Nedachi Taku, Kanzaki Makoto
TUBERO/Tohoku University Biomedical Engineering Research Organization, Tohoku University 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
Am J Physiol Endocrinol Metab. 2006 Oct;291(4):E817-28. doi: 10.1152/ajpendo.00194.2006. Epub 2006 May 30.
It is well established that insulin stimulation of glucose uptake in skeletal muscle cells is mediated through translocation of GLUT4 from intracellular storage sites to the cell surface. However, the established skeletal muscle cell lines, with the exception of L6 myocytes, reportedly show minimal insulin-dependent glucose uptake and GLUT4 translocation. Using C(2)C(12) myocytes expressing exofacial-Myc-GLUT4-enhanced cyan fluorescent protein, we herein show that differentiated C(2)C(12) myotubes are equipped with basic GLUT4 translocation machinery that can be activated by insulin stimulation ( approximately 3-fold increase as assessed by anti-Myc antibody uptake and immunostaining assay). However, this insulin stimulation of GLUT4 translocation was difficult to demonstrate with a conventional 2-deoxyglucose uptake assay because of markedly elevated basal glucose uptake via other glucose transporter(s). Intriguingly, the basal glucose transport activity in C(2)C(12) myotubes appeared to be acutely suppressed within 5 min by preincubation with a pathophysiologically high level of extracellular glucose (25 mM). In contrast, this activity was augmented by acute glucose deprivation via an unidentified mechanism that is independent of GLUT4 translocation but is dependent on phosphatidylinositol 3-kinase activity. Taken together, these findings indicate that regulation of the facilitative glucose transport system in differentiated C(2)C(12) myotubes can be achieved through surprisingly acute glucose-dependent modulation of the activity of glucose transporter(s), which apparently contributes to obscuring the insulin augmentation of glucose uptake elicited by GLUT4 translocation. We herein also describe several methods of monitoring insulin-dependent glucose uptake in C(2)C(12) myotubes and propose this cell line to be a useful model for analyzing GLUT4 translocation in skeletal muscle.
胰岛素刺激骨骼肌细胞摄取葡萄糖是通过葡萄糖转运蛋白4（GLUT4）从细胞内储存位点转位到细胞表面介导的，这一点已得到充分证实。然而，据报道，除了L6肌细胞外，已建立的骨骼肌细胞系显示出最小程度的胰岛素依赖性葡萄糖摄取和GLUT4转位。使用表达细胞外-Myc-GLUT4-增强型青色荧光蛋白的C2C12肌细胞，我们在此表明，分化的C2C12肌管配备了基本的GLUT4转位机制，该机制可被胰岛素刺激激活（通过抗Myc抗体摄取和免疫染色测定评估增加约3倍）。然而，由于通过其他葡萄糖转运蛋白的基础葡萄糖摄取显著升高，用传统的2-脱氧葡萄糖摄取测定法很难证明这种胰岛素对GLUT4转位的刺激作用。有趣的是，通过与病理生理水平的细胞外高葡萄糖（25 mM）预孵育，C2C12肌管中的基础葡萄糖转运活性在5分钟内似乎被急性抑制。相反，通过一种未知机制，急性葡萄糖剥夺可增强这种活性，该机制独立于GLUT4转位，但依赖于磷脂酰肌醇3-激酶活性。综上所述，这些发现表明，分化的C2C12肌管中促进性葡萄糖转运系统的调节可通过葡萄糖转运蛋白活性的惊人的急性葡萄糖依赖性调节来实现，这显然有助于掩盖由GLUT4转位引起的胰岛素对葡萄糖摄取的增强作用。我们在此还描述了几种监测C2C12肌管中胰岛素依赖性葡萄糖摄取的方法，并提出该细胞系是分析骨骼肌中GLUT4转位的有用模型。
