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

我们已经证明，交感神经递质去甲肾上腺素(NA)或β3肾上腺素能激动剂(BRL37344)通过涉及β3肾上腺素能受体和cAMP的机制促进培养的棕色脂肪细胞和L6心肌细胞对葡萄糖的摄取，而不会引起GLUT1或GLUT4葡萄糖转运体从细胞内池到质膜的转位。为了确定哪一种GLUT异构体负责NA诱导的葡萄糖转运的激活，我们用膜不通透性的光活性双甘露糖衍生物ATB-[~3H]BMPA标记了GLUT1和GLUT4的表面葡萄糖结合部位。与胰岛素的作用不同，NA不增加GLUT4的表面光亲和标记。但NA可增加细胞表面GLUT1的ATB-[~(3 H)]BMPA标记，但不增加GLUT1的免疫反应量。这些结果表明，NA通过cAMP依赖的机制激活细胞表面的GLUT1，从而刺激葡萄糖在棕色脂肪细胞中的转运。我们进一步分析了NA选择性激活GLUT1的机制，假设某些调节蛋白(S)可能与GLUT1在细胞质区域相互作用，抑制其内在活性。为了分离这种胞浆蛋白(S)，我们制备了谷胱甘肽S转移酶(GST)-GLUT1C末端的融合蛋白(GST-G1C)，并将其固定在CNBr-Sepharose小球上，制备了GST-G1C亲和柱。棕色脂肪细胞胞浆蛋白经亲和层析后，可与GST-G1C特异性结合。NA处理的脂肪细胞胞浆蛋白的回收率高于对照组和胰岛素处理的脂肪细胞。这些结果表明，33 kDa的蛋白可以通过NA处理而解离，从而从推测的抑制相互作用中释放GLUT1。

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.