24-Norursodeoxycholic acid ameliorates experimental alcohol-related liver disease and activates hepatic PPARγ

Background & Aims Alcohol-related liver disease (ALD) is a global healthcare challenge with limited treatment options. 24-Norursodeoxycholic acid (NorUDCA) is a synthetic bile acid with anti-inflammatory properties in experimental and human cholestatic liver diseases. In the present study, we explored the efficacy of norUDCA in experimental ALD. Methods NorUDCA was tested in a preventive and therapeutic setting in an experimental ALD model (Lieber–DeCarli diet enriched with ethanol). Liver disease was phenotypically evaluated using histology and biochemical methods, and anti-inflammatory properties and peroxisome proliferator-activated receptor gamma activation by norUDCA were evaluated in cellular model systems. Results NorUDCA administration ameliorated ethanol-induced liver injury, reduced hepatocyte death, and reduced the expression of hepatic pro-inflammatory cytokines including tumour necrosis factor (Tnf), Il-1β, Il-6, and Il-10. NorUDCA shifted hepatic macrophages towards an anti-inflammatory M2 phenotype. Further, norUDCA administration altered the composition of the intestinal microbiota, specifically increasing the abundance of Roseburia, Enterobacteriaceae, and Clostridum spp. In a therapeutic model, norUDCA also ameliorated ethanol-induced liver injury. Moreover, norUDCA suppressed lipopolysaccharide-induced IL-6 expression in human peripheral blood mononuclear cells and evoked peroxisome proliferator-activated receptor gamma activation. Conclusions NorUDCA ameliorated experimental ALD, protected against hepatic inflammation, and affected gut microbial commensalism. NorUDCA could serve as a novel therapeutic agent in the future management of patients with ALD. Impact and implications Alcohol-related liver disease is a global healthcare concern with limited treatment options. 24-Norursodeoxycholic acid (NorUDCA) is a modified bile acid, which was proven to be effective in human cholestatic liver diseases. In the present study, we found a protective effect of norUDCA in experimental alcoholic liver disease. For patients with ALD, norUDCA could be a potential new treatment option.


Introduction
Alcohol overconsumption is estimated to cause 5.3% of all deaths worldwide 1,2 and is still the most common indication for liver transplantation in Europe. 3The hepatic manifestation of alcohol overconsumption is alcohol-related liver disease (ALD), comprising a spectrum from hepatic steatosis, hepatitis (alcoholic hepatitis) to fibrosis, cirrhosis, and hepatocellular carcinoma.The pathogenesis of ALD is multilayered and involves hepatic ethanol toxicity resulting in the generation of reactive oxygen species, 4 as well as the increased translocation of pathogen-associated molecular patterns (such as lipopolysaccharide [LPS]) from the leaky gut into the systemic circulation.][7][8] Furthermore, alcohol consumption perturbates bile acid metabolism. 9,10For example, ethanol downregulates farnesoid X receptor (FXR/NR1H4), 11,12 alters bile acid conjugation, 13 and affects enterohepatic bile acid circulation. 13,14Chronic alcohol consumption results in an increased bile acid pool and decreased excretion of bile acids.Increased exposure to toxic bile acids might thereby aggravate hepatic injury. 14All these factors facilitate the production of pro-inflammatory cytokines such as IL-6, tumour necrosis factor (TNF), and IL-1b along with the infiltration of pro-inflammatory cells, 15 which culminates in cell necrosis, liver injury, and fibrogenesis. 16,17n addition to inflammatory mediators, numerous immune cell populations are involved in the pathogenesis of ALD.9][20] Ethanol is known to promote the polarisation of M1 macrophages through NF-jB signalling. 21][24] 24-Norursodeoxycholic acid (NorUDCA) is a side chainshortened ursodeoxycholic acid (UDCA) with potent choleretic properties. 25,266][27] NorUDCA ameliorated the pathology in Mdr2-deficient mice, a model of primary sclerosing cholangitis (PSC), 28,29 and showed potent antiinflammatory effects in mouse models of non-cholestatic liver injury such as hepatic schistosomiasis, 28 experimental nonalcoholic steatohepatitis, 27 and acute non-cytolytic lymphocytic choriomeningitis virus infection. 30Importantly, when compared with placebo, norUDCA was effective in reducing serum levels of alkaline phosphatase and transaminases in patients with PSC. 31 Furthermore, in a recent placebo-controlled clinical trial, 32 treatment of patients with non-alcoholic fatty liver disease (NAFLD) with norUDCA resulted in a dose-dependent reduction of serum alanine aminotransferase (ALT) within 12 weeks, suggesting efficacy in human NAFLD.
Here, we explored the therapeutic efficacy of norUDCA in experimental ALD.NorUDCA treatment ameliorated experimental liver disease, as demonstrated by reduced liver injury, reduced hepatocyte death, and reduced expression of pro-inflammatory cytokines.NorUDCA enhanced PPARg activity independent from ethanol exposure and increased abundance of potentially beneficial bacteria.

