Enhancement of Insulin Responsiveness by Nitric Oxide-mediated Inactivation of Protein-tyrosine Phosphatases
Enhancement of Insulin Responsiveness by Nitric Oxide-mediated Inactivation of Protein-tyrosine Phosphatases
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DOI：
10.1074/jbc.m109.057513
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发表时间：
2010-03-12
影响因子：
4.8
通讯作者：
Meng, Tzu-Ching
中科院分区：
文献类型：
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作者：
Hsu, Ming-Fo;Meng, Tzu-Ching
NO合成是胰岛素靶向组织中适当胰岛素敏感性的先决条件;然而，该过程的分子基础仍不清楚。使用内皮型一氧化氮合酶（eNOS）转染的COS-7细胞的功能获得模型，我们已经显示了NO在胰岛素反应性中的关键作用，如通过胰岛素受体及其下游效应物胰岛素受体底物-1和PKB/AKT的酪氨酸磷酸化水平的NO依赖性增加所证明的。我们推测，NO诱导的内源性蛋白酪氨酸磷酸酶（PTPs）的失活将增强胰岛素受体介导的信号转导。为了验证这一假设，我们设计了一种新的方法的PTP标记使用半胱氨酸巯基反应的探针。在本研究中采用的酸性条件下，探针识别内源性PTP的还原和活性形式，但不识别S-亚硝基化和非活性形式。我们的数据表明，磷酸酶SHP-1，SHP-2和PTP 1B，但不是TC-PTP，可能是S-亚硝基化的活性位点半胱氨酸残基伴随着一个突发的NO生产的信号响应胰岛素刺激。这些结果进一步证实了磷酸酶活性测定。我们进一步研究了NO作为胰岛素信号转导的调节剂的作用，通过RNA干扰消除内皮MS-1细胞中内源性eNOS的表达。我们已经证明eNOS依赖的NO产生对于胰岛素信号的激活是必不可少的。我们的研究结果表明，NO介导的胰岛素反应性的增强，通过抑制胰岛素受体磷酸酶。
NO synthesis is a prerequisite for proper insulin sensitivity in insulin-targeted tissues; however, the molecular basis for this process remains unclear. Using a gain-of-function model of endothelial nitric-oxide synthase (eNOS)-transfected COS-7 cells, we have shown a critical role of NO in insulin responsiveness, as evidenced by an NO-dependent increase of tyrosine phosphorylation levels of the insulin receptor and its downstream effectors insulin receptor substrate-1 and PKB/AKT. We hypothesized that NO-induced inactivation of endogenous protein-tyrosine phosphatases (PTPs) would enhance insulin receptor-mediated signaling. To test this hypothesis, we devised a new method of the PTP labeling using a cysteine sulfhydryl-reacted probe. Under the acidic conditions employed in this study, the probe recognized the reduced and active forms but not the S-nitrosylated and inactive forms of endogenous PTPs. Our data suggest that phosphatases SHP-1, SHP-2, and PTP1B, but not TC-PTP, are likely S-nitrosylated at the active site cysteine residue concomitantly with a burst of NO production in signaling response to insulin stimulation. These results were further confirmed by phosphatase activity assays. We investigated further the role of NO as a regulator of insulin signaling by RNA interference that ablates endogenous eNOS expression in endothelial MS-1 cells. We have shown that eNOS-dependent NO production is essential for the activation of insulin signaling. Our findings demonstrate that NO mediates enhancement of insulin responsiveness via the inhibition of insulin receptor phosphatases.