Effect Of Pericentrin On Pancreatic ?-Cell Insulin Secretion And The Molecular Mechanism

Background:The regulation of insulin secretion included the proximal step(second messenger)and the final step(exocytosis).At present,the study on the regulation mechanism of the proximal step was relatively clear,but there were few studies on the mechanism of insulin secretion in final step,and the mechanism is not clear enough.The recent studies showed that pericentrin(PCNT)was rich in the cytoplasm of pancreatic beta cells,and inhibition of PCNT lead to lower content of insulin in beta cell cytoplasm.The PCNT might regulate insulin secretion at final step through cytoskelet.However the effect of PCNT on the insulin secretion of beta cell was still unclear,and the mechanism of regulating insulin Vesicles secretion was still to be explored.In this study,we constructed transgenic mice to inhibit the expression of PCNT in pancreatic beta cells for further investigating the effect and mechanism of PCNT on insulin secretion and its role in the development of impaired glucose tolerance.We also explored if any drug could regulate insulin secretion through PCNT.Objectives:Through the ?PCNTk? mice and IR mice modle,the present study aims to:1)To observe the effect of PCNT on impaired glucose tolerance and insulin secretion;2)To investigate the change of insulin vesicle distribution in pancreatic beta cells and the mechanism of abnormal insulin secretion after inhibition of PCNT;3)To evaluate the role of PCNT on impaired glucose tolerance and insulin secretion with the extension of high-fat feeding.4)To explore the mechanism of insulin secretion regulated by GLP-1 in final step through interaction with PCNT.Methods:Two kinds of mouse models and MIN-6 cells were used.Mouse model of PCNT reduction specifically in ? cells(?PCNT?)was built by Tet-on induction system;impaired glucose tolerance model(IR)was built by high-fat feeding.Glucose tolerance,insulin secretion,the distribution of intracellular insulin granules,pancreas PCNT,insulin and F-actin expressions were compared among control,?PCNT? and IR mice at baseline,4 weeks,12 weeks by WB,IF,ELISA and transmission electron microscope.MIN6 cells were divided into control(LG,HG),exenatide group(Exn100 nm)and repaglinide group(Rep50nm),Si-PCNT group and Si-PCNT+Exn group.PCNT,Insulin,F-actin expressions were observed by confocal microscopy at baseline,15 min,30 min,2 h and 6 h.Results:1)Compared with control,?PCNTP mice had lower level expression of PCNT in the pancreatic islets,similar fasting glucose level,but higher fasting insulin level at baseline.But there was no difference in the level of cell proliferation between the two groups.2)While after intraperitoneal glucose injection(IPGTT+GSIS),?PCNT? mice showed higher blood glucose area under the curve(AUCGlu0-15)(P<0.01)and lower insulin area under the curve(AUCINS0-15)(P<0.05)at 0-15 min.3)Lower F-actin level,and reduced insulin granules density(0-300 nm under the cyto-membrane)were detected in ?PCNT? mice(P<0.01).4)The expression of PCNT decreased gradually with the extension of high-fat feeding.After fed with high-fat diet for 4 weeks,compared with control,PCNT expression of IR mice reduced and AUCGLU0-15 increased significantly(P<0.05).But compared with ?PCNT?+IR mice,PCNT expression of IR mice and AUCINS0-15(P<0.01)increased while AUCGLU0-15 reduced significantly(P<0.05).After fed with high-fat diet for 12 weeks,the PCNT expression of IR mice dropped to that of?PCNT? mice level,and there were no difference in AUCs between these two groups.5)Compared with HG and LG control,the PCNT,insulin and F-actin expressions of MIN-6 cells in Exn group decreased obviously at 15min,30min and 2h.While this three proteins in Exn group dramatically increased after 6 h compared with HG control.No difference could be found between Rep group and HG control.6)The RNA interference experiment showed that obvious decrease in intracellular F-actin and INS level were observed in siPCNT compared with the control(P<0.01).Exenatide could not change the PCNT?insulin and F-actin expressions of Si-PCNT group.Conclusions:1)PCNT could adjust insulin secretion(GSIS 0-15min)by changing insulin granules density(0-300 nm under the cyto-membrane)through F-actin.2)The decrease of PCNT expression in pancreatic ? cell is closely related to the occurrence and development of impaired glucose toleranc.3)The PCNT expression decreased in pancreatic ? cell with the extension of high-fat feeding.PCNT played an important role in the development of glucose tolerance induced by high-fat diet.4)GLP-1 had two-way regulating effect on beta cells that GLP-1 promote insulin secretion by suppressing the expression of PCNT at early stage(<2 h)and prevent excessive insulin secretion by promoting PCNT expression at late stage(>6 h).