Regulation Of Berberine On3T3-L1Readipocyte Differentination And The Relationship With Insig-2

Objective:To investigate the effect of Berberine on3T3-L1preadipocyteproliferation and differentiation, and the effects on Insig-2and other relatedgene expression.Methods:1. Effects of Berberine on proliferation and differentiation of3T3-L1preadipocytes: Cell proliferation was determined by MTT assay. Celldifferentiation was examined by Oil red O staining. The marker genes ofadipocyte differentiation were examined by real-time quantitative PCR.2. Effects of Berberine on the mRNA expression of Insig-2and otherrelated gene: The mRNA expressions of Insig-2, Insig-1, SEBP1c, FAS,PPARγ and VDR in differentiated adipocyte were examined by real-timequantitative PCR.3. Construction and identification of the human Insig-2promoterluciferase reporter plasmid pGL3-Insig-2: The Insig-2promoter about1400bp was amplified by PCR from human genome DNA and was insertedinto the vector pGL3-basic. Then，recombinant plasmid pGL3-Insig-2was identificated by restriction digestion and DNA sequencing.4. Dual luciferase reporter gene detection system to detect the Insig-2promoter activity: the recombinant vector pGL3-Insig-2and control vectorpRL-TK were transiently co-transfected into HEK293、HepG2and3T3-L1cells, then the promoter activity was detected by Dual-Luciferase RepoterAssay.Results:1.MTT assay results showed that Berberine can inhibit theproliferation of3T3-L1preadipocyte in dose-and time-dependent; Oil redO staining showed that Berberine can inhibit the differentiation of3T3-L1preadipocytes and the gene expression of adipocyte differentiation markergenes.2. Berberine increased the expression of Insig-2and VDR gene, butinhibited the expressiong of Insig-1, SEBP1c, FAS and PPARγ gene.3. The Insig-2promoter recombinant plasmid was successfullyconstructed, and the results of restriction digestion and DNA sequencingwere completely correct.4. The luciferase activity assay showed the recombinant vectorpGL3-Insig-2had strong promoter activity in HEK293, HepG2and3T3-L1cells. Berberine increased the promoter activity in HepG2and3T3-L1cells,and the Insig-2gene promoter activity in3T3-L1cells was higher thanHepG2cells. The effect of10μM Berberine was strongest. Conclusion:Berberine can inhibit the proliferation and differentiation of3T3-L1preadipocytes, and lipid formation, its mechanism maybe upregulate theInsig-2gene expression, then inhibit the expression and activation ofSREBPs, thereby block the expression of adipocyte differentiationtranscription factors and lipid synthesis enzyme. The human Insig-2promotor luciferase reporter gene vector was successfully constructed.Berberine can increase the Insig-2gene promoter activity and promotetranscription.