Characterization of the variability in the extent of nonalcoholic fatty liver induced by a high-fat diet in the genetically diverse Collaborative Cross mouse model

Abstract

Interindividual variability and sexual dimorphisms in the development of nonalcoholic fatty liver disease (NAFLD) are still poorly understood. In the present study, male and female strains of Collaborative Cross (CC) mice were fed a high-fat and high-sucrose (HF/HS) diet or a control diet for 12 weeks to investigate interindividual- and sex-specific variations in the development of NAFLD. The severity of liver steatosis varied between sexes and individual strains and was accompanied by an elevation of serum markers of insulin resistance, including increases in total cholesterol, low-density lipoproteins, high-density lipoproteins, phospholipids, and glucose. The development of NAFLD was associated with overexpression of the critical fatty acid uptake and de novo lipogenesis genes Pparg, Mogat1, Cd36, Acaab1, Fabp2, and Gdf15 in male and female mice. The expression of Pparg, Mogat1, and Cd36 was positively correlated with liver triglycerides in male mice, and Mogat1 and Cd36 expression were positively correlated with liver triglycerides in female mice. Our results indicate the value of CC mice in combination with HF/HS diet-induced alterations as an approach to study the susceptibility and interindividual variabilities in the pathogenesis of nonalcoholic fatty liver and early nonalcoholic steatohepatitis at the population level, uncovering of susceptible and resistant cohorts, and identifying sex-specific molecular determinants of disease susceptibility.

Keywords: Cd36; collaborative cross mice; gene expression; nonalcoholic fatty liver disease.

Figure 1.. Body weight and liver-to-body weight ratio in Collaborative Cross (CC) mice fed a high-fat and high-sucrose diet or a control diet.

(A) Population averages for body weight and (B) liver-to-body weight ratios in male and female mice fed a control diet or HF/HS diet. (C) Average change in body weight and (D) liver-to-body weight ratios in HF/HS diet-fed mice (black dot) as compared to control diet-fed mice (white dot) in the individual mouse strains. Asterisks * denote significant difference (p < 0.05).

Figure 2.. Effect of a high-fat and high-sucrose diet on metabolic alterations and extent of steatosis across the Collaborative Cross mouse strains.

(A, B) Population averages for the percentage of osmium tetroxide staining in liver sections, liver triglycerides, liver cholesterol, and plasma ALT activity in male (A) and female (B) mice fed a control diet or HF/HS diet. (C) Strain-specific changes in osmium staining in HF/HS diet-fed mice (black dot) as compared to control diet-fed mice (white dot). Asterisks * denote significant difference (p < 0.05). (D) Representative hematoxylin and eosin and osmium staining of liver sections from CC011 and CC042 male mice and CC013 and CC060 female mice fed control or HF/HS diet.

Figure 3.. Population averages and correlation analysis of serum biochemical indicators of metabolic alterations and extent of steatosis across the Collaborative Cross mouse strains.

(A, C) Population averages for the serum triglycerides, non-esterified fatty acids (NEFA), total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), phospholipids, glucose, and insulin in male (A) and female (C) mice fed control diet or HF/HS diet. Asterisks * denote significant difference (p < 0.05). Spearman’s rank-order correlation matrix of the phenotypes in male (B) and female (D) mice. Each circle represents a significant correlation (p < 0.05) that was positive (red) or negative (blue). Color bar shows Spearman correlation.

Figure 4.. Expression of lipid metabolism genes in the livers of Collaborative Cross mice fed high-fat and high-sucrose or control diet.

(A) Schematic of hepatic lipid metabolism pathways and genes selected for qRT-PCR gene expression analysis. Heat map fold change in the expression of lipid accumulation genes in individual mouse strains of male (B) and female (C) mice. Gene expression was measured by qRT-PCR and the relative amount of each mRNA transcript was determined using the 2^−ΔΔCt method. Significant differentially expressed genes in the livers of mice fed a HF/HS diet compared to a control diet were identified by Student’s t-test (p < 0.05 considered significant). Red color denotes > 1.5-fold increase in gene expression in the livers of HF/HS diet-fed mice; blue color denotes > 0.67 decrease in gene expression in the livers of HF/HS diet-fed mice. Grey boxes denote non-significant change in gene expression. (D) Averages of genes significantly altered (p < 0.05) at the population level in the livers of male and female mice fed a HF/HS diet compared to mice fed control diet. (E) Heat map of Pearson correlation coefficients (r) of genes with population wide significant changes in gene expression of male and female mice fed a HF/HS diet in relation to osmium staining and liver triglyceride levels. p<0.05 was considered a significant correlation. Red color denotes positive correlation, blue color denotes negative correlation, grey boxes statistically not significant. Pearson r values are indicated.

Figure 5.. Expression of fibrosis marker genes in the livers of Collaborative Cross mice fed a high-fat and high-sucrose or control diet.

(A, C) Population averages of fibrosis marker-gene expression with significant changes (p < 0.05) in the livers of HF/HS diet-fed male (A) and female (C) compared to control diet-fed mice. (B, D) Strain-specific changes in expression of fibrosis marker-genes in the livers of HF/HS diet-fed mice (black dot) as compared to control diet-fed mice (white dot). Asterisks * denote significant difference (p < 0.05)

Figure 5.. Expression of fibrosis marker genes in the livers of Collaborative Cross mice fed a high-fat and high-sucrose or control diet.

(A, C) Population averages of fibrosis marker-gene expression with significant changes (p < 0.05) in the livers of HF/HS diet-fed male (A) and female (C) compared to control diet-fed mice. (B, D) Strain-specific changes in expression of fibrosis marker-genes in the livers of HF/HS diet-fed mice (black dot) as compared to control diet-fed mice (white dot). Asterisks * denote significant difference (p < 0.05)