Involvement of vesicular H+-ATPase in insulin-stimulated glucose transport in 3T3-F442A adipocytes
Involvement of vesicular H+-ATPase in insulin-stimulated glucose transport in 3T3-F442A adipocytes
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DOI：
10.1507/endocrj.k07-090
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发表时间：
2007-10-01
影响因子：
2
通讯作者：
Jang, Yeon Jin
中科院分区：
文献类型：
--
作者：
Choi, Young Ok;Park, Jong-Ha;Jang, Yeon Jin
在分泌细胞中，分泌颗粒的渗透膨胀被认为是分泌颗粒与质膜胞外融合的中间步骤。对于颗粒的渗透膨胀，液泡型H+-ATPase(V-ATPase)产生的H+梯度可能是K+通过与H+交换而积累的驱动力，同时伴随着Cl-和H2O的积累。在这里，我们研究了在胰岛素刺激下，3T3-F442a脂肪细胞GLUT4向质膜的募集是否与类似的化学渗透机制有关。在高渗介质中孵育的细胞显著增加2-脱氧葡萄糖(2-DG)摄取和质膜GLUT4含量(可能是通过诱导含有GLUT4的囊泡(G4V)的渗透肿胀)，并增强胰岛素刺激的2-DG摄取。使用电子中和型K+/H+交换离子载体黑素促进G4V膜离子通透性，增加2-DG摄取量和质膜GLUT4含量。然而，与黑素和胰岛素联合治疗并没有显示出相加的效果。诊断特异性的V-ATPase抑制剂Bafilmycin A可抑制胰岛素和黑素刺激的2-DG摄取。免疫吸附和免疫印迹表明，GLUT4和V-ATPase共定位于同一细胞内膜。这些结果表明，G4V膜上的V-ATPase可能通过参与囊泡的渗透性肿胀，在胰岛素刺激的G4V与质膜的胞外融合中发挥重要作用。
In secretory cells, osmotic swelling of secretory granules is proposed to be an intermediate step in exocytic fusion of the granules with the plasma membrane. For osmotic swelling of the granule, a H+ gradient generated by vacuolar-type H+-ATPase (V-ATPase) may be a driving force for accumulation of K+ via its exchange with H+, concurrent with accumulation of Cl- and H2O. Here, we investigated whether a similar chemiosmotic mechanism is involved in the insulin-stimulated recruitment of GLUT4 to the plasma membrane in 3T3-F442A adipocytes. Incubating cells in a hypeosmotic medium significantly increased 2-deoxy glucose (2-DG) uptake and the plasma membrane GLUT4 content (possibly via induction of osmotic swelling of GLUT4-containing vesicles (G4V)) and also potentiated the insulin-stimulated 2-DG uptake. Promotion of the G4V membrane ionic permeability using nigericin, an electroneutral K+/H+ exchange ionophore, increased 2-DG uptake and the plasma membrane GLUT4 content. However, co-treatment with nigericin and insulin did not show an additive effect. Bafilomycin A, a diagnostically specific inhibitor of V-ATPase, inhibited insulin- and nigericin-stimulated 2-DG uptake. Immunoadsorption plus immunoblotting demonstrated that GLUT4 and V-ATPase co-localize in the same intracellular membranes. Together, these results indicate that V-ATPases in the G4V membrane may play all important role in the insulin-stimulated exocytic fusion of G4V with the plasma membrane via its participation in osmotic swelling of the vesicle.