A preclinical model of severe NASH-like liver injury by chronic administration of a high-fat and high-sucrose diet in mice

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a progressive liver disease, affecting 38% of adults globally. If left untreated, NAFLD may progress to more advanced forms of the disease, including non-alcoholic steatohepatitis (NASH), liver cirrhosis, and fibrosis. Early NAFLD detection is critical to prevent disease progression. Using an obesogenic high-fat and high-sucrose (HF/HS) diet, we characterized the progression of NAFLD in male and female Collaborative Cross CC042 mice after 20-, 40-, and 60-week intervals of chronic HF/HS diet feeding. The incidence and severity of liver steatosis, inflammation, and fibrosis increased in both sexes over time, with male mice progressing to a NASH-like disease state faster than female mice, as indicated by earlier and more pronounced changes in liver steatosis. Histopathological indication of macrovesicular steatosis and gene expression changes of key lipid metabolism genes were found to be elevated in both sexes after 20 weeks of HF/HS diet. Measurement of circulating markers of inflammation (CXCL10 and TNF-α), histopathological analysis of immune cell infiltrates, and gene expression changes in inflammation-related genes indicated significant liver inflammation after 40 and 60 weeks of HF/HS diet exposure in both sexes. Liver fibrosis, as assessed by Picosirius red and Masson's trichrome staining and changes in expression of key fibrosis related genes indicated significant changes after 40 and 60 weeks of HF/HS diet exposure. In conclusion, we present a preclinical animal model of dietary NAFLD progression, which recapitulates human pathophysiological and pathomorphological changes, that could be used to better understand the progression of NAFLD and support development of new therapeutics.

Keywords: Collaborative cross; Non-alcoholic fatty liver disease (NAFLD); Non-alcoholic steatohepatitis (NASH); Obesogenic diet; Preclinical mouse model.

Figure 1.. Body and liver weight in CC042 mice fed a chronic HF/HS diet for 20, 40, or 60 weeks.

Average body weight of CC042 male (a) mice and female (b) mice fed a control diet or HF/HS diet for up to 60 weeks. Error bars denote standard error of the mean. * Denotes significant difference from control diet-fed mice determined by Holm-Sidak multiple comparisons test p<0.05. Average percent weight gain in CC042 male (c) and female (d) mice fed a control diet or HF/HS diet for 20, 40, and 60 weeks. Percent weight gain was calculated by subtracting body weight of individual animal at the beginning of the experiment (0 weeks) from the body weight of that animal at the specified time point. Average liver weight in CC042 male (e) and female (f) mice fed a control or HF/HS diet for 20, 40, or 60 weeks. Average ratio of body weight/liver weight in CC042 male (g) and female (h) mice fed a control of HF/HS diet for 20, 40, or 60 weeks. N=6/sex/diet/time point. Error bars denote standard deviation. * Denotes significant difference (p < 0.05) from control diet-fed mice at the same time point.

Figure 2.. Progression of serum biochemical indicators of a NASH-like phenotype in CC042 mice after feeding a HF/HS diet.

Averages for the serum alanine transaminase (ALT) (a), aspartate transaminase (AST) (b), triglycerides (c), cholesterol (d), glucose (e), insulin (f), high-density lipoprotein (HDL-C) (g), low-density lipoprotein (LDL-C) (h), and inflammatory cytokines CXCL10 (i) and TNFα (j) in male and female mice fed control diet or HF/HS diet for 20, 40, or 60 weeks. N=6/sex/diet/time point. Error bars denote standard deviation. * Denotes significant difference (p < 0.05) from control diet-fed mice at the same time point.

Figure 3.. Progression of NASH-like histopathological alterations in the livers of CC042 mice fed a HF/HS diet.

Representative images of hematoxylin and eosin staining in liver sections in CC042 male (a), and female (b) mice fed a control diet or HF/HS diet for 20, 40, or 60 weeks. Hepatic macrovesicular steatosis (large, clear-staining vacuoles) indicated by arrows and inflammation indicated by arrowheads.

Figure 4.. Hepatic profile of esterified fatty acid composition in the livers of CC042 mice fed a HF/HS diet.

Principal component analysis illustrating the differences in the composition of esterified fatty acids in the liver of CC042 male (a) and female (b) mice fed a HF/HS diet or a control diet for 20, 40, or 60 weeks. Percent composition of esterified fatty acids in the liver of CC042 male (c) and female (d) mice fed a HF/HS diet or a control diet. Quantified fatty acids include oleic/vaccenic acid (C18:1), palmitic acid (C16:0), linoleic acid (C18:2), palmitoleic/7-hexadecenoic acid (C16:1), stearic acid (C18:0), ω-3/ω-6-arachidonic acid (C20:4), docosahexaenoic acid (C22:6), and other fatty acids (myristic acid (C14:0), heptadecanoic acid (C17:0), α/γ-linolenic acid (C18:3), 11-eicosenoic acid (C20:1), eicosatrienoic acid (C20:3), eicosapentanoic acid (C20:5), and docosapentaenoic acid (C22:5)). The ratio of esterified polyunsaturated fatty acids (PUFA) to monounsaturated fatty acids (MUFA) in CC042 male (e) and female (f) mice fed a control diet or HF/HS diet for 20, 40, or 60 weeks. Error bars denote standard deviation. * Denotes significant difference (p < 0.05) from control diet-fed mice at the same time point.

Figure 5.. Expression of genes involved in lipid metabolism, inflammation, and fibrogenesis in the livers of CC042 mice fed a HF/HS diet.

Analysis of genes involved in (a) hepatic lipid metabolism pathways, (b) NASH-related inflammation, and (c) fibrosis in CC042 male and female mice fed a control diet or HF/HS diet for 20, 40, or 60 weeks. Gene expression was measured by qRT-PCR and the relative amount of each mRNA transcript was determined using the 2^−ΔΔCt method. N=6/sex/diet/time point. Error bars denote standard deviation. * Denotes significant difference (p < 0.05) from control diet-fed mice at the same time point.