Hyslop P A, Kuhn C E, Sauerheber R D
Biochem Pharmacol. 1987 Jul 15;36(14):2305-10. doi: 10.1016/0006-2952(87)90595-8.
Aspects of the mechanism by which insulin stimulates the membrane glucose transport system were examined by assessing the influence of the bilayer lipid structure on transport stimulation characteristics, and considering the form of the insulin dose-response curve. We tested the effects of membrane lipid perturbation on the insulin stimulation process. Benzyl alcohol, at concentrations (25 mM) that grossly fluidize lipids forming the adipocyte membrane bilayer matrix, caused 50% inhibition of intrinsic transporter activity. However, this membrane perturbation had no significant effect on either the insulin dose-response curve (conducted at 37 degrees) or the time-course of the insulin stimulation of hexose transport (conducted at 32 degrees). These data are difficult to rationalize in terms of a model in which transport stimulation involves interaction of transporters and hormone-bound receptors that is limited by lateral diffusion of these proteins in the fluid lipid bilayer. Curve-fitting experimental insulin dose-response data for stimulation of 2-deoxy-D-glucose and D-glucose uptake provided an estimate of an insulin "association constant" for transport regulation that may be compared with recent insulin receptor binding data. Similar magnitude constants were obtained whether estimated directly from plots of transport velocity versus arithmetic hormone dose, or by extrapolation from linear segments of sigmoidal velocity versus log dose plots, or from inverse (Lineweaver-Burk-type) plots of the insulin dose-response data. Insulin apparently regulates transport by associating with a binding site, having an apparent dissociation constant which is determinable through kinetic measurements of hexose uptake (KDapp approx. 17-40 pM). This is in good agreement with the dissociation constant, KD, determined from Scatchard plots of recent binding data to adipocytes, for a class of receptors representing the "high affinity" binding sites for insulin. Insulin dose-response curve simulations also indicated that the stimulation process may be classified in pharmacologic terms as a typical graded biologic response and may involve insulin association with a site that regulates transport rates in a manner kinetically analogous to allosteric modulation of a V-series enzyme by a noncompetitive ligand. From the results we suggest that a relatively close association occurs between transport and receptor proteins in the membrane, where the relative activation of transport depends on the fractional occupancy of functional high affinity receptors by insulin, and the insulin stimulation of transport involves regions of the membrane that are not influenced significantly by
通过评估双层脂质结构对转运刺激特性的影响，并考虑胰岛素剂量反应曲线的形式，研究了胰岛素刺激膜葡萄糖转运系统的机制。我们测试了膜脂质扰动对胰岛素刺激过程的影响。苄醇浓度为25 mM时，会使构成脂肪细胞膜双层基质的脂质严重流化，导致内在转运体活性受到50%的抑制。然而，这种膜扰动对胰岛素剂量反应曲线（在37℃下进行）或胰岛素刺激己糖转运的时间进程（在32℃下进行）均无显著影响。就转运刺激涉及转运体与激素结合受体相互作用的模型而言，这些数据难以解释，因为这种相互作用受这些蛋白质在流体脂质双层中的侧向扩散限制。对刺激2-脱氧-D-葡萄糖和D-葡萄糖摄取的实验胰岛素剂量反应数据进行曲线拟合，得出了一个用于转运调节的胰岛素“缔合常数”估计值，可与近期的胰岛素受体结合数据进行比较。无论是直接从转运速度与算术激素剂量的图中估计，还是从S形速度与对数剂量图的线性段外推，或者从胰岛素剂量反应数据的倒数（Lineweaver-Burk型）图中估计，都得到了相似大小的常数。胰岛素显然通过与一个结合位点结合来调节转运，该结合位点具有一个表观解离常数，可通过己糖摄取的动力学测量来确定（KDapp约为17 - 40 pM）。这与从近期脂肪细胞结合数据的Scatchard图中确定的解离常数KD非常一致，该解离常数代表了一类胰岛素“高亲和力”结合位点的受体。胰岛素剂量反应曲线模拟还表明，刺激过程在药理学上可归类为典型的分级生物学反应，可能涉及胰岛素与一个位点的结合，该位点以动力学上类似于非竞争性配体对V系列酶的变构调节的方式调节转运速率。从结果中我们认为，膜中的转运蛋白和受体蛋白之间存在相对紧密的联系，其中转运的相对激活取决于胰岛素对功能性高亲和力受体的占据分数，并且胰岛素对转运的刺激涉及膜的一些区域，这些区域不受……的显著影响
