Onoufriou, Maria Helena; (2024) Metabolomics Study of In Vitro Models of Hepatic Steatosis in HepG2 Cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Non-alcoholic fatty liver disease (NAFLD) is one of the leading causes of liver disease with a current global prevalence of 25%. NAFLD consists of a spectrum of diseases ranging from mild steatosis to non-alcoholic steatohepatitis (NASH) and fibrosis and can be diet or drug-induced. The early stages of NAFLD and drug-induced fatty liver disease (DIFLD) are reversible. However, the symptoms of steatosis are vague and there is a lack of specific and sensitive biomarkers. The overall aim of this project was to identify metabolite biomarkers for mild steatosis using NMR-based metabolomics; HepG2 monolayers and spheroids were used to create dietary and drug-induced in vitro models of mild steatosis. To create the steatotic models HepG2 cells were dosed with various doses of either a 2:1 mixture of oleic and palmitic acid, tetracycline or valproate and incubated for 24 hours. To ensure doses were not cytotoxic MTS and LDH assays were conducted. Steatosis was confirmed using Oil Red O staining and a triglyceride assay. Cell extracts were obtained from all models and analysed by 1H NMR and multivariate statistical methods. While a mixture of oleic and palmitic acid induce an increase in lipid accumulation and upregulation of beta-oxidation, tetracycline and valproate affect lipid metabolism by reducing beta-oxidation with both eventually leading to NASH. Therefore, changes in the TCA cycle metabolites were observed as well as changes in the methylation pathways. In particular, there were changes in levels of lactate, choline, homocysteine, and arachidonic acid in all models. These metabolites could potentially be useful in a panel of biomarkers for the early detection of steatosis. Literature has suggested that HepG2 monolayers cells have poor CYP expression compared to spheroids therefore, this study analysed CYP 2D6, 2E1 and 3A4 expression in both monolayers and spheroids. The resulting Western blots indicated changes in all CYPs in both monolayers and spheroids in response to steatosis. The results from this project provide a good overview of the major metabolic pathways affected in steatosis, regardless of the cause. The use of HepG2 spheroids in druginduced steatosis offers a novel in vitro model for biomarker research that has not been reported before in the literature.

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