Primers used in RT-PCR and quantitative real-time PCR (mouse).

<p>Primers used in RT-PCR and quantitative real-time PCR (mouse).</p

Inhibition of retinol metabolism by 4-MP suppresses HSC activation in CCl₄-induced liver fibrosis.

<p>Mice were concurrently treated with CCl_<b>4</b> and 4-MP for 2 weeks and HSCs were isolated from the treated mice. (A) Representative Western blot images for α-SMA, TGF-β1, pSmad3, and ADH3 in isolated HSCs and quantification of respective images. Semi-quantitative PCR (B) and qPCR (C) for α-SMA, TGF-β1, COL1A1, MCP-1, and IL-6 in isolated HSCs. (D) Retinol and their metabolites, all trans-retinaldehyde (atRald) and retinoic acid (atRA), in isolated HSCs. Treatment with 4-MP increased retinol levels and reduced its metabolites significantly. The data are representative of three independent experiments and are expressed as mean ± SEM. *P < 0.05, **P < 0.01 compared with the respective controls.</p

Treatment with 4-MP increased production of IFN-γ by NK cells in CCl₄-treated mice.

<p>Hepatic fibrosis was induced with CCl_<b>4</b> and the mice were concurrently treated with 4-MP for 2 weeks. (A and B) FACS analyses of isolated mononuclear cells to assess the frequency of NK cells and IFN-γ production, respectively. (C) Serum levels of IFN-γ. (D) qPCR for IFN-γ in isolated NK cells. (E) Fluorescent TUNEL staining to demonstrate apoptosis in activated HSCs after treatment with 4-MP and the respective quantified data (scale bar = 25 μm). The data are representative of three independent experiments and are presented as mean ± SEM. *P < 0.05, **P < 0.01 when compared with the respective controls.</p

Treatment with 4-MP during CCl₄-induced liver fibrosis in mice.

<p>Liver fibrosis was induced by intraperitoneal injection of CCl_<b>4</b> and different doses of 4-MP (10 to 100 μg/g) was administered for 2 weeks. (A) Experimental protocol of the injections with CCl_<b>4</b> and 4-MP. (B) Alteration of animal body weight during the study. (C) Serum levels of ALT and AST were measured. (D, E) Hematoxylin and eosin staining of liver and lung sections after administration of CCl_<b>4</b> and 4-MP (scale bar = 500 μm). The data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 compared with the respective controls.</p

Transcriptional regulation of bile acid enzyme genes by <i>Crebh.</i>

<p>(<b>A</b>) Mice (n = 5) or primary human hepatocytes (n = 3) were infected with indicated adenoviruses for 96 hrs. Liver tissues were obtained and protein and total RNA was extracted for western blot and qPCR analyses, respectively. *<i>p</i><0.05 vs. Ad-GFP group. (<b>B</b>) HepG2 cells were co-transfected with CREBH-N and different CYP7A1-Luc and CYP27A1-Luc promoter constructs, and luciferase assay was performed. (<b>C–D</b>) HepG2 cells were transfected with wild type (wt) or CREBH-mutant (mut) constructs of CYP7A1-Luc or CYP27A1-Luc followed by 2-AG-ether treatment for 12 hrs and luciferase assay was performed (D) or immunoprecipitation of HepG2 chromatin from cells exposed to DMSO (control) or 2-AG-ether was performed with IgG or Crebh antibody (E). Promoter regions were amplified by PCR, as depicted. Percentage of DNA immunoprecipitated with Crebh antibody relative to input chromatin was quantified by qPCR. *<i>p</i><0.05 vs. control. Data represents mean ± SE.</p

<i>Crebh</i> knockdown restores bile acid homeostasis in alcohol-exposed insulin-resistant mice model.

<p>(<b>A</b>) B6, STZ-treated and <i>db/db</i> mice (n = 4–5) were treated with EtOH as indicated. Liver tissues were obtained and protein and total RNA was extracted for western blot and qPCR analyses, respectively. *<i>p</i><0.01 vs. B6 control group, **<i>p</i><0.01 vs. STZ-treated and <i>db/db</i> group. (<b>B</b>) <i>db/db</i> mice (n = 5) were infected with the indicated adenoviruses for 96 hrs followed by treatment with EtOH. Liver tissues were obtained, protein and total RNA was extracted for western blot and qPCR analyses, respectively. Liver tissues and serum were further utilized for total bile acid measurement. *<i>p</i><0.01 vs. Ad-USi group, **<i>p</i><0.01 vs. Ad-USi+EtOH group. Data (A-B) represents mean ± SE. (<b>C</b>) Proposed model for the regulation of hepatic bile acid homeostasis by acute alcohol exposure via CB1R and CREBH. Acute alcohol exposure and endocannabinoid signaling induce <i>Crebh</i> gene expression and generates active <i>Crebh</i> (CREBH-N). Active <i>Crebh</i> regulates the expression of <i>Cyp7a1</i> and <i>Cyp27a1</i> directly and that of <i>Cyp7b1</i> and <i>Cyp8b1</i> indirectly, which leads to elevated hepatic bile acid concentrations. This, in turn, causes liver injury. On the other hand, insulin receptor signaling maintains bile acid homeostasis via inhibition of <i>Crebh</i> gene expression and activity.</p

Acute alcohol and endocannabinoid regulates key bile acid genes expression.

<p>(<b>A–B</b>) Mice (n = 4) or primary human hepatocytes (n = 3) were treated with EtOH or 2-AG-ether. Liver tissues were obtained and protein and total RNA was extracted for western blot and qPCR analyses, respectively. *<i>p</i><0.01 vs. control group. (<b>C</b>) Mice (n = 4) were treated with AM251 or AM251+EtOH as indicated. Liver tissues were obtained and protein and total RNA was extracted for western blot and qPCR analyses, respectively. *<i>p</i><0.01 vs. control group, **<i>p</i><0.01 vs. EtOH-treated group. Data represents mean ± SE.</p

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