Investigating NAFLD from RNA-seq data (human cell line Huh7) for samples under three treatments: low fat low sugar, low fat high sugar and high fat high sugar. Wild type samples are compared against DGAT2 knocked out.

Non-alcoholic fatty liver disease (NAFLD) is a major public health burden and it covers a spectrum of diseases. NAFLD starts with the accumulation of lipid droplets (LDs) within hepatocytes (steatosis). Part of the challenge of studying the mechanistic processes involved in LD accumulation and their implications on the pathogenesis of human NAFLD is due to the available models. Investigating hepatic LDs in humans is challenging and relies on liver biopsies, meaning only cross-sectional data be obtained. On the other hand, LD patterns in in vitro models are poorly defined and rarely reported. Diacylgylcerol acyltransferase (DGAT)2 is one of two enzymes that carry out the final committed step in triacylglycerol (TAG) synthesis. It is unclear whether the enzymes are able to compensate for each other or whether they have distinct roles. It has been hypothesised that DGAT1 primarily utilises exogenous fatty acids and DGAT2 uses de novo-derived fatty acids. Given the important role of this enzyme in TAG synthesis and accumulation, the aims of this study are first to create a cellular model of intrahepatocellular TAG accumulation by manipulating nutritional substrates and to investigate intracellular metabolism in wildtype and DGAT2 knockout cells under these conditions. The experimental workflow for this study is as follows: Huh7 cells (either wild type or knockout) were grown in media containing 11 mM glucose and 2% human serum (HS) for seven days before additional sugars and fatty acids (FAs) were added for a further seven days. All treatments contained 11 mM glucose and 2% HS, either with 200 µM FAs (low fat low sugar; LFLS), 5.5 mM fructose + 200 µM FAs (low fat high sugar; LFHS) or 5.5 mM fructose + 800 µM FAs (high fat high sugar; HFHS). FA metabolism, lipid droplet characteristics and transcriptomic signatures were investigated.