Regulation of glucose transporters in skeletal muscle

描述（由申请人提供）：GLUT 1（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）的调节尚未完全阐明影响GLUT 1细胞表面丰度、GLUT 1运输和GLUT 1对其在骨骼肌中的两种底物的活性的因素。初步数据表明，共济失调毛细血管扩张突变（ATM）可能在调节骨骼肌中的基础葡萄糖转运和GLUT 1丰度中发挥作用。此外，其他研究者报道了p38在增加GLUT 1对葡萄糖转运的内在活性中的潜在作用。GLUT 1中有三个C-末端磷酸化位点; S490是ATM的已知靶点，S473和T478分别与ATM和p38的共有靶基序匹配。该项目的目标是确定这些GLUT 1磷酸化位点（或这些位点的潜在激酶的激活）在培养的骨骼肌细胞或小鼠骨骼肌中GLUT 1丰度、定位和活性调节中的作用。该项目的具体目标1是确定GLUT 1的S473和S490是否在GLUT 1丰度或细胞表面定位的调节中发挥作用。一般的假设是，这些位点的磷酸化将保持GLUT 1水平和细胞表面定位，而在这些位点未磷酸化的GLUT 1将更倾向于内化和降解。将确定S473和S490突变对GLUT 1丰度、运输和对葡萄糖和DHA的转运活性的影响，还将确定ATM（两个位点的潜在激酶）活化对GLUT 1的影响。具体目标2是确定T478是否在GLUT 1的底物特异性或内在活性调节中发挥作用。假设T478磷酸化将刺激GLUT 1对葡萄糖的活性增加，并降低对DHA的活性。将确定T478突变对GLUT 1对葡萄糖和DHA的内在活性的影响，但也将检查对GLUT 1丰度和运输的潜在影响。将确定p38（T478的潜在激酶）活化对GLUT 1活性和运输的影响。此外，还将确定改变DHA转运的因素是否也与活性氧水平的变化有关和/或影响肌肉细胞中的胰岛素作用。该项目提供的信息可用于制定策略，以增加骨骼肌中的基础葡萄糖转运或增加DHA转运，以支持骨骼肌的抗氧化状态。 公共卫生相关性：该项目将研究可以调节GLUT 1的因素，GLUT 1是一种允许糖整天进入骨骼肌的蛋白质（而不是另一种负责在饭后或运动后将糖转移到肌肉中的糖转运蛋白）。此外，GLUT 1在提供肌肉维生素C（一种破坏自由基的关键抗氧化剂）的构建模块中的作用将被研究。该项目的目标是提供可能有助于改善血糖控制或维持肌肉抗氧化防御的信息。

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.