Luc and treated with ACEA (10 μM) for 3 h with or without GSK5182 (10 μM). (B–D) HepG2 cells, AML12 cells, and mouse primary hepatocytes (MPH) were treated with ACEA (10 μM) for 3 h with or without GSK5182 (10 μM). (E and G) GSK5182 (40 mg/kg) was administrated to male C57BL/6J mice (n = 3–4 per group) daily by intraperitoneal injection for 4 days. ACEA (10 mg/kg) was also given by intraperitoneal injection daily during the final 3 days. (A–D) <i>FGF21</i> and <i>ERRγ</i> mRNA levels were measured by qPCR analysis and normalized to <i>actin</i> mRNA levels. (F) AML12 cells were treated with ACEA (10 μM) for 3 h with or without GSK5182 (10 μM). Culture media was recovered for FGF21 secretion analysis. (G) Male C57BL/6J mice (n = 3) were treated with ACEA (10 mg/kg) with and without GSK5182 (40 mg/kg) daily for 3 days. Serum was analyzed for FGF21 secretion. (H) Male C57BL/6J mice (n = 5 per group) were fed an alcohol-containing diet for 4 weeks and GSK5182 (40mg/kg once daily) was given by oral gavage for the final 2 weeks of alcohol feeding. (I) Schematic diagram of ERRγ-mediated <i>FGF21</i> gene expression. GSK5182 inhibits activation of <i>FGF21</i> gene expression and FGF21 secretion mediated by increased ERRγ caused by activation of the hepatic CB1 receptor. All data are the means ± standard errors for at least three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 by one-way ANOVA.</p

ERRγ overexpression induces <i>FGF21</i> gene expression.

<p>(A–C) HepG2 cells, AML12 cells, and mouse primary hepatocytes (MPH) were infected with Ad-GFP and Ad-ERRγ. (D) Ad-GFP or Ad-ERRγ was injected into male C57BL/6J mice via the tail vein. Mice were sacrificed at 5 days after injection. (A–D) <i>FGF21</i> and <i>ERRγ</i> mRNA levels were measured by quantitative qPCR analysis and normalized to <i>actin</i> mRNA levels. (E) Culture media of adenovirus-infected AML12 cells was obtained for FGF21 secretion analysis. (F) Ad-GFP or Ad-ERRγ was injected via the tail vein into male C57BL/6J mice. Serum from these mice was analyzed for FGF21 secretion. All data are the means ± standard errors of at least three independent experiments. ***p < 0.001 by Student’s t-test.</p

ACEA increases FGF21 protein levels.

<p>(A–C) Whole cell lysates of ACEA-treated HepG2 cells and AML12 cells and livers of ACEA-treated intact mice were harvested for western blot analysis. (D–E) AML12 cells and rat primary hepatocytes were treated with ACEA (10 μM) for the indicated time periods. Culture media were collected for FGF21 secretion analysis. (F) Mice were treated with ACEA (10 mg/kg) for the indicated number of days. Serum was obtained for FGF21 secretion analysis. All data are the means ± standard errors of at least three independent experiments. **p < 0.01; ***p < 0.001 by one-way ANOVA.</p