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Mouse models of non-alcoholic fatty liver disease (NAFLD) are required to define therapeutic targets, but detailed time-resolved studies to establish a sequence of events are lacking. Here, we fed male C57Bl/6N mice a Western or standard diet over 48 weeks. Multiscale time-resolved characterization was performed using RNA-seq, histopathology, immunohistochemistry, intravital imaging, and blood chemistry; the results were compared to human disease. Acetaminophen toxicity and ammonia metabolism were additionally analyzed as functional readouts. We identified a sequence of eight key events: formation of lipid droplets; inflammatory foci; lipogranulomas; zonal reorganization; cell death and replacement proliferation; ductular reaction; fibrogenesis; and hepatocellular cancer. Functional changes included resistance to acetaminophen and altered nitrogen metabolism. The transcriptomic landscape was characterized by two large clusters of monotonously increasing or decreasing genes, and a smaller number of 'rest-and-jump genes' that initially remained unaltered but became differentially expressed only at week 12 or later. Approximately 30% of the genes altered in human NAFLD are also altered in the present mouse model and an increasing overlap with genes altered in human HCC occurred at weeks 30-48. In conclusion, the observed sequence of events recapitulates many features of human disease and offers a basis for the identification of therapeutic targets.

Little is known about how liver fibrosis influences lobular zonation. To address this question, we used three mouse models of liver fibrosis, repeated CCl 4 administration for 2, 6 and 12 months to induce pericentral damage, as well as bile duct ligation (21 days) and mdr2 - / - mice to study periportal fibrosis. Analyses were performed by RNA-sequencing, immunostaining of zonated proteins and image analysis. RNA-sequencing demonstrated a significant enrichment of pericentral genes among genes downregulated by CCl 4 ; vice versa, periportal genes were enriched among the upregulated genes. Immunostaining showed an almost complete loss of pericentral proteins, such as cytochrome P450 enzymes and glutamine synthetase, while periportal proteins, such as arginase 1 and CPS1 became expressed also in pericentral hepatocytes. This pattern of fibrosis-associated 'periportalization' was consistently observed in all three mouse models and led to complete resistance to hepatotoxic doses of acetaminophen (200 mg/kg). Characterization of the expression response identified the inflammatory pathways TGFβ, NFκB, TNFα, and transcription factors NFKb1, Stat1, Hif1a, Trp53, and Atf1 among those activated, while estrogen-associated pathways, Hnf4a and Hnf1a, were decreased. In conclusion, liver fibrosis leads to strong alterations of lobular zonation, where the pericentral region adopts periportal features. Beside adverse consequences, periportalization supports adaptation to repeated doses of hepatotoxic compounds., Competing Interests: the authors declare no conflicts of interest.