Program in Cell Biology, The Hospital for Sick Children, 555 University Ave., Toronto, ON, M5G 1X8, Canada.
Am J Physiol Endocrinol Metab. 2010 May;298(5):E1058-71. doi: 10.1152/ajpendo.00773.2009. Epub 2010 Feb 16.
Muscle contraction stimulates glucose uptake acutely to increase energy supply, but suitable cellular models that faithfully reproduce this complex phenomenon are lacking. To this end, we have developed a cellular model of contracting C(2)C(12) myotubes overexpressing GLUT4 with an exofacial myc-epitope tag (GLUT4myc) and explored stimulation of GLUT4 traffic by physiologically relevant agents. Carbachol (an acetylcholine receptor agonist) induced a gain in cell surface GLUT4myc that was mediated by nicotinic acetylcholine receptors. Carbachol also activated AMPK, and this response was sensitive to the contractile myosin ATPase inhibitor N-benzyl-p-toluenesulfonamide. The gain in surface GLUT4myc elicited by carbachol or by the AMPK activator 5-amino-4-carboxamide-1 beta-ribose was sensitive to chemical inhibition of AMPK activity by compound C and partially reduced by siRNA-mediated knockdown of AMPK catalytic subunits or LKB1. In addition, the carbachol-induced gain in cell surface GLUT4myc was partially sensitive to chelation of intracellular calcium with BAPTA-AM. However, the carbachol-induced gain in cell surface GLUT4myc was not sensitive to the CaMKK inhibitor STO-609 despite expression of both isoforms of this enzyme and a rise in cytosolic calcium by carbachol. Therefore, separate AMPK- and calcium-dependent signals contribute to mobilizing GLUT4 in response to carbachol, providing an in vitro cell model that recapitulates the two major signals whereby acute contraction regulates glucose uptake in skeletal muscle. This system will be ideal to further analyze the underlying molecular events of contraction-regulated GLUT4 traffic.
肌肉收缩会刺激葡萄糖摄取，从而在短期内增加能量供应，但目前缺乏能够真实再现这一复杂现象的合适细胞模型。为此，我们构建了一个过表达质膜 GLUT4 并带有细胞外 myc 表位标签（GLUT4myc）的可收缩 C(2)C(12)肌管细胞模型，探索了生理相关试剂对 GLUT4 转运的刺激作用。乙酰胆碱受体激动剂卡巴胆碱（carbachol）诱导 GLUT4myc 增加，该过程由烟碱型乙酰胆碱受体介导。卡巴胆碱还激活了 AMPK，这一反应对肌球蛋白 ATP 酶抑制剂 N-苄基对甲苯磺酰胺敏感。卡巴胆碱或 AMPK 激活剂 5-氨基-4-羧基-1-β-D-核糖（5-amino-4-carboxamide-1β-ribose）引起的 GLUT4myc 表面增加对 AMPK 活性的化学抑制敏感，可被化合物 C 抑制，并部分被 AMPK 催化亚基或 LKB1 的 siRNA 敲低所减少。此外，细胞内钙螯合也可部分抑制 GLUT4myc 表面增加。然而，尽管表达了这种酶的两种同工型，且细胞内钙水平因卡巴胆碱而升高，钙调蛋白依赖性激酶抑制剂 STO-609 却不能使卡巴胆碱引起的 GLUT4myc 表面增加敏感化。因此，有两个独立的 AMPK 和钙依赖信号参与调节 GLUT4 的动员，该系统提供了一个体外细胞模型，可再现急性收缩调节骨骼肌葡萄糖摄取的两个主要信号。这个系统非常适合进一步分析收缩调节 GLUT4 转运的潜在分子事件。
