Effects Of ZAG On Triglyceride Deposition In Palmitic Acid - Induced HepG2 Cells And Its Mechanism

Objective: To construct recombinant eukaryotic expression vectorpIRES2-ZsGreen1-hZAG containing ZAG gene and transiently transfected intoHepG2cells. To investigate the effect of overexpression of ZAG on palmitic acidinduced deposition of triglycerides in vitro.Methods: The total RNA was extracted from HepG2cells. ZAG sequence wasamplified by RT-PCR. The confirmed PCR products were inserted into eukaryoticexpression plasmid ((pIRES2-ZsGreen1) by DNA ligation after enzyme by XhoI andSacII. After identification by detecting nucleotide sequence, the human ZAGeukaryotic expression plasmid (pIRES2-ZsGreen1-hZAG) containing human ZAGsequence was cloned and transfected into HepG2cells by lipofection2000.Theprotein expression of ZAG was detected by western-blotting. HepG2cells withover-expression ZAG were treated with or without palmitate acids for24hours.Intracellular liqid accumulation was observed by Oil red O. The levels of intracellulartriglyceride were measured by ELISA.Results: The recombinant eukaryotic expression vector of pIRES2-ZsGreen1-hZAGwas contructed. The protein expression of ZAG increased inpIRES2-ZsGreen1-hZAG transfection group compared with control group. Aftertreatment with palmitate acids, concentration of triglyceride was decreased inpIRES2-ZsGreen1-hZAG transfection group, and lipid droplets became fewer andsmaller compared with control group.Conclusions: The recombinant eukaryotic expression vector ofpIRES2-ZsGreen1-hZAG was successfully contructed and transfected into HepG2 cells, inducing transient over-expression of ZAG in HepG2cells. Over-expression ofZAG in HepG2cells alleviated palmitic acid induced deposition of triglycerides. Objective: To explore the mechanism of ZAG regulates palmitic acid inducedtriglycerides deposition in HepG2cells.Methods: The HepG2cells with overexpression of ZAG were cultured and treatedwith or without palmitic acids. The protein expression levels ofmetabolic nuclear receptors (SREBP1c, PPAR alpha, LXR alpha and FXR), synthetic(ACC, FAS) and the beta oxidation genes were detected by Western-blotting.Results: Compared with control group, the protein levels of SREBP-1c, LXR alpha,ACC and FAS were increased while the levels of PPAR alpha and FXR weredecreased after treatment with palmitic acids. Compared with vector group, theprotein levels of FXR and CPT-1A were increased while the levels of SREBP-1c weredecreased in ZAG overexpression group. Compared with vector group, the proteinlevels of FXR, PPAR alpha and CPT1A were increased while the levels of SREBP-1c,LXR alpha, ACC and FAS were decreased in ZAG overexpression group aftertreatment with palmitic acids.Conclusions: Palmitic acid can contribute to deposition of triglycerides byup-regulation of the expression of SREBP-1c, LXR alpha, ACC and FAS anddown-regulation of expression of PPAR alpha and FXR. Over-expression ZAG canincrease the expression of FXR and CPT-1A and decrease the expression ofSREBP-1c. Over-expression ZAG could alleviate palmitic acid induced deposition oftriglycerides by up-regulation of the expression of FXR, PPAR alpha and CPT1A anddown-regulation of expression of SREBP-1c, LXR alpha, ACC and FAS.