Comparison of hepatoma cell lines and primary human hepatocytes as models to study the development of hepatic steatosis

Non-alcoholic fatty liver disease (NAFLD) is a worldwide health burden. The first stage is the accumulation of fat within hepatocytes (hepatic steatosis). Dietary and genetic factors determine steatosis, with particular emphasis on dietary fat, dietary sugars, and patatin-like phospholipase domain containing 3 (PNPLA3) genotype. Primary human hepatocytes (PHH) are currently the gold standard in vitro human model of steatosis but hepatoma cell lines are often used. This thesis aimed to compare the lipid accumulation and metabolism of PHH and hepatoma cell lines, and to develop a system to study the PNPLA3 genotype in relation to nutritional stimuli. Lipid accumulation in response to 48 hours treatment with fatty acids (0µM or 200µM of palmitic, oleic and linoleic acids in the ratio 2:2:1), glucose (5mM or 11mM) and fructose (0mM, 2mM or 8mM) was measured in HepG2, Huh7, McA-RH7777 and PHH using a Nile Red staining assay normalised to DNA. Insulin sensitivity across the cell lines, and lipid accumulation in response to insulin treatment in HepG2 and PHH, were also measured. RT-qPCR and microarray analysis was used to compare the gene expression profiles of HepG2 and PHH. Human DNA was used to produce lentiviruses to overexpress wild-type or polymorphic PNPLA3. All cell types accumulated lipid in response to fatty acid treatment (P<0.001), with HepG2 cells and Huh7 cells reaching very similar maximum fold changes of 4.717 and 4.560 respectively. McA-RH7777 cells reached a higher fold change maximum of 7.707, whereas the PHH ranged between 1.403 and 4.822 depending on the liver. Only PHH showed an increase in lipid accumulation in response to fructose (P