Ludvigsen C, Jarett L
Diabetes. 1980 May;29(5):373-8. doi: 10.2337/diab.29.5.373.
Specific D-glucose transport in plasma membranes prepared from control and insulin-treated rat adipocytes was measured using a recently developed dual isotope ([3H]-D-glucose and [14C]-L-glucose), rapid filtration assay which allowed measurements of initial rates at 1 s and 37 degrees. Plasma membranes from insulin-treated adipocytes showed an increase in glucose transport compared with control cells. Saturation kinetic data revealed that the plasma membranes from insulin-treated and control cells had the same Km (26 mM) for glucose transport, whereas insulin treatment increased the Vmax from 4433 pmol/mg protein/s to 9465 pmol/mg proteins/s. Arrhenius plots showed no difference in the energy of activation between control and insulin-stimulated glucose transport states. The optimum pH of both control and insulin-stimulated glucose transport was 7.4. Lower or higher pHs progressively decreased both control and insulin-stimulated glucose transport proportionately. Calcium in the transport assay media did not affect basal or insulin-stimulated glucose transport. However, omission of calcium from the adipocyte incubation media significantly lowered the insulin stimulation by 24% while basal levels were not significantly affected. Insulin specifically bound to the plasma membrane was carried through the fractionation procedure, but removal of this insulin did not alter the stimulated glucose transport. Glucose transport by plasma membranes from control or insulin-treated adipocytes was equally (percentage) inhibited by N-ethylmaleimide, dithiothreitol, reduced glutathione, or cytochalasin B. No inhibition of control or insulin-stimulated transport was seen with cytochalasin D or oxidized glutathione. The data presented are consistent with insulin causing the formation of new transport sites similar to the existing basal sites.
利用最近开发的双同位素（[3H]-D-葡萄糖和[14C]-L-葡萄糖）快速过滤分析法，测量了从对照和胰岛素处理的大鼠脂肪细胞制备的质膜中特异性D-葡萄糖转运，该方法能够在1秒和37摄氏度下测量初始速率。与对照细胞相比，胰岛素处理的脂肪细胞质膜显示出葡萄糖转运增加。饱和动力学数据表明，胰岛素处理细胞和对照细胞质膜对葡萄糖转运的Km值相同（26 mM），而胰岛素处理使Vmax从4433 pmol/mg蛋白质/秒增加到9465 pmol/mg蛋白质/秒。阿累尼乌斯曲线表明，对照和胰岛素刺激的葡萄糖转运状态之间的活化能没有差异。对照和胰岛素刺激的葡萄糖转运的最佳pH值均为7.4。较低或较高的pH值会成比例地逐渐降低对照和胰岛素刺激的葡萄糖转运。转运测定培养基中的钙不影响基础或胰岛素刺激的葡萄糖转运。然而，从脂肪细胞孵育培养基中去除钙会使胰岛素刺激显著降低24%，而基础水平没有受到显著影响。特异性结合到质膜上的胰岛素在分级分离过程中被保留，但去除这种胰岛素并没有改变刺激的葡萄糖转运。对照或胰岛素处理的脂肪细胞质膜的葡萄糖转运同样（百分比）受到N-乙基马来酰亚胺、二硫苏糖醇、还原型谷胱甘肽或细胞松弛素B的抑制。细胞松弛素D或氧化型谷胱甘肽对对照或胰岛素刺激的转运没有抑制作用。所呈现的数据与胰岛素导致形成类似于现有基础位点的新转运位点一致。
