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 年首次得到证实，是与改善健康相关的最佳记录的运动益处之一，但其机制的识别一直难以捉摸。瘦弱的正常人的运动对近端胰岛素信号传导几乎没有影响，这表明运动会改变远端部位。进展陷入停滞，直到发现已知对葡萄糖转运 (GT) 至关重要的最远端信号传导步骤中的 PEX 持续增加：160 kDa 的 Akt 底物 (AS160) 磷酸化。磷酸化 AS160 (pAS160) 的持续 PEX 增加与瘦大鼠中胰岛素诱导的 GT 升高密切相关，并且在瘦人 PEX 的肌肉中也发现了持续的 pAS160。 pAS160 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者。广泛的长期目标是充分阐明胰岛素敏感性 PEX 增强的机制。 3 个具体目标是： 1) 确定运动后瘦大鼠骨骼肌中 AS160 磷酸化持续增加的机制。 2) 通过在大鼠骨骼肌中表达 AS160（经过基因改造以防止关键位点磷酸化），确定 AS160 在运动引起的瘦大鼠 GT 变化中的作用。 3) 确定瘦大鼠和肥胖大鼠在运动诱导胰岛素刺激 GT 改善的具体机制方面的异同。有证据表明 pAS160 中 PEX 的持续增加并非归因于激酶的持续激活。一个待测试的新想法是 pAS160 持续升高是因为 Ser/Thr 蛋白磷酸酶减弱了 AS160 去磷酸化。由于初步数据表明蛋白磷酸酶 2A (PP2A) 参与 AS160 去磷酸化，因此目标 1 将使用多种方法来探测 PP2A 对 pAS160 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根肌纤维来测量 GT，但野生型 AS160 或经过基因改造以防止关键位点磷酸化的 AS160 的表达有所不同，以了解瘦大鼠中 PEX 胰岛素介导的 GT 的改善是否归因于 pAS160 的增强。目标 3 将确定众所周知的运动对瘦大鼠和肥胖大鼠的胰岛素敏感性的益处是否是通过不同的机制产生的。肥胖大鼠的运动预计会降低肌肉中高水平的胰岛素抵抗介质（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？激活），从而增强胰岛素信号传导和 GT。然而，在瘦大鼠的肌肉中，预计运动不会极大地改变其已经很低的介质水平或近端胰岛素信号的正常水平。据报道，在瘦人中观察到的 pAS160 PEX 持续增加在肥胖人中较少。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较高的PEX胰岛素介导的GT是否可归因于瘦大鼠中较高的pAS160 PEX。拟议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，这对瘦人和肥胖者来说都是一个主要的健康益处。 公共健康相关性：拟议的研究与公共健康相关，因为骨骼肌处理葡萄糖的胰岛素抵抗是 2 型糖尿病的一个基本缺陷，也许是主要缺陷，而运动可以极大地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，阐明改善胰岛素抵抗个体胰岛素敏感性的机制至关重要。了解正常个体改善胰岛素敏感性的机制也很重要，因为久坐的瘦人的胰岛素敏感性水平可能无法带来最佳的健康状况，而且因为确定健康个体的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.