Hickson G R, Chamberlain L H, Maier V H, Gould G W
Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Davidson Building, Glasgow, G12 8QQ, United Kingdom.
Biochem Biophys Res Commun. 2000 Apr 21;270(3):841-5. doi: 10.1006/bbrc.2000.2525.
Insulin-stimulates glucose transport in peripheral tissues by stimulating the movement ('translocation') of a pool of intracellular vesicles containing the glucose transporter Glut4 to the cell surface. The fusion of these vesicles with the plasma membrane results in a large increase in the numbers of Glut4 molecules at the cell surface and a concomitant enhancement of glucose uptake. It is well established that proteins of the VAMP- (synaptobrevin) and syntaxin-families play a fundamental role in the insulin-stimulated fusion of Glut4-containing vesicles with the plasma membrane. Studies have identified key roles for vesicle associated membrane protein-2 (VAMP2) and syntaxin-4 in this event, and more recently have also implicated SNAP-23 and Munc18c in this process. In this study, we have quantified the absolute levels of expression of these proteins in murine 3T3-L1 adipocytes, with the objective of determining the stoichiometry of these proteins both relative to each other and also in comparison with previous estimates of Glut4 levels within these cells. To achieve this, we performed quantitative immunoblot analysis of these proteins in 3T3-L1 membranes compared to known amounts of purified recombinant proteins. Such analyses suggest that in 3T3-L1 adipocytes there are approximately 374,000 copies of syntaxin 4, 1.15 x 10(6) copies of SNAP23, 495,000 copies of VAMP2, 4.3 x 10(6) copies of cellubrevin and 452,000 copies of Munc18c per cell, compared to previous estimates of 280,000 copies of Glut4. Thus, the main SNARE proteins involved in insulin-stimulated Glut4 exocytosis (syntaxin 4 and VAMP2) are expressed in approximately equimolar amounts in adipocytes, whereas by contrast the endosomal v-SNARE cellubrevin is present at approximately 10-fold higher levels and the t-SNARE SNAP-23 is also present in an approximately 3-fold molar excess. The implications of this quantification for the mechanism of insulin-stimulated Glut4 translocation are discussed.
胰岛素通过刺激包含葡萄糖转运蛋白Glut4的细胞内囊泡池向细胞表面移动（“易位”），从而促进外周组织中的葡萄糖转运。这些囊泡与质膜融合导致细胞表面Glut4分子数量大幅增加，同时葡萄糖摄取增强。众所周知，VAMP-（突触小泡蛋白）和 syntaxin家族的蛋白质在胰岛素刺激的含Glut4囊泡与质膜融合中起基本作用。研究已经确定了囊泡相关膜蛋白-2（VAMP2）和 syntaxin-4在此过程中的关键作用，最近还发现SNAP-23和Munc18c也参与其中。在本研究中，我们定量了这些蛋白质在小鼠3T3-L1脂肪细胞中的绝对表达水平，目的是确定这些蛋白质彼此之间的化学计量关系，以及与这些细胞中先前估计的Glut4水平进行比较。为了实现这一目标，我们对3T3-L1膜中的这些蛋白质进行了定量免疫印迹分析，并与已知量的纯化重组蛋白进行比较。此类分析表明，在3T3-L1脂肪细胞中，每个细胞约有374,000个 syntaxin 4拷贝、1.15×10⁶个SNAP23拷贝、495,000个VAMP2拷贝、4.3×10⁶个细胞ubrevin拷贝和452,000个Munc18c拷贝，而先前估计Glut4为280,000个拷贝。因此，参与胰岛素刺激的Glut4胞吐作用的主要SNARE蛋白（syntaxin 4和VAMP2）在脂肪细胞中的表达量大致相等，而相比之下，内体v-SNARE细胞ubrevin的水平约高10倍，t-SNARE SNAP-23的摩尔过量约为3倍。本文讨论了这种定量分析对胰岛素刺激的Glut4易位机制的影响。
