McClenaghan N H, Barnett C R, Ah-Sing E, Abdel-Wahab Y H, O'Harte F P, Yoon T W, Swanston-Flatt S K, Flatt P R
School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, U.K.
Diabetes. 1996 Aug;45(8):1132-40. doi: 10.2337/diab.45.8.1132.
A novel insulin-secreting cell line (BRIN-BD11) was established after electrofusion of RINm5F cells with New England Deaconess Hospital rat pancreatic islet cells. Wells of cell fusion mixture with insulin output 5-10 times greater than parent RINm5F cells were subcultured with eventual establishment of clones, including BRIN-BD11. Morphological studies established that these cells grow as monolayers with epithelioid characteristics, maintaining stability in tissue culture for > 50 passages. Culture of these cells for 24 h at 5.6-33.3 mmol/l glucose revealed a 1.8- to 2.0-fold increase of insulin output compared with 1.4 mmol/l glucose. Dynamic insulin release was recorded in response to 16.7 mmol/l glucose, resulting in a rapid threefold insulin secretory peak followed by a sustained output slightly above basal. In acute 20-min tests, 4.2-16.7 mmol/l glucose evoked a stepwise two- to three-fold stimulation of insulin release. 3-Isobutyl-1-methylxanthine (1 mmol/l) served to increase basal and glucose-stimulated insulin release, shifting the threshold from 4.4 to 1.1 mmol/l glucose. Stimulation of insulin secretion with 16.7 mmol/l glucose was abolished by mannoheptulose or diazoxide (15 or 0.5 mmol/l). In contrast, glyceraldehyde (10 mmol/l) and 25 mmol/l K+ evoked 1.7- to 9.0-fold insulin responses. L-Alanine (10 mmol/l) evoked a twofold secretory response, which was potentiated 1.4-fold by increasing the Ca2+ concentration from 1.28 to 7.68 mmol/l. Forskolin (25 mumol/l) and phorbol 12-myristate 13-acetate (10 nmol/l) both increased insulin secretion in the presence of L-alanine (1.4- and 1.8-fold, respectively). Western blotting confirmed that BRIN-BD11 cells expressed the GLUT2 glucose transporter. This, coupled with a high glucokinase/hexokinase ratio in the cells, confirms an intact glucose sensing mechanism. High-performance liquid chromatography analysis demonstrated that insulin was the major product secreted under stimulatory conditions. Collectively, these data indicate that the BRIN-BD11 cell line represents an important stable glucose-responsive insulin-secreting beta-cell line for future studies.
将RINm5F细胞与新英格兰女执事医院大鼠胰岛细胞进行电融合后，建立了一种新型胰岛素分泌细胞系（BRIN-BD11）。将胰岛素分泌量比亲代RINm5F细胞高5至10倍的细胞融合混合物孔进行传代培养，最终建立了包括BRIN-BD11在内的克隆。形态学研究表明，这些细胞以具有上皮样特征的单层形式生长，在组织培养中保持了50代以上的稳定性。在5.6至33.3 mmol/l葡萄糖浓度下培养这些细胞24小时，与1.4 mmol/l葡萄糖相比，胰岛素分泌量增加了1.8至2.0倍。记录了对16.7 mmol/l葡萄糖的动态胰岛素释放情况，结果显示胰岛素分泌迅速出现三倍峰值，随后持续分泌量略高于基础水平。在急性20分钟试验中，4.2至16.7 mmol/l葡萄糖引起胰岛素释放呈逐步的两倍至三倍刺激。3-异丁基-1-甲基黄嘌呤（1 mmol/l）可增加基础和葡萄糖刺激的胰岛素释放，将阈值从4.4 mmol/l葡萄糖转变为1.1 mmol/l葡萄糖。甘露庚酮糖或二氮嗪（15或0.5 mmol/l）可消除16.7 mmol/l葡萄糖对胰岛素分泌的刺激。相比之下，甘油醛（10 mmol/l）和25 mmol/l K+引起1.7至9.0倍的胰岛素反应。L-丙氨酸（10 mmol/l）引起两倍的分泌反应，将Ca2+浓度从1.28 mmol/l增加到7.68 mmol/l可使其增强1.4倍。福斯高林（25 μmol/l）和佛波醇12-肉豆蔻酸酯13-乙酸酯（10 nmol/l）在存在L-丙氨酸的情况下均增加胰岛素分泌（分别为1.4倍和1.8倍）。蛋白质印迹法证实BRIN-BD11细胞表达GLUT2葡萄糖转运蛋白。这与细胞中高的葡萄糖激酶/己糖激酶比率相结合，证实了完整的葡萄糖传感机制。高效液相色谱分析表明，胰岛素是刺激条件下分泌的主要产物。总体而言，这些数据表明BRIN-BD11细胞系代表了一种重要的稳定的葡萄糖反应性胰岛素分泌β细胞系，可用于未来的研究。