Mouse experiments
Two different models of experimental ALD were used to study the role of norUDCA in ALD.All experiments were aligned to ethical principles according to Austrian laws (2020-0.152.544) and were carried out in the animal facility of the Medical University of Innsbruck.(1) To study the effect of norUDCA in a preventive setting, 7-to 8-week-old female wild-type (wt/ wt; C57BL/6) mice were fed a Lieber-DeCarli diet containing 1-5 vol% (ethanol-fed) ad libitum for 15 days (Safe, Rosenberg, Germany).Control groups were fed a pair diet containing isocaloric maltose.Half of the ethanol-fed and pair-fed groups were treated with norUDCA (1 mg/ml diet, 5% wt/wt 30 ) by supplementing norUDCA into the diet.(2) To study possible therapeutic effects of norUDCA in experimental ALD, 7-to 8week-old female wild-type mice were fed a Lieber-DeCarli diet containing 1-5 vol% (ethanol-fed) ad libitum for 15 days.NorUDCA treatment was conducted from Day 10 to Day 15.

Histology
Liver tissue samples were fixed in formaldehyde (Sigma, St. Louis, MO, USA) immediately after sacrifice.Samples were embedded in paraffin and further prepared at the Department of Pathology at the Medical University of Innsbruck.Liver sections were deparaffinised in xylene and dehydrated in an ethanol gradient.Dehydration, embedding, and H&E staining 33 were carried out at the Department of Pathology at the Medical University of Innsbruck, before stained slides were evaluated by an experienced pathologist (GO, INNPATH).Up to 20 high-power fields (1 mm 2 ) per slide were analysed.

Microbiome studies
The microbial community composition in collected caecal stool samples was analysed by ribosomal small subunit (SSU rRNA/16S rRNA) gene amplicon sequencing 34 at the Joint Microbiome Facility (Vienna) under the Project ID at the Joint Microbiome Facility (Vienna).DNA extraction from stool samples using the QIAamp DNA Fast stool Kit was automated on the QiaCube Connect.For microbial community profiling, the 16S rRNA genes were amplified by PCR applying primers that cover most bacterial and archaeal clades (515F, 806R). 35After PCR amplification of the marker gene region, the amplicons were barcoded, multiplexed, and sequenced on the Illumina MiSeq platform at the Joint Microbiome Facility. 34Negative controls were performed during sampling, DNA extraction, and barcoding.Further details on amplicon sequence data processing are provided in the Supplementary methods.

Data availability
SSU rRNA gene amplicon datasets are deposited in Sequence Read Archive (SRA) under the BioProject accession number PRJNA907184.

Statistical analysis
For analysing the present data, we used GraphPad Prism 5 (La Jolla, CA, USA).Unpaired two-tailed Student's t test, the Kruskal-Wallis test followed by Dunn's multiple comparison test, and one-way ANOVA followed by the post hoc Newman-Keuls test were used where appropriate.Two or more independent experiments were performed for each modality.Results are shown as mean ± SEM.Statistical significance was considered at p <0.05.
Further information on materials and methods are provided in the Supplementary materials.

