Bevan A P, Krook A, Tikerpae J, Seabright P J, Siddle K, Smith G D
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QR, United Kingdom.
J Biol Chem. 1997 Oct 24;272(43):26833-40. doi: 10.1074/jbc.272.43.26833.
Insulin signal transduction, initiated by binding of insulin to its receptor at the plasma membrane, activates the intrinsic receptor tyrosine kinase and leads to internalization of the activated ligand-receptor complex into endosomes. This study addresses the role played by the activated insulin receptor within hepatic endosomes and provides evidence for its central role in insulin-stimulated events in vivo. Rats were treated with chloroquine, an acidotrophic agent that has been shown previously to inhibit endosomal insulin degradation, and then with insulin. Livers were removed and fractionated by density gradient centrifugation to obtain endosomal and plasma membrane preparations. Chloroquine treatment increased the amount of receptor-bound insulin in endosomes at 2 min after insulin injection by 93% as determined by exclusion from G-50 columns and by 90% as determined by polyethylene glycol precipitation (p < 0.02). Chloroquine treatment also increased the insulin receptor content of endosomes after insulin injection (integrated over 0-45 min) by 31% when compared with controls (p < 0.05). Similarly, chloroquine increased both insulin receptor phosphotyrosine content and its exogenous tyrosine kinase activity after insulin injection (64%; p < 0.01 and 96% and p < 0. 001, respectively). In vivo chloroquine treatment was without any observable effect on insulin binding to plasma membrane insulin receptors, nor did it augment insulin-stimulated receptor autophosphorylation or kinase activity in the plasma membrane. Concomitant with its effects on endosomal insulin receptors, chloroquine treatment augmented insulin-stimulated incorporation of glucose into glycogen in diaphragm (p < 0.001). These observations are consistent with the hypothesis that chloroquine-dependent inhibition of endosomal insulin receptor dissociation and subsequent degradation prolongs the half-life of the active endosomal receptor and potentiates insulin signaling from this compartment.
胰岛素信号转导由胰岛素与质膜上的受体结合引发，激活内在受体酪氨酸激酶，并导致活化的配体-受体复合物内化进入内体。本研究探讨了活化的胰岛素受体在肝内体中所起的作用，并为其在体内胰岛素刺激事件中的核心作用提供了证据。用氯喹（一种先前已证明可抑制内体胰岛素降解的酸营养剂）处理大鼠，然后注射胰岛素。取出肝脏并通过密度梯度离心进行分级分离，以获得内体和质膜制剂。通过G-50柱排除法测定，氯喹处理使胰岛素注射后2分钟内体内受体结合胰岛素的量增加了93%，通过聚乙二醇沉淀法测定增加了90%（p<0.02）。与对照组相比，氯喹处理还使胰岛素注射后（0-45分钟内积分）内体的胰岛素受体含量增加了31%（p<0.05）。同样，氯喹增加了胰岛素注射后胰岛素受体磷酸酪氨酸含量及其外源酪氨酸激酶活性（分别为64%；p<0.01和96%；p<0.001）。体内氯喹处理对胰岛素与质膜胰岛素受体的结合没有任何可观察到的影响，也没有增强胰岛素刺激的质膜受体自身磷酸化或激酶活性。与对内体胰岛素受体的作用一致，氯喹处理增强了胰岛素刺激的膈肌中葡萄糖掺入糖原的过程（p<0.001）。这些观察结果与以下假设一致，即氯喹依赖性抑制内体胰岛素受体解离和随后的降解延长了活性内体受体的半衰期，并增强了来自该区室的胰岛素信号传导。
