Demaegdt H, Smitz L, De Backer J-P, Le M T, Bauwens M, Szemenyei E, Tóth G, Michotte Y, Vanderheyden P, Vauquelin G
Department of Molecular and Biochemical Pharmacology, Vrije Universiteit Brussel, Brussels, Belgium.
Br J Pharmacol. 2008 Jun;154(4):872-81. doi: 10.1038/bjp.2008.117. Epub 2008 Apr 21.
Insulin-regulated aminopeptidase (IRAP) and the insulin-dependent glucose transporter GLUT4 colocalize in specific intracellular vesicles (that is, GLUT4 vesicles). These vesicles move slowly to the cell surface, but their translocation is markedly enhanced by insulin, resulting in higher glucose uptake. Previous studies of the insulin-mediated translocation of IRAP to the cell surface have been hampered by the laborious detection of IRAP at the cell surface. We aimed to develop a more direct and faster method to detect IRAP. To this end, we used model systems with well-characterized IRAP: CHO-K1 cells expressing endogenous IRAP and recombinant HEK293 cells expressing human IRAP. A more widespread application of the method was demonstrated by the use of 3T3-L1 adipocytes.
After stimulation of the cells with insulin, internalization of IRAP was inhibited by the addition of phenyl arsine oxide (PAO). Then, cell-surface IRAP was detected by the high-affinity binding of radiolabelled angiotensin (Ang) IV (either 125I or 3H).
We monitored the time- and concentration dependence of insulin-mediated translocation of IRAP in both cell lines and 3T3-L1 adipocytes. A plateau was reached between 6 and 8 min, and 10(-7) M insulin led to the highest amount of IRAP at the cell surface.
Based on the capacity of the IRAP apoenzyme to display high affinity for radiolabelled Ang IV and on the ability of PAO to inhibit IRAP internalization, we developed a more direct and faster method to measure insulin-mediated translocation of IRAP to the cell surface.
胰岛素调节氨肽酶（IRAP）与胰岛素依赖型葡萄糖转运体GLUT4共定位于特定的细胞内囊泡（即GLUT4囊泡）。这些囊泡缓慢移动至细胞表面，但胰岛素可显著增强其转位，从而导致更高的葡萄糖摄取。此前关于胰岛素介导的IRAP转位至细胞表面的研究因在细胞表面费力检测IRAP而受阻。我们旨在开发一种更直接、更快的方法来检测IRAP。为此，我们使用了具有明确特征的IRAP模型系统：表达内源性IRAP的CHO-K1细胞和表达人IRAP的重组HEK293细胞。通过使用3T3-L1脂肪细胞证明了该方法更广泛的应用。
用胰岛素刺激细胞后，添加苯胂氧化物（PAO）抑制IRAP的内化。然后，通过放射性标记的血管紧张素（Ang）IV（125I或3H）的高亲和力结合来检测细胞表面的IRAP。
我们监测了IRAP在两种细胞系和3T3-L1脂肪细胞中胰岛素介导的转位的时间和浓度依赖性。在6至8分钟之间达到平台期，10-7 M胰岛素导致细胞表面IRAP的量最高。
基于IRAP脱辅基酶对放射性标记的Ang IV显示高亲和力的能力以及PAO抑制IRAP内化的能力，我们开发了一种更直接、更快的方法来测量胰岛素介导的IRAP转位至细胞表面。
