Regulation of glucose transporters in skeletal muscle

描述（由申请人提供）：GLUT1是骨骼肌的基础葡萄糖转运蛋白和主要的脱氢抗坏血酸（DHA）转运蛋白，其调控尚未完全阐明影响GLUT1细胞表面丰度、GLUT1运输和GLUT1在骨骼肌中对其两种底物活性的因素。初步研究表明，ataxia毛细血管扩张突变（ATM）可能参与骨骼肌基础葡萄糖转运和GLUT1丰度的调节。此外，其他研究人员报道了p38在增加GLUT1对葡萄糖运输的内在活性方面的潜在作用。GLUT1有三个c端磷酸化位点；S490是ATM的已知靶标，S473和T478分别符合ATM和p38的共识靶标基序。该项目的目标是确定这些GLUT1磷酸化位点（或激活这些位点的潜在激酶）在调节培养骨骼肌细胞或小鼠骨骼肌中GLUT1的丰度、定位和活性中的作用。该项目的具体目的1是确定GLUT1的S473和S490是否在调节GLUT1丰度或细胞表面定位中发挥作用。一般的假设是，这些位点的磷酸化将保持GLUT1水平和细胞表面定位，而在这些位点未磷酸化的GLUT1将更容易内化和降解。将确定S473和S490突变对GLUT1丰度、葡萄糖和DHA的运输和转运活性的影响，以及ATM（两个位点的潜在激酶）活化对GLUT1的影响。特异性目的2是确定T478是否在调节GLUT1的底物特异性或内在活性方面发挥作用。假设是T478磷酸化会刺激GLUT1对葡萄糖的活性增加，并降低对DHA的活性。T478突变对GLUT1对葡萄糖和DHA的内在活性的影响将被确定，尽管对GLUT1丰度和运输的潜在影响也将被研究。p38 （T478的一种潜在激酶）的激活对GLUT1活性和运输的影响将被确定。此外，还将确定改变DHA运输的因素是否也与活性氧水平的变化有关和/或是否可以影响肌肉细胞中的胰岛素作用。该项目提供的信息可能用于制定增加骨骼肌基础葡萄糖运输或增加DHA运输以支持骨骼肌抗氧化状态的策略。

DESCRIPTION (provided by applicant): Regulation of GLUT1, the basal glucose transporter of skeletal muscle and the predominant dehydroascorbic acid (DHA) transporter, has not been fully-elucidated in terms of factors that influence GLUT1 cell surface abundance, GLUT1 trafficking, and GLUT1's activity toward its two substrates in skeletal muscle. Preliminary data suggest that ataxia telangiectasia mutated (ATM) may play a role in regulation of basal glucose transport and GLUT1 abundance in skeletal muscle. In addition, other investigators have reported a potential role for p38 in increasing intrinsic activity of GLUT1 toward glucose transport. There are three C-terminal phosphorylation sites in GLUT1; S490 is known target of ATM, and S473 and T478 match consensus target motifs for ATM and p38, respectively. The goal of the project is to determine the roles of these GLUT1 phosphorylation sites (or activation of the potential kinases for these sites) in regulation of GLUT1 abundance, localization, and activity in cultured skeletal muscle cells or mouse skeletal muscle. Specific Aim 1 of the project is to determine whether S473 and S490 of GLUT1 play roles in regulation of GLUT1 abundance or cell surface localization. The general hypotheses are that phosphorylation of these sites will preserve GLUT1 levels and cell surface localization, while GLUT1 that is not phosphorylated at these sites will be more prone toward internalization and degradation. Effects of S473 and S490 mutations will be determined for GLUT1 abundance, trafficking, and transport activity toward glucose and DHA, and effects of activation of ATM (a potential kinase for both sites) on GLUT1 will also be determined. Specific Aim 2 is to determine whether T478 plays a role in regulation of substrate specificity or intrinsic activity of GLUT1. The hypothesis is that T478 phosphorylation will stimulate increased GLUT1 activity toward glucose and decrease activity toward DHA. Effects of T478 mutations will be determined for intrinsic activity of GLUT1 toward glucose and DHA, though potential effects on GLUT1 abundance and trafficking will also be examined. Effects of activation of p38 (a potential kinase of T478) on GLUT1 activity and trafficking will be determined. Additionally, it will be determined whether factors that alter DHA transport also are associated with changes in levels of reactive oxygen species and/or can influence insulin action in muscle cells. Information provided by this project might be used to develop strategies to increase basal glucose transport in skeletal muscle or to increase DHA transport to support skeletal muscle antioxidant status. PUBLIC HEALTH RELEVANCE: This project will investigate factors that could regulate GLUT1, a protein that allows movement of sugar into skeletal muscle all day long (as opposed to another sugar-transporting protein that is responsible for moving sugar into muscle after meals or exercise). Additionally, the role of GLUT1 in providing the building block for muscle vitamin C, a key antioxidant that destroys free radicals, will be investigated. The goal of the project is to provide information that might be useful in improving blood sugar control or in maintaining antioxidant defenses in muscle.