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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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[1] Ghzili H, Grumolato L, Thounnon E, et al. Role of PACAP in the physiology and pathology of the sympathoadrenal system. Front Neuroendocrinol, 2008, 29(1):128-141.
[2] Winzell M S,Ahren B. Role of VIP and PACAP in islet function. Peptides, 2007, 28 (9):1805-1813.
[3] Ma Y, Luo T, Xu W, Ye Z, et al. A new recombinant pituitary adenylate cyclase-activating peptide-derived peptide efficiently promotes glucose uptake and glucose-dependent insulin secretion. Acta Biochim Biophys Sin, 2012, 44(11): 948-956.
[4] Ma Y, Ma M, Dai Y, et al. Expression, identification and biological effects of a novel VPAC2-specific agonist with high stability and bioactivity. Acta Biochimica et Biophysica Sinica, 2010,42(1):21-29.
[5] Ma Y, Yu RJ, Zeng L, et al. Cloning and expression of the new gene recombinant RMBAY against Type-2 diabetes and its production optimization. Chin Biotech, 2009, 29(4):17.
[6] 王丽静,张尉,刘小莺,等.地塞米松诱导3T3-L1脂肪细胞胰岛素抵抗模型的建立.福建医科大学学报,2007,41(3):282-284. Wang L J, Zhang W, Liu X Y, et al. The development of dexamethasone induced insulin resistance in 3T3-L1 adipocyte. Journal of Fujian Medical University, 2007, 41(3):282-284.
[7] 汪忠煌,杨明炜,陈立,等.小蘖碱与梓醇及其配伍对胰岛素抵抗3T3-L1脂肪细胞Glut-4、IRS-1、IRS-1 Ser307磷酸化蛋白表达的影响.中国药师,2008,11(10):1142-1145. Wang ZH Y, Yang M W, Chen L, et al. Effect of berberine, catalpol and their combination on the expression of GLUT4, IRS-1, IRS-1 Ser307, phosphorylation in insulin resistsnt 3T3-L1 adipocyte. China Pharmacist, 2008, 11(10):1142-1144.
[8] 杨桂枝,欧可群,赵佳,等.罗格列酮改善胰岛素抵抗细胞摄取葡萄糖的途径研究.四川大学学报(医学版), 2007,38(5):816-818. Yang G ZH, Gao X P, Yan J F, et al. Mechanism of rosiglitasone to improve glucose-uptake of 3T3-L1 adipocyte with insulin resistance induced by dexamethasone and insulin. J Sichuan Univ ( Med Sci Edi), 2007, 38(5):816-818.
[9] Tsutsumi M, Claus T H, Liang Y. A potent and highly selective VPAC2 agonist enhances glucose-induced insulin release and glucose disposal, a potential therapy for type 2 diabetes. Diabetes, 2002, 51(5): 1453-1460.
[10] Yung S L, Dela Cruz F, Hamren S, et al. Generation of highly selective VPAC2 receptor agonists by high throughput mutagenesis of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide. J Biol Chem, 2003, 278(12):10273-10281.
[11] Morino K, Petersen K F, Dufour S, et al. Reduced mitochondrial density and increased IRS-1 serine phosphorylation in muscle of insulin-resistant offspring of type 2 diabetic parents. The Journal of Clinical Investigation, 2005, 115 (12):3587-3595.
[12] Danielsson A, Ost A, Lystedt E, et al. Insulin resistance in human adipocytes occursdownstream of IRS1 after surgical cell isolation but at thelevel of phosphorylation of IRS1 in 1 type 2 diabetes. The FEBS Journal, 2005, 272(1):141–151.
[13] Li S Q, Zhou Y, Wang Y, et al.Upregulation of IRS-1 Expression in Goto-Kakizaki Rats Following Roux-en-Y Gastric Bypass Surgery: Resolution of Type 2 Diabetes? Tohoku J Exp Med, 2011, 225(3):179-186.
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