Overexpression Of P-glycoprotein Affects The Biological Phenotype Of Islet β Cells-a Preliminary Study

Objective Our previous study has shown that overexpression of P-glycoprotein(P-gp)in the pancreatic beta cell(INS-1)promotes high glucose-stimulated insulin secretion(GSIS)and did not affect biosynthesis of insulin,but the underlying mechanism is not clear.In this study,the mechanism is preliminarily evaluated by analysis of beta cell differentiation,proliferation and apoptosis,as well as GSIS,both in vitro and in vivo.Methods(1)INS-1 cells and SD rats were used in our experiment.(2)Adenovirus containing P-gp coding gene,abcb1 b,was transfected to INS-1 cells in a serum-free medium at a concentration of 50×10~6 /m L.After 4 hours,the culture media were replaced to normal medium(1640 medium supplemented with 10% fetal bovine serum,100 U/m L penicillin,100 U/m L streptomycin,50?μm b-mercaptoethanol).The cells were incubated for further 44 hours.(3)Total protein,cytoplasmic protein and plasma membrane protein of the cells were extracted for western blot to detect P-gp,calcium channel(Cav1.2),caveolin-1(CAV1),PKA,p-CREB,CREB,SNAP25,STX1 A,SYT1,GAPDH or tubulin,respectively.(4)Potential interaction between CREB and Cav1.2 gene promoter was detected by chromatin immunoprecipitation(Ch IP).(5)Interaction between P-gp and CAV1,P-gp and SNAP25 were detected by co-immunoprecipitation(Co-IP).(6)Pancreatic beta-cell conditional transgenic rat overexpressing P-gp was constructed by random insertion of the gene(Ins2-Abcb1b).(7)Islets of rat were hand-picked from the pancreas digested with collagenase in vitro,and GSIS test was performed after overnight culture of the islets in the normal culture media without β-mercaptoethanol.Insulin was measured by ELISA.(8)Total RNA of the cells and islets was extracted for real-time quantitative fluorescent PCR to detect the expression of different genes.(9)Pathologic section of pancreas was prepared to detect the expression levels of P-gp,caspase3,caspase8,caspase9,bcl-2,ki67 by immunohistochemistry application.TUNEL was used to examine apoptosis of the islets.Results(1)Compared to the control group(transfected by adenovirus only,termed as Ad-control),expression of total amount of the calcium channel protein Cav1.2 was increased in the P-gp overexpression group(transfected by adenovirus containing P-gp coding gene abcb1 b,termed as Ad-abcb1b),but the cell membrane/cytoplasmic ratio was not changed significantly(P>0.05).(2)Expression of PKA and the phosphate level of CREB in the Ad-abcb1 b group was significantly increased than that of the Ad-control(P<0.05).(3)There were interactions between CREB and Cav1.2 gene promoter in INS-1 cells,and the binding level was increased responded to overexpressed P-gp(P<0.05 vs.Ad-control group).(4)The expression levels of CAV1 and SNAP25 were increased in the Ad-abcb1 b group(P<0.05 vs.Ad-control group),whereas STX1 A and SYT expression was not influenced(P>0.05 vs.Ad-control group).P-gp interacts with CAV1 and SNAP25,and the interaction between P-gp and CAV1 was increased(P<0.05),while the interaction with SNAP25 was decreased(P<0.05).(5)Compared to the Ad-control group,the transcription levels of Ngn3 and Mafb were up-regulated in INS-1 cells(P>0.05),whereas Mafa was down-regulated dramatically(P>0.05).No significance was detected in other transcription factors.Unlike INS-1 cells,none of transcription factors detected from the isolated islets were affected with the expression level of P-gp(P>0.05).(6)In vivo study had shown that there was no significance in GSIS between transgenic rats and wild type rats(P>0.05).Immunohistochemistry results indicated that the expression levels of caspase3,caspase8,caspase9,bcl-2 and ki67 of islet were not changed in the transgenic rats(P>0.05 vs.wild type).TUNEL apoptosis analysis showed a consistent result as above(P>0.05).Conclusion P-gp enhances insulin secretion of pancreatic beta cell line(INS-1)associated with up-regulating expression of L-type calcium channels protein Cav1.2,but not disturb this channel protein’s membrane trafficking.P-gp may mediate Cav1.2 transcription via PKA/CREB pathway.On the other hand,P-gp not only synchronizes with expression of CAV1,SNAP25,but also interacts with CAV1 and SNAP25,respectively.It is reasonable speculated that P-gp binds CAV1 in higher affinity might replace the interacted SNAP25 on P-gp,and to expose the binding domains of SNAP25 which are critical for insulin granule priming and docking.Overexpression of P-gp may dedifferentiate INS-1 cells,but has not in islets isolated from rat.Meanwhile,islets isolated from transgenic mice do not present similar phenotype concerning beta cell proliferation,differentiation and apoptosis,as well as insulin secretion.Even though the transgenic rat should have unforeseeable shortages,uncoordinated findings between cell line and isolated islets still warn us to be cautious when we try to apply experiments in vitro to explore pathophysiology of insulin secretion in vivo.