Huang Jie, Imamura Takeshi, Babendure Jennie L, Lu Juu-Chin, Olefsky Jerrold M
Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093-0673, USA.
J Biol Chem. 2005 Dec 23;280(51):42300-6. doi: 10.1074/jbc.M510920200. Epub 2005 Oct 20.
Although the cytoskeletal network is important for insulin-induced glucose uptake, several studies have assessed the effects of microtubule disruption on glucose transport with divergent results. Here, we investigated the effects of microtubule-depolymerizing reagent, nocodazole and colchicine, on GLUT4 translocation in 3T3-L1 adipocytes. After nocodazole treatment to disrupt microtubules, GLUT4 vesicles were dispersed from the perinuclear region in the basal state, and insulin-induced GLUT4 translocation was partially inhibited by 20-30%, consistent with other reports. We found that platelet-derived growth factor (PDGF), which did not stimulate GLUT4 translocation in intact cells, was surprisingly able to enhance GLUT4 translocation to approximately 50% of the maximal insulin response, in nocodazole-treated cells with disrupted microtubules. This effect of PDGF was blocked by pretreatment with wortmannin and attenuated in cells pretreated with cytochalasin D. Using confocal microscopy, we found an increased co-localization of GLUT4 and F-actin in nocodazole-treated cells upon PDGF stimulation compared with control cells. Furthermore, microinjection of small interfering RNA targeting the actin-based motor Myo1c, but not the microtubule-based motor KIF3, significantly inhibited both insulin- and PDGF-stimulated GLUT4 translocation after nocodazole treatment. In summary, our data suggest that 1) proper perinuclear localization of GLUT4 vesicles is a requirement for insulin-specific stimulation of GLUT4 translocation, and 2) nocodazole treatment disperses GLUT4 vesicles from the perinuclear region allowing them to engage insulin and PDGF-sensitive actin filaments, which can participate in GLUT4 translocation in a phosphatidylinositol 3-kinase-dependent manner.
尽管细胞骨架网络对于胰岛素诱导的葡萄糖摄取很重要，但多项研究评估了微管破坏对葡萄糖转运的影响，结果却不尽相同。在此，我们研究了微管解聚试剂诺考达唑和秋水仙碱对3T3-L1脂肪细胞中葡萄糖转运蛋白4（GLUT4）转位的影响。用诺考达唑处理破坏微管后，基础状态下GLUT4囊泡从核周区域分散开来，胰岛素诱导的GLUT4转位被部分抑制了20%-30%，这与其他报告一致。我们发现，血小板衍生生长因子（PDGF）在完整细胞中不会刺激GLUT4转位，但令人惊讶的是，在微管被破坏的诺考达唑处理的细胞中，它能够将GLUT4转位增强至最大胰岛素反应的约50%。wortmannin预处理可阻断PDGF的这种作用，而细胞松弛素D预处理的细胞中该作用减弱。使用共聚焦显微镜，我们发现与对照细胞相比，PDGF刺激后，诺考达唑处理的细胞中GLUT4与F-肌动蛋白的共定位增加。此外，显微注射靶向基于肌动蛋白的马达蛋白Myo1c而非基于微管的马达蛋白KIF3的小干扰RNA，在诺考达唑处理后显著抑制了胰岛素和PDGF刺激的GLUT4转位。总之，我们的数据表明：1）GLUT4囊泡在核周的正确定位是胰岛素特异性刺激GLUT4转位的必要条件；2）诺考达唑处理使GLUT4囊泡从核周区域分散，使其能够与胰岛素和PDGF敏感的肌动蛋白丝结合，后者可以以磷脂酰肌醇3激酶依赖的方式参与GLUT4转位。
