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Molecular Mechanism of activation of glucose transporters by noradrenaline in primary cultures of brown adipocytes and L6 myocytes.

棕色脂肪细胞和 L6 肌细胞原代培养物中去甲肾上腺素激活葡萄糖转运蛋白的分子机制。

基本信息

批准号：
08457052
负责人：
SHIMAZU Takashi
金额：
$ 4.03万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

We have shown that the sympathetic neurotransmitter, noradrenaline (NA), or the beta_3-adrenergic agonist (BRL37344) enhances glucose uptake into cultured brown adipocytes and L6 myocytes by a mechanism involving beta_3-adrenergic receptors and cAMP,without causing translocation of either GLUT1 or GLUT4 glucose transporters from an intracellular pool to the plasma membrane. In order to determine which isoform of GLUT is responsible for the NA-induced activation of glucose transport, we labelled the exofacial glucose binding sites of GLUT1 and GLUT4 with a membrane-impermeable, photoactive bismannose derivative, ATB- [ ^3H ]BMPA.In contrast to the action of insulin, NA did not increase the exofacial photoaffinity labelling of GLUT4. However, NA was shown to increase ATB- [ ^3H ]BMPA labelling of cell surface GLUT1, without an increase in the amount of immunoreactive GLUT1. These results demonstrate that NA stimulates glucose transport in brown adipocytes by activation of the cell surface GLUT1 through a cAMP-dependent mechanism.We have further analyzed the mcchanism of selective activation of GLUT1 by NA,assuming that certain regulatory protein (s) may interact with GLUT1 at their cytoplasmic domain and inhibit their intrinsic activity. To isolate such cytosolic protein (s) , we made glutathione S-transferase (GST) -fusion protein corresponding to the C-terminus of GLUT1 (GST-G1C) , and immobilized it on CNBr-Sepharose beads to prepare the GST-G1C affinity column. When cytosol proteins from brown adipocytes were subjected to affinity chromatography on this column, a 33 kDa protein was detected to bind specifically with the GST-G1C.The recovery of the 33 kDa protein was higher from the cytosol proteins of NA-treated adipocytes than those from control and insulin-treated adipocytes. These results suggest that the 33 kDa protein can be dissociated by treatment with NA,thereby releasing GLUT1 from the presumed inhibitory interaction.

我们发现交感神经递质去甲肾上腺素（NA）或β_3-肾上腺素能激动剂（BRL 37344）通过β_3-肾上腺素能受体和cAMP的机制增强培养的棕色脂肪细胞和L 6肌细胞对葡萄糖的摄取，而不引起GLUT 1或GLUT 4葡萄糖转运蛋白从细胞内转运到质膜。为了确定哪种GLUT亚型负责NA诱导的葡萄糖转运激活，我们用一种膜不可渗透的光敏双甘露糖衍生物ATB- [ ^3H ]BMPA标记了GLUT 1和GLUT 4的外表面葡萄糖结合位点，与胰岛素的作用相反，NA并没有增加GLUT 4的外表面光亲和标记。然而，NA可增加细胞表面GLUT 1的ATB- [ ^3H ]BMPA标记，而免疫反应性GLUT 1的量未增加。这些结果表明NA通过cAMP依赖性激活细胞表面GLUT 1来促进棕色脂肪细胞的葡萄糖转运，我们进一步分析了NA选择性激活GLUT 1的机制，并假设某些调节蛋白可能在其胞质结构域与GLUT 1相互作用并抑制其内在活性。为了分离这样的胞质蛋白，我们制备了对应于GLUT 1的C-末端的谷胱甘肽S-转移酶（GST）-融合蛋白（GST-G1 C），并将其固定在CNBr-Sepharose珠上以制备GST-G1 C亲和柱。当棕色脂肪细胞的胞浆蛋白在该柱上进行亲和层析时，检测到33 kDa蛋白与GST-G1 C特异性结合，NA处理的脂肪细胞的胞浆蛋白中33 kDa蛋白的回收率高于对照和胰岛素处理的脂肪细胞的胞浆蛋白。这些结果表明，33 kDa的蛋白质可以通过用NA处理而解离，从而从假定的抑制性相互作用中释放GLUT 1。