Effects Of Different Composition Of Fatty Acids On Hepatocyte Steatosis, Endoplasmic Reticulum Stress And Its Mechanisms

BackgroundElevated serum free fatty acid levels contribute to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Whereas adipocytes have a unique capacity to store excess fatty acids in the form of triglyceride, nonadipose tissues, including liver, do not. Accumulation of lipids in nonadipose tissues can lead to cell dysfunction and cell death, a phenomenon known as lipotoxicity. Previous study in pancreaticβ-cell suggested that saturated and unsaturated fatty acids differed significantly in their contributions to lipotoxicity. In the present study, we examined the effects of different composition of fatty acids on hepatocyte steatosis, endoplasmic reticulum stress and its mechanisms.Methods1. The optimized concentration and treatment time was selected by MTT assay. Steatosis model was induced by adding oleate acid (unsaturated fatty acids) or palmitic acid (saturated fatty acids) to human hepatocyte line L02.2. The experiment was grouped as follows: normal control group; DMSO control group; Palmitic acid-treated group; Oleic acid-treated group.3. Lipid accumulation in the hepatocytes with Oil Red O staining was observed by optical microscopy.4. The levels of TG, MDA, AST and ALT were measured by biochemical assays.5. The apoptosis was evaluated by ANNEXINⅤFITC/PI and Acridine Orange/Ethidium Bromide(AO/EB).6. The Expression of GRP78 was detected by RT-PCR and Western blot.Results1. In normal control groups and DMSO groups, only a little orange lipids were seen; Compared with normal control groups and DMSO control groups, there were much more visible orange lipid accumulations in both palmitic acid groups and oleic acid groups. Integration with lipid drops could be observed along with the prolonging of the culture time in oleic acid groups.2. Compared with the normal control group and DMSO control group, the contents of TG, MDA, AST and ALT gradually increased in the cultural supernatant of palmitic acid group or oleic acid group (P<0.05).3. AO / EB staining or ANNEXINⅤFITC / PI double staining showed that the ratio of hepatic apoptosis in Oleic acid groups or palmitic acid groups was gradually increased accompanied with the prolonging of the culture time in vitro, but it was significantly increased at 48h, 72h in palmitic acid groups (P<0.05), the rate of hepatic apoptosis in palmitic acid group was higher than that in oleic acid group (P<0.05).4. The expression of GRP78 protein and mRNA in palmitic acid groups was more significantly increased than that in the control groups, DMSO control groups and oleic acid groups at 24, 48, 72 h (P <0.05).Conclusions1. Steatosis which was characterized by increased triglyceride in hepatocytes was induced by either saturated fatty acids or unsaturated fatty acids, Palmitic acid more significantly increased than Oleic acid on the levels of AST, ALT, MDA and the ratio of apoptosis in hepatic steatosis ,suggesting that lipotoxicity is made more severe by saturated fatty acids.2. The data demonstrate that steatosis characterized by increases saturated fatty acids could up-regulate GRP78 expression, endoplasmic reticulum stress, and liver cell injury. Oleic acid (unsaturated fatty acids)does not affect the expression of GRP78.