Molecular networks affected by fatty acids and glucose in human hepatic spheroids investigated by proteomics and lipidomics analysis

Non-alcoholic fatty liver disease (NAFLD) is characterised by lipids accumulating in the liver. It starts with simple steatosis (fat accumulation), causing an inflammatory response leading to non-alcoholic steatohepatitis (NASH). Constant inflammation leads to fibrosis which can cause decreased liver function, cirrhosis and hepatocellular carcinoma. NAFLD is a highly prevalent disease affecting 25% of the world’s population; however, at this moment, no approved drug treatment is available. Additionally, diagnosis is challenging due to NAFLD being asymptomatic until late stages and the invasiveness of the liver biopsy used for diagnosis. We hypothesise that an in-depth analysis of the temporal changes of the molecular proteins and lipid networks underlying this pathogenesis would advance the development of effective interventions, drugs, and non-invasive biomarkers that could detect the disease at its early stages and prevent or slow it down. 

Following disease progression in vivo is invasive and complicated to perform. Therefore rodent models capable of recapitulating the hallmarks of NAFLD have been developed. These models have significantly contributed to understanding molecular and cellular events leading to NAFLD. However, extensive scale experiments using animals are time-consuming, costly and very often, not all results can be directly translated into the response of human organs and tissues. An alternative and widely adopted method is using the ex vivo culture of organ-specific human cells. Recent technology advancements allow us to culture human cells in 3- dimensional assemblies called spheroids. This creates a micro-environment that closely resembles the in vivo tissue and improves organs specific function of the cells, their viability and sensitivity to environmental clues. The 3D culture also allows for culturing cells for extended periods, which was impossible with traditional cell culture techniques. 

Thus, in this thesis, I combine 3-dimensional spheroid cultures of human liver cells (C3A/HepG2 hepatocytes) with advanced proteomics and lipidomics techniques to identify and quantify changes of proteins and lipids upon the exposure of the cells to NAFLD risk factors (high levels of glucose and fatty acids). Hepatic spheroids developed upon fatty acid exposure lipidome changes similar to in vivo. Fatty acids did not cause an immense difference in protein expression. Still, an increase in proteins was seen in NAFLD and enrichment of signalling related to epithelial-to-mesenchymal transition (EMT). Glucose or fatty acids and glucose exposure of hepatic spheroids affected the function and proteome a lot more than only fat. Glucose exposure resulted in enormous changes of proteome related to metabolism and increased motility, as seen in EMT. Additional fatty acids in a high glucose background cause redirecting of metabolism to ATP production and higher induction of EMT markers.