In situ detection of the activation of Rac1 and RalA small GTPases in mouse adipocytes by immunofluorescent microscopy following in vivo and ex vivo insulin stimulation
In situ detection of the activation of Rac1 and RalA small GTPases in mouse adipocytes by immunofluorescent microscopy following in vivo and ex vivo insulin stimulation
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体内和离体胰岛素刺激后，通过免疫荧光显微镜原位检测小鼠脂肪细胞中 Rac1 和 RalA 小 GTP 酶的激活
DOI：
10.1016/j.cellsig.2017.08.004
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发表时间：
2017
影响因子：
4.8
通讯作者：
Satoh Takaya
中科院分区：
文献类型：
--
作者：
Takenaka Nobuyuki;Nihata Yuma;Ueda Sho;Satoh Takaya
Rac1通过骨骼肌中葡萄糖转运体GLUT4的质膜易位参与胰岛素依赖性葡萄糖摄取。尽管脂肪组织中的胰岛素也能刺激葡萄糖的摄取，但Rac1在脂肪细胞胰岛素信号传导中的作用仍存在争议。为了揭示Rac1在脂肪细胞中的作用，我们打算建立免疫荧光显微镜来检测活化的Rac1在细胞内的分布。Rac1特异性靶标的表位标记的Rac1结合域被用作特异性识别Rac1激活形式的探针。利用免疫荧光显微镜成功地观察了Rac1在原代脂肪细胞培养和小鼠白色脂肪组织中对体内胰岛素刺激的反应。这些Rac1激活是由磷酸肌苷3-激酶介导的。另一种小的GTPase RalA也与胰岛素刺激的骨骼肌和脂肪组织的葡萄糖摄取有关。与Rac1类似，使用激活的RalA特异性多肽探针的免疫荧光显微镜使我们能够检测胰岛素激活的RalA在脂肪细胞中的细胞内分布。这些可视化脂肪细胞中小gtpase激活状态的新方法将在很大程度上有助于理解信号转导机制，特别是胰岛素作用。
Rac1 has been implicated in insulin-dependent glucose uptake by mechanisms involving plasma membrane translocation of the glucose transporter GLUT4 in skeletal muscle. Although the uptake of glucose is also stimulated by insulin in adipose tissue, the role for Rac1 in adipocyte insulin signaling remains controversial. As a step to reveal the role for Rac1 in adipocytes, we aimed to establish immunofluorescent microscopy to detect the intracellular distribution of activated Rac1. The epitope-tagged Rac1-binding domain of a Rac1-specific target was utilized as a probe that specifically recognizes the activated form of Rac1. Rac1 activation in response toex vivoandin vivoinsulin stimulations in primary adipocyte culture and mouse white adipose tissue, respectively, was successfully observed by immunofluorescent microscopy. These Rac1 activations were mediated by phosphoinositide 3-kinase. Another small GTPase RalA has also been implicated in insulin-stimulated glucose uptake in skeletal muscle and adipose tissue. Similarly to Rac1, immunofluorescent microscopy using an activated RalA-specific polypeptide probe allowed us to detect intracellular distribution of insulin-activated RalA in adipocytes. These novel approaches to visualize the activation status of small GTPases in adipocytes will largely contribute to the understanding of signal transduction mechanisms particularly for insulin action.