Lipotoxicity of free fatty acid mixture and effect of free fatty acid mixture on lipid metabolism-related genes in L-02 cells

AIM:To investigate the lipotoxicity of free fatty acid(FFA) mixture and the effect of the FFA mixture on lipid metabolism-related genes in L-02 cells.METHODS;A normal human hepatocytes-derived cell line L-02 was treated with 0.5,1 and 2 mmol/L FFA mixture(oleate and palmitate,2=1) for 24 h.The cellular total lipid accumulation was determined after Nile red staining by confocal laser scanning microscopy and flow cytometry.Intracellular triglyceride(TG) content was measured using an enzymatic kit.The viability of L-02 cells was determined by MTT assay and the apoptosis-inducing effect of FFA mixture was evaluated by annexin V/PI staining.The levels of alanine aminotransferase(ALT) and aspartate,aminotransferase(AST) in the culture medium were detected by ALT and AST kits.The mRNA levels of lipid metabolism-related genes,adipose differentiation-related protein(ADRP) and sterol regulatory element-binding protein 1(SREBP-1),were examined by quantitative real-time PCR.RESULTS:All the different concentrations of FFA mixture increased intracellular lipid accumulation and TG content in a dose-dependent manner.FFA mixture at concentration of 1 mmol/L increased intracellular TG by 2.6 folds,which matched with the change in non-alcoholic fatty liver disease patients.Treatment for 24 h with 0.5 mmol/L and 1 mmol/L FFA mixture did not trigger apparent cell death and apoptosis,while treatment with 2 mmol/L FFA mixture resulted in a marked decrease in cell viability and induced early and late stages of apoptosis in L-02 cells.The levels of ALT and AST in the culture supernatant had no significant difference between control group and FFA treatment group.Treatment with 1 mmol/L FFA mixture up-regulated the expression of ADRP and SREBP-1 by 2.660 and 2.758 folds,respectively.CONCLUSION:FFA mixture induces the hepatic steatosis and 2 mmol/L FFA mixture causes mild cells damage in L-02 cells.The up-regulation of ADRP and SREBP-1 may be involved in FFA-induced hepatic steatosis.