Immunoelectron microscopic demonstration of insulin-stimulated translocation of glucose transporters to the plasma membrane of isolated rat adipocytes and masking of the carboxyl-terminal epitope of intracellular GLUT4.
Immunoelectron microscopic demonstration of insulin-stimulated translocation of glucose transporters to the plasma membrane of isolated rat adipocytes and masking of the carboxyl-terminal epitope of intracellular GLUT4.
复制标题
免疫电子显微镜演示了胰岛素刺激的葡萄糖转运蛋白易位至分离的大鼠脂肪细胞的质膜以及细胞内 GLUT4 的羧基末端表位的掩蔽。
DOI：
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发表时间：
1991
影响因子：
11.1
通讯作者：
L. Jarett
中科院分区：
文献类型：
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作者：
Robert M. Smith;M. J. Charron;N. Shah;H. Lodish;L. Jarett
在免疫电镜研究中使用了GLUT4转运蛋白的氨基或羧基末端肽序列的多克隆抗体，以证明在完整的分离大鼠脂肪细胞中GLUT4的位置和胰岛素诱导的易位。用氨基末端抗体标记未处理的脂肪细胞，发现95%的GLUT4在细胞内，与质膜内陷或与细胞膜相邻或在细胞膜75 nm内的囊泡相关。胰岛素治疗增加质膜标记约13倍，总转运蛋白的52%，并降低细胞内标记的比例。相比之下，用羧基末端抗体或结合GLUT 4羧基末端的单克隆抗体（1F8）标记未处理的脂肪细胞，检测到的转运蛋白较少，其中只有约40%在细胞内。在胰岛素处理的细胞中，质膜标记增加约20倍，但标记的转运蛋白的总数也增加约13倍。细胞内转运蛋白的数量没有改变。胰岛素诱导的质膜标记增加是可逆的。因此，在未处理的脂肪细胞中，绝大多数GLUT4转运蛋白在细胞内的内陷或囊泡中附着或靠近质膜。胰岛素治疗导致转运蛋白易位至质膜，这涉及转运蛋白从内陷流至细胞表面以及质膜下囊泡与质膜的可能融合。通过肽抗体标记细胞内转运蛋白的差异表明细胞内转运蛋白的羧基末端表位被掩蔽。羧基端在转运至质膜过程中的暴露可能是胰岛素刺激大鼠脂肪细胞葡萄糖转运机制的一部分。
Polyclonal antibodies to the amino- or carboxyl-terminated peptide sequences of the GLUT4 transporter protein were used in immunoelectron microscopic studies to demonstrate the location and insulin-induced translocation of GLUT4 in intact isolated rat adipocytes. Labeling of untreated adipocytes with the amino-terminal antibody revealed 95% of GLUT4 was intracellular, associated with plasma membrane invaginations or vesicles contiguous with or within 75 nm of the cell membrane. Insulin treatment increased plasma membrane labeling approximately 13-fold, to 52% of the total transporters, and decreased intracellular labeling proportionately. In contrast, labeling of untreated adipocytes with the carboxyl-terminal antibody or with a monoclonal antibody (1F8) that binds to the carboxyl terminus of GLUT4 detected fewer transporters, only approximately 40% of which were intracellular. In insulin-treated cells, plasma membrane labeling increased approximately 20-fold, but the total number of labeled transporters also increased approximately 13-fold. The number of intracellular transporters was not changed. The insulin-induced increase in plasma membrane labeling was reversible. Thus, the vast majority of GLUT4 transporters in untreated adipocytes are intracellular in invaginations or vesicles attached or close to the plasma membrane. Insulin treatment causes translocation of transporters to the plasma membrane, which involves flow of transporters from invaginations to the cell surface and possible fusion of subplasma membrane vesicles with the plasma membrane. Differences in the labeling of intracellular transporters by peptide antibodies suggested the carboxyl-terminal epitope of intracellular transporters was masked. The unmasking of the carboxyl terminus during translocation to the plasma membrane may be part of the mechanism by which insulin stimulates glucose transport in rat adipocytes.