Regulatory Effect Of FGF21 On Endoplasmic Reticulum Stress In 3T3-L1 Cell

Endoplasmic reticulum(ER)is an important organelle of cellular protein synthesis,folding,modification,processing and quality monitoring,and ER homeostasis is essential for maintaining normal cell function.The ER stress(ERS)can be induced when it is difficult for ER to undertake the load of the misfolded proteins.Under the condition of rich nutrition,excessive protein synthesis and fat accumulation can lead to ERS in adipose tissue.Unfolded protein response(UPR)activated by ERS is an important feature of insulin resistance and type 2 diabetes mellitus at the cellular and molecular level.FGF21 is discovered recently as a new adipokine,which plays important roles in regulating lipid metabolism balance of liver,adipose tissue and body and improving insulin resistance and type 2 diabetes.Therefore,in this study,we stimulate 3T3-L1 preadipocyte with nutrient palmitic acid(PA)to explore whether FGF21 is involved in regulating ERS,consequently controlling inflammation,as a result,improving insulin resistance.Our research results will provide theoretical and experimental basis for preventing and fighting the metabolism-related diseases.The detail of our results are as follows:1.3T3-L1 cells were treated with different concentrations of PA,and the effect of PA on cell viability was detected by MTT method.According to the results of MTT Cell Viability Assay,cell survival rate is about 80%at 200μM PA induced 12h,which was thought to be the appropriate concentration and time of PA stimulation for the further experiments.2.After 3T3-L1 cells were treated with 200μM PA in different time,morphological changes were observed using laser confocal scanning microscopy through universal ER staining method.The expression levels of ATF4 and eIF2a phosphorylated proteins were significantly increased in 3T3-L1 cells with 200μM PA for 6h treatment.The expression levels of GRP78,XBP1 and CHOP genes mRNA were remarkably up-regulated in 3T3-L1 cells with 200μM PA for 12h stimulation.These results showed that Palmitic acid can induce 3T3-L1 cells ERS.3.Under ERS conditions,PA-induced FGF21 protein and mRNA levels were increased significantly,indicating that ERS can activate FGF21 expression.4.In FGF21-overexpression 3T3-L1 cells or in 3T3-L1 cells treated with FGF21 recombinant protein,the mRNA and protein levels of ERS marker genes were obviously suppressed.The results showed that FGF21 reduced chaperone protein GRP78 and pro-apoptosis factor CHOP mRNA and protein expression,decreased ATF4 protein levels and inhibited eIF2a phosphorylation,indicating that FGF21 improved ERS and protect cells from apoptosis via eIF2a-ATF4-CHOP pathway.In conclusion,our studies demonstrated that PA can cause cells ERS at the cellular or molecular level,and FGF21 is an essential physiological component of the cellular UPR program,which exerts beneficial feedback regulation counteracting ERS through lipid metabolism.