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

Huggett, Zoe (2020) Comparison of hepatoma cell lines and primary human hepatocytes as models to study the development of hepatic steatosis. PhD thesis, University of Nottingham.

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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<0.001) overall but this was not consistent across livers. Both McA-RH7777 cells and PHH accumulated lipid with 11mM glucose treatment when fatty acids were present, but again this was not consistent across livers. HepG2 and Huh7 cells were found to be insulin resistant compared to the insulin sensitive McA-RH7777 cells, but both HepG2 and PHH accumulated lipid in response to insulin treatment. Gene expression patterns varied between HepG2 and PHH, for example lipogenic gene expression was decreased by fatty acids in HepG2 cells but not PHH. Microarray comparisons showed different baseline levels of gene expression reflecting different phenotypes. PNPLA3 lentiviruses were successfully produced but protein overexpression could not be measured by the commercial antibodies tested. The human cell lines were confirmed to be homozygous for the polymorphic variant of PNPLA3 but all of the livers used for PHH isolation were either homozygous wild type or heterozygous.

The data from this thesis suggests that HepG2 cells are not a suitable model to replace PHH. Further work on the interactions between genotype and dietary factors in different individuals is required.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Salter, Andrew
Brameld, John
Bennett, Andrew
Keywords: Hepatic steatosis, Non-alcoholic fatty liver disease, Lipid accumulation, Hepatic lipid metabolism
Subjects: Q Science > QP Physiology > QP501 Animal biochemistry
Faculties/Schools: UK Campuses > Faculty of Science > School of Biosciences
Item ID: 60701
Depositing User: Huggett, Zoe
Date Deposited: 28 Jul 2020 10:31
Last Modified: 24 Jul 2022 04:30

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