The Effect Of Recombinant Adenovirus-Mediated Adiponectin Rnai In Adipocytes

PART 1 AD-ACRP30-1 INHIBIT THE ADIPONECTIN EXPRESSION IN 3T3-L1 ADIPOCTTESOBJECTIVE: To observe the change of adiponectin expression of murine 3T3-L1 adipocytes after infected by Adenovirus-Mediated RNA Interference Ad-Acrp30-1.METHODS:Murine 3T3-L1 cell line was transfected with the Acrp30-siRNA adenovirus expression vector. The mRNA expression and protein levels of Acrp30 in those cells were evaluated by semi-quantitative RT-PCR and ELISA.RESULTS: The Acrp30 mRNA expression and protein levels were obviously decreased in 3T3-L1 adipocytes with Ad-Acrp30-1, and the inhibition ratio of acrp30 was about 60%.CONSTRUCTION: The shRNA adenoviral vector against Acrp30 expression inhibits the expression of Acrp30 in 3T3-L1 adipocytes successfully and effectively.PART 2 THE CHANGE OF CELL METABOLISM AFTER INHIBITING ACRP30 EXPRESSION IN ADIPOCYTESOBJECTIVE:To investigate the effects of Acrp30 deficiency on glucose turnover, metabolism related gene and CORS-26mRNA expression in the 3T3-L1 adipocytes.METHODS:Murine adipocytes were infected with recombinant adenovirus Ad-Acrp30-1.An evaluation about the rates of glucose disappear and the uptake rates of glucose under basal states were made after treating with recombinant adenovirus. The changes of glucose-lipid metabolism related gene was analyzed by RT-PCR and Dot blot under basal states and insulin stimulation.RESULTS:The rates of glucose disappear and the uptake rates of glucose were significantly decreased (P<0.01 and P<0.05, respectively)under basal states after Acrp30 deficiency. And GLUT-1, GLUT-4, HSL, IRS-1 gene mRNA expression was decreased (P<0.01,P<0.05, P<0.01 and P<0.05 , respectively), but The level of ATGL, PPAR-αand PPAR-γmRNA expression was no difference. The protein of GLUT-4 was also down-regulated ( P < 0.01). After insulin stimulation, GLUT-1, GLUT-4, HSL, IRS-1 and PPAR-αgene mRNA expression were decreased (P<0.05, P<0.01 , P<0.01 and P<0.01,respectively). The protein of GLUT-4 and the protein tyrosine phosphorylation of IRS-1 was also significantly decreased (P<0.01,P<0.01).The level of ATGL and PPAR-γmRNA expression was no difference. The level of GLUT-1,HSL and IRS-1 mRNA expression was lower down-regulated(52%vs35%, 60%vs43%, 48%vs28%)comparing with basel state. The protein of GLUT-4 was also lower down-regulated (23%vs16%). adenovirus Ad-Acrp30-1 treatment was also down-regulated visfatin mRNA and protein expression (P<0.01)under basal states and insulin stimulation. But the CORS-26 mRNA expression was up-regulated in basal state(P<0.05).CONSTRUCTION:Acrp30 deficiency exerts a negative effect on glucose turnover and lipid metabolism maybe through down-regulating GLUT-1, GLUT-4, IRS-1 and HSL expression, and also results in marked alteration of other adipocytokine.