In Vivo Stimulation of Beta-Cell Proliferation by Exendin-4 Is Mediated Through the Camp Pathway and Cyclin A2.

Glucagon-like peptide-1 (GLP-1) and its analog exendin-4 (E4) promote beta-cell proliferation and stimulate the cyclic AMP (cAMP) signaling pathway via the GLP-1 receptor. Cyclin A2 (ccna2) activates cyclin-dependent kinases essential for mitosis at two critical steps of the cell cycle. Further, the ccna2 immediate 5-prime UTR contains a cAMP-response element (CRE) site. To determine the role the cAMP signaling pathway may have on cyclin A2 expression, male C57BI/6 mice were exposed to E4 (n=4; 10 nmol/kg/day i.p.) or PBS (n=4) for 5 days. Western blot analysis of extracted islets showed a 3-fold increase in cyclinA2 immunoreactivity in the Ex4-treated islets as compared to control islets. We also examined islets from mice with constitutively upregulated nuclear cAMP signaling (CBP-S436A mutant mice). CREB-CBP interaction is disrupted by phosphorylation of CBP at serine 436. The mutated phosphorylation site of CBP-S436A mice allows CBP to remain bound to CREB, increasing basal activity of the cAMP pathway at the nuclear level. 4-month-old wild type (WT, n=2) and heterozygous (n=2) CBP-S436A female littermates were used for islet isolation. Western immuno-blot showed 2.3 fold increased cyclin A2 in heterozygous CBP-S436A islets as compared to WT islets, Immunofluorescence histochemistry of 2 WT and mutant CBP-S436A animals each showed a 2-fold increase (0A5/WT-islet (44 islets examined) versus 0.99/mut-islet (44 islets examined) in Ki67 and insulin-double positive cells in mutant islets. To verify the effect of GLP- l/Ex4 on ccna2 regulation, a 800 bp fragment of the 5-prime untranslated region of the ccna2 gene, which contains a CRE was inserted into a luciferase reporting vector (CCNA2-LUC) and used for transient transfection assays in Min6 cells (p33). Ex4 treatment for 4 hours stimulated reporter activity by 3.7+ 1.3 (mean+SEM) in MIN6 cells. In contrast, a CRE mutant of the reporter construct showed 0.7+0.2 fold acitivity upon E4 exposure. CCNA2-LUC co-transfected with CREB into Min6 cells showed a 9.3+0.8 fold activation over control, whereas co-transfection with dominant negative A-CREB reduced luciferase expression to 0.6+0.2 fold. Knockdown of cyclin A2 by siRNA in MIN6 cells reduced (approx 50%) cyclin A2 protein on Western immunoblots. This resulted in an approx. 7-11% increase in cell doubling time. We conclude that in vivo GLP-1/Ex4 simulated beta-cell proliferation is mediated by cyclic AMP pathway resulting in increased ccna2 expression. [ABSTRACT FROM AUTHOR]