Studies On The Effect And Mechanism Of Wnt Signaling Pathway In Pancreatic NIT-1 Beta Cells

PartⅠEffect of Wnt3a in NIT-1 beta cells cultured in vitroObjective To establish whether Wnt signaling pathway plays a role in miceβ-cell function and survival in vitro.Methods Mice NIT-1 beta cells were cultured in glucose concentrations of 33.3 mmol/L and the cytokines interleukin-1β(IL-1β10ng/ml), interferon-y (IFN-γ50 ng/ml) and tumor necrosis factor-α(TNF-α50 ng/ml) with or without the addition of purified Wnt3a protein (100 ng/ml) in vitro for 24 h. Subsequently,β-cell apoptosis by Tunnel, FCM andβ-cell proliferation by BrdU were analyzed. Insulin secretion was determined by Radioimmunoassay.Results Incubations of NIT-1 cells with high glucose and cytokines resulted a dramatically increase inβ-cell apoptosis and decrease inβ-cell proliferation after recombinant Wnt3a protein stimulation compared to the control group. (P＜0.001).Conclusion Wnt3a regulates the proliferation of pancreatic NIT-1 beta-cell, and protected beta-cell from glucotoxicity and cytokine toxicity with respect to proliferation and apoptosis, and improve the beta-cell insulin secretion. PartⅡThe study of Wnt/beta-catenin/TCF signaling pathway in NIT-1 beta cellsObjective To explore the mechanisms of protectiveeffectof Wnt/beta-catenin/TCF signaling pathway in NIT-1.Methods Recombinant Wnt3a protein was applied to NIT-1 beta-cells to activate Wnt signaling pathway. Real-time PCR and Western Bloting were applied to study the expression levels of some components of Wnt signaling pathway, such as LRP-5,GSK3β,β-catenin,TCF7L2, and cell cycle regulators like Pitx2,CyclinD2, insulin transcriptor factor PDX-1 and glucose sensing molecules such as GLUT2,Glucokines (GK), IRS2,IRS1 and Bcl-2 were also detected.Results After the stimulation of Wnt3a protein, the mRNA expression of TCF7L2 and freeβ-catenin protein expression was increased. The mRNA expression of Bcl-2, pitx2, CyclinD2, PDX-1 and GK were also up-regulated; both the mRNA and protein level about IRS-2 were increased. By contrast, IRS-1 and GLUT2 expression was not increased by wnt3a.Conclusion Wnt3a induced the activation of canonical Wnt/β-catenin/TCF signaling pathway, stimulated the target gene expression downstream the Wnt signaling pathway, and stimulated the beta cell survival and function directly or indirectly. PartⅢThe role of IRS-2/PI3K/AKT in mediating beta-cell growth and survival induced by Wnt/beta-catenin/TCFObjective To study the role of IRS-2/PI3K/AKT involved in the stimulation of Wnt3a on NIT-1 beta cells survival and function, and to explore the biological mechanisms of Wnt signaling pathway on beta cells.Methods Wnt signaling pathway in NIT-1βcells was activated by Wnt3a protein and cell proliferation and apoptosis were determined by flow cytometry. IRS2, Phosphorylated AKT and GSK3βwere detected by Western Bloting. Then inhibitors DKK1 and Wortmannin were applied to the cells to block Wnt pathway or PI3K pathway. Cell proliferation and apoptosis were determined by flow cytometry again. The expression and Phosphorylation levels of target genes in Wnt/beta-catenin/TCF signaling pathway and IRS-2/PI3K/AKT signaling pathway were determined by real-time PCR and Western Bloting.Results Wnt3a rapidly activated Wnt/β-catenin/TCF signaling pathway, promoted Irs2 expression and stimulated AKT phosphorylation and GSK3βin NIT-1 cells. These effects were completely abrogated by inhibition, Dkkl or Wortmannin. Wnt3a also stimulated NIT-1 cell proliferation, and inhibited cytokines-induced beta-cell apoptosis, promoted the GSIS in beta cells. Both of these effects were also prevented by Dkkl and Wortmannin treatment.Conclusions These results identify IRS-2 as an essential mediator linking the Wnt/beta catenin/pathway signal to the PI3K/Akt pathway that modulates beta-cell growth and survival, and the effects of Wnt signaling on beta-cell is PI3K/Akt dependent.