NorUDCA affects bile acid metabolism
As expected, norUDCA treatment altered bile acid metabolism.An increased gallbladder weight could be observed in both pairfed (p <0.001; Fig. 2F) and ethanol-fed mice (p <0.001; Fig. 2F) treated with norUDCA, in line with its potent choleretic effects.Similarly, plasma bile acid concentration was significantly higher in norUDCA-treated mice in both the pair-fed (p <0.001; Fig. 2G) and ethanol-fed (p <0.001; Fig. 2G) groups, whereas no difference in gallbladder weight or bile acid concentration could be observed between control groups (Fig. 2F and G).Furthermore, norUDCA significantly increased the hepatic mRNA expression of NorUDCA impacts on hepatic lipid metabolism Hepatic steatosis was quantified on H&E-stained liver slides by an experienced board-certified liver pathologist based on a previously established scoring system. 37Ethanol feeding led to enhanced hepatic lipid accumulation (p <0.001; Fig. 3A and B), whereas norUDCA treatment significantly decreased the hepatic steatosis score in ethanol-fed mice (p <0.001; Fig. 3A and B).NorUDCA mainly tended to reduce microvesicular and mediovesicular hepatic steatosis in ethanol-fed mice (Fig. S3A and B), whereas macrovesicular steatosis was unchanged by norUDCA treatment compared with that in ethanol-fed controls (Fig. S3C).This could explain the tendency towards increased accumulation of triglycerides in the liver of ethanol-fed, norUDCA-treated mice compared with controls (Fig. 3C).In a next step, we performed quantitative PCR analysis of several genes involved in hepatic fatty acid metabolism.Interestingly, in ethanol-fed mice, nor-UDCA significantly increased the hepatic expression of Pparg (p <0.001; Fig. 3D), carnitine palmitoyltransferase 1 (Cpt-1) (p <0.01; Fig. 3D), and sterol regulatory element-binding protein 1 (Srebp1c) (p <0.001; Fig. 3D).

NorUDCA shows potent anti-inflammatory properties via PPARg activation
PPARg (mainly isoform PPARg1) levels were increased upon norUDCA administration in ethanol-fed mice (Fig. 3E and F).As PPARg was shown to exhibit anti-inflammatory properties and to induce an anti-inflammatory (M2) macrophage phenotype, we assessed the activation of PPARg upon norUDCA stimulation.Using human immortalised hepatocytes and a luciferase assay, we could demonstrate an activation of PPARg by norUDCA (Fig. S3D).In a second step, immortalised human hepatocytes were stimulated with norUDCA (500 lM), rosiglitazone (PPARg agonist, 10 lM), and control for 48 h.Notably, PPARg was enhanced after norUDCA stimulation comparable with rosiglitazone in nuclear extracts (Fig. 3G).To further study the antiinflammatory potential of norUDCA as indicated by a decreased expression of pro-inflammatory cytokines and an increased Pparg expression, we performed an in vitro experiment, stimulating peripheral blood mononuclear cells (PBMCs) of healthy donors with LPS and norUDCA (50 and 500 lM) for 24 h.IL-6 levels were significantly decreased in PBMCs stimulated with 500 lM norUDCA (p <0.05; Fig. 3H).Furthermore, we observed a trend towards increased Pparg expression in HepG2 cells upon norUDCA and LPS treatment (Fig. S3E).

Therapeutic norUDCA treatment ameliorates experimental ALD
To further assess the potential therapeutic effect of norUDCA in already established experimental ALD, mice were fed a Lieber-DeCarli diet for 15 days, and norUDCA supplementation was started at Day 10 (Fig. 5A).Ethanol feeding resulted in significantly increased ALT levels compared with that in pair-fed control mice, whereas norUDCA treatment decreased the ALT by 45% in ethanol-fed mice (p = 0.06; Fig. 5B).Serum ethanol concentration was not significantly different between the groups (Fig. S4A).Gallbladder weight, as a surrogate marker for increased bile flow, was significantly higher in norUDCA-treated mice in both the ethanol-and pair-fed groups (Fig. S4B).Eventually, the hepatic expression of Mrp4 was increased in ethanolfed mice, but not pair-fed mice, upon norUDCA administration (Fig. S4C).NorUDCA increased the hepatic expression of Pparg (p <0.001; Fig. 5C) and Cpt-1 (p <0.001; Fig. 5C) in both pair-fed and ethanol-fed mice, whereas norUDCA decreased fatty acid synthase (Fasn) expression only in ethanol-fed mice (p <0.01; Fig. 5C).On the protein level, CPT-1 was augmented in ethanolfed, norUDCA-treated mice (Fig. S4D and E).
NorUDCA induced a reduction of hepatic Tnf (p <0.05) and Il-6 (p <0.05) expression in ethanol-fed mice (Fig. 5D).Similar to that in the preventive setting, norUDCA treatment increased the number of F4/80 + cells in ethanol-and pair-fed mice (Fig. 5E and  F).Myeloperoxidase-positive neutrophils were increased by ethanol feeding compared with those in controls but were lowered upon norUDCA treatment in ethanol-fed mice (p <0.001; Fig. 5G and H).

