Effects Of Human Islet Amyloid Polypeptide On Autophagy And Underlying Mechanism Of Autophagy Acitivation By Rosiglitazone In ? Cells

Objective:Islet amyloid deposition is a common pathological feature in patients with type 2 diabetes mellitusand closely related to ? cell failure.Human islet amyloid polypeptide(hIAPP)is the main component of islet amyloid.In this study,we explored the effects and underlying mechanisms of hIAPP on autophagy in rat pancreatic INS-1 cells,the relationship between autophagy and hIAPP-induced toxcity.Furthermore,we investigated the role of autophagy in the protective effects of rosiglitazone that alleviated hIAPP-induced toxicity in ? cell.This study will enrich our understanding of the underlying mechanisms of hIAPP-induced cytotoxicity and protective effects of thiazolidinediones on ? cell.Methods:1.Effects of hIAPP on autophagy in INS-1 cells: 1)To examinethe effects of hIAPP on autophagy in INS-1 cell,transmission electron microscopy analysiswas preformed to observe autophagic vacuoles and western blot was used to measure the levels of autophagic marker LC3-? and p62 in INS-1 cells.2)To evaluated the role of ROS in hIAPP-induced autophagy,as well as the effects of hIAPP on AMPK phosphorylation and whether ROS involved in this process.We measured intracellular ROS levels by DCFH-DA assay and the protein contents of LC3-? and phospho-AMPK(Thr 172)by western blot.INS-1 cells were separately treated with AMPK activator AICAR or inhibitor compound C.Then the protein levels of LC3-? and phospho-AMPK(Thr172)were measured to evaluate the role of AMPK in hIAPP-induced autophagy.2.Effects of autophagy on hIAPP-induced cytotoxicity: To investigatethe effects of autophagy on hIAPP-induced cytotoxicity and oxidative damage in INS-1 cells,autophagy was inhibited by 3-MA or modulated through AMPK signaling pathway by compound C or AICAR.Then we examined cell viability by MTT assay,ROS by DCFH-DA assay and MDA by TBA method.3.Rosiglitazone alleviated hIAPP-induced toxicitythrough activation of autophagyin INS-1 cells.1)Cell viability was determined by MTT assay to explore the effects of rosiglitazone on hIAPP-induced toxicity.2)To valuated effects of rosiglitazone on autophagy in INS-1 cells,the number of Intracellular autophagosomes was counted by electron microscopy and the amount of LC3-? and p62 were measured by western blot.3)AMPK was inhibited by compound C,then LC3-? and phospho-AMPK(Thr172)were measured by western blot to evaluate the role of AMPK in rosiglitazone-induced autophagy.4)Autophagy of INS-1 cell was inhibited by 3-MA or compound C,MTT assay was carried out to evaluate the role of autophagy in the protection of rosiglitazone.Results:1.Treatment with hIAPP resulted in increase of the number of autophagsomes,upregulation of LC3-?,decrease of p62 in INS-1 cells.NAC decreased ROS generation and protein levels of LC3-? and phospho-AMPK(Thr172)in hIAPP-treated INS-1 cells.AICAR upregulated the levels of LC3-? and phospho-AMPK(Thr172)in INS-1 cells.AICAR or hIAPP induced LC3-? upregulation were both inhibited by compound C.2.Incubation with hIAPP(10um)for 24 hours significantly reduced the INS-1 cell viability to 55.18 + 7.73%(P<0.01,versus control group).3-MA enhanced cell viability loss(P<0.05),cellular ROS(P<0.01)and MDA(P<0.05)accumulation in hIAPP-treated INS-1 cells.AICAR suppressed hIAPP-induced cell viability loss(P<0.05),the increase of ROS(P<0.01)and MDA(P<0.05)in INS-1 cells,these protective effects of AICAR were reversed by inhibition of autophagy.3.Rosiglitazone alleviated cell viability loss(P<0.05)in hIAPP-treated INS-1 cells.Rosiglitazone increased the number of intracellular autophagsomes,the amount of LC3-? and phospho-AMPK(Thr172)in INS-1 cells.Compound C suppressed the levels of LC3-? and phospho-AMPK(Thr172)which were both upregulated by rosiglitazone.Inhibition of autophagy by 3-MA or compound C suppressedprotective effects of rosiglitazone on hIAPP-treated INS-1 cell.Conclusion:1.Autophagy is activated by treatment of hIAPP in INS-1 cells.ROS-mediated AMPK activation is involved in hIAPP-induced autophagy.2.Inhibit of autophagy aggravates hIAPP-induced toxicity and oxidative stress in INS-1 cells.3.Rosiglitazone protects INS-1 cells from hIAPP-induced cytotoxicity.Rosiglitazone induces autophagy through AMPK activation in INS-1 cells.The protective effects of rosiglitazone on hIAPP-treated INS-1 cells are autophagy-dependent.