Moss A L, Ward W F
Department of Physiology, University of Texas Helath Science Center, San Antonio 78284-7756.
J Cell Physiol. 1991 Nov;149(2):319-23. doi: 10.1002/jcp.1041490220.
In a companion report (Moss and Ward: J. Cell. Physiol 149:313-318, 1991) evidence was presented for multiple pathways for insulin internalization based on differences between the internalization of insulin and that of two other ligands, asialofetuin (Afet) and epidermal growth factor (EGF), in the presence of several perturbations of endocytosis. In the present study we have explored the characteristics of three internalization pathways and the contribution of each to overall insulin uptake. Freshly isolated hepatocytes were incubated with radiolabeled ligands in the presence of hyperosmolar sucrose, treatment that is thought to inhibit the coated pit pathway of endocytosis. Insulin internalization was decreased approximately 39%, but much greater decreases were observed with Afet (86%) and EGF (62%). Competition between uptake of radiolabeled and unlabeled insulin was observed in hyperosmolar-treated cells, suggestive of endocytosis by a receptor-mediated noncoated-pit pathway. Uptake of radiolabeled insulin that persisted in the presence of hyperosmolarity and high concentrations of unlabeled insulin suggested a third uptake pathway: fluid-phase endocytosis. A rate of fluid-phase endocytosis of 7.2 microL/hr/10(6) cells was determined from the uptake of the fluid-phase marker lucifer yellow. At high insulin concentrations (greater than or equal to 250 ng/ml), fluid-phase endocytosis appears to be the predominant pathway for insulin uptake, but at lower insulin concentrations (physiological) the coated pit and noncoated pit pathways are the primary routes for insulin internalization.
在一篇相关报告中（莫斯和沃德：《细胞生理学杂志》149:313 - 318，1991年），基于胰岛素与另外两种配体——去唾液酸胎球蛋白（Afet）和表皮生长因子（EGF）在几种内吞作用扰动存在时内化情况的差异，提出了胰岛素内化的多种途径的证据。在本研究中，我们探究了三种内化途径的特征以及每种途径对整体胰岛素摄取的贡献。将新鲜分离的肝细胞与放射性标记的配体在高渗蔗糖存在的情况下孵育，这种处理被认为会抑制内吞作用的被膜小窝途径。胰岛素内化减少了约39%，但Afet（86%）和EGF（62%）的减少幅度更大。在高渗处理的细胞中观察到放射性标记胰岛素与未标记胰岛素摄取之间的竞争，这表明存在一种受体介导的非被膜小窝途径的内吞作用。在高渗和高浓度未标记胰岛素存在时仍持续的放射性标记胰岛素摄取表明存在第三种摄取途径：液相内吞作用。根据液相标记物路西法黄的摄取情况确定液相内吞作用的速率为7.2微升/小时/10⁶个细胞。在高胰岛素浓度（大于或等于250纳克/毫升）时，液相内吞作用似乎是胰岛素摄取的主要途径，但在较低胰岛素浓度（生理浓度）时，被膜小窝和非被膜小窝途径是胰岛素内化的主要途径。
