James D E, Pilch P F
Department of Biochemistry, Boston University School of Medicine, MA 02118.
Biochem J. 1988 Dec 15;256(3):725-32. doi: 10.1042/bj2560725.
We subfractionated intracellular vesicles from rat adipocytes in order to examine the subcellular distribution of endocytic vesicles or endosomes with respect to insulin-regulatable glucose-transporter (GT)-containing vesicles [James, Lederman & Pilch (1987) J. Biol. Chem. 262, 11817-11824]. Vesicles mediating fluid-phase endocytosis sedimented as a single major peak of greater density than the single distinct peak of GT-containing vesicles. This difference was also apparent during cellular insulin exposure and after insulin removal. Endocytosis of insulin and IGF (insulin-like growth factor) II was also examined. In sucrose gradients, IGF II-containing vesicles were less dense than those containing internalized insulin. Receptor-mediated endocytic vesicles were distinct from fluid-phase endocytic vesicles, but overlapped with the GT-containing vesicles. Vesicles containing internalized ligand were further fractionated by agarose-gel electrophoresis after various times of internalization. At least three different vesicle subpopulations containing the iodinated ligands were resolved after 5 min of internalization. Endocytic vesicles containing rapidly internalized insulin (1.5 min at 37 degrees C) consistently co-migrated with GT-containing vesicles. These data indicate that fluid-phase and receptor-mediated endocytosis occur via different pathways in adipocytes. Furthermore, whereas the intracellular GT-containing vesicles are distinct from fluid-phase vesicles, a rapidly labelled pool of insulin-containing vesicles consistently co-fractionated with GT-containing vesicles when separation techniques based on size, density and charge were used. This suggests that the insulin receptor may directly interact with the intracellular GT-containing vesicles after insulin-induced endocytosis.
为了研究内吞小泡或内体相对于含胰岛素可调节葡萄糖转运蛋白（GT）的小泡在亚细胞水平的分布情况，我们对大鼠脂肪细胞的细胞内小泡进行了亚分级分离[詹姆斯、莱德曼和皮尔希（1987年）《生物化学杂志》262卷，11817 - 11824页]。介导液相内吞作用的小泡沉降为一个单一的主峰，其密度大于含GT小泡的单一明显峰。这种差异在细胞暴露于胰岛素期间以及去除胰岛素后也很明显。我们还研究了胰岛素和胰岛素样生长因子（IGF）II的内吞作用。在蔗糖梯度中，含IGF II的小泡比含内化胰岛素的小泡密度小。受体介导的内吞小泡与液相内吞小泡不同，但与含GT的小泡重叠。在不同内化时间后，通过琼脂糖凝胶电泳对含内化配体的小泡进行进一步分级分离。内化5分钟后，至少分辨出三个不同的含碘化配体的小泡亚群。含快速内化胰岛素（37℃下1.5分钟）的内吞小泡始终与含GT的小泡共同迁移。这些数据表明，液相和受体介导的内吞作用在脂肪细胞中通过不同途径发生。此外，虽然细胞内含GT的小泡与液相小泡不同，但当使用基于大小、密度和电荷的分离技术时，一个快速标记的含胰岛素小泡池始终与含GT的小泡共同分级分离。这表明胰岛素受体在胰岛素诱导的内吞作用后可能直接与细胞内含GT的小泡相互作用。
