The Preliminary Study On The Underlying Molecular Mechanisms Of P-glycoprotein Regulating Insulin Secretion In Rat Pancreatic Beta Cells

Objective Previous studies have indicated that rat P-glycoprotein(encoded by abcb1b)promoted release of insulin granules.It has been suggested to be a regulatory protein together with chloride channel protein 3(ClC-3)and calcium channel protein mediating insulin granules acidification,priming and release.In order to prove the hypothesis,we used several cellular and molecular biology techniques to explore the biological functions of P-gp regulating insulin secretion.Methods(1)Rat islets were isolated from pancreas by collagenase V digestion.(2)A insulinoma cells(INS-1)was cultured.(3)Use the designed abcb1b siRNA to down-regulated the expression of P-gp.Transfection efficiency was evaluated by fluorescence microscope.Real-time PCR and western blot were applied to determine the effects of siRNA.(4)A Carl Zeiss confocal microscope and whole cell patch clamp were used to investigate the calcium influx.The INS-1 cells were stained by dye DND-160 to evaluate the insulin granular pH with a PTI set up.Whole cell patch clamp were employed to measure the insulin granules release.An apoptosis kit was used to detect cell apoptosis by a fluorescence microscope.(5)Islets were treated by CsA(1 ?g/ml)for 24 hours.After incubations Rhodamine123 uptake experiments was used to evaluate the P-gp's efflux function.(6)After the incubations of INS-1 cells with different concentrations of glucose and insulin,real-time PCR,western blot and Rhodamine123 uptake were explored to analyze P-gp transcription,expression and efflux functions.Results(1)90%islets and INS-1 cells showed red-(islet)and green-(INS-1 cells)fluorescence 48 hours after the oligonucleotides(the silencer to the abcb1b and the control oligonucleotides)transfection,respectively,which indicated the RNAi was successful.The expression of P-gp was down-regulated both in the mRNA and protein level.(2)Compared to the blank group and negative control group,the[Ca2+]i of the group of siRNA was decreased significantly and do not interfered by the isradipine further.The pH of insulin granules was increased in the siRNA group,and both the first and the second phase of insulin secretion reduced significantly compared to those of the other groups.The fluorescence microscope showed that no significant apoptosis were observed in the siRNA group.(3)The efflux function of P-gp was decreased after the incubation of CsA(1.0?g/ml)for 24 hours,but the expression of P-gp was not changed accordingly.(4)The expression of abcb1b mRNA remarkably decreased following the increase of glucose concentrations in a time-dependent manner,in particularly the groups incubating with 16.7mmol/l and 22.2mmol/l.The expression of abcb1b mRNA changed with insulin concentrations,it decreased gradually with the increase of insulin concentrations when the culture medium containing 5.6mmol/l glucose,but was increased when the medium containing 11.1mmol/l glucose.The expression of P-gp was consistent with the changes in the transcription level in the glucose incubations,but not showed in the insulin incubations.The efflux function of P-gp decreased opposite to the increase of glucose inoculation,but not to insulin inoculation.Conclusion(1)After down-regulated the expression of P-gp RNAi,the calcium influx was decreased and the L-type calcium channel proteins may involved in these processes.P-gp might be a regulatory unit to the ClC-3 protein mediating insulin granular acidification which plays a crucial role to the second phase of insulin granules secretion.The decrease of insulin secretion regulated by P-gp is not due to apoptosis.But in our study,we lack direct evidences of the interaction between the P-gp,L-type calcium channel and the chloride channel protein 3(CIC-3).These needs further investigation.CsA can depress the efflux function of P-gp,but not influence the expression of P-gp,whether CsA decrease the second phase of insulin secretion through P-gp pathway is not excluded in this study.Glucose,but not insulin can inhibit the expression and the efflux function of P-gp in dose-,time-manner.Although the molecular mechanism needs further investigation,we can speculate that P-gp may contribute to the pathogenesis of type 2 diabetes.