Intracellular Pattern Recognition Receptors Induce Pancreatic Beta Cell Dysfunction

Objective:Intracellular pattern recognition receptors(mainly TLR3 and RIG-I)involve in the multiple pathogenetic processes of inflammatory-associated diseases including Type 2 diabetes.Recent studies have suggested that TLR3 and RIG-I are closely related to the insulin resistance of periphery tissue in type 2 diabetes.However,role of TLR3 and RIG-I in the central tissue pancreatic beta cells remains unclear.This study aims to explore the mechanism underlying TLR3 and RIG-I which induce the proliferation inhibition of pancreatic ? cells and their functional decompensation.Methods:High carbohydrate and high fat(16.7 mM glucose + 0.4 mM palmitic acid)or inflammatory-related factors were utilized in mouse pancreatic ? cell line MIN-6 and NIT-1.And islets of db/db mice were extracted to build up pancreatic ? cells as diabetes models in vivo.Quantitative real-time RT-PCR and western blot were applied to detect the variation of TLR3 and RIG-I in the inRNA and protein levels after stimulation of glucolipotoxicity or inflammatory-related factors.Poly(I:C)and retinoic acid were utilized to activate TLR3 and up-regulate RIG-I function respectively.The growth activity of pancreatic ? cells,cell proliferation and the variation of the cell cycle were measured by MTT assay,EdU fluorescence labeling and flow cytometry,respectively.Quantitative real-time RT-PCR and western blot were applied respectively to test the variation of cyclin D1,D2,D3 and E,p18,p21 and p27 as well as the change of the signal molecular Skp2 in the mRNA and protein levels.Results:Glucolipotoxicity or inflammatory-related factors could dramatically up-regulate TLR3 and RIG-I of pancreatic ? cells in protein level.The growth activity and proliferation ability of pancreatic beta cells decreased accompany with up-regulation of TLR3 or RIG-I function.Simultaneously,the cell cycle arrested in G1 phase which could be obviously reversed by the specific siRNA targeting down-regulation of TLR3 or RIG-I function.The activation of TLR3 could induce the remarkable decrease of cyclin D1 and D2,and this effect could be antagonized by the pretreatment of proteasome inhibitor MG132.Up-regulating of RIG-I function could dramatically promote the P27 protein level which could be reversed by the functional enhancement of ubiquitin-ligating enzyme Skp2.Conclusions:Glucolipotoxicity and inflammatory-related factors which are considered as the risk factors of Type 2 diabetes can obviously up-regulate the innate immunity signals TLR3 and RIG-I in pancreatic ? cells.They can down-regulate the growth activity of pancreatic ? cells,induce the cell cycle arrest in G1 phase and inhibit the cell proliferation to various degrees.TLR3 also can induce the down-regulation of cyclin D1 and D2 which are closely associated with the inhibition of ? cell proliferation.The decrease of cyclin Dl and D2 mainly lies in the ubiquitin-proteasome mediated protein degradation instead of their inhibition in the transcription level.The inhibition of pancreatic ? cells proliferation induced by RIG-I suggests the closely relationship with the up-regulation of cycling protein P27 function.The P27 induced cell cycle arrest can be regulated by down-regulation of Skp2.This study suggests that intracellular pattern recognition receptors TLR3 and RIG-I involve in dysfunction of pancreatic(3 cells in the organism by inhibition of islet ? cells proliferation,which can induce their relative depletion in numbers and functional decompensation.The studies provide new drug targets for clinical treatment to Type 2 diabetes.