Expression Of Amylin Receptors In Pancreatic β Cells And Exploration Of Their Possible Function

Purpose:Amylin,also named islet amyloid polypeptide（IAPP）,one of the major peptidal hormones in pancreaticβcell,is considered as an important regulator of local islet function.The direct target of amylin onβcells remains unclear.Three high-affinity amylin receptors which are composed of calcitonin receptor（CTR）and one of three receptor activity modifying proteins（RAMPs）have been identified.They are supposed to be the target of amylin’s effect.This study was designed to explore the expression and co-localization of three amylin receptors’components and their possible contributions to the inhibitory effect of amylin on insulin secretion and the cytotoxic effect of aggregated human amylin,discussing the possible mechanism,providing potential therapeutic target in the treatment of glycemic disorders.Methods:Expression of amylin receptor components in ratβcell line INS-1were determined by RT-PCR and western blot.Immunofluorescent staining was used to verified the colocalization of CTR and RAMP proteins on INS-1 cell membrane and the location of CTR and RAMPs in normal rat pancreaticβcells.Glucose stimulated insulin release and m RNA transcription were measured after short-time treatment of amylin or amylin receptor antagonist AC253 by ELISA and real time RT-PCR respectively.Laser scanning confocal microscope was used to monitor the intracellular fluorescence intensity changes which represent Ca²⁺concentration changes upon glucose、sulfonylurea or KCl stimulation.Amylin fibril aggregation evaluated by thioflavin-T binding and transmission electron microscopy,cell viability,Caspase 3 activity were determined in this process.INS-1 cells were treated with amylin or AC253 together or alone for a long time.Amylin fibril aggregation,cell viability,activated Caspase 3 production and Caspase 3 activity were determined in order to observe the participation of amylin receptor in cytotoxic effect.The influence of amylin and AC253 on m RNA expression of three RAMPs was observed by real time RT-PCR.Changes of RAMP1 protein expression and distribution on cell membrane were observed by western blot and immunofluorescent staining respectively.Cells were treated with RAMP1 antibody at different concentrations together with amylin for 24 hours and then MTT test was carried out so as to explore the relatively more important type of RAMP.Results:（1）Both RT-PCR and western blot analysis demonstrated that all the components of amylin receptors were expressed in INS-1 cells.Colocalization of CTR and three RAMPs on INS-1 cell membrane were further determined by double immunofluorescent staining.CTR and three RAMPs were also expressed in normal rat pancreaticβcells.（2）Glucose stimulated insulin secretion was decreased from（2.08±0.35）ng/ml to（0.73±0.24）ng/ml（P<0.01）,m RNA transcription was decreased to 0.39±0.09（P<0.01）by short exposure to amylin.Insulin release was promoted to（1.41±0.32）ng/ml,m RNA was increased to 0.62±0.08 by addition of AC253,statistically different from using amylin alone（P<0.05）.AC253 attenuated amylin’s inhibition on glucose induced increase of Ca²⁺,with average of Ca²⁺concentration and peak value increased from 650.155±4.818 and1.154±0.011 to 769.795±4.956 and1.589±0.013 respectively（P<0.01）.Similarly,the inhibitory effects of amylin on calcium influx induced by sulfonylurea or KCl were all attenuated in the presence of AC253.No amyloid fibrils were detected within 20 min,while after 2 hours,a few amyloid fibrils emerged in the solution.However,cell survival was not changed and Caspase 3 activity,which is an important apoptosis related enzyme,was not activated after incubation with amylin for two hours.（3）No changes were detected in RAMP m RNA expression after short term incubation with amylin.RAMP2 and RAMP3m RNA transcription were decreased by 23%（P<0.05）and 34%P<0.05)respectively,while,RAMP1 m RNA was slightly increased by 25%（P<0.05）after 24 hours’treatment with human amylin.Cell membrane distribution of RAMP1 was also slightly increased.AC253 had no effect on RAMP expression and it did not interfere with amylin’s influences on RAMPs.Treatment with human amylin for 24 hours significantly evoked cell death with cell viability decreased to（69.69±12.63）%（P<0.01）.Application of amylin receptor antagonist AC253 significantly promoted cell viability to（93.89±15.94）%,statistically different from cells that incubated with human IAPP alone（P<0.01）.While for cells treated for 48 hours,no obvious increase of cell survival was detected.Large quantity of amylin fibrils were detected in solution and production of activated Caspase 3 and its bioactivity were increased after24 hours’incubation with human amylin.AC253 inhibited production of activated Caspase 3 and activation of Caspase 3 activity by amylin.It did not interfere with amylin fibrils aggregation.Application of RAMP1 antibody diluted at 1:500 and1:1000 with human IAPP also significantly promoted cell viability to（94.49±12.16）%and（95.76±10.68）%respectively compared with incubation with human amylin alone（P<0.01）.Isotype Ig G had no such effect.Conclusions:Three amylin receptors were expressed in rat pancreaticβcells.Amylin receptors were involved in the inhibitory effects on insulin secretion,synthesis and regulation of intracellular Ca²⁺.Amylin receptor,especially RAMP1,participated in cytotoxicity of human amylin.