Formation Of IAPP-derived Islet Amyloid And Cytotoxicity Of Three Different Islet Amyloid Type To Cat Islet Cells In Vitro

Objectives:Islet amyloid (IA) deposits and P -cell continuate decrease are the common characters in type 2 diabetes. Islet aniyloid polypeptide (IAPP), also named as "amylin" is the component peptide of amyloid deposits found in islet. A growing of reports show that IA plays an important role in 'the pathogenesis of type 2 diabetes: the formation of IA is a diabetogenic factor, and IA is a consequence of insulin resistance and a cause of insulin insufficiency as well. IA plays a "center role"in P -cell failure of type 2 diabetes. Amyloid deposits do not appear to be inert in vivo, but rather are in a dynamic state of turnover and can even regress if the formation of fibril is halted. Studies on the formation of IA and its cytotoxicity to P -cells are particularly promising in a clinical point of view. Using fihine pancreatic islet cells in vitro, this study involved in the correlation between the formation of IA and the ratio of IAPP/insulin (IAPPIIns), the mechanisms of cytotoxicity of different IA products, including amorphous (Am), IAf (islet amyloid fiber) and IAPP modified with advanced glycatin and products (AGEs-IAPP).Methods:1. Isolated pancreatic islet cells from cat were cultured as a form of monolayer or tissue predigest in vitro. The cells of glucose-induced IA formation were detected with thT fluorescent dye, Congo red stain and transmission electric microscope, respectively. The quantity of IA formation was evaluated as thT fluorescence intensity monitored by ACAS57O. The level of IAPP and insulin in culture medium were detected with radioimmunoassay and the molar ratio of IAPP/Ins was calculated. 2. The function of Am formed cells maintained in high glucose medium for 10 days were identified by glucose load test. F-actin and [Cali were measured with phalloidine rhodamin and Fluo-3 assays, respectively. The membrane fluidity and [Ca2~ji were monitored with fluorescence probe of DPH (l,6-dihenyl?,3,5-hexatriene) and Fluo-3. Themechanisms of [Ca2~]i changes were also explored. There groups were set as follow: Calcium channel blocker group, non-Ca2~ medium group and soluble cholesterol therapy group with 1011 m IAPP. With preparation and identified AGEs-IAPP, MTT assay was used to detect the mitochondria activity after islet cells exposed to serial dilution of various AGEs-IAPP 24 hours.Results:!. The high glucose-induced IA was identified in cultured cells for 10 days with three different assays. The transmission electric microscope approved the product of IA. is Am. The fluorescence intensity of thT depended on the molar ratio of IAPP/Ins, but not glucose concentration. 2. The secrete function of Am formed cells were significantly insufficient compared with the other two controls (p