Khoursandi Saeed, Scharlau Daniel, Herter Peter, Kuhnen Cornelius, Martin Dirk, Kinne Rolf K H, Kipp Helmut
Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany.
Am J Physiol Cell Physiol. 2004 Oct;287(4):C1041-7. doi: 10.1152/ajpcell.00197.2004. Epub 2004 Jun 16.
We recently reported that a considerable amount of the sodium-d-glucose cotransporter SGLT1 present in Caco-2 cells, a model for human enterocytes, is located in intracellular compartments attached to microtubules. A similar distribution pattern was also observed in enterocytes in thin sections from human jejunum, highlighting the validity of the Caco-2 cell model. Fluorescent surface labeling of live Caco-2 cells revealed that the intracellular compartments containing SGLT1 were accessible by endocytosis. To elucidate the role of endosomal SGLT1 in the regulation of sodium-dependent d-glucose uptake into enterocytes, we compared SGLT1-mediated D-glucose uptake into Caco-2 cells with the subcellular distribution of SGLT1 after challenging the cells with different stimuli. Incubation (90 min) of Caco-2 cells with mastoparan (50 microM), a drug that enhances apical endocytosis, shifted a large amount of SGLT1 from the apical membrane to intracellular sites and significantly reduced sodium-dependent alpha-[(14)C]methyl-D-glucose uptake (-60%). We also investigated the effect of altered extracellular D-glucose levels. Cells preincubated (1 h) with d-glucose-free medium exhibited significantly higher sodium-dependent alpha-[(14)C]methyl-D-glucose uptake (+45%) than did cells preincubated with high d-glucose medium (100 mM, 1 h). Interestingly, regulation of SGLT1-mediated d-glucose uptake into Caco-2 cells by extracellular D-glucose levels occurred without redistribution of cellular SGLT1. These data suggest that, pharmacologically, d-glucose uptake can be regulated by a shift of SGLT1 between the plasma membrane and the endosomal pool; however, regulation by the physiological substrate d-glucose can be explained only by an alternative mechanism.
我们最近报道，在人肠上皮细胞模型Caco-2细胞中存在的大量钠-葡萄糖协同转运蛋白SGLT1定位于附着于微管的细胞内区室。在人空肠薄片的肠上皮细胞中也观察到类似的分布模式，突出了Caco-2细胞模型的有效性。活Caco-2细胞的荧光表面标记显示，含有SGLT1的细胞内区室可通过内吞作用进入。为了阐明内体SGLT1在调节钠依赖性葡萄糖摄取进入肠上皮细胞中的作用，我们在用不同刺激物刺激细胞后，比较了SGLT1介导的葡萄糖摄取进入Caco-2细胞与SGLT1的亚细胞分布。用mastoparan（50 microM）孵育Caco-2细胞90分钟，mastoparan是一种增强顶端内吞作用的药物，它使大量SGLT1从顶端膜转移到细胞内位点，并显著降低钠依赖性α-[(14)C]甲基-D-葡萄糖摄取（-60%）。我们还研究了细胞外葡萄糖水平改变的影响。用无葡萄糖培养基预孵育1小时的细胞比用高葡萄糖培养基（100 mM，1小时）预孵育的细胞表现出显著更高的钠依赖性α-[(14)C]甲基-D-葡萄糖摄取（+45%）。有趣的是，细胞外葡萄糖水平对SGLT1介导的葡萄糖摄取进入Caco-2细胞的调节发生在细胞SGLT1没有重新分布的情况下。这些数据表明，在药理学上，葡萄糖摄取可通过SGLT1在质膜和内体池之间的转移来调节；然而，生理底物葡萄糖的调节只能用另一种机制来解释。
