Metabolic Regulation Roles Of Zinc-α2-Glycoprotein On Triglyceride In Adipocytes

Objective:To construct pcDNA3.1(-)-mZAG recombinant mammalian expression vector,which were transfected into3T3-L1preadipocytes cells and to explore themechanism of Zinc-α2-Glycoprotein (ZAG) regulate triglyceride metabolism inadipocytes.Methods:1. The total RNA from mouse liver tissue was extracted. The reverse-transcript(RT)-PCR method was used to amplify the complete domain sequence of mZAG.The confirmed PCR products were inserted into expression plasmid by DNAligation. After identification by RT-PCR and DNA sequencing, mouse ZAGexpression plasmids (pcDNA3.1(-)-mZAG) containing mouse ZAG cDNA werecloned and transfected into3T3-L1preadipocytes by lipofection. The stabletransfected3T3-L1cell line was established after selection with G418. Theprotein expression of ZAG protein and HSL, ATGL were detected by WesternBlot. The mRNA expression of HSL and ATGL were detected by RT-PCR. Thelevel of FFA in culture medium was measured by ELISA.2.3T3-L1adipocytes were cultured and differentiated into mature adipocytes invitro and treated with or without recombinant ZAG protein (20μg/mL).Intracellular lipid accumulation was observed by Oil Red O. Change inmitochondrial number in adipocyte differentiation assayed by transmissionelectron microscopy. The level of FFA in culture medium was measured byELISA.Results:1. The prokaryotic expression vector pcDNA3.1(-)-mZAG was constructed andconfirmed by DNA sequencing. 2．Compared with control group and empty plasmid pcDNA3.1(-) group, mRNA andprotein expression of HSL and ATGL increased in pcDNA3.1(-)-mZAG group(P<0.05).3. Compared with control group and empty plasmid pcDNA3.1(-) group, FFAconcentration in culture supernatants had no significant difference inpcDNA3.1(-)-mZAG group (P>0.05).4. Compared with the control group, FFA concentration in mature adipocytes culturesupernatants were decreased after treatment with recombinant ZAG protein for48h(P<0.05).In addition, lipid droplets became fewer amd smaller,mitochondrion amount were increased significantly.Conclusion:1. Expression plasmid pcDNA3.1(-)-mZAG was successfully constructed in thisstudy. The stable3T3-L1cell line expression ZAG was successfully established.2. ZAG can promote lipolysis by up-regulation the expression of ATGL and HSLin adipocytes.3. Recombinant ZAG protein can enhance lipolysis and increase the mitochondriaAmount of adipocytes.