The Molecular Mechanisms Of P-glycoprotein Regulates Insulin Secretion In ? Cells-interaction Of Lipid Rafts Associated Protein

Objective The association between P-glycoprotein and blood sugar steady has been researched more and more recent years at home and abroad.In this paper,we aimed to analysis the impact of P-glycoprotein on insulin secretion in pancreatic?cells and the underlying mechanism-interaction between lipid rafts associated proteins.MethodsTo confirm the above views,the experiments was performed at the tissue and cellular levels as follows:?1?An insulinoma cell line?INS-1 832/13 cells?and rat pancreatic islet were cultured.?2?Optimization of adenovirus infection:INS-1832/13 cells were infected with control virus or various concentrations of adenovirus containing abcb1b?5.0×10⁶,10×10⁶,50×10⁶,100×10⁶pfu/mL?for 24 h,or with the concentration of50×10⁶pfu/mL for 12h,24 and 48h.The conventional MTT assays were used to determine the toxicity of P-gp to cell.The mRNA and protein expression were analysed by Q-PCR and western blot respectively.The efflux function of P-gp was determined by rhodamine-123 accumulation assays.The expression of Bcl2 and Cleved-caspase3were detected by western blot.?3?The genes of mdr1 and insulin synthesis were analysed by Q-PCR,including pdx1,ins1 andins2.?4?Expression levels of apoptotic proteins?Bcl2 and Cleved-caspase3?,P-AMPK,P-PKA,P-CREB and lipid rafts associated proteins,including caveolin-1,L-type calcium channels protein?Cav1.2?,the K_ATPTP channel subunit6.2?Kir6.2?and adenylate cyclase8?AC8?were detected by western blot.?5?The activity of cAMPand was insulin secretion in glucose stimulated insulin secretion test were detected by ELISA methods.?6?The relationships between proteins were detected by co-immunoprecipitation.?7?Determination of calciumsignals in INS-1 832/13 cells were analysed by Confocal laser technology.Results?1?MTT results showed that cell activity with different concentrations of abcb1b after 24 h was not affected obviously?p>0.05?compared to the control group.The expression of P-gp was significantly increased compared to control group both at mRNA and protein levels?P<0.01?,and the P-gp expression in cell membrane was also increased.The efflux function of P-gp in the Ad-Abcb1b group was enhanced?P<0.01?.?2?The expression of Bcl2 and Cleved-caspase3 had no change?p>0.05?,indicating that the impact of P-gp on insulin secretion is most unlikely to relate to apoptosis.?3?Q-PCR showed that the genes of insulin synthesis including pdx1,ins1 and ins2 had no change?p>0.05?.Results showed that adenovirus-mediated overexpression of P-gp in the 832/13 cells markedly enhanced GSIS in INS-1 832/13cells?P<0.05?,however,there has no significanct influence on basal insulin secretion and cellular insulin content.?4?The protein expression of Kir6.2 had no change compared to the control group?P>0.05?,while,the expression of Cav1.2 and Cav-1were significantly increased,and the Cav1.2 expression in cell membrane was also increased.?5?Moreover,we found a direct interaction between p-glycoprotein and caveolin-1,caveolin-1 and AC-8,caveolin-1 and Kir6.2,one of the K_ATPTP channel subunit.,which leads us to speculate P-gp might regulate insulin secretion through lipid raft associated proteins.?6?The activity of AC8,cAMP,PKA and CREB were increased,while the activity of AMPK was reduced?P<0.05?,which leads us to assume that P-gp regulates insulin secretion might through cAMP-PKA-CREB-Cav1.2 signal path.?7?Confocal laser technology showed that calcium signals in abcb1b group were enhanced?P<0.05?.Conclusion Adenovirus-mediated overexpression of P-gp in the 832/13 cells markedly enhances GSIS in INS-1 832/13 cells,which is characterized by stimulated insulin secretion enhancement,but no change in the basal insulin secretion and the synthesis of insulin.It is reasonable to speculate that P-gp mediates insulin secretion partially via the effect of lipid raft associated protein on Cav1.2 cell membrane transport as well as the activation of cAMP-PKA-CREB-Ca²⁺1.2 pathways.