Identification of the differential and synergic lipotoxic patterns of oleic acid, palmitic acid, and their mixture in 3D HepG2/C3A tissue using liver-on-chip technology.

The metabolic dysfunction-associated steatotic liver disease (MASLD, previously formerly known as non-alcoholic fatty liver disease, NAFLD) is rapidly expanding worldwide in parrallel with the obesity pandemic. Dietary fatty acids including oleic (OA) and palmitic acids (PA) contribute to the hepatic intracellular triglyceride accumulation, and are therefore thought to play key roles in disease development and progression. Taking advantage of the cutting-edge organ-on-chip technology that mimics the 3D organ dynamic environment, we aimed at investigating the role of OA, PA and a 2:1 OA/PA mixture on the growth and function of the HepG2/C3A, a liver cell line model, over 2 and 7 days. OA supported sustained cell growth, leading to dense 3D tissues, whereas PA and OA exposure did not affect cell proliferation. PA treatment downregulated the GLUT2, INSRA, SREBP1, FASN, mRNA levels indicating a lipid metabolism perturbation in our model. The cell dysfunction caused by OA, PA, and OA/PA was associated with an increase in reactive oxygen species (ROS) production over time. Intracellular lipid monitored by oil red O was higher in cells exposed to OA than in the control ones and cells cultured with PA. Our data confirm the role of fatty acids on the growth and dysfunction of HepG2/C3A cells, and highlight distinct mechanisms through which OA and PA exert their effects.