Horuk R, Matthaei S, Olefsky J M, Baly D L, Cushman S W, Simpson I A
J Biol Chem. 1986 Feb 5;261(4):1823-8.
We have studied the biochemical mechanism of insulin action on glucose transport in the rat adipocyte. Plasma membranes and low-density microsomes were prepared by differential ultracentrifugation of basal and insulin-stimulated cells. The photochemical cross-linking agent hydroxysuccinimidyl-4-azidobenzoate was used to covalently bind [3H]cytochalasin B to the glucose transporter which migrated as a 45-50-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing of the eluted 40-55-kDa proteins revealed two peaks of D-glucose-inhibitable [3H]cytochalasin B radioactivity focusing at pH 6.4 and 5.6 when low-density microsomes were used as the starting material. In contrast, only one D-glucose inhibitable peak, focusing at pH 5.6, was found in plasma membranes. Pretreatment of the cells with insulin led to a marked redistribution of the pH 5.6 form of the glucose transporter from low-density microsomes to plasma membranes with no effect on the pH 6.4 form of the glucose transporter. Following isolation from the isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels, both glucose transporter isoforms were shown to cross-react with an antiserum raised against the purified human erythrocyte glucose transporter. Following incubation of [3H]cytochalasin B-labeled low-density microsomal and plasma membranes with neuraminidase, the pH 5.6 transporter isoform was shifted on isoelectric focusing to a more basic pH, while the pH 6.4 isoform was not affected. These data demonstrate that: there is a heterogeneity of glucose transporter species in the intracellular pool while the plasma membrane transporters are more uniform in structure. The pH 5.6 glucose transporter isoform is translocated by insulin from the low-density microsomes to the plasma membrane but the pH 6.4 isoform is not sensitive to insulin. Differential sensitivity of the glucose transporter isoforms to neuraminidase suggests that the heterogeneity is at least partially due to differences in glycosylation state.
我们研究了胰岛素对大鼠脂肪细胞葡萄糖转运作用的生化机制。通过对基础状态和胰岛素刺激后的细胞进行差速超速离心制备质膜和低密度微粒体。光化学交联剂4-叠氮苯甲酸羟基琥珀酰亚胺酯用于将[³H]细胞松弛素B共价结合到葡萄糖转运体上，该转运体在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上迁移为45 - 50 kDa的蛋白质。当以低密度微粒体为起始材料时，对洗脱的40 - 55 kDa蛋白质进行等电聚焦，发现两个D-葡萄糖可抑制的[³H]细胞松弛素B放射性峰聚焦在pH 6.4和5.6处。相比之下，在质膜中仅发现一个聚焦于pH 5.6的D-葡萄糖可抑制峰。用胰岛素预处理细胞导致葡萄糖转运体pH 5.6形式从低密度微粒体显著重新分布到质膜，而对葡萄糖转运体pH 6.4形式无影响。从等电聚焦和十二烷基硫酸钠-聚丙烯酰胺凝胶中分离后，两种葡萄糖转运体同工型均显示与针对纯化的人红细胞葡萄糖转运体制备的抗血清发生交叉反应。用神经氨酸酶孵育[³H]细胞松弛素B标记的低密度微粒体和质膜后，pH 5.6转运体同工型在等电聚焦时向更碱性的pH值移动，而pH 6.4同工型不受影响。这些数据表明：细胞内池中葡萄糖转运体种类存在异质性，而质膜转运体在结构上更为均匀。葡萄糖转运体pH 5.6同工型被胰岛素从低密度微粒体转运到质膜，但pH 6.4同工型对胰岛素不敏感。葡萄糖转运体同工型对神经氨酸酶的不同敏感性表明这种异质性至少部分是由于糖基化状态的差异。
