Involvement Of Zinc In The Deposition Of Islet Amyloid Polypeptide

Objective:Diabetes is one of the endocrine and metabolic disorder characterized by chronic hyperglycemia.Diabetes is caused by various factor,and the pathogenesis of diabetes is complicated.In recent years,islet amyloidal polypeptide?IAPP?was found in the autopsy material of patients with type 2 diabetes,within 90%of these patients had pancreatic islet fibrous deposition accompanied by a significant reduction in the number of beta cells.Thus,it has been suggested that the pathological deposition of IAPP may be the important reason which affects beta cell survival and insulin secretion.Therefore,to explore the mechanism on pathological deposition of IAPP in pancreatic beta cells may be has important theoretical and practical significance.Several studies have confirmed that zinc ion was closely related to the synthesis and metabolism of IAPP.It has been reported that zinc ion could bind to the 18 points histidine residue of IAPP,so that promote IAPP monomer adjacent crosslinked,eventually forming a fibrous amyloid aggregation.These phenomena prompted us that the imbalance of zinc metabolism in islet beta cells may be the important reason of amyloid deposition.Regulating the homeostasis of zinc in DM patients and maintaining the dynamic balance of generation and degradation of IAPP,may become the new strategy of DM therapy.In this study,we observed the effects of zinc on islet amyloid deposition use mice and cell models,and examined the effects of zinc on IAPP synthesis and metabolism.The role of zinc on apoptosis in beta cells was also detected.Our aim was to investigate the mechanism of zinc on pathological deposition of amyloid peptide,and in order to provide experimental basis for the selection of new targets on the treatment of type 2 diabetes.Methods:In this study,hIAPP transgenic mice and INS-1 cells transfected with hIAPP gene were used to study the effects of zinc and zinc chelator on amyloid deposition,IAPP metabolism and islet beta cell apoptosis.1.One month old homozygous hIAPP transgen-ic mice were used in this study.The mice were feed with high fat and high sugar diet in order to induce type 2 diabetes animal model.The model mice were given zinc and zinc chelator?CQ?.The general state of mice was observed,the body weight and blood gluco-se were detected.Deposition of zinc was observed by AMG staining.Effect of zinc on the expression of IAPP and insulin were detected by immunofluorescence staining.Western blot was used to detect the protein levels of IAPP,insulin and IAPP related enzymes.2.INS-1 cells stably transfected with hIAPP gene were cultured.ZnSO₄ and TPEN was added into cells respectively.Zinquin staining was used to detecting zinc content in different groups.The expression levels of IAPP and insulin were observed by immunofluorescent staining.ELISA was used to detect the secretion amount of insulin in hIAPP-INS-1 cells.Effect of zinc on apoptosis of hIAPP-INS-1 cells was detected by flow cytometry.The protein levels of IAPP,insulin,enzymes related to IAPP anabolic and protein related to apoptosis signaling pathway were detected by Western blot.Results:1.Zinc affects body weight and blood glucose of hIAPP transgenic mice.The hIAPP transgenic mice fed with high glucose and high rouge appear diabetic symptoms,their blood glucose increased and body weight lost.Compared with vehicle group,the mice with excess zinc showed significant body weight loss,and the blood glucose level increased rapidly.Zinc chelator?CQ?treatment could improve the symptoms of diabetes in hIAPP transgenic mice.2.CQ can chelating zinc in islets.AMG staining results showed that a large amount of zinc deposition was observed in the islets of mice with high concentration of zinc treatment,while the expression of zinc in the pancreas of Zn+CQ group was significantly reduced.3.Zinc promoted IAPP deposition and reduced insulin expression in islets of hIAPP transgenic mice.Results of immunofluorescence double-staining showed that IAPP and insulin were co-expressed in cytoplasm of islet?-cells.IAPP fluorescence staining in islets of mice with excess zinc was increased,while insulin fluorescence staining was weakened.Compared with Zn group,in the Zn+CQ group,IAPP deposition decreased and insulin expression increased.The results of Western blot were consistent with immunofluorescence staining.High concentration of zinc increased the content of IAPP protein and decreased the level of insulin.CQ could reverse this phenomenon.4.Zinc affects the synthesis of IAPP in pancreas of hIAPP transgenic mice.Western blot results showed that compared with the vehicle group,zinc increased the content of carboxypeptidase?CPE?in the pancreas of hIAPP transgenic mice,and CQ treatment decreased the level of CPE,but prohormone convertase 1/3?PC1/3?and prohormone convertase 2?PC2?were not affected by zinc.5.Results of MTT colorimetric assay showed that ZnSO₄?50?M?and TPEN?1?M?incubated for 12h could not affect the activity of hIAPP-INS-1 cells.6.Zinquin fluorescence staining showed that ZnSO₄?50?M?increased fluorescence intensity of zinc significantly,but TPEN?zinc chelator?reduced fluorescence intensity of zinc.7.High concentration of zinc increased the expression of IAPP and decreased the expression of insulin in hIAPP-INS-1 cells.The results of immunofluorescence double-staining showed that the expression of IAPP was enhanced but the expression of insulin was decreased in the hIAPP-INS-1cells treated with ZnSO₄.But the expression of IAPP in hIAPP-INS-1 cells treated with zinc chelator was significantly lower than cells treated with ZnSO₄,and the level of insulin was increased significantly.Western blot results confirmed that excess zinc reduced intracellular insulin secretion and enhanced IAPP protein expression.Zinc chelator could attenuate the effect of zinc.ELISA experiment was also confirmed that excessive zinc reduced the ability of insulin secretion in hIAPP-INS-1 cells.8.Zinc affects the level of IAPP synthesis enzymes in hIAPP-INS-1 cells.CPE,PC1/3 and PC2are key enzymes in IAPP synthesis.Western blot results showed that high concentration of zinc significantly increaseed the level of CPE,but zinc chelator reduce the level of CPE.However,zinc and zinc chelator had no significant effect on protein content of PC1/3 and PC2.9.Flow cytometry result confirmed that zinc promoted apoptosis of hIAPP-INS-1 cells,but zinc chelators could inhibit zinc-induced apoptosis.10.ERK/JNK signaling pathway was involved in zinc-induced apoptosis.We used Western blot to detect the protein levels of P-ERK,ERK,P-JNK,JNK,P-c-jun,c-jun,and caspase-3 in hIAPP-INS-1 cells treated with zinc or zinc chelator.These results indicate that zinc activated ERK/JNK signal pathway,and increased the expression of caspase-3,so that induce islet beta cell apoptosis.TPEN could inhibit zinc-induced apoptosis.Conclusions:1.High concentration of zinc could significantly increase the synthesis of IAPP,reduce insulin secretion,and aggravate the symptoms of diabetes in hIAPP transgenic mice and hIAPP-INS-1 cells.2.Zinc treatment can significantly enhance the activity of carboxypeptidase,but does not affect the level of prohormone converting enzyme.Abnormal IAPP intermediates was increased and amyloid deposition was aggravated.3.Zinc chelator inhibits zinc-induced apoptosis.4.ERK/JNK signaling pathway was involved in zinc-induced islet cell apoptosis.