Martin S, Reaves B, Banting G, Gould G W
Department of Biochemistry, University of Glasgow, Scotland, U.K.
Biochem J. 1994 Jun 15;300 ( Pt 3)(Pt 3):743-9. doi: 10.1042/bj3000743.
The exposure of isolated adipocytes to insulin results in an approximately 20-fold increase in the rate of glucose transport into the cell. This increase is mediated by the movement of a pool of intracellular vesicles containing the so-called insulin-responsive glucose transporter (GLUT4) to the cell surface. In the resting state, most of the GLUT4 molecules are sequestered inside the adipocyte in an as yet unidentified intracellular compartment. TGN38 is an integral membrane protein which has been shown to be predominantly localized to the trans Golgi network [Luzio, Brake, Banting, Howell, Braghetta and Stanley (1990) Biochem. J. 270, 97-102]. Here we investigate whether GLUT4 and TGN38 are co-localized in the murine 3T3-L1 adipocyte cell line. Immuno-adsorption of intracellular vesicles containing GLUT4 with an anti-peptide antibody specific for this isoform did not deplete the low-density microsomal fraction of TGN38 in these cells; moreover, no TGN38 was detected in the GLUT4-containing vesicles by immunoblotting with a TGN38-specific antiserum. Immuno-adsorption of TGN38-containing vesicles and subsequent analysis of the proteins in these vesicles revealed that a detectable amount of GLUT4 (5-10%) did co-localise with TGN38. The amount of GLUT4 in the TGN38-containing vesicles did not change in response to insulin. Immunofluorescence analysis of TGN38 and GLUT4 in these cells revealed markedly different staining patterns. Reversal of insulin-stimulated glucose transport and subsequent analysis of the TGN38-containing vesicles demonstrated that during the re-cycling of GLUT4 to the intracellular storage site there was no increase in the amount of GLUT4 co-localized with TGN38. Taken together, these results suggest that the trans Golgi network is not the major site of the intracellular GLUT4 pool within 3T3-L1 adipocytes.
将分离的脂肪细胞暴露于胰岛素会导致葡萄糖进入细胞的速率增加约20倍。这种增加是由一群含有所谓胰岛素反应性葡萄糖转运蛋白（GLUT4）的细胞内囊泡移动到细胞表面介导的。在静止状态下，大多数GLUT4分子被隔离在脂肪细胞内一个尚未确定的细胞内区室中。TGN38是一种整合膜蛋白，已被证明主要定位于反式高尔基体网络[卢齐奥、布雷克、班廷、豪厄尔、布拉盖塔和斯坦利（1990年）《生物化学杂志》270卷，97 - 102页]。在此我们研究GLUT4和TGN38在小鼠3T3 - L1脂肪细胞系中是否共定位。用针对该异构体的抗肽抗体对含有GLUT4的细胞内囊泡进行免疫吸附，并未耗尽这些细胞中TGN38的低密度微粒体部分；此外，用TGN38特异性抗血清进行免疫印迹时，在含有GLUT4的囊泡中未检测到TGN38。对含有TGN38的囊泡进行免疫吸附并随后分析这些囊泡中的蛋白质，结果显示可检测到一定量的GLUT4（5 - 10%）与TGN38共定位。含有TGN38的囊泡中GLUT4的量不会因胰岛素而改变。对这些细胞中TGN38和GLUT4进行免疫荧光分析，结果显示染色模式明显不同。胰岛素刺激的葡萄糖转运逆转并随后分析含有TGN38的囊泡表明，在GLUT4再循环至细胞内储存位点的过程中，与TGN38共定位的GLUT4量没有增加。综上所述，这些结果表明反式高尔基体网络不是3T3 - L1脂肪细胞内GLUT4池的主要位点。
