Gibbs E M, Lienhard G E, Appleman J R, Lane M D, Frost S C
J Biol Chem. 1986 Mar 25;261(9):3944-51.
Fluid phase endocytosis by monolayers of 3T3-L1 adipocytes has been followed by measuring [14C]sucrose uptake, a well characterized pinocytic marker. Insulin, at a maximal stimulatory concentration, increased the pinocytic rate by 2-fold within 5 min of its addition; this activation persisted for at least 2 h. The dose-response curve for the enhancement of fluid-phase endocytosis by insulin was identical with that for the stimulation of hexose transport, as measured by the uptake of 2-deoxyglucose. The concentration of insulin eliciting half-maximal effects was 6 nM. These results suggest that activation of endocytosis and hexose uptake by insulin are triggered by the same signalling event. Insulin-activated pinocytosis was not dependent upon the increased metabolism of D-glucose that occurs in response to the hormone, since the stimulation of fluid-phase endocytosis occurred in the absence of 5 nM glucose. Fluid-phase exocytosis was examined by loading cells with [14C]sucrose for various times and then measuring tracer efflux. The rate of sucrose release was biphasic; a portion of the internalized sucrose was rapidly released from the cell (t1/2 approximately 5 min), whereas the remainder was released slowly (t1/2 approximately to 5 h). These results are consistent with a sequential two-compartment model in which the [14C] sucrose first enters a compartment from which about 70% of the sucrose is rapidly released back into the medium and the remaining 30% is transferred to a second compartment. Therefore, the true rate of endocytosis is much greater than the observed accumulation rates, except after short uptake times. Insulin increases the rate of sucrose efflux from both compartments as well as the rate of transfer from the first compartment to the second compartment by about 2-fold. Furthermore, insulin increased the apparent size of the first and second compartments by 1.6- and 3-fold, respectively. The lysosomotropic agent chloroquine (200 muM) had only a small effect on fluid movements in these cells. The rapid and prolonged stimulation of fluid-phase endocytosis and exocytosis by insulin are hitherto unrecognized effects of this hormone.
通过测量[14C]蔗糖摄取来追踪3T3-L1脂肪细胞单层的液相内吞作用，[14C]蔗糖是一种特征明确的胞饮标志物。胰岛素在最大刺激浓度下，在添加后5分钟内使胞饮速率增加了2倍；这种激活持续至少2小时。胰岛素增强液相内吞作用的剂量反应曲线与刺激己糖转运的曲线相同，己糖转运通过2-脱氧葡萄糖摄取来测量。引起半数最大效应的胰岛素浓度为6 nM。这些结果表明，胰岛素激活内吞作用和己糖摄取是由相同的信号事件触发的。胰岛素激活的胞饮作用不依赖于因该激素而发生的D-葡萄糖代谢增加，因为在没有5 nM葡萄糖的情况下也发生了液相内吞作用的刺激。通过用[14C]蔗糖加载细胞不同时间，然后测量示踪剂流出，来检测液相外排作用。蔗糖释放速率是双相的；一部分内化的蔗糖迅速从细胞中释放出来（半衰期约5分钟），而其余部分则缓慢释放（半衰期约5小时）。这些结果与一个顺序两室模型一致，其中[14C]蔗糖首先进入一个室，约70%的蔗糖迅速释放回培养基中，其余30%转移到第二个室。因此，除了短摄取时间后，真正的内吞速率远大于观察到的积累速率。胰岛素使两个室的蔗糖流出速率以及从第一个室到第二个室的转移速率增加约2倍。此外，胰岛素使第一个和第二个室的表观大小分别增加了1.6倍和3倍。溶酶体促渗剂氯喹（200 μM）对这些细胞中的液体运动只有很小的影响。胰岛素对液相内吞作用和外排作用的快速和长期刺激是该激素迄今未被认识的作用。