Discussion
We investigated the efficacy of norUDCA in experimental ALD.Although ALD is one of the most frequent liver diseases, with alcohol-related liver cirrhosis being associated with 22.2 million disability adjusted life years in 2016, therapeutic options are still limited. 38Different mechanisms contribute to the development of ALD, including ethanol toxicity, gut barrier dysfunction, and endotoxaemia. 5,6,8,39Notably, bile acids may also play an important role in the pathogenesis of ALD. 9 In this study, we demonstrated that the administration of norUDCA protected wild-type mice from ethanol-induced liver injury in a preventive and therapeutic setting.Furthermore, we unravelled several potential mechanisms how norUDCA, a side chain shortened homologue of UDCA, ameliorates experimental ALD.Specifically, we demonstrated that norUDCA treatment ameliorated serum concentrations of ALT, reduced hepatic cell death, and decreased hepatic expression of pro-inflammatory cytokines, such as Tnf, Il-6, and Il-1b.The amelioration of pro-  inflammatory pathways could potentially be explained by an increased expression of anti-inflammatory Pparg in norUDCAtreated mice.In addition, norUDCA could alter the intestinal microbiota in both ethanol-and pair-fed mice.NorUDCA, was primarily tested in the treatment of cholestatic liver diseases such as PSC.In an experimental PSC model, Mdr2 -/- mice treated with norUDCA showed reduced hepatic inflammation. 25Furthermore, norUDCA was proven to affect cells of the innate and adaptive immune system such as macrophages and CD8 + T cells in models of experimental cholestatic liver diseases. 30In hepatocyte-specific NF-jB essential modulator (NEMO)-deficient mice, a genetic model for NAFLD, norUDCA attenuated liver damage, depicted by decreased transaminases, histological improvement, and reduced hepatic fibrosis. 27The anti-inflammatory properties of norUDCA were further observed in a model of Schistosoma mansoni-induced liver injury, whereas norUDCA treatment attenuated liver inflammation by reducing the expression of MHC class II molecules on antigen-presenting cells including macrophages. 28ikewise, in ALD, macrophages represent a cornerstone in the pathogenesis of the disease. 40Under numerous stimulating factors in their microenvironment, macrophages may polarise into either pro-inflammatory M1 or anti-inflammatory M2 phenotype.Whereas the macrophage phenotype M1 is induced by LPS and interferon gamma, IL-13 and IL-4 activate M2 macrophages. 41Ethanol administration likewise induces the polarisation of M1 macrophages through NF-jB signalling. 20,21,42n our study, we found increased numbers of F4/80 + macrophages after norUDCA administration in ethanol-fed mice as well as in pair-fed mice.Interestingly, macrophages showed a pronounced M2 phenotype depicted by an increased Arg/iNos expression only in norUDCA-treated, ethanol-fed mice.We were further able to demonstrate the anti-inflammatory properties of norUDCA on macrophages in vitro.NorUDCA treatment attenuated pro-inflammatory cytokine production upon LPS stimulation of PBMCs.In conclusion, we could show an amelioration of the ethanol-induced liver injury by norUDCA, possibly mediated by enhanced macrophage M2 polarisation.
PPARg is a nuclear receptor with potent effects in metabolic and inflammatory pathways that may be present in different isoforms, 43 namely, PPARg1 and PPARg2.Whereas PPARg1 shows anti-inflammatory properties and is expressed in macrophages, 44,45 PPARg2 is mainly involved in lipid storage and is found in adipocytes as well as in hepatocytes. 46In the course of exploring new therapeutic agents for fatty liver disease, PPARg agonists (such as rosiglitazone) were tested.8][49][50] Although chronic ethanol consumption was associated with an activation of PPARg2, 51 PPARg2 -/-mice had decreased ethanol-induced liver injury. 52Nevertheless, the role of PPARg1 in ALD is currently unclear.
Notably, in our study, we found a significantly enhanced expression of Pparg upon norUDCA administration, both in the preventive as well as therapeutic setting and in pair-fed as well as ethanol-fed mice.Moreover, we could demonstrate a strong induction of anti-inflammatory PPARg1 in livers of norUDCAtreated, ethanol-fed mice.The induction of PPARg by norUDCA was further confirmed by two different in vitro assays.Interestingly, glitazones were tested in a clinical trial to treat alcohol addiction, 53 but the trial was stopped early because of increased craving in pioglitazone-treated patients as a result of augmented neuroendocrine stress response to LPS.In line with these data, PPARg agonists were not influencing IL-6 and TNF levels in macrophages in vitro or in vivo. 54Therefore, an alternative strategy to target PPARg without facing the adverse effects of its synthetic ligands could be norUDCA.
Similarly, Beraza et al. 27 observed tendency towards increased Pparg expression after UDCA administration compared with that in controls, whereas norUDCA decreased the expression in NEMO -/-mice.Interestingly, PPARg is an important regulator of both cell differentiation and polarisation of macrophages induced by NF-jB and IL-4/IL-13, 20,22,55 which also might explain the increase of anti-inflammatory M2 macrophage polarisation in our study.Wagner et al. 56 found improved endothelial barrier and reduced inflammatory parameters in mice treated with ethanol, LPS and rosiglitazone compared to ethanol-and LPStreated controls.In conclusion, norUDCA induces PPARg activation, and this effect might contribute to its anti-inflammatory mode of action in the liver, mainly by ameliorating the hepatic cytokine response and induction of an M2 macrophage phenotype.
Changes within the intestinal microbiota composition and alcohol-induced liver disease are closely associated.Alcohol overconsumption results in microbial changes, but different bacterial strains may influence disease progression.In weighted UniFrac principal coordinates analysis plots, all four observed groups were significantly different, suggesting that not only ethanol feeding but also norUDCA treatment changed the intestinal microbiota composition in our experiments.Comparing norUDCA treatment groups with controls, we could observe a decreased abundance of Muribaculaceae-and Fecalibactulumrelated ASVs.Muribaculaceae, a not well-described strain, was increased by UDCA treatment in high-fat-diet mice. 57Faecalibacteria are known as beneficial butyrate producers in the human gut.A recent study found increased Faecalibacterium prausnitzii in patients treated with UDCA. 58Llopis et al. 59 found decreased Faecalibacterium abundance in patients with ALD.In our study, we observed an increased abundance of Roseburiarelated ASVs.The administration of Roseburia in a model of ALD resulted in the improvement of hepatic steatosis and inflammation, 60 suggesting a beneficial role of this strain in ALD.
In summary, we could demonstrate efficacy of norUDCA in experimental ALD, which might be caused by an increase in hepatic PPARg1 and enhanced macrophage polarisation towards an anti-inflammatory M2 phenotype.Future clinical trials for norUDCA in ALD are now warranted to prove its efficacy in ALDs in humans.

