Thr649Ala-AS160 knock-in mutation does not impair contraction/AICAR-induced glucose transport in mouse muscle
Thr649Ala-AS160 knock-in mutation does not impair contraction/AICAR-induced glucose transport in mouse muscle
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DOI：
10.1152/ajpendo.00379.2011
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发表时间：
2012-05-01
影响因子：
5.1
通讯作者：
Chen, Shuai
中科院分区：
文献类型：
--
作者：
Ducommun, Serge;Wang, Hong Yu;Chen, Shuai
张文，王海艳，张文，张文，张文. Thr（649）Ala-AS 160敲入突变不损害小鼠肌肉中的收缩/AICAR诱导的葡萄糖转运。Am J Physiol Endocrinol Metab 302：E1036-E1043，2012.首次发表于2012年2月7日; doi：10.1152/ajpendo.00379.2011.- AS 160及其密切相关的蛋白质TBC 1D 1已成为胰岛素和收缩刺激的肌肉葡萄糖摄取的关键介质，通过调节GLUT 4运输。胰岛素可增加AS 160在多个Akt/PKB共有位点的磷酸化，包括Thr（649），并通过磷酸化Thr促进其与14-3-3蛋白的结合（649）。我们最近提供的遗传证据表明，AS 160-Thr（649）磷酸化/14-3-3结合在介导胰岛素刺激的肌肉葡萄糖摄取中起关键作用。收缩也被提出通过AMPK增加AS 160和TBC 1D 1的磷酸化，这可以通过通用磷酸化Akt底物（PAS）抗体检测。在此，用位点特异性磷酸化抗体分析肌肉提取物中的AS 160免疫沉淀物，结果显示收缩和AICAR不会导致主要PAS识别位点AS 160-Thr的磷酸化水平升高，而是略有降低（649）。与此一致，收缩未能增强14-3-3与AS 160的结合。与以前的报告一致，我们还观察到，在原位收缩刺激的PAS抗体免疫反应蛋白的信号强度类似于150-160 kDa的肌肉提取物。使用TBC 1D 1缺失突变小鼠，我们发现TBC 1D 1蛋白占收缩肌肉提取物中PAS抗体免疫反应性信号的大部分，类似于150-160 kDa。与所提出的AS 160-Thr（649）磷酸化/14-3-3结合在介导葡萄糖摄取中的作用一致，AS 160-Thr（649）Ala敲入小鼠在收缩时显示正常的葡萄糖摄取，并且在分离的肌肉中显示AICAR。我们的结论是，以前报道的PAS抗体免疫反应条带类似于150-160 kDa，在收缩时增加，不代表AS 160，但TBC 1D 1，和AS 160-Thr（649）Ala取代损害胰岛素，但既不收缩，也不AICAR刺激的小鼠骨骼肌中的葡萄糖摄取。
Ducommun S, Wang HY, Sakamoto K, MacKintosh C, Chen S. Thr(649)Ala-AS160 knock-in mutation does not impair contraction/AICAR-induced glucose transport in mouse muscle. Am J Physiol Endocrinol Metab 302: E1036-E1043, 2012. First published February 7, 2012; doi:10.1152/ajpendo.00379.2011.-AS160 and its closely related protein TBC1D1 have emerged as key mediators for both insulin-and contraction-stimulated muscle glucose uptake through regulating GLUT4 trafficking. Insulin increases AS160 phosphorylation at multiple Akt/PKB consensus sites, including Thr(649), and promotes its binding to 14-3-3 proteins through phospho-Thr(649). We recently provided genetic evidence that AS160-Thr(649) phosphorylation/14-3-3 binding plays a key role in mediating insulin-stimulated glucose uptake in muscle. Contraction has also been proposed to increase phosphorylation of AS160 and TBC1D1 via AMPK, which could be detected by a generic phospho-Akt substrate (PAS) antibody. Here, analysis of AS160 immunoprecipitates from muscle extracts with site-specific phospho-antibodies revealed that contraction and AICAR caused no increase but rather a slight decrease in phosphorylation of the major PAS recognition site AS160-Thr(649). In line with this, contraction failed to enhance 14-3-3 binding to AS160. Consistent with previous reports, we also observed that in situ contraction stimulated the signal intensity of PAS antibody immunoreactive protein of similar to 150-160 kDa in muscle extracts. Using a TBC1D1 deletion mutant mouse, we showed that TBC1D1 protein accounted for the majority of the PAS antibody immunoreactive signals of similar to 150-160 kDa in extracts of contracted muscles. Consistent with the proposed role of AS160-Thr(649) phosphorylation/14-3-3 binding in mediating glucose uptake, AS160-Thr(649)Ala knock-in mice displayed normal glucose uptake upon contraction and AICAR in isolated muscles. We conclude that the previously reported PAS antibody immunoreactive band similar to 150-160 kDa, which were increased upon contraction, does not represent AS160 but TBC1D1, and that AS160-Thr(649)Ala substitution impairs insulin-but neither contraction- nor AICAR-stimulated glucose uptake in mouse skeletal muscle.