Key Lab of Hormone and Development (Tianjin and Ministry of Health of the People's Republic of China), Institution of Endocrinology, Tianjin Metabolic Diseases Hospital, Tianjin Medical University, Tianjin 300070, China.
Chin Med J (Engl). 2009 Nov 5;122(21):2587-92.
A 65-kD mdr1 (multi-drug resistance protein 1, P-glycoprotein)-like protein has been suggested to be the regulatory protein to the chloride channel protein 3 (ClC-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.
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 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. Biphasic insulin release was measured in static incubations of rat islets with radioimmunology assay.
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 (16.7 mmol/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.
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.
一种 65kDa mdr1（多药耐药蛋白 1，P-糖蛋白）样蛋白被认为是调节氯离子通道蛋白 3（ClC-3）的调节蛋白，介导小鼠胰腺β细胞中胰岛素颗粒的酸化和释放。但该蛋白尚未得到深入研究。在这项研究中，我们在大鼠胰岛中鉴定出了该 65kDa 蛋白的存在，并初步探讨了其生物学功能。
以大鼠肾脏总 RNA 为阳性对照，分别提取胰腺、胰岛和 INS-1 细胞进行逆转录 PCR（RT-PCR）。使用从编码 P-糖蛋白的 abcb1b mRNA 中选择的特异性引物运行 cDNA。所有 PCR 产物均在琼脂糖凝胶电泳中可视化，并进行测序。大鼠胰岛和 INS-1 细胞匀浆进行 SDS-PAGE。用特异性单克隆抗体 C219 检测 P-糖蛋白。用放射免疫测定法在静态孵育中测量大鼠胰岛的双相胰岛素释放。
与阳性对照相比，在胰岛、INS-1 细胞和胰腺中检测到 P-糖蛋白 mRNA 片段的表达。序列分析证实 PCR 产物与 P-糖蛋白的 mRNA 匹配。Western blot 中，胰岛匀浆中识别出 65kDa 蛋白，但在 INS-1 细胞匀浆中未识别出。相反，INS-1 细胞的匀浆含有被抗体识别的 160kDa 蛋白。大鼠胰岛的胰岛素分泌受高葡萄糖（16.7mmol/L）刺激，并在 60 分钟孵育期间呈双相曲线。与环孢素 A（CsA）共孵育后，P-糖蛋白的特异性抑制剂，第二相胰岛素分泌显著减少，而第一相不受影响。
在大鼠胰岛中表达的 65kDa 蛋白很可能是一种小型 P-糖蛋白。它可能在调节双相胰岛素释放中起关键作用。
