Regulation of the GLUT1 glucose transporter in cultured myocytes: total number and subcellular distribution as determined by photoaffinity labelling.
Regulation of the GLUT1 glucose transporter in cultured myocytes: total number and subcellular distribution as determined by photoaffinity labelling.
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培养的肌细胞中 GLUT1 葡萄糖转运蛋白的调节：通过光亲和标记确定的总数和亚细胞分布。
DOI：
10.1042/bj3010035
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
C. Wilson
中科院分区：
文献类型：
--
作者：
I. M. El;S. Roser;R. Pollet;S. Cushman;C. Wilson
我们使用不渗透的光亲和标记物2-N-4-（1-azi-2，2，2-三氟乙基）苯甲酰基-[2- 3 H] 1，3-双-（D-甘露糖-4-基氧）-2-丙胺（ATB-[2- 3 H]BMPA）来鉴定和量化BC 3 H-1细胞表面的葡萄糖转运蛋白，BC 3 H-1细胞是一种连续培养的骨骼肌细胞系，缺乏细胞融合所需的MyoD转录因子。ATB-[2- 3 H]BMPA与GLUT 1葡萄糖转运蛋白（这些细胞中表达的唯一亚型）的免疫沉淀联合使用。总细胞GLUT 1含量也测定光标记和免疫沉淀后，细胞渗透毛地黄皂苷（0.025%）。在葡萄糖饥饿的细胞中，85%的葡萄糖转运蛋白在基础状态下存在于细胞表面，对胰岛素（200 nM）的反应几乎没有变化，这与缺乏对胰岛素的额外2-脱氧葡萄糖摄取有关。用葡萄糖（25 mM）喂养细胞24 h导致总GLUT 1含量相对于饥饿细胞减少80%，其中仅25%存在于细胞表面上。这与2-脱氧葡萄糖摄取减少85%有关。此外，用胰岛素或佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）急性刺激饲养细胞导致细胞表面GLUT 1增加，并且相应地，2-脱氧葡萄糖摄取增加约10%。2-和4倍。我们的结论是，exofacial光亲和标记的葡萄糖转运ATB-[2- 3 H]BMPA在毛地黄皂苷的存在和不存在下，随后通过特异性免疫沉淀，提供了一个准确的测量总的和细胞表面的葡萄糖转运蛋白在分化的BC 3 H-1肌细胞。该技术表明，葡萄糖预喂养（1）减少了GLUT 1的总数，（2）将大部分剩余的转运蛋白重新分布到细胞内位点，在那里它们现在可以响应胰岛素和PMA而易位到细胞表面。
We have used the impermeant photoaffinity label 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-[2-3H] 1,3-bis-(D-mannos-4-yloxy)-2-propylamine (ATB-[2-3H]BMPA) to identify and quantify the glucose transporters on the surface of BC3H-1 cells, a continuously cultured skeletal-muscle cell line lacking the MyoD transcription factor required for cell fusion. ATB-[2-3H]BMPA was used in combination with immunoprecipitation of the GLUT1 glucose transporter, the only isoform expressed in these cells. The total cellular GLUT1 content was also determined by photolabelling and immunoprecipitation after cell permeabilization with digitonin (0.025%). In glucose-starved cells, 85% of the glucose transporters were present at the cell surface in the basal state, with little change in response to insulin (200 nM), correlating with lack of additional 2-deoxyglucose uptake in response to insulin. Feeding the cells with glucose (25 mM) for 24 h resulted in an 80% decrease in the total GLUT1 content relative to starved cells, of which only 25% were present on the cell surface. This was associated with an 85% decrease in 2-deoxyglucose uptake. In addition, acute stimulation of the fed cells with insulin or phorbol 12-myristate 13-acetate (PMA) led to an increase in GLUT1 at the cell surface, and, in correspondence, an increase in 2-deoxyglucose uptake by approx. 2- and 4-fold respectively. We conclude that exofacial photoaffinity labelling of glucose transporters with ATB-[2-3H]BMPA in the presence and absence of digitonin, followed by specific immunoprecipitation, provides an accurate measure of total and cell-surface glucose transporters in differentiated BC3H-1 muscle cells. This technique demonstrates that glucose pre-feeding (1) decreases the total number of GLUT1 and (2) redistributes the majority of the remaining transporters to an intracellular site, where they can now be translocated to the cell surface in response to insulin and PMA.