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首页> 美国卫生研究院文献>Biochemical Journal >Actin filaments play a critical role in insulin-induced exocytotic recruitment but not in endocytosis of GLUT4 in isolated rat adipocytes.
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Actin filaments play a critical role in insulin-induced exocytotic recruitment but not in endocytosis of GLUT4 in isolated rat adipocytes.

机译：肌动蛋白丝在胰岛素诱导的胞吞募集中起关键作用但在离体大鼠脂肪细胞中GLUT4的胞吞作用中不发挥关键作用。

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Actin-based cytoskeletons have been implicated in insulin-stimulated glucose transport and translocation of the insulin-regulated glucose transporter, GLUT4, from the intracellular pool to the plasma membrane. However, most previous studies were done using adherent cell systems such as L6 myotubes and 3T3-L1 adipocytes, and very little information is available on the significance of the actin filaments to the insulin action in isolated adipocytes, a widely used experimental system. In the present study, we investigated the physiological role of actin filaments in the subcellular trafficking of GLUT4 in isolated rat adipocytes. We first compared the effects of two actin-disrupting reagents, latrunculin A and cytochalasin D, on the organization of the actin filaments as well as on the insulin action on glucose transport by laser confocal microscopy combined with biochemical analysis of the insulin action. Treatment of the cells with latrunculin A induced dose- and time-dependent disappearance of the filamentous actin, which correlated very well with inhibition of the insulin effect on glucose transport. Although cytochalasin D at 50 microM significantly inhibited insulin-stimulated glucose transport, it was not effective in disassembly of the actin filaments; rather, many intense punctate signals were observed in cytochalasin D-treated cells. In the actin-disrupted adipocytes treated with latrunculin A, insulin-induced GLUT4 translocation was inhibited completely. In addition, latrunculin A remarkably inhibited both insulin-induced glucose transport and GLUT4 translocation in the presense of D(k)-(62-85), a potent inhibitor of GLUT4 endocytosis, suggesting that intactness of the actin filaments was necessary for insulin-induced exocytosis of the GLUT4-containing vesicles. On the other hand, latrunculin A showed little inhibitory effect on either endocytosis of the trypsin-cleaved 35-kDa fragment of GLUT4 or decay of the glucose transport activity after addition of wortmannin in insulin-stimulated cells. The results of our experiment show clearly that, in rat adipocytes, (i) latrunculin A may be a more suitable tool than cytochalasin D for disruption of actin filaments, and (ii) actin filaments play a crucial role in exocytotic recruitment of GLUT4 to the plasma membrane from the intracellular pool, but not in its endocytosis.
机译：基于肌动蛋白的细胞骨架与胰岛素刺激的葡萄糖转运以及胰岛素调节的葡萄糖转运蛋白GLUT4从细胞内池到质膜的转运有关。但是，大多数以前的研究都是使用贴壁细胞系统（例如L6肌管和3T3-L1脂肪细胞）完成的，关于肌动蛋白丝对分离的脂肪细胞（广泛使用的实验系统）中胰岛素作用的重要性的信息很少。在本研究中，我们调查了肌动蛋白丝在离体大鼠脂肪细胞中GLUT4的亚细胞运输中的生理作用。我们首先通过激光共聚焦显微镜结合胰岛素作用的生化分析，比较了两种破坏肌动蛋白的试剂，即latrunculin A和细胞松弛素D，对肌动蛋白丝的组织以及胰岛素对葡萄糖转运的作用。用latrunculin A处理细胞会导致丝状肌动蛋白剂量和时间依赖性消失，这与抑制胰岛素对葡萄糖转运的作用非常相关。尽管50 microM的细胞松弛素D显着抑制了胰岛素刺激的葡萄糖转运，但在肌动蛋白丝的拆卸中并不有效。相反，在细胞松弛素D处理的细胞中观察到许多强烈的点状信号。在用latrunculin A处理的肌动蛋白破坏的脂肪细胞中，胰岛素诱导的GLUT4易位被完全抑制。此外，在D（k）-（62-85）（一种强力的GLUT4内吞作用抑制剂）的存在下，latrunculin A显着抑制了胰岛素诱导的葡萄糖转运和GLUT4易位，这表明肌动蛋白丝的完整性对于胰岛素诱导含有GLUT4的囊泡的胞吐作用。另一方面，在胰岛素刺激的细胞中加入渥曼青霉素后，latrunculin A对胰蛋白酶切割的35-kDa GLUT4片段的内吞作用或葡萄糖转运活性的衰减几乎没有抑制作用。我们的实验结果清楚地表明，在大鼠脂肪细胞中，（i）latrunculin A可能比细胞松弛素D更适合破坏肌动蛋白丝，并且（ii）肌动蛋白丝在GLUT4的胞外募集中起关键作用。质膜来自细胞内池，但未在其内吞作用。

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