Exendin-4 Protects Against Dexamethasone-induced Cell Death In Insulin Secreting MIN-6 Cells Via AKT/PKB And PKA

OBJECTIVE: To explore the possible effect and mechanism of Extendin-4 on dexmethasone-induced Apoptosis in murine MIN6 pancreaticβ-cells.METHODS : To establish the cell apoptosis model by choosing the Murine MIN6 pancreaticβ-cells as the object of the study, which was cultured in vitro and induced by dexamethasone at various concentration and for different time period. Cells were pretreated with Exendin-4 (100 nmol/L) prior to exposure to dexamethasone. The percentage of cell viability was determined through MTT assay.The percentage of apoptosis was determined through fluorsecence microsopic analysis after hochest/PI staining and flow cytometric assay after Annexin-Ⅴ-FITC-PI staining. The content of caspase-3 was measured by the caspase-3 activity assay kit. The expression of cyt-c,bcl-2,bax,AKT,p-AKT was detected by western-blot.RESULTS: The 4 times analysis results of of MTT assay reveal that after exposure to dexamethasone with concentration ranging from 50~800nmol/L for 1-4d, the survival rate of MIN6 cells was significantly reduced. Hochest/PI staining proved that when treated by dexamethasone with concentration over 100nmol/L for 48 hours, the apoptotic rate of MIN6 cells was significant increased, reaching up to 30.44±4.52%,(P<0.01 compared with control group). So did the analysis result of flow cytometric assay after Annexin-Ⅴ-FITC-PI staining. After exposure to dexamethasone(100nmol/L)for 72h the activity of caspase-3 increse significantly,(P<0.01 compared with control group). After exposure to dexamethasone at a concentration ranging from 50~800nmol/L for 48h,With the concentration increased, the expression of cyt-c increase,bcl-2 and p-AKT decrease; while bax and T-AKT remained unchanged. Exendin-4 protects against glucocorticoid-induced apoptosis, an effect mimicked by forskolin and reversed by PKA inhibitors H89 and PI3K inhibitors wortmannin.CONCLUSION:Murine MIN6 pancreaticβ-cells apoptosis could be induced by dexmethasone, probably via activating pathway of AKT and down regulating the expression of BCL; while such dexmethasone-induced Murine MIN6 pancreaticβ-cells apoptosis could be reversed by exendin-4, probably via activating signaling pathway of PI3-K-AKT and CAMP-PKA and up regulating the expression of bcl-2.