Skeletal Muscle Glucose Transport: Exercise and Insulin

描述（由申请人提供）：运动后（PEX）胰岛素敏感性增加于1982年首次得到证实，是与改善健康相关的最佳记录运动益处之一，但其机制的确定一直难以捉摸。瘦，正常人的运动对近端胰岛素信号的影响很小，这表明运动改变了远端部位。进展停滞，直到发现在已知对葡萄糖转运（GT）至关重要的最远端信号步骤中持续的PEX增加：160 kDa Akt底物（AS 160）的磷酸化。在瘦大鼠中，磷酸化AS 160（pAS 160）的持续PEX增加与胰岛素诱导的GT升高密切相关，并且在瘦人类PEX的肌肉中也发现了持续的pAS 160。pAS 160增加已成为解释胰岛素敏感性PEX改善的一个有吸引力的候选因素。广泛的，长期的目标是充分阐明增强胰岛素敏感性PEX的机制。本研究的3个具体目的是：1）明确运动后瘦大鼠骨骼肌中AS 160磷酸化持续增加的机制。2)通过在大鼠骨骼肌中表达经遗传修饰以防止关键位点磷酸化的AS 160，确定AS 160在瘦大鼠中运动诱导的GT变化中的作用。3)确定瘦大鼠和肥胖大鼠在各自运动诱导的胰岛素刺激GT改善的具体机制方面的相似性和差异。证据表明pAS 160中的持续PEX增加并不归因于激酶的持续激活。一个有待检验的新观点是，pAS 160的持续升高是由于Ser/Thr蛋白磷酸酶减弱了AS 160的去磷酸化作用。由于初步数据表明蛋白磷酸酶2A（PP 2A）参与AS 160去磷酸化，因此将使用多种方法研究Aim 1以探测PP 2A对pAS 160的调节。目标2将使用一种创新方法，通过与纤维类型匹配的单一肌纤维测量GT，但野生型AS 160或经遗传修饰以防止关键位点磷酸化的AS 160的表达不同，以了解瘦大鼠中改善的PEX胰岛素介导的GT是否可归因于更大的pAS 160。目的3将确定是否众所周知的运动对瘦和肥胖大鼠胰岛素敏感性的好处是通过不同的机制累积的。肥胖大鼠的运动预计会降低高肌肉水平的胰岛素抵抗介质（诱导型一氧化氮合酶;蛋白S-亚硝基化;脂质代谢物; JNK，IKK和PKC？然而，在来自瘦大鼠的肌肉中，预测运动不会极大地改变它们已经低水平的介体或正常水平的近端胰岛素信号传导。据报告，在瘦人中观察到的pAS 160 PEX持续增加在肥胖人群中较少。因此，将进行实验以测试先前在瘦大鼠与肥胖大鼠中发现的更大PEX胰岛素介导的GT是否可归因于瘦大鼠中更大的pAS 160 PEX。完成拟议的研究将为改善胰岛素敏感性的具体机制提供新的见解，这对瘦和肥胖个体都是一个主要的健康益处。 公共卫生相关性：这项拟议的研究与公共卫生有关，因为骨骼肌对葡萄糖处理的胰岛素抵抗是2型糖尿病的一个基本缺陷，可能是主要缺陷，运动可以大大改善胰岛素介导的葡萄糖摄取。由于肥胖、胰岛素抵抗和糖尿病的相关流行，阐明胰岛素抵抗个体中改善胰岛素敏感性的机制至关重要。了解正常人胰岛素敏感性改善的机制也很重要，因为在久坐不动的瘦个体中发现的胰岛素敏感性水平可能不会带来最佳健康，并且因为在健康个体中识别机制有可能激发开发有益于胰岛素抵抗人群的新疗法。

DESCRIPTION (provided by applicant): Increased post-exercise (PEX) insulin sensitivity, first demonstrated in 1982, is among the best documented exercise benefits linked to improved health, but identification of the mechanism has been elusive. Exercise by lean, normal individuals has little effect on proximal insulin signaling, suggesting exercise alters a distal site. Progress stalled until the discovery of a sustained PEX increase in the most distal signaling step known to be crucial for glucose transport (GT): phosphorylation of Akt Substrate of 160 kDa (AS160). The sustained PEX increase in phosphorylated AS160 (pAS160) tracks closely with elevated insulin-induced GT in lean rats, and sustained pAS160 is also found in muscle of lean humans PEX. Increased pAS160 has emerged as an attractive candidate to explain improved insulin sensitivity PEX. The broad, long-term goal is to fully elucidate the mechanisms that underlie enhanced insulin sensitivity PEX. The 3 Specific Aims are: 1) Identify the mechanism for the sustained increase in AS160 phosphorylation in skeletal muscle of lean rats after exercise. 2) By expressing in rat skeletal muscle AS160 that is genetically modified to prevent phosphorylation on key sites, determine AS160's role in exercise-induced changes in GT in lean rats. 3) Identify the similarities and differences between lean and obese rats in the specific mechanisms for their respective exercise-induced improvements in insulin-stimulated GT. Evidence indicates the sustained PEX increase in pAS160 is not attributable to persistent activation of kinases. A novel idea to be tested is that the sustained elevation in pAS160 is because of attenuated AS160 dephosphorylation by Ser/Thr protein phosphatases. Because preliminary data implicate protein phosphatase 2A (PP2A) in AS160 dephosphorylation, multiple approaches will be used for Aim 1 to probe PP2A's regulation of pAS160. Aim 2 will use an innovative method to measure GT by single muscle fibers that are matched for fiber type, but differ by expression of wildtype AS160 or AS160 genetically modified to prevent phosphorylation on key sites to learn if the improved PEX insulin-mediated GT in lean rats is attributable to greater pAS160. Aim 3 will determine if the well-known exercise benefits on insulin sensitivity for lean and obese rats are accrued by distinct mechanisms. Exercise by obese rats is predicted to reduce high muscle levels of mediators of insulin resistance (inducible nitric oxide synthase; protein S- nitrosylation; lipid metabolites; JNK, IKK & PKC? activation) leading to enhanced insulin signaling and GT. However, in muscles from lean rats, exercise is predicted to not greatly alter their already low levels of mediators or normal levels of proximal insulin signaling. The sustained increase in pAS160 PEX observed in lean humans was reported to be less in obese people. Accordingly, experiments will be performed to test if the greater PEX insulin-mediated GT previously found for lean vs. obese rats is attributable to greater pAS160 PEX in lean rats. Completion of the proposed research will provide novel insights into the specific mechanisms that account for improved insulin sensitivity, a major health benefit for both lean and obese individuals. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because insulin resistance for glucose disposal by skeletal muscle is an essential and perhaps primary defect for Type 2 diabetes, and exercise can greatly improve insulin-mediated glucose uptake. Because of the related epidemics of obesity, insulin resistance and diabetes, it is crucial to elucidate mechanisms for improved insulin sensitivity in insulin resistant individuals. It is also important to understand mechanisms for improved insulin sensitivity in normal individuals because the level of insulin sensitivity found in sedentary lean individuals may not confer optimal health and because identifying mechanisms in healthy individuals has the potential to inspire the development of new therapies that benefit insulin-resistant people.