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A study on the Intracellular dynamics of insulin granules using a phogrin-GFP fused protein

使用 phogrin-GFP 融合蛋白研究胰岛素颗粒的细胞内动力学

基本信息

批准号：
13470006
负责人：
SENDA Takao
金额：
$ 7.94万
依托单位：
Fujita Health University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470006/
关键词：
insulin secretion MIN6 cells pancreatic B cells phogrin glucose calmodulin apoptosis caveolin-1 γ-チュブリンインスリン顆粒カルシウムイオン CaMマウストルブタミド神経型NO合成酵素 MIN6 顆粒運動 GFP

项目摘要

1.We constructed an expression vector in which phogrin is bound to the C-terminus of GFP. Using the vector, we successfully expressed. GFP-phogxin in MIN6 cells.2.We got several new findings concerning regulation mechanisms of the insulin granule traffic.(1)Glucose accelerated the insulin granule traffic in MIN6 cells. The calmodulin-inhibitor W-7 inhibited the glucose-induced acceleration of the granule traffic, and myosin light chain kinase inhibited the glucose-induced acceleration of the granule traffic.(2)The blockers to Ca^<2+> channels (nifedipine and nitrendipine) did not inhibit the glucose-induced acceleration of the granule traffic.(3)The K_<ATP>-opener (diazoxide) did not inhibit the glucose-induced acceleration of the granule traffic.(4)The glucose stimulation increased phosphatized myosin light chain in MIN6 cells.3.We investigated the mechanism of B cell loss in transgenic mice with elevated levels of B cell calmodulin.(1)The transgenic mice experienced a sudden rise in blood glucose levels, which was associated with developed of severe hypoinsulinemia and loss of B cells from the islets.(2)Ultrastructural analysis revealed that compromised granule formation and apoptotic changes in the transgenic B cells preceded the onset of hyperglycemia.(3)Intraperitoneal injection of tolbutamide, an antidiabetic sulfonylurea, decreased blood glucose levels but increased the number of apoptotic B cells.(4)Injection of the mice with N^ω-nitro-L-arginine methyl ester, which inhibits nitric oxide synthase activity, prevented hyperglycemia and lessened the changes in number and size of B cells.4.We found that caveolin-1 was expressed in MIN6 cells, and colocalized with γ-tubulin in microtubule-organizing center. The signal of caveolin-1 decreased after glucose stimulation.

1.构建了荧光蛋白与绿色荧光蛋白C端结合的表达载体。使用该载体，我们成功地表达了。2.在胰岛素颗粒运输的调控机制方面有了新的发现。（1）葡萄糖可加速MIN 6细胞胰岛素颗粒的运输。钙调素抑制剂W-7抑制葡萄糖诱导的颗粒运输加速，肌球蛋白轻链激酶抑制葡萄糖诱导的颗粒运输加速。（2）钙通道阻断剂硝苯地平和尼群地平不能抑制葡萄糖引起的颗粒运输加速。（3）K_<ATP>-开放剂（二氮嗪）不能抑制葡萄糖诱导的颗粒运输加速。（4）葡萄糖刺激可增加MIN 6细胞肌球蛋白轻链磷酸化水平。3.我们研究了B细胞钙调素水平升高的转基因小鼠B细胞丢失的机制。（1）转基因小鼠血糖水平突然升高，这与严重的低胰岛素血症和胰岛B细胞的损失有关。（2）超微结构分析显示，转基因B细胞在高血糖发生前出现颗粒形成障碍和凋亡改变。（3）腹腔注射磺脲类降糖药甲苯磺丁脲可降低血糖水平，但增加凋亡B细胞数量。（4）注射N^ω-硝基-L-精氨酸甲酯可抑制一氧化氮合酶的活性，降低高血糖的发生率，并减轻B细胞数量和大小的改变。葡萄糖刺激后小窝蛋白-1信号减弱。