Skeletal Muscle Glucose Transport: Exercise and Insulin

描述（由申请人提供）：运动后（PEX）胰岛素敏感性的增加，于1982年首次被证明，是与改善健康有关的最佳运动益处之一，但其机制的识别一直难以捉摸。瘦削的正常人运动对近端胰岛素信号几乎没有影响，这表明运动改变了远端部位。直到在已知对葡萄糖转运（GT）至关重要的最远信号步骤（Akt底物160 kDa （AS160）的磷酸化）中发现PEX持续增加，研究进展才停滞不前。在瘦肉大鼠中，磷酸化AS160 （pAS160）的持续PEX增加与胰岛素诱导的GT升高密切相关，并且在瘦肉人的PEX肌肉中也发现了持续的pAS160。pAS160的增加已成为解释胰岛素敏感性改善PEX的一个有吸引力的候选者。广泛的长期目标是充分阐明胰岛素敏感性增强PEX的机制。3个具体目的是：1)明确运动后瘦大鼠骨骼肌AS160磷酸化持续升高的机制。2)通过在大鼠骨骼肌中表达AS160，通过基因修饰防止关键位点磷酸化，确定AS160在运动诱导的瘦大鼠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胰岛素刺激的GT改善的具体机制上的异同。有证据表明，pAS160的持续PEX增加不是由于激酶的持续激活。一个有待验证的新观点是，pAS160的持续升高是由于丝氨酸/苏氨酸蛋白磷酸酶减弱了AS160的去磷酸化。由于初步数据暗示蛋白磷酸酶2A （PP2A）参与AS160的去磷酸化，因此Aim 1将使用多种方法来探测PP2A对pAS160的调控。Aim 2将使用一种创新的方法来测量单个肌肉纤维的GT，这些肌肉纤维类型匹配，但不同的是野生型AS160或转基因AS160的表达，以防止关键位点的磷酸化，以了解瘦大鼠中PEX胰岛素介导的GT改善是否归因于更高的pAS160。目的3将确定众所周知的运动对瘦大鼠和肥胖大鼠胰岛素敏感性的益处是否通过不同的机制积累。肥胖大鼠运动可降低肌肉中胰岛素抵抗介质（诱导型一氧化氮合酶、蛋白质S-亚硝基化、脂质代谢物、JNK、IKK和PKC）的高水平。然而，在瘦老鼠的肌肉中，运动预计不会大大改变它们已经很低水平的介质或正常水平的近端胰岛素信号。据报道，在瘦人体内观察到的pAS160 PEX持续增加在肥胖人群中较少。因此，将进行实验来测试先前在瘦大鼠与肥胖大鼠中发现的更高的PEX胰岛素介导的GT是否归因于瘦大鼠更高的pAS160 PEX。这项研究的完成将为改善胰岛素敏感性的具体机制提供新的见解，这对瘦人和肥胖者都是一个主要的健康益处。

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.