Understanding the early onset of intracellular lipid accumulation induced by oleicand palmitic acids in HepG2 cells

Increasing hepatic lipid accumulation is the primary cause of non-alcoholic fatty liver disease (NAFLD), which has become an emerging health concern globally. Many studies have used the HepG2 cell-based in vitro model of NAFLD to investigate intracellular lipid accumulation after several hours of exposure to free fatty acids (FFAs). However, the molecular mechanisms underlying the early onset of lipid accumulation are yet to be unveiled. In this study, we examined oleic acid (OA) and palmitic acid (PA)- induced lipid accumulation in HepG2 cells at early time points, i.e., in minutes. Using Oil Red O staining and fluorescence microscopy imaging, we observed a time-dependent increase in intracellular lipid accumulation in cells treated with 0, 0.25, 0.5, and 1.0 mM FFAs. Notably, significant lipid droplet formation was detected within 15 min of OA treatment at 0.5 and 1.0 mM concentrations compared to the control, whereas PA did not elicit such an early response. Gene expression analysis revealed upregulation of genes related to lipid metabolism (SREBF1, PDK4, and G6PC), beta-oxidation (CPT1a) and cholesterol synthesis (HMGCR) at the early time point. Additionally, immunoblot analysis showed increased expression of Fatty acid synthase (FASN), which is a well-known marker of lipogenesis. In summary, our findings indicate that OA induces lipid accumulation more robustly than PA at early time points, providing insights into the molecular changes at the onset of NAFLD progression.