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Analysis of intracellular mechanisms contributing to insulin secretion from pancreatic β-cells

胰腺β细胞分泌胰岛素的细胞内机制分析

基本信息

批准号：
11670097
负责人：
ISHIKAWA Tomohisa
金额：
$ 2.18万
依托单位：
University of Shizuoka
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670097/
关键词：
Islets of Langerhans β-cells rat NO NO synthase Ca^<2+>glucose DAF-2 NOC 7 インスリン

项目摘要

METHODS : Pancreatic islets and β-cells were isolated from male Wistar rats (9-12 weeks old) by collagenase digestion technique. (1) Double immunostaining with antiserum to NOS1 or NOS3 and that to insulin or glucagon was performed in the isolated islets, and the fluorescent images were analyzed in a confocal laser scanning microscope. (2) The production of NO in islet cells was measured with a fluorescent NO indicator, DAF-2 DA in a confocal laser scanning microscope. (3) [Ca^<2+>]_i was measured in fura 2-loaded β-cells by Argus-50/CA system. (4) The secretion of insulin from isolated islets was measured by the batch incubation method and EIA.RESULTS AND DISCUSSION : (1) Double immunostaining showed that both NOS1- and NOS3-immunoreactivity could be detected in rat β-cells. (2) The amount of NO in β-cells was elevated by glucose in a concentration-dependent manner. (3) In the presence of L-NNA, an NO donor, NOC 7, at 0.5 μM increased the amplitude of [Ca^<2+>]_i oscillations induced by 11.1 mM glucose, and at 10 μM terminated them. A soluble guanylyl cyclase inhibitor suppressed the stimulatory action of NOC7 at low concentrations, but did not affect the inhibitory one at high concentrations, suggesting that the former is mediated by cGMP while the latter is not. (4) Insulin secretion induced by 11.1 mM glucose was facilitated by 0.5 μM NOC7, while it was suppressed by 10 μM NOC7.CONCLUSION : NO is an endogenous regulator of insulin secretion, which facilitates and inhibits glucose-induced insulin secretion at low and high concentrations, respectively.

方法：采用胶原酶消化法分离9-12周龄雄性Wistar大鼠胰岛和胰岛β细胞。(1)用抗NOS 1或NOS 3抗体和抗胰岛素或胰高血糖素抗体对分离的胰岛进行双重免疫染色，并在共聚焦激光扫描显微镜下分析荧光图像。(2)在激光共聚焦扫描显微镜下，用NO荧光指示剂<$A-2 DA测定胰岛细胞中NO的产生。(3)用Argus-50/CA系统测定Fura 2负载的β细胞内[Ca^<2+>] i。(4)结果与讨论：（1）免疫组化双重染色结果显示，大鼠胰岛β细胞中可检测到NOS 1-和NOS 3-的免疫反应性。(2)葡萄糖浓度依赖性地增加β细胞内NO含量。(3)在L-NNA存在下，0.5 μM的NO供体NOC 7可增加11.1mM葡萄糖诱导的[Ca^<2+>] i振荡幅度，而在10 μM时可终止该振荡。可溶性鸟苷酸环化酶抑制剂在低浓度下抑制NOC 7的刺激作用，但在高浓度下不影响抑制作用，表明前者是由cGMP介导的，而后者不是。(4)0.5 μM的NOC 7促进11.1mM葡萄糖诱导的胰岛素分泌，10 μM的NOC 7则抑制胰岛素分泌。结论：NO是胰岛素分泌的内源性调节剂，在低浓度和高浓度时分别促进和抑制葡萄糖诱导的胰岛素分泌。