Background A 65-kD mdrl （multi-drug resistance protein 1, P-glycoprotein）-Iike protein has been suggested to be the regulatory protein to the chloride channel protein 3 （CIC-3） mediating insulin granules acidifica...Background A 65-kD mdrl （multi-drug resistance protein 1, P-glycoprotein）-Iike protein has been suggested to be the regulatory protein to the chloride channel protein 3 （CIC-3） mediating insulin granules acidification and release in mouse pancreatic 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 Total RNAs of rat kidneys served as positive controls, and pancreas, 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 abcblb 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. Biphasic insulin release was measured in static incubations of rat islets with radioimmunology assay. Results Compared with positive control, expression of the P-glycoprotein mRNA segments were detected in the islets, INS-1 cells and pancreas. Sequence analysis confirmed that the PCR products were matched with mRNA of P-glycoprotein. A 65-kda protein was recognized by the antibody in the islets homogenate but not in that of INS-1 cells in Western-blotting. Instead, the homogenate of INS-1 cells contained a 160-kda protein recognized by the antibody. Insulin secretion of rat islets were stimulated by high glucose （16.7mmol/L）, and showed biphasic curve during 60-minute incubation. After co-incubation with cyclosporine A （CsA）, specific inhibitor to P-glycoprotein, the second phase of insulin secretion was reduced significantly while the first phase was not influenced. Conclusions 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.展开更多
基金Acknowledgements： This study was supported by EFSD Grant Award for Collaborative Diabetes Research between China and Europe supported by Bristol-Myers-Squibb to LI Dai-qing and Tianjin Municipal Natural Science Foundation to LI Dai-qing （No. 08JCZDJC25100 ）.
文摘Background A 65-kD mdrl （multi-drug resistance protein 1, P-glycoprotein）-Iike protein has been suggested to be the regulatory protein to the chloride channel protein 3 （CIC-3） mediating insulin granules acidification and release in mouse pancreatic 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 Total RNAs of rat kidneys served as positive controls, and pancreas, 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 abcblb 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. Biphasic insulin release was measured in static incubations of rat islets with radioimmunology assay. Results Compared with positive control, expression of the P-glycoprotein mRNA segments were detected in the islets, INS-1 cells and pancreas. Sequence analysis confirmed that the PCR products were matched with mRNA of P-glycoprotein. A 65-kda protein was recognized by the antibody in the islets homogenate but not in that of INS-1 cells in Western-blotting. Instead, the homogenate of INS-1 cells contained a 160-kda protein recognized by the antibody. Insulin secretion of rat islets were stimulated by high glucose （16.7mmol/L）, and showed biphasic curve during 60-minute incubation. After co-incubation with cyclosporine A （CsA）, specific inhibitor to P-glycoprotein, the second phase of insulin secretion was reduced significantly while the first phase was not influenced. Conclusions 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.
