Endocrine Abstracts

Introduction: Non-alcoholic fatty liver disease (NAFLD) has become the most common form of chronic liver disease in children in association with the increasing prevalence of obesity. The underlying mechanisms are incompletely understood, however the accumulation of cholesterol and fatty acid lipotoxins plays an important role. 5-hydroxymethylcytosine (5hmC) is an epigenetic modification generated from 5-methylcytosine (5mC) by the Ten-eleven translocase isoenzymes (Tets). Tet function is modified by glucose metabolites suggesting that Tet enzymes may be a novel modulator of energy homeostasis. We hypothesised that Tet-mediated DNA hydroxymethylation impacts on NAFLD pathogenesis.

Methods: C57Bl6/j mice were fed 58% saturated fat (HFD) or control diet for 17 weeks before intraperitoneal glucose tolerance testing and analysis of liver histology. Genome-wide profiling of 5hmC was undertaken using DNA immunoprecipitation and semiconductor proton sequencing. Hepatic transcriptomic analysis was performed using Illumina WG6 beadchip microarrays. A parallel HFD-fed mouse cohort with was switched to control diet for 12 weeks to reverse the phenotype. Human embryonic stem cell-derived hepatocytes were challenged with energy substrates lactate, pyruvate and octanoate for 48 hours, with 5hmC and transcriptome analysis.

Results: HFD induced obesity, glucose intolerance, insulin resistance and hepatic steatosis; strikingly these changes were reversed after switching to control diet. Whilst the overall global 5hmC profile was not altered by HFD, there was significant genic enrichment of 5hmC in upregulated mediators of cholesterol synthesis and transport (Lss, Sc4mol, Fdps, Hsd17b7, Cyp17a1, Mvd, Cyp1a2, Dhcr7 and Apoa4), with no enrichment in genes with other pathological functions. 5hmC enrichment was abolished following reversal of the NAFLD phenotype. These findings were largely replicated in human hepatocytes exposed to high-energy substrates with specific 5hmC enrichment in genes synthesising and transporting lipotoxic molecules (PLIN2, CIDEC, APOA4, ACADVL, HMGCS2, APOA5, CYP2J2, IGFBP1, PPAP2C, ACSL1, APOC3, ANGPTL4, NRG1) and no enrichment in upregulated genes of alternative function.

Conclusion: In these models of NAFLD, overnutrition induces genic hydroxymethylation with concurrent transcriptional activation specifically in genes driving the synthesis and transport of lipotoxic molecules. Such changes are reversible strengthening the functional association. We suggest that Tet-mediated hydroxymethylation may be an important mechanism in NAFLD pathogenesis and progression.