Rab10 in insulin-stimulated GLUT4 translocation

In fat and muscle cells, insulin stimulates the movement to and fusion of intracellular vesicles containing GLUT4 with the plasma membrane, a process referred to as GLUT4 translocation. Previous studies have indicated that Akt [also known as PKB (protein kinase B)] phosphorylation of AS 160, a GAP (GTPase-activating protein) for Rabs, is required for GLUT4 translocation. The results suggest that this phosphorylation suppresses the GAP activity and leads to the elevation of the GTP form of one or more Rabs required for GLUT4 translocation. Based on their presence in GLUT4 vesicles and activity as AS 160 GAP substrates, Rabs 8A, 8B, 10 and 14 are candidate Rabs. Here, we provide further evidence that Rab10 participates in GLUT4 translocation in 3T3-L1 adipocytes. Among Rabs 8A, 8B, 10 and 14, only the knockdown of Rab10 inhibited GLUT4 translocation. In addition, we describe the subcellular distribution of Rab10 and estimate the fraction of Rab10 in the active GTP form in vivo. Approx. 5% of the total Rab10 was present in GLUT4 vesicles isolated from the low-density microsomes. In both the basal and the insulin state, 90% of the total Rab10 was in the inactive GDP state. Thus, if insulin increases the GTP form of Rub 10, the increase is limited to a small portion of the total Rab10. Finally, we report that the Rab10 mutant considered to be constitutively active (Rab10 Q68L) is a substrate for the AS160 GAP domain and, hence, cannot be used to deduce rigorously the function of Rab10 in its GTP form.

在脂肪细胞和肌肉细胞中，胰岛素刺激含有葡萄糖转运蛋白4（GLUT4）的细胞内囊泡向细胞膜移动并与之融合，这一过程被称为GLUT4转位。先前的研究表明，Akt[也称为PKB（蛋白激酶B）]对AS160（一种Rab蛋白的GTP酶激活蛋白，GAP）的磷酸化作用是GLUT4转位所必需的。研究结果表明，这种磷酸化作用抑制了GAP活性，并导致GLUT4转位所需的一种或多种Rab蛋白的GTP形式增加。基于它们存在于GLUT4囊泡中以及作为AS160 GAP底物的活性，Rab8A、Rab8B、Rab10和Rab14是可能的Rab蛋白。在此，我们提供了进一步的证据表明Rab10参与3T3 - L1脂肪细胞中GLUT4的转位。在Rab8A、Rab8B、Rab10和Rab14中，只有Rab10的敲低抑制了GLUT4转位。此外，我们描述了Rab10的亚细胞分布，并估计了体内处于活性GTP形式的Rab10的比例。从低密度微粒体中分离出的GLUT4囊泡中约有5%的总Rab10存在。在基础状态和胰岛素刺激状态下，总Rab10中有90%处于无活性的GDP状态。因此，如果胰岛素增加Rab10的GTP形式，这种增加也仅限于总Rab10的一小部分。最后，我们报道被认为具有组成型活性的Rab10突变体（Rab10 Q68L）是AS160 GAP结构域的底物，因此不能用于严格推断Rab10处于其GTP形式时的功能。