Exercise, denervation and insulin resistance in muscle

描述（由申请人提供）：AMP活化蛋白激酶（AMPK）发挥作用 在许多细胞对改变其能量的压力的反应中起主要作用 状态在骨骼肌中，AMPK活性在运动过程中增加 （收缩），大量的证据表明，这调节 观察到葡萄糖转运、脂肪酸氧化和UCP 3增加 表情这些知识大部分是利用AICAR（一种AMPK）获得的 其特异性受到质疑的激活剂。在这里，我们将利用 AICAR和调节AMPK活性的替代方法，以探索 AMPK在肌肉中的生理作用及其作用的生化基础 对葡萄糖转运、糖原合成和UCP 3表达的影响。以下是 我们的目的：（1）表征药理学和其他手段， AMPK活性，并确定它们是否刺激葡萄糖 通过一氧化氮运输。预先缺氧、与一种 无葡萄糖培养基、二甲双胍和α-肾上腺素能刺激，所有这些 增加AMPK活性，将与AICAR进行比较。另外我们 将评估新的小分子量AMPK抑制剂的特异性。（二） 为了确定骨骼肌中AMPK激活的生化途径， 肌肉导致GLUT 4易位和UCP-3基因转录。体外 将使用离体肌肉的磷酸化测定和代谢标记 确定AMPK与葡萄糖转运蛋白相关的磷蛋白靶点 易位我们还将介绍组成主动和主导 阴性AMPK构建体进入培养的心肌细胞，以确定 AMPK依赖性的UCP 3 mRNA诱导，我们将试图确定 转录因子来改变这个过程。(3)探索基础， 抑制胰岛素刺激的糖原合成 趾长伸肌（趾长伸肌）及其温育修复 AICAR。我们将确定AICAR的效果是否被其他药物所模仿。 AMPK激活器，AMPK激活恢复的过程以及AICAR为什么不恢复 在失神经支配的比目鱼肌中也有类似的效果。(4)以确定是否 在体内用AICAR或二甲双胍治疗可预防或减弱 基因表达的改变（例如GLUT 4、UCP-3、肌细胞生成素等），和脂质 在大鼠肌肉失神经支配后6-72小时发生。我们还将评估， 用这些药物治疗刺激GLUT 4募集，我们将使用新的 测试运动/收缩/AMPK反应性的假设的方法 葡萄糖转运蛋白库与胰岛素应答库的不同之处在于 生物化学组成和与糖原颗粒结合的能力。

DESCRIPTION (provided by applicant): AMP-activated protein kinase (AMPK) plays a major role in the response of many cells to stresses that alter their energy state. In skeletal muscle, AMPK activity is increased during exercise (contraction), and a large body of evidence indicates that this regulates observed increases in glucose transport, fatty acid oxidation and UCP3 expression. Much of this knowledge has been obtained utilizing AICAR, an AMPK activator whose specificity has been questioned. Here, we will make use of AICAR and alternative approaches to modulate AMPK activity, to explore the physiological roles of AMPK in muscle and the biochemical basis for its effects on glucose transport, glycogen synthesis and UCP3 expression. The following are our aims: (1) To characterize pharmacological and other means for increasing AMPK activity in incubated muscles and to determine if they stimulate glucose transport via nitric oxide. The effects of prior hypoxia, incubation with a glucose-free medium, metformin and alpha-adrenergic stimulation, all of which increase AMPK activity, will be compared with that of AICAR. In addition, we will assess the specificity of a new small molecular weight AMPK inhibitor. (2) To determine the biochemical pathway(s) by which AMPK activation in skeletal muscle leads to GLUT4 translocation and UCP-3 gene transcription. In vitro phosphorylation assays and metabolic labeling of isolated muscle will be used to identify the phosphoprotein targets of AMPK related to glucose transporter translocation. We will also introduce constitutively active and dominant negative AMPK constructs into cultured myocytes to ascertain the AMPK-dependency of the UCP3 mRNA induction, and we will attempt to identify the transcription factors that modify this process. (3) To explore the basis for the inhibition of insulin-stimulated glycogen synthesis in the denervated extensor digitorum longus (EDL) muscle and its restoration by incubation with AICAR. We will determine whether the effects of AICAR are mimicked by other AMPK activators, what processes AMPK activation restores and why AICAR does not have a similar effect in the denervated soleus. (4) To determine whether treatment with AICAR or metformin in vivo prevents or attenuates the alterations in gene expression (e.g. GLUT4, UCP-3, myogenin, etc.), and lipids that occur in rat muscle 6-72 hrs after denervation. We will also assess if treatment with these agents stimulates GLUT4 recruitment, and we will use new methodology to test the hypothesis that exercise/contraction/AMPK responsive glucose transporter pools differ from insulin-responsive pools in their biochemical composition and ability to associate with glycogen particles.