The Experimental Study Of Apoptosis Evoked By Amylin In Primary Culture Rat Islet Cells

[objective] Type 2 diabetes mellitus is characterized by islet amyloid deposits and decreased islet 6-cell mass. Amylin which secreted from the pancreatic beta-cell is the major and unique component of islet amyloid. While it is now widely accepted that abnormal aggregation of amylin has a role in 6-cell death in type 2 diabetes mellitus,the mechanism remains unknown.The main objective of this study include:to obseve the propensity of pure synthetic human amylin to aggregate and form fibrils in vitro;to confirm the apoptosis evoked by huaman amylin in primary cultured rat islet cells;to clarify the role of Ca2+ in the path way of apoptosis induced by huan amylin in islet cells;to observe the effect of some agents on huaman amylin cytotoxcity in islet cells.[method] (l)The feature of fibrils derived from the human amylin were viewed and photographed with TEM;125I-HA precipitation assay was developed to determinate the time-dependent aggregation of human amylin. (2) The cell viability were determined by the reduction of MTT and the calcein-AM / ethidium homodimer-1 (EthD-1) double-staining method independently.(3) The cellular morphologic changes were determind by electron microscope,DNA fragmentation was analyzed with agarose gel electrophoresis and hochest-staiming.(4) The fura-2 /AM-staining and the uptake of 45CaCl2 were used to clarify the role of Ca in the apoptosis .(5) The effects of some agents on the apoptosis were also analyzed by MTT reduction.[results] ( 1 ) Human amylin can aggregate into amyloid fibrils spontaneously in aqueous HA solutions,the extent of fibril formation shows time-dependent.(2) The exposure of islet cells to synthetic HA evoked both concentration- and time-dependent cell apoptosis.(3)With EM,following 1 h of HA exposure,islet cells displayed a noticeable decrease in the abundance of cell surface microvilli and there was evidence of cell surface blebbing for the first time. After 6 -12h of HA exposure,the cultured cells exhibited chromatin margination,at the same time DNA laddering becomes evident. After 22 h of exposure to HA,cells showed cytoplasmic vacuolization,and margination of chromatin with nuclear shrinkage and convolution of the nuclear membran. Agglutination and margination of chomatin can also be find by Hochest- staining.(4)Amylin evoked apoptosis in islet cells with neither the changes of [Ca2+];nor the increased uptake of excelluar Ca2+;both A23187 and Tg inducedapoptosis in islet cells either with the presence or theabsence of HA,confirming that increased [Ca2+];itself can elicit apoptosis in islet cells. Ca2+ channel blocker verapamil,the Ca2+ chelator EGTA cytosolic Ca2+ buffer,-BAPTA/AM failed to inhibit apoptosis evoke-d by HA.(5)The protein synthesis inhibitor-cycloheximed,the RNA synthesis inhibitor-actinomycin D and the endonuclease inhibitor- aurintricarboxylic acid (ATA) all inhibited amylin islet toxicity. We failed to find significant effect of sexsteroids,dexameth,glucagons,glibenclamide and glucose of high Concentration on apoptosis in islet cells induced by human amylin.[conclusion] (l)Human amylin formed into fibrils in vitro,evoked time-andConcentration-dependent apoptosis in rat primary cultured islet cells.(2)Amylin do not induce apoptosis in rat islet cells through the Ca-path way.(3)Human amylin evoked apoptosis in rat islet cells by the actvition of non-Ca-dependent endonuclease and synthesis of protein and RNA.(4) Sex steroids,dexameth,glucagons,glibenclamide and glucose of high Concentration all have no significant effect on apoptosis in islet cells induced by human amylin.