ER-Stress-Induced Autophagy Is A Protective Response To Intermittent-Hypoxia-Caused Pancreatic β-Cell Apoptosis

ObejectiveIntermittent hypoxia（IH）,the most important pathophysiologic pathway of obstructive sleep apnea（OSA）,is believed to be a potential major factor causing pancreaticβ-cells apoptosis which may play the key role in the development and progression of glucose metabolism disorder.Autophagy is a phenomenon that widely presents in eukaryotic cells,and it plays an important role in numerous hypoxic-excitotoxic conditions.Endoplasmic reticulum（ER）stress,a common cellular stress response that is triggered by a variety of conditions that disturb cellular homeostasis,is not only associated with pancreaticβ-cell apoptosis,but is also related to autophagy activation.However,the role of ER stress and autophagy in IH-caused pancreaticβ-cell apoptosis is still unclear.Thus,this study aimed to investigate the contributions of ER stress and autophagy to IH-induced pancreaticβ-cell apoptosis.Besides,the types and molecular mechanisms of autophagy induced by ER stress under IH condition were explored.This study will provide theoretical evidence for elucidating the mechanism of IH-caused pancreaticβ-cell apoptosis.Methods1.The IH models were established in vitro and in vivo.The chambers of SD rats were alternately flushed with nitrogen（30s）or compressed air（90s）,which sustained6 weeks,8 h/day（9AM to 5PM）.The chambers of INS-1 cells were alternately flushed with 1.5%O₂（5min）and 21%O₂（10min）,which sustained 4h,8h and 12h.2.Immunoblotting and immunofluorescence were used to evaluate the levels of biomarkers for autophagy and ER stress in pancreaticβ-cell,which treated by IH,TUDCA or 4-PBA.The number of autophagic vacuoles was observed by transmission electron microscopy（TEM）.3.GSK2606414 and siRNA PERK were used in INS-1 cells before IH treatment,and the expression levels of p-PERK/PERK,p-eIF2α/eIF2α,ATF4 and LC3-Ⅱwere detected by immunoblotting.4.TEM was used to observe the formation of“ER-containing autophagosomes”.Immunoblotting was performed to detect the expression level of FAM134B in INS-1cells after IH treatment and TUDCA/4-PBA treatment.5.The pancreaticβ-cell apoptosis was detected by immunoblotting and TUNEL.Pharmacology（Chloroquine,Rapamycin）and siRNA（Atg5,FAM134B）were used to intervene autophagy,and the expression level of Cleaved caspase3 was detected by immunoblotting.Results1.Compared with the normoxia control group,the expression levels of LC3-II,GRP78,CHOP and caspase12 were increased significantly,and the level of SQSTM1was decreased in IH group.The number of autophagic vacuoles was also increased after IH treatment.The ER stress was mitigated by TUDCA/4-PBA,which was accompanied with autophagy reduction.2.Treatment with the PERK inhibitor and transfection with siRNA PERK successfully reduced the level of p-PERK,p-eIF2α,and ATF4,as well as IH-induced LC3-II.3.The formation of“ER-containing autophagosomes”was observed,and the expression level of FAM134B was increased significantly after IH treatment.The level of FAM134B was descreased with TUDCA/4-PBA treatment.4.The apoptotic protein Cleaved caspase 3 and the percentage of apoptotic cells were increased with the duration of IH.The level of cleaved caspase 3 was further increased by chloroquine treatment,while it was decreased by rapamycin treatment.Knockdown of Atg5 and FAM134B also suppressed the expression of LC3-II,and promoted the INS-1 cell apoptosis caused by IH.Conclusions1.IH induces ER stress and autophagy activation in pancreaticβ-cell.2.IH activates autophagy through the ER-stress-related PERK/eIF2α/ATF4signaling pathway.3.IH-induced autophagy contains selective ER-phagy via FAM134B protein.4.Autophagy activation ameliorates pancreaticβ-cell apoptosis caused by IH.