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Preparation of the Novel Recombinant VPAC2 Receptor Agonist RD and Its Molecular Mechanism of Promoteing Insulin Fuction |
MA Yi, LUO Tian-jie, HONG An |
Department of Cell Biology of Jinan University, Institute of Biological Medicine of Jinan University, National Engineering Research Center of Genetic Medicine, Guangzhou 510632, China |
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Abstract Using genetic recombinant, prokaryotic expression, purification by Chitin-Beads column and HPLC, and identification by mass spectrometry technologies, a new VPAC2-specific agonist RD with the function of anti-type 2 diabetes was prepared, and its molecular mechanism of the effectively promoting insulin signal transduction in treating diabetes was studied. The results show that the molecular weight of the prepared RD is 3.785kDa. and its purity is 96%. Treating normal and insulin resistant 3T3-L1 adipocytes (IR model cells) with recombinant RD,the 1μmol/L and 5μmol/L RD treated normal 3T3-L1 adipocytes groups can significantly promote the expression of the IRS-1 protein, with an expression increase of 36% and 42% respectively. After the IR model cells were treated with the 1μmol/L and 5μmol/L RD, the IRS-1 expressions were increased by 55% and 63% respectively. After the IR model cells were treated with 1,5 and 10μmol/L RD, the pIRS-1(Ser307) expressions were decreased by 5.9%, 10.7% and 32.7% respectively. 5μmol/L and 10μmol/L RD treated IR model cells groups can decrease the expression of the IRS-2 protein by 12.8% and 40.6% respectively, and 1,5 and 10μmol/L MHDBAY treated IR model cells groups, the expressions of the pIRS-2 protein were decreased by 35.1%, 40.8% and 48.5% respectively. The expressions of the Akt protein in the IR model cells in 5μM and 10μM MHDBAY treated group were significantly increased, with an expression increase of 74% and 77% respectively. In IR model cells treated with 1, 5, 10μM RD, the phosphorylation levels of Akt Ser473 were decreased by,33.9%,64.0% and 71.1% respectively, and the phosporylation levels of Akt Thr308 were increased by 13.5%,78.6% and 83.3% respectively. This study established the technical preparation of the recombinant VPAC2 receptor agonist RD, and its biological effects in cell level were detected (significantly promote IRS-1 expression in normal 3T3-L1 adipocytes and IR model cell, decrease pIRS1 (Ser307), IRS-2, pIRS2 expression in IR cell model, and promote Akt expression and Akt Thr308 phosphorylation level, etc.). That may provide the experimental basis for illuminating molecular mechanism of RD in treating diabetes and its pharmaceutical research and development.
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Received: 13 August 2014
Published: 25 November 2014
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