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. 2021 Oct;45(10):1980-1993.
doi: 10.1111/acer.14700. Epub 2021 Sep 29.

A Western diet with alcohol in drinking water recapitulates features of alcohol-associated liver disease in mice

Michael Schonfeld  1 , Maura O'Neil  2   3 , Maria T Villar  4 , Antonio Artigues  4 , Janice Averilla  1 , Sumedha Gunewardena  5 , Steven A Weinman  1   3   6 , Irina Tikhanovich  1   3
Affiliations

A Western diet with alcohol in drinking water recapitulates features of alcohol-associated liver disease in mice

Michael Schonfeld et al. Alcohol Clin Exp Res. 2021 Oct.

Abstract

Background: Mouse models of alcohol-associated liver disease vary greatly in their ease of implementation and the pathology they produce. Effects range from steatosis and mild inflammation with the Lieber-DeCarli liquid diet to severe inflammation, fibrosis, and pyroptosis seen with the Tsukamoto-French intragastric feeding model. Implementation of all of these models is limited by the labor-intensive nature of the protocols and the specialized skills necessary for successful intragastric feeding. We thus sought to develop a new model to reproduce features of alcohol-induced inflammation and fibrosis with minimal operational requirements.

Methods: Over a 16-week period, mice were fed ad libitum with a pelleted high-fat Western diet (WD; 40% calories from fat) and alcohol added to the drinking water. We found the optimal alcohol consumption to be that at which the alcohol concentration was 20% for 4 days and 10% for 3 days per week. Control mice received WD pellets with water alone.

Results: Alcohol consumption was 18 to 20 g/kg/day in males and 20 to 22 g/kg/day in females. Mice in the alcohol groups developed elevated serum transaminase levels after 12 weeks in males and 10 weeks in females. At 16 weeks, both males and females developed liver inflammation, steatosis, and pericellular fibrosis. Control mice on WD without alcohol had mild steatosis only. Alcohol-fed mice showed reduced HNF4α mRNA and protein expression. HNF4α is a master regulator of hepatocyte differentiation, down-regulation of which is a known driver of hepatocellular failure in alcoholic hepatitis.

Conclusion: A simple-to-administer, 16-week WD alcohol model recapitulates the inflammatory, fibrotic, and gene expression aspects of human alcohol-associated steatohepatitis.

Keywords: HNF4α; fibrosis; gender differences; liver inflammation; steatohepatitis.

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Conflict of interest statement

