Longitudinal characterization of diet-induced genetic murine models of non-alcoholic steatohepatitis with metabolic, histological, and transcriptomic hallmarks of human patients

Abstract

Non-alcoholic steatohepatitis (NASH) is a fast-growing liver disease in the Western world. Currently, only a few animal models show both the metabolic and histological features of human NASH. We aimed to explore murine NASH models in a time dependent manner that exhibit metabolic, histological and transcriptomic hallmarks of human NASH. For this, the murine strains C57BL/6J, ob/ob, and KK-A^y were used and three types of nutritional regimes were administered: normal chow diet (NCD); high-fat, high-fructose, and high-cholesterol diet (fast food diet; FFD); or choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD), for 2, 4, 8, 12, 18, 24, and 30 weeks. All strains under the FFD and CDAHFD regimes developed steatohepatitis. Among the strains treated with FFD, the non-alcoholic fatty liver disease (NAFLD) activity score, fibrosis progression and metabolic abnormalities such as hyperinsulinemia and obesity were more pronounced in ob/ob mice than in C57BL/6J and KK-A^y mice. In ob/ob mice fed FFD, the development of hepatic crown-like structures was confirmed. Furthermore, molecular pathways involved in steatohepatitis and fibrosis showed significant changes from as early as 2 weeks of starting the FFD regime. Ob/ob mice fed FFD showed metabolic, histological, and transcriptomic dysfunctions similar to human NASH, suggesting their potential as an experimental model to discover novel drugs for NASH.

Keywords: Fast food; Fibrosis; Hyperinsulinemia; Non-alcoholic steatohepatitis; Obesity.

Fig. 1.

Body weight and food intake changes in C57BL/6J, ob/ob and KK-A^y mice under NCD, FFD, or CDAHFD regimes for 30 weeks. Body weight (A–C) and food intake (D–F). Values are mean±s.e.m., n=5. *P<0.05, **P<0.01 versus NCD (Student's t-test, two-tailed).

Fig. 2.

Plasma insulin, plasma ALT levels and hepatic cholesterol levels in C57BL/6J, ob/ob and KK-A^y mice under NCD, FFD, or CDAHFD regimes for 2, 4, 8, 12, 18, 24 and 30 weeks. Plasma insulin levels (A–C), plasma ALT levels (D–F) and hepatic cholesterol levels (G–I). Values are mean±s.e.m., n=3–5 (the exact number of animals are shown in the figure). *P<0.05, **P<0.01 versus NCD (Student's t-test, two-tailed).

Fig. 3.

Histological changes in C57BL/6J, ob/ob and KK-A^y mice under NCD, FFD, or CDAHFD regimes for 2, 4, 8, 12, 18, 24 and 30 weeks. (A) Hematoxylin and Eosin staining of liver sections at 30 weeks. Scale bars: 200 µm. (B) Sirius Red staining of liver sections at 30 weeks. Scale bars: 50 µm. NAFLD activity score (NAS) (C–E) and Sirius Red-positive area (%) (F–H). Values are mean±s.e.m., n=4–5 (the exact number of animals are shown in the figure). *P<0.05, **P<0.01 versus NCD (NAS: Wilcoxon's test, Sirius Red-positive area: Student's t-test, two-tailed).

Fig. 4.

Histological changes of hCLS in ob/ob mice under NCD or FFD regimes. (A,B) F4/80 staining of liver sections at 12 weeks, (C) hCLS numbers (cells/field) at 2, 4, 8, 12, 18, 24 and 30 weeks. Values are mean±s.e.m., n=4–5 (the exact number of animals are shown in the figure). *P<0.05, **P<0.01 versus NCD (Student's t-test, two-tailed). Scale bars: 50 µm.

Fig. 5.

Hepatic transcriptomic pathway analysis in ob/ob mice under NCD or FFD regimes. (A) Heat map visualization of biological categories related to NASH disease progression for 30 weeks. Values are expressed as–log (P-values). (B) Top 50 canonical pathways at 18 weeks. Red stars, green stars, purple stars, and blue stars indicate pathways involved in lipid metabolism, inflammatory processes, fibrosis and cancer and oxidative stress, respectively.

Fig. 6.

Hepatic gene expression analysis in ob/ob mice under NCD or FFD regimes for 2, 4, 8, 12, 18, 24 and 30 weeks. (A) Heat map of genes involved in inflammation and fibrosis. Red, upregulated; green, downregulated. (B–F) Expression levels of selected genes. Values are mean±s.e.m., n=5. *P<0.05, **P<0.01 versus NCD (Student's t-test, two-tailed).