Proteomics of lipid accumulation and DGAT inhibition in HepG2 liver carcinoma cells

Non-alcoholic fatty liver disease (NAFLD) is a manifestation of the metabolic syndrome in the liver. It is marked by hepatocyte accumulation of triacylglycerol (TAG) rich lipid droplets. In some patients, the disease progresses to non-alcoholic steatohepatitis (NASH), characterized by cellular damage, inflammation and fibrosis. In some cases, cirrhosis and liver failure may occur. However, the pathogenesis of NAFLD is still unclear. The present project is based on the hypothesis that hepatocytes are equipped with mechanisms that allow them to manage lipid accumulation to a certain extent. Continued or increased lipid accumulation beyond this triggers molecular mechanisms such as oxidative stress, lipid peroxidation and cell death that aggravate the condition and cause disease progression. The aim of this project is to study the effects of lipid accumulation on the cells using proteomics approach to identify proteins involved in the disease progression. A cell culture model was used in the study. HepG2 cells, a human liver carcinoma cell line, were treated with a mixture of fatty acids (FA) to induce lipid accumulation. The lipid accumulation in HepG2 cells was measured with Oil red O assay and the effect of lipid accumulation on the proliferation of the cells was measured using an MTT cell proliferation assay. HepG2 cells treated with 1 mM FA mixture for 6 hours induced lipid accumulation 1.4 times of control with 90% of cell proliferation capacity of the control cells. The final and the only committed step in TAG biosynthesis is catalysed by acyl-CoA diacylglycerol acyltransferase (DGAT) enzymes. To investigate if limiting lipid accumulation in HepG2 cells would prevent molecular mechanisms of pathogenesis, inhibition of DGAT by small molecule inhibitors was performed. Among the three DGAT inhibitors (A922500, PF06424439 and PF04620110) tested, PF04620110 reduced the lipid accumulation to 1.2 fold of the control cells when they were treated with 100 μM of the inhibitor in the presence of 1 mM FA mixture for 6 h. Proteomic analyses were carried out for the control, FA-treated and inhibitor-treated cell groups to identify protein changes in the abundance. Functional analyses of the changed proteins identified suggest that lipid accumulation tends to adversely affect the functioning of the ER and the mitochondria. A complex interplay between the two organelles, possibly mediated by Ca2+ signalling may be vital in ensuring cell survival. PF04620110 was able to counter the FA induced changes in the abundance of some proteins involved in the metabolic processes but it had limited effect on the ER chaperones whose abundance in the inhibitor-treated sample was comparable to that of the FA-treated sample. These data provided important information for future discoveries of biomarkers and molecular mechanisms involved in the progression of NAFLD