Financial support
HT is supported by the excellence initiative VASCage (Centre for Promoting Vascular Health in the Ageing Community), an R&D K-Centre (COMET program -Competence Centers for Excellent Technologies) funded by the Austrian Ministry for Transport, Innovation and Technology, the Austrian Ministry for Digital and Economic Affairs, and the federal states Tyrol, Salzburg, and Vienna.CG is supported by the Austrian Society of Gastroenterology and Hepatology (ÖGGH) and received speaker fees from Abbvie.TEA is supported by the Austrian Science Fund (FWF P33070) and by the European Union (ERC-STG Grant agreement No. 101039320).JS is supported by the Austrian Society of Gastroenterology and Hepatology (ÖGGH) and the German Society of Inflammatory Bowel Disease (DACED).MT is supported by the Austrian Science Fund (FWF) F7310.He received speaker fees from Falk Foundation, Gilead, Intercept, and MSD; he advised for Abbvie, Albireo, BiomX, Boehringer Ingelheim, Falk Pharma GmbH, Genfit, Gilead, Hightide, Intercept, Janssen, MSD, Novartis, Phenex, Pliant, Regulus, Siemens, and Shire.He further received travel grants from Abbvie, Falk, Gilead, and Intercept and research grants from Albireo, Alnylam, Cymabay, Falk, Gilead, Intercept, MSD, Takeda, and UltraGenyx.He is also the co-inventor of patents on the medical use of norUDCA filed by the Medical Universities of Graz and Vienna.

