Table 1.
Composition of the high fat, fructose and cholesterol “western” alcohol-induced steatohepatitis diet (WASH) compared to the standard Lieber-DeCarli diet.
Fig 1.
Ethanol combined with fat, cholesterol and fructose promotes hepatomegaly and whole-body adiposity.
A. Total body weight of mice placed on the Lieber De-Carli (LD) or Western/Alcohol Steatohepatitis diet (WASH) with (LD-Ethanol, WASH-Ethanol) or without 4.5% ethanol for 5 weeks (left panel) or 7 weeks (right panel) B. Liver to body weight rations of mice fed the LD and WASH ethanol or cognate control diets for 5 or 7 weeks. C. Percent fat mass of mice fed LD and WASH diets for 5 weeks or D. 7 weeks E. Percentage fat-free lean mice in mice fed the LD and WASH diets for 5 or F.7 weeks. Body composition measured using BrükerSpin mini spec rodent NMR. Sample size is n = 8 per group. Means compared using One-Way ANOVA. *p<0.05. +/- standard error of the mean (S.E.M).
Fig 2.
Ethanol induced liver toxicity is exacerbated by the WASH diet.
A. Serum triglyceride (TG) levels in mice placed on the LD and WASH diets +/- ethanol for 5 or B. 7 weeks. C. Total circulating cholesterol in mice fed the WASH or LD diets+/- ethanol for 5 or D. 7 weeks E. Serum alanine amino transferase (ALT) levels in mice fed the WASH and LD diets +/- ethanol for 5 weeks or F. 7 weeks. G. Serum aspartate amino transferase (ALT) levels in mice fed the WASH and LD diets +/- ethanol for 5 weeks or H. 7 weeks. Serum was isolated from cardiac blood. Serum levels of lipids and proteins were determined using a Cobas C11 clinical chemistry bioanalyzer (Roche Diagnostics). (n = 8 mice per group). Mean levels were compared using One-Way ANOVA. * p<0.05, **p<0.01 ***p<0.001 +/- S.E.M.
Fig 3.
Hepatic lipid content is elevated in mice fed the WASH-ethanol diet.
A. Hematoxylin and eosin (H&E) staining of liver sections from fed the LD and WASH diets +/- ethanol for 5 weeks B. 7 weeks C. BODIPY neutral lipid staining of liver samples from mice receiving the LD and WASH ethanol diet +/- ethanol for 5 or D. 7 weeks. Representative images from sections analyzed from 8 mice (n = 8). Left (H&E) or right (BODIPY) lateral lobes were stained as described in the materials and methods section.
Fig 4.
WASH-ethanol diet fed mice show worsened liver pathology and amplified fibrosis.
A. Masson Trichome staining of liver sections isolated from mice exposed to the WASH or LD diet +/- ethanol for 5 or B. 7 weeks. Trichrome staining was performed on left lateral lobe liver sections that were formalin fixed paraffin embedded. Representative image (n = 8). C. Immunoblot showing expression of latent and mature (TGFB-L and TGFB-M respectively), IL6 and HSP90 in mice placed on their respective diets for 7 weeks. D. Fold protein expression of TGFB-L, TGFB-M and IL6 in mice placed on the LD or WASH control or ethanol diets for 7 weeks. Target proteins normalized to HSP90 and represented as fold increase relative to LD- and WASH-Diet Pair-fed controls. OD quantified in ImageJ. E. Immunoblot showing collagen expression (COL1A1) in LD and WASH control versus ethanol-fed mice at 7 weeks. F. Quantified expression of COL1A1 normalized to HSP90 in the samples shown in E. Means compared using student’s t-test *p<0.05 **p<0.01. +/- S.E.M.
Fig 5.
Once-daily high fat, fructose and cholesterol meals (HFFC) significantly altered ethanol induced liver pathology.
A. H&E staining of liver sections of mice fed the LD-diet +/- ethanol with or without a once daily meal of two 400mg high fat fructose and cholesterol (HFFC) or control pellets between 1200 and 1400 h daily. B. BODIPY-neutral lipid staining of liver sections showing the lipid content of the livers from mice on the LD-diet +/- ethanol with HFFC pellets or control chow pellets. Representative slides (n = 10). Mice we allowed to ingest HFFC pellets of chow pellet controls for 2 h before being transferred to a fresh cage.
Fig 6.
The WASH-ethanol diet model can be used to evaluate the effect of novel drugs on liver, toxicity, pathology and fibrogenesis.
A. H&E staining of liver sections of mice (n = 10) placed on the WASH-ethanol diet and dosed with 30mg/kg SR9238 or vehicle control (10:10:80 DMSO:Tween80:PBS) Representative images (n = 10). Mice were placed on the WASH diet +/- ethanol for 2 weeks before dosing began. SR9238 or vehicle was administered via i.p. once daily for 4 weeks. B. Immunohistochemistry (IHC) showing expression IL6 in the livers of vehicle versus SR9238 dosed mice. Representative images (n = 10). C. IHC expression of TGFB in vehicle and SR9238 treated mice. Representative images (n = 10). D. PicoSirius Red staining showing hepatic collagen deposits in livers of mice treated with SR9238 or vehicle. (Lower panel) Percent area of collagen positive staining in WASH-diet + ethanol-fed mice treated with SR9238 or vehicle. PicoSirius Red staining was quantified using image J. Representative image (n = 10) D. Liver-transaminases (ALP, AST and ALT) in mice fed the WASH-ethanol diet and dosed with SR9238. Liver transaminase levels were quantified using the Cobas C11 bioanalyzer and cognate assay kits (n = 8). E. RT-QPCR showing expression of the LXR isotypes Nr1h3, Nr1h2, Fatty-acid synthase (Fasn) and Sterol-regulatory element binding protein-1c (Srebp1c). F. Hepatic mRNA expression of Tnfa and Tgfb in vehicle and SR9238 dosed mice. Total mRNA was isolated from flash frozen liver sections and gene expression was quantified using cognate primers. Mean values were compared using student’s-t test. *p<0.05 +/- S.E.M.