Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells
Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells
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DOI：
10.1161/01.cir.101.13.1539
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发表时间：
2000-04-04
期刊：
影响因子：
37.8
通讯作者：
Quon, MJ
中科院分区：
文献类型：
--
作者：
Zeng, GY;Nystrom, FH;Quon, MJ
背景-以前，我们证明胰岛素刺激内皮细胞产生一氧化氮（NO）。然而，具体的胰岛素信号通路介导的生产NO.Methods和结果，我们开发的方法转染人脐静脉内皮细胞（HUVECs）和直接测量NO开始定义胰岛素信号通路相关的NO生产。用增强型绿色荧光蛋白（eGFP）和另一种感兴趣的基因共转染HUVEC。通过选择表达eGFP的细胞获得>95%的转染效率。在HUVEC中胰岛素受体的过表达导致响应于胰岛素的NO的产生增加约3倍。相反，过度表达酪氨酸激酶缺陷突变型胰岛素受体的HUVEC具有与对照细胞相似的剂量-反应曲线。过度表达的抑制性突变体的磷脂酰肌醇3-激酶（PI 3 K）或Akt导致几乎完全抑制胰岛素刺激的生产NO。过度表达的抑制性突变体的RNS有一个小得多的effect. Conclusions受体激酶活性是必要的，通过胰岛素受体介导NO的生产。PI 3 K和Akt对这一过程都有重要贡献，而Ras的贡献很小。
Background-Previously, we demonstrated that insulin stimulates production of nitric oxide (NO) in endothelial cells. However, specific insulin-signaling pathways mediating production of NO have not been elucidated.Methods and Results-We developed methods for transfection of human umbilical vein endothelial cells (HUVECs) and direct measurement of NO to begin defining insulin-signaling pathways related to NO production. HUVECs were cotransfected with enhanced Green Fluorescent Protein (eGFP) and another gene of interest. Transfection efficiencies >95% were obtained by selecting cells expressing eGFP. Overexpression of insulin receptors in HUVECs resulted in an approximate to 3-fold increase in production of NO in response to insulin. In contrast, HUVECs overexpressing a tyrosine kinase-deficient mutant insulin receptor had a dose-response curve similar to that of control cells. Overexpression of inhibitory mutants of either phosphatidylinositol 3-kinase (PI3K) or Akt resulted in nearly complete inhibition of insulin-stimulated production of NO. Overexpression of an inhibitory mutant of Rns had a much smaller effect.Conclusions-Receptor kinase activity is necessary to mediate production of NO through the insulin receptor. Both PI3K and Akt contribute importantly to this process, whereas the contribution of Ras is small.