Ethanol measurement
Ethanol concentration in serum samples was measured using the EnzyChrom assay

ELISA:
IL-6 concentration was measured using commercially available ELISA kits from R&D systems (Minneapolis, MN, USA) according to the manufacturer's Instructions.

Microbiome-Studies:
The obtained sequence data was quality-filtered and demultiplexed, followed by amplicon sequencing variant (ASV) inference with DADA2 [2](4), enabling analysis at the highest possible taxonomic resolution.Resulting ASV sequences were taxonomically classified using SINA [3] with the newest release of the the SILVA SSU rRNA database [4].If necessary, contaminants were removed in silico using the decontam software package [5].
Abundance measurements (counts) of ASVs, as well as ASV sum counts at higher taxonomic levels were statistically evaluated, to test for significant differences in microbial community composition between the subject groups.Detection of significantly more abundant amplicon sequence variants in the case over the controls weas performed, and adjusted P-values were calculated using the Benjamini-Hochberg method and differences supported with P-values < 0.05 were considered significant.
Statistical analysis was performed with the metagenomeSeq software, which has been proven optimal for amplicon datasets [6].MetagenomeSeq normalized the abundance data to address varying depths of sequencing coverage across samples, and then a zero-inflated log-normal mixture model was applied to calculated the fold changes between the case and control group for each taxonomic level [7].

In-vitro studies:
Inflammation analysis: Peripheral Blood Mononuclear Cells (PBMCs) were collected from five healthy volunteers.Blood samples were collected in heparinized tubes, followed by density gradient centrifugation of whole blood samples on Lymphoprep solution according to the manufacturer's instructions (Axis Shields, Oslo, Norway) [8].
After isolation, PBMCs were stimulated with lipopolysaccharide (LPS) (1 pg/mL; Invivogen, San Diego, CA, USA) for 24 hours and 50µM as well as 500µM norUDCA.DMSO served as negative control.24 hours after stimulation, cells supernatant was harvested and IL-6 was measured by ELISA.
washed with 5 ml of HB buffer with Triton and centrifuged for 10 min at 1,200 rpm, the pellet was washed with 5 ml of HB buffer without Triton and centrifuged at 1,200 rpm for 10 min.Finally, pellets were suspended in 50 µl of HB buffer modified with 360mM KCl and incubated at 4°C for 30 min, before a final centrifugation for 5 min at 13,000 rpm.The supernatants (nuclear fractions) were kept at -80°C before use in gel shift.

Fig. 1 .
Fig. 1.NorUDCA protects from experimental alcoholic liver disease.(A) Graphical illustration of experimental design.(B) Serum concentrations of ALT and (C) number of TUNEL + cells were significantly decreased in norUDCA-treated, EtOH-fed mice compared with Ctrl.(D) Representative images and quantification of TUNEL + liver cells per HPF based on immunoreactivity (brown indicates TUNEL + cells, black arrows indicate positivity of TUNEL+ cells).(E) NorUDCA treatment significantly decreased the mRNA expression of Tnf, Il-1b, Il-6, and Il-10 in EtOH-fed mice compared with house-keeping gene b-actin.Data are shown as mean ± SEM. *p <0.05, **p <0.01, and ***p <0.001 according to one-way ANOVA with Bonferroni post hoc analysis or the Kruskal-Wallis test with Dunn's post hoc analysis.b-actinwas used as a housekeeping gene (E).ALT, alanine aminotransferase; Ctrl, control; EtOH, ethanol; HPF, high-power field; norUDCA, 24-norursodeoxycholic acid; Tnf, tumour necrosis factor; TUNEL + , terminal deoxynucleotidyl transferase dUTP nick end labelling-positive.

