Non-alcoholic fatty liver disease-associated DNA methylation and gene expression alterations in the livers of Collaborative Cross mice fed an obesogenic high-fat and high-sucrose diet

ABSTRACT

Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent chronic liver disease, and patient susceptibility to its onset and progression is influenced by several factors. In this study, we investigated whether altered hepatic DNA methylation in liver tissue correlates with the degree of severity of NAFLD-like liver injury induced by a high-fat and high-sucrose (HF/HS) diet in Collaborative Cross (CC) mice. Using genome-wide targeted bisulphite DNA methylation next-generation sequencing, we found that mice with different non-alcoholic fatty liver (NAFL) phenotypes could be distinguished by changes in hepatic DNA methylation profiles. Specifically, NAFL-prone male CC042 mice exhibited more prominent DNA methylation changes compared with male CC011 mice and female CC011 and CC042 mice that developed only a mild NAFL phenotype. Moreover, these mouse strains demonstrated different patterns of DNA methylation. While the HF/HS diet induced both DNA hypomethylation and DNA hypermethylation changes in all the mouse strains, the NAFL-prone male CC042 mice demonstrated a global predominance of DNA hypermethylation, whereas a more pronounced DNA hypomethylation pattern developed in the mild-NAFL phenotypic mice. In a targeted analysis of selected genes that contain differentially methylated regions (DMRs), we identified NAFL phenotype-associated differences in DNA methylation and gene expression of the Apoa4, Gls2, and Apom genes in severe NAFL-prone mice but not in mice with mild NAFL phenotypes. These changes in the expression of Apoa4 and Gls2 coincided with similar findings in a human in vitro cell model of diet-induced steatosis and in patients with NAFL. These results suggest that changes in the expression and DNA methylation status of these three genes may serve as a set of predictive markers for the development of NAFLD.

KEYWORDS:

• DNA methylation
• Collaborative Cross mice
• gene expression
• non-alcoholic fatty liver disease
• GLS2

List of abbreviation

CC: Collaborative Cross; HCC: hepatocellular carcinoma; HF/HS: high fat and high sucrose; NAFL: nonalcoholic fatty liver; NAFLD: nonalcoholic fatty liver disease; NASH: nonalcoholic steatohepatitis; DMRs: differentially methylated regions; NGS: next-generation sequencing.

Disclosure statement

Arun J. Sanyal is the President of Sanyal Biotechnology, LLC, and has stock options in Genfit, Akarna, Tiziana, Indalo, and Durect. He has served as a consultant to AbVie, Astra Zeneca, Nitto Denko, Ardelyx, Conatus, Nimbus, Amarin, Salix, Tobira, Takeda, Fibrogen, Jannsen, Gilead, Boehringer, Lilly, Zafgen, Novartis, Novo Nordisk, and Pfizer. He has served as an unpaid consultant to Exhalenz, Intercept, Echosens, Immuron, Galectin, Fractyl, Northsea Pharma, Gencia, Syntlogic, Affimune, Chemomab, Nordic Bioscience Zydus, and Bristol Myers Squibb. His institution has received grant support from Gilead, Salix, Tobira, Bristol Myers, Shire, Intercept, Merck, Astra Zeneca, Malinckrodt, Cumberland, and Novartis. He receives royalties from Elsevier and UptoDate. Virginia Commonwealth University has ownership interests in Sanyal Biotechnology, LLC.

Data availability statement

The data that support the findings of this study are available in Gene Expression Omnibus depository at https://www.ncbi.nlm.nih.gov/geo/, reference numbers GSE149863 and GSE126848. These data were derived from the following resources available in the public domain: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE149863 and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE126848. Any additional data that support the findings of this study are available from the corresponding author, IP, upon request.

Additional information

Funding

This work was supported by the FDA-NCTR Intramural Research Program, the FDA-Office of Minority Health and Health Equity Intramural Challenge Grants, NCI-NCTR Interagency Agreement, and, in part, by a grant from NIEHS R01 ES029911.