Mechanism Underlying Functions Of Pancreatic Beta Cell Regulated By PKR Subunits

Objective:Intracellular pattern recognition receptor PKR is involved in the pathogenesis of inflammatory diseases such as type 2 diabetes mellitus.The studies showed that PKR was closely associated with insulin resistance of peripheral tissues in type 2 diabetes mellitus.However,it is not clear that the regulation of pancreatic beta cell proliferation and its related functions.The aim of this study was to investigate the role of PKR in the regulation of cell proliferation and related functions of islet beta cells.Methods:Islet beta cells were stimulated by high glucose and high fat or inflammatory factors,and the islet beta cell model of type 2 diabetes mellitus was constructed.PKR,PKR-K296R or GyrB-PKR-K296H fusion gene plasmid was used to transfect mouse pancreatic beta cell line Min6 to construct cell models of PKR specific catalytic and substrate binding activity.Low concentration of BEPP,glucolipotoxicity,or proinflammatory factor TNF-α treatment can upregulate related functions of PKR.Western blot detects the levels of protein and its phosphorylation.Co-Immunoprecipitation analysis the changes of protein combination;thiazole blue(MTT)method,EdU fluorescence labeling and flow cytometry evaluate islet beta cell growth,cell proliferation and cell cycle,respectively.Realtime quantitative RT-PCR was used to measure the mRNA levels The luciferase reporter gene(Luciferase Assay)was used to study the transcriptional activity.At the same time,transfection of specific small interfering RNA,could downregulate PKR upstream signaling molecules,and explore the mechanisms underlying PKR molecular structure and function.Results:the predisposing factors of type 2 diabetes can lead to the increase of PKR substructural function---catalytic activity and substrate-binding function.In the PKR beta cells,low concentrations of BEPP,TNF-α and glucolipotoxicity can specifically induce PKR activation and eIF-2a phosphorylation,inhibition of beta cell proliferation and the cell cycle in the G1 phase in kinase activity dependent manner.Increased PKR activity can lead to an increase in P53 protein levels and its interaction with Ubc9.Ubc9 can be used as SUMO E2 ligase to induce SUMO modification of P53.Ceramide synthase inhibitor FUMO B1 or PKR inhibitor 2-AP can inhibit PKR activation by downregulating ceramide accumulation,and attentuate the SUMO modification of P53 and its related biological functions,and then promote islet cell survival,G1/S transition and cell proliferation.Substrate binding function of inactive PKR could increase the mRNA and protein levels of c-Myc;TRAF2 gene silencing can downregulate c-Myc and beta cell proliferation induced by protein binding function of PKR protein with obvious antagonistic function.Conclusion:the risk factors of type 2 diabetes mellitus---glucolipotoxicity or inflammatory can significantly increase PKR kinase activity and its protein binding function in pancreatic beta cells.The different substructure can regulate growth and proliferation of pancreatic beta cells.PKR induces the increase of SUMO dependent protein level and the inhibition of cell proliferation by P53,and is closely related to the second messenger ceramide.Its protein binding function leads to the enhancement of c-Myc function in TRAF2 dependent manner,and promotes the proliferation of pancreatic beta cells.The study reveals the mechanism underlying pattern recognition receptor PKR and its intracellular regulation of beta cell proliferation and its related functions through different submolecular structure,and clarifys that the in vivo activation of PKR can reduce the number of beta cells and induced relative decompensation,leading to the overall function of islet beta cell disorder.The study also explains that PKR is involved in tumor cell proliferation of pancreatic islet,and provides a new way to improve the treatment of type 2 diabetes mellitus.