Fig. 3 .
Fig. 3. NorUDCA alters hepatic lipid metabolism by induction of PPARg.(A) NorUDCA treatment was associated with decreased hepatic steatosis scoring in EtOH-fed mice.(B) Representative H&E-stained liver sections.(C) EtOH feeding resulted in increased triglyceride accumulation, although no significant difference between EtOH-fed, norUDCA-treated mice and Ctrl could be observed.(D) NorUDCA treatment significantly increased the hepatic expression of Pparg in both EtOH-fed and pair-fed mice, and Cpt-1 and Srebp1c in EtOH-fed mice.(E) Western blot analysis of PPARg1/2 and GAPDH and (F) quantification of PPARg1 and PPARg2.(G) Human primary immortalised hepatocytes were stimulated with norUDCA (500 lM), rosiglitazone (PPARg agonist, 10 lM), and Ctrl.PPARg was

(
ECET-100, BioAssay Systems, Hayward, CA, USA) according to the manufacturer instructions.Western blot Hepatic protein was isolated using T-PER tissue protein extraction reagent.The reagent was supplemented with HALT proteinase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA).Protein concentrations were measured by Bradford Protein Assay (BioRad, 5000006), separated by SDS-PAGE (Hercules, Bio Rad, CA, USA) and blotted onto Hybond-P PVDF membranes (GE Healthcare, Chicago, IL, USA).Blocking of membranes was performed with 5% skim milk.Membranes were incubated overnight with the primary antibody.Following antibodies were used for detection: PPARg, Cell Signaling Technology #2443; PPARg A3409A, Invitrogen; CPT1A, Cell Signaling Technology #97361; GAPDH, Cell Signaling Technology #2118.Visualization of immunoreactivity was carried out by using HRP-conjugated secondary antibodies (Cell Signalling Technology, #7074) and ECL Select Western Blotting Detection Reagent (Amersham, RPN2235).GAPDH (GAPDH, glyceraldehyde 3-phosphate dehydrogenase) was used as a reference protein.Quantitation of the western blot signals was performed using the Biorad ChemiDoc MP (Hercules, CA, USA).Densitometry of immunoblots was performed with BioRad Image Lab software.

GATCCTAGAATATAGGTCAGGGAAG- 3 )
Protein concentrations were measured by the bicinchoninic assay (ThermoFisher, were end labeled with γ-32P ATP (Hartmann Analytic, Braunschweig, Germany) using T4-polynucleotide kinase (New England Biolabs, Frankfurt am Main, Germany) and purified by column elution using the QIAquick nucleotide removal kit (Qiagen, Hilden, Germany).Oligonucleotides and cytosolic and nuclear extracts (1 μg) were mixed in a DNA binding buffer containing in a 20µl final volume (10mM Tris pH 7.5, 150mM NaCl, 1 mMDTT, 1mM EDTA, 5% glycerol (Sigma-Aldrich, Vienna, Austria), for 10 minutes at room temperature before the radiolabeled probe (0.5 ng) was added.Binding reactions were further incubated for 10 minutes with PPARg antibody (sc-7273X, Santa-Cruz biotechnology, Heidelberg, Germany) and resolved by 4 % non-denaturing polyacrylamide gel electrophoresis in 0.25X Tris-Borate-EDTA (TBE) buffer at room temperature.After run, gel was dried for 1h at 60°C and transferred into developing cassette (Biomax, Kodak) for overnight film exposure at -80C.Quantification of alanine aminotransferase (ALT) in mouse serumThe quantification of murine ALT (alanine aminotransferase) in serum samples was performed by using an enzymatic assay (BQ-Kit, San Diego, CA, USA) according to the manufacturer instructions.