Longitudinal analysis of a dietary mouse model of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH)

Figure 1.. Characterization of mice fed a control or NASH diet for up to 30 weeks. Schematic of experimental design (A). Effect of feeding male mice with either a control diet (10% kcal from fat) or NASH diet (40% kcal from fat, 20% kcal fructose, and 2% cholesterol) on body weight (B) and body composition (C) for up to 30 weeks. Effect of the diets on serum levels of alanine aminotransferase (ALT) and cholesterol (D). Significant differences in body weight were determined by ANOVA. Significant differences in body composition and serum levels of ALT and cholesterol were determined using Student’s t-tests. Changes were considered significant at p < 0.05. * p < 0.05, ** p < 0.01, **** p<0.0001.

Figure 2.. Increased food consumption in NASH diet-fed mice. Instrumented metabolic caging was used to quantify food consumption (A-C), water consumption (D-F), energy expenditure (G-I), net energy balance (J-L), locomotor activity (M-P), and respiratory exchange ratio (Q-R) over a three-day period in mice fed a control or NASH diet for 10 weeks. n = 8; * p < 0.05.

Figure 3.. Biochemical and histological analysis of livers from mice fed a NASH diet for up to 30 weeks. Effect of feeding male mice with either a control diet (10% kcal from fat) or NASH diet (40% kcal from fat, 20% kcal fructose, and 2% cholesterol) on liver weight (as a percent of body weight) (A), as well as on levels of glycogen and triglycerides in the liver (B) at 10, 20, and 30 weeks on the respective diets. Representative hematoxylin and eosin (H&E) and Masson’s Trichrome stained sections of liver harvested after 10, 20, or 30 weeks on a control or NASH diet (C). Scale bar = 100 μM. Bar graphs summarizing steatosis and fibrosis scores after 10, 20, or 30 weeks on a control or NASH diet. Scores were determined by an independent, blinded pathologist contracted by HistoWiz. Significant differences in liver weight, as well as liver glycogen and triglycerides were determined by Student’s t-tests at individual time points. Changes were considered significant at p < 0.05. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4.. Hepatic transcriptome from mice fed a NASH diet for 10 weeks. Effect of feeding male mice either a control diet (10% kcal from fat) or NASH diet (40% kcal from fat, 20% kcal fructose, and 2% cholesterol) for 10 weeks on hepatic gene expression. Heat map of the top 40 differentially expressed genes (DEGs; A). Pathways related to immune response, including TREM1 Signaling, Th1 Pathway, and Neuroinflammation Signaling Pathway, are activated in the liver of animals fed a Western-style diet for 10 weeks compared to a control diet (B). Orange bars denote activated pathways (positive z-score > 2), whereas blue bars denote inhibited pathways (negative z-score <− 2.0). Putative upstream regulators of the DEGs at this time point include immune-related molecules such as interferon gamma, interleukin 2, interleukin 4, and tumor necrosis factor (C). In the liver of mice fed a NASH diet, the gene set for adaptive immune response is significantly enriched (D). Genes related to the adaptive immune response, including Ccl2, Ccr2, Icam1, Tlr4, Tnf, and Vcam1 are significantly upregulated in the liver of mice fed the NASH diet for 10 weeks, compared to the control diet (E). Statistical significance…

Figure 5.. Hepatic transcriptome from mice fed a NASH diet for 20 weeks. Effect of feeding male mice either a control diet (10% kcal from fat) or NASH diet (40% kcal from fat, 20% kcal fructose, and 2% cholesterol) for 20 weeks on hepatic gene expression. Heat map of the top 40 differentially expressed genes (DEGs; A). Pathways related to fibrosis, including Hepatic Fibrosis Signaling Pathway and Pulmonary Fibrosis Idiopathic Signaling Pathway, are activated in the liver of animals fed a NASH diet for 20 weeks compared to a control diet (B). Orange bars denote activated pathways (positive z-score > 2), whereas blue bars denote inhibited pathways (negative z-score <− 2.0). Putative upstream regulators of the DEGs at this time point include fibrosis-related molecules such as TGFB1 and AGT (C). In the liver of mice fed a NASH diet, the gene set for collagen fibril organization is significantly enriched (D). Genes related to fibrosis, including Aebp1, Col1a1, Col1a2, Mmp2, Mmp13, and Timp1, are significantly upregulated in the liver of mice fed the NASH diet for 20 weeks, compared to the control diet (E). Statistical significance was determined by t-test, with p < 0.05 considered significant. *…

Figure 6.. Hepatic transcriptome from mice fed a NASH diet for 30 weeks. Effect of feeding male mice either a control diet (10% kcal from fat) or NASH diet (40% kcal from fat, 20% kcal fructose, and 2% cholesterol) for 30 weeks on hepatic gene expression. Heat map of the top 40 differentially expressed genes (DEGs; A). Pathways related to fibrosis and immune response, including Hepatic Fibrosis Signaling Pathway, Pulmonary Fibrosis Idiopathic Signaling Pathway, and IL-8 signaling, are activated in the liver of animals fed a Western-style diet for 30 weeks compared to a control diet (B). Orange bars denote activated pathways (positive z-score > 2), whereas blue bars denote inhibited pathways (negative z-score <− 2.0). Putative upstream regulators of the DEGs at this time point include fibrosis- and immune-related molecules such as TGFB1 and TNF (C). In the liver of mice fed a NASH diet, the gene set for cytokine secretion is significantly enriched (D), including genes in the inflammasome pathway. Genes related to the inflammasome, including Aim2, Casp1, and Nlrp3, are upregulated in an animal-dependent manner. Other inflammasome-related genes, including Pycard, Tlr4, and Tlr9, are…

Figure 7.. Longitudinal transcriptomic analysis from mice fed a NASH diet for up to 30 weeks. Venn diagrams report the numbers of both up- and down-regulated genes (A and B, respectively) in mice fed a NASH diet or a control diet for up to 30 weeks. Pathway analysis of differentially expressed genes that are common to all time points or specific to the 30-week time point alone are also reported (C and D, respectively).

Figure 8.. Microbiome analysis from mice fed a NASH diet for up to 20 weeks. Effect of feeding mice a NASH diet or a control diet for up to 20 weeks on beta diversity of the microbiome (A). PC1 accounts for 43.12% of the variance in beta diversity and PC2 accounts for 15.48% of the variance in beta diversity due to diet (B). Plot of relative abundance of bacterial species in the 20-week cohort after feeding a NASH or control diet for 20 weeks (C); selected species are exhibited in D. Functional impact of microbiome cellular processes (E). Statistical significance was determined by t-test, with p < 0.05 considered significant. * p < 0.05, ** p <0.01, **** p< 0.0001.

Figure 9.. Microbiome analysis from mice fed a NASH diet for up to 30 weeks. Effect of feeding mice a NASH diet or a control diet for up to 30 weeks on beta diversity of the microbiome (A). PC1 accounts for 31.68% of the variance in beta diversity, and PC2 accounts for 20.87% of the variance in beta diversity due to diet (B). Plot of relative abundance of bacterial species in the 20-week cohort after feeding a NASH or control diet for 20 weeks (C); selected species are exhibited in D. Functional impact of microbiome cellular processes (E). Statistical significance was determined by t-test, with p < 0.05 considered significant. * p < 0.05, ** p <0.01, *** p < 0.001, **** p< 0.0001.