Conflict of Interest statement: The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1.
Figure 1.. Alcohol in water in combination with western diet feeding model.
A-F. Male and female C57BL6/J mice were fed ad libitum western diet and either given plain water or alcohol in water at indicated concentrations. A. Liquid intake and average alcohol intake of mice during the first 4 weeks of feeding. B. Liquid intake and average alcohol intake of mice during the last 2 weeks of feeding. C. Feeding scheme. Mice were slowly adapted to alcohol in water during the first 2 weeks of feeding (1%, 3%, 10% v/v at equal intervals). The 20/10% group received 20% v/v alcohol 4 days per week (Thursday-Monday) and 10% v/v alcohol 3 days per week (Monday-Thursday). This group had access only to 20% v/v alcohol during the last 2 weeks of feeding. D. Blood alcohol concentration in mice from 6 groups at 12 weeks after the start of the feeding. E. Average food intake in these mice. F. Weight gain during 16 weeks of feeding in male and female mice. Both male and female groups had N=11 (control), N=5 (10%) and N=11 (20/10%) mice per group.
Figure 2.
Figure 2.. Alcohol in water in combination with western diet feeding produces a liver injury phenotype in male and female mice.
A. Examples of livers from control and alcohol (20/10%) groups and corresponding spleens. B. Liver/body weight ratios of these mice. Both male and female groups had N=11 (control), N=5 (10%) and N=11 (20/10%) mice per group. **, P < 0.01. C. Representative images of H&E staining from 6 groups. D. Images showing features of ALD in the liver. E. Steatosis percentage for male and female mice on the WD and 20/10% WDA diet. F. Serum ALT and AST levels in these mice at indicated time points. **, P < 0.01, *, P < 0.05.
Figure 3.
Figure 3.. Alcohol in water in combination with western diet feeding results in liver fat accumulation.
A-C. Whole liver extracts were analyzed by TMT mass spectrometry. N=3 mice per group. Relative abundance of proteins (A) fold change by alcohol (B) and GO TERM enrichment (C) in WD control and WD alcohol mouse livers. D. Liver triglycerides in livers of male mice in control (water) and alcohol (20/10%) groups. N=5. **, P < 0.01. E-F. Relative liver mRNA in these mice. N=5. *, P < 0.05.
Figure 4.
Figure 4.. Alcohol in water in combination with western diet feeding results in liver fibrosis.
A. Representative images of Sirius Red staining of liver sections from 6 groups. B. Sirius Red positive area. Both male and female groups had N=5 (---, water), N=5 (10%) and N=8 (20/10%). *, P < 0.05; **, P < 0.01. C. Trichrome staining of liver sections in control (water, WD) and alcohol (20/10% alcohol, WDA) groups. D-E. Relative liver mRNA in these mice. N=5 (---, water), N=5 (10%) and N=8 (20/10%). *, P < 0.05; **, P < 0.01.
Figure 5.
Figure 5.. Alcohol in water in combination with western diet feeding results in liver immune cell accumulation and changes in the immune environment.
A. Relative liver mRNA in mice from 6 groups. N=6 (chow), N=5 (---, water), N=5 (10%) and N=8 (20/10%). *, P < 0.05. B. Representative images of F4/80 staining of liver sections in control (water) and alcohol (20/10%) groups. Right. Average number of F4/80 positive cells per high power field. N=5 per group. **, P < 0.01. C-F. Relative liver mRNA in mice from 6 groups. Corresponding mRNA levels in chow fed, age matched controls are indicated. *, P < 0.05; **, P < 0.01.
Figure 6.
Figure 6.. Alcohol in water in combination with western diet feeding results in changes in the immune cell gene expression.
A-C. CD45 positive cells were isolated from control and alcohol (20/10% group) fed mouse livers (n=3 mice per group) and subjected to single cell RNA-seq analysis (3 mice per group). A. Volcano plot of differentially regulated genes in B-cells, T-cells, Granulocytes and Macrophages. B. Ratio of cell numbers for individual cell population. C-D. GO TERM enrichment in top upregulated and downregulated genes.
Figure 7.
Figure 7.. Alcohol in water in combination with western diet feeding results in HNF4α dysregulation.
A. Relative liver mRNA in mice from 6 groups. N=6 (chow), N=5 (---, water), N=5 (10%) and N=8 (20/10%). *, P < 0.05, **, P < 0.01. B. Representative images of HNF4α protein staining in liver sections in control (water) and alcohol (20/10%) groups. C. Western blot analysis of HNF4α protein levels in liver extracts from control (water) and alcohol (20/10%) fed mice. Right. Densitometry analysis of HNF4α protein expression. N=3 mice per group. *, P < 0.05. D. INR and Prothrombin time of mice from chow fed (chow), WD fed (western diet + water) and WDA fed (western diet + 20/10% alcohol) mice. N≥4. *, P < 0.05, **, P < 0.01.E. Relative liver mRNA in mice fed Lieber-DeCarli alcohol or control liquid diet for 4 weeks. F. Relative liver mRNA in mice from 6 groups as in Figure 1. N=5 (---, water), N=5 (10%) and N=8 (20/10%). *, P < 0.05. Right. Correlation between Hnf4a p2 mRNA and Tgfb1 mRNA in mice fed western diet with (○) or without (●) alcohol. r=0.75, N=64 mice, P < 0.00001.
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