The Study Of The Expression And Functions Of The P-glycoprotein In Rat Islets

Objective A 65-kda mdrl (multi-drug resistance protein 1, P-glycoprotein)-like protein has been suggested to be a regulatory protein to chloride channel protein 3 (C1C-3) mediating insulin granules acidification and release in mouse beta cells. But the protein has not been deeply investigated. In this study, we identified existence of the 65-kda protein in rat islets and preliminarily explored its biological functions.Methods (1) Rat pancreas were digested by collagenase V through bile-duct injection and islets were isolated manually. A insulinoma cells (INS-1) were cultured. Total RNAs of the rat kidneys served as positive controls, pancreata, islets and INS-1 cells were extracted for reverse transcript PCR (RT-PCR), respectively. The cDNAs were run with specific primers selected from the mRNA of abcb1b encoding P-glycoprotein. All PCR products were visualized in agarose gel electrophoresis and sequenced. Homogenates of rat islets and INS-1 cells were applied to SDS-PAGE. P-glycoprotein was detected by a specific monoclonal antibody (C219). (2) Rat Islets were incubated with designed siRNAs to down-regulate expression of the abcb1b gene. Transfection efficiency was evaluated by fluorescence microscope, quantitative PCR and western blot. The transfected islets were incubated with high glucose (16.7mM) buffer and biphasic insulin release was measured by radioimmunology (RIA) assay. Meanwile the expression of ins1, ins2 and glucagon were also determined by quantitative PCR (3) Rat islets were incubated in RPMI1640 (with 33.3mM glucose) for 48 hours to induce apoptosis. The apoptosis were assessed both in the silenced islets and in the islets incubated with the 33.3mM glucose media by quantitative PCR. (4) Rat islets were treated by cyclosporine which is a specific inhibitor to P-glycoprotein. Comparison of insuling release between the treated islets and non-treated islets was carried out by RIA.Results (1) Compared with the positive control, expression of the P-glycoprotein mRNA segments were detected in the islets, INS-1 cells and pancreas. Sequence analysis confirmed the PCR products were matched with mRNA of P-glycoprotein. In western blot, a 65-kda protein was recognized by the antibody in the islets homogenate but not in that of INS-1 cells. Instead, the homogenate of INS-1 cells contained a 160-kda protein recognized by the antibody. (2)90% islets showed red-fluorescence 48 hours after the oligonucleotides (the silencers to the abc1b1 and the control oligonucleotides) transfection. The expression of 65kDa protein and the mRNA of abcb1b were down-regulated in the silenced islets. All of them suggested the RNAi was successful. Insulin secretion of rat islets stimulated by high glucose (16.7mM) showed biphasic curve during 60-min incubation. After the RNAi, both the first and the second phase of insulin secretion was reduced significantly. The expression of insl, ins2 and glucagon were not interfered with the RNAi. (3) Several key genes mediating apoptosis did not show any differences between pre and post-RNAi in the rat islets, whereas the apoptosis genes were changed significantly by the high glucose incubation accordingly. (4) After incubation with cyclosporine A, the second phase of insulin secretion was reduced significantly but the first phase was not influenced.Conclusion The 65-kda protein expressed in rat islets is most likely a mini-P-glycoprotein. It may play a key role regulating biphasic insulin release. The regulation may not induce by apoptosis or insulin synthesis. It might regulate the insulin granule acidification and the priming. Characterization of the structures and functions of the mini-P-glycoprotein may provide potentials to develop new anti-diabetic drugs.