Aging, Calorie Restriction and Insulin Sensitivity

胰岛素抵抗与许多最普遍、最具破坏性的年龄相关疾病有关，包括 2型糖尿病、心血管疾病和认知功能障碍。骨骼肌占高达85% 胰岛素介导的血糖清除和葡萄糖摄取(GU)是肌肉的速率控制步骤 葡萄糖代谢。卡路里限制(CR)增强老年大鼠骨骼肌胰岛素介导的GU 和人类，但对细胞机制知之甚少。广泛的、长期的目标是推进 了解改善胰岛素敏感性的机制，从而导致健康衰老。具体目标是：1) 确定AS160‘S在CR诱导的慢性粒细胞白血病改善中所起作用的具体机制 骨骼肌胰岛素介导的GU；2)发现新的CR反应的磷酸蛋白，即胰岛素-GU。 骨骼肌的调节和Akt依赖；3)测定CR对AMP活化蛋白的影响 骨骼肌中肌动蛋白(AMPK)异三聚体的特异性活性以及AMPK在多大程度上 活化方通过肌肉增强CR对胰岛素介导的GU的作用。新创建的AS160零大鼠 用腺相关病毒传递的野生型或磷酸化突变的AS160表达将被用来揭示 AS160位点选择性磷酸化对于胰岛素介导的更多的GU与CR是必不可少的。尽管阿克特- 依赖的AS160磷酸化对于胰岛素对GU的全面作用是重要的，可能是其他Akt- 底物也有助于CR对胰岛素敏感性的影响。然而，目前还没有确定任何一种。 因此，我们将使用基于定量质谱学的磷酸蛋白质组学来分析肌肉(来自 老年AL与CR大鼠)处理±胰岛素和±选择性Akt-抑制剂以发现新的蛋白磷酸化位点 受CR、胰岛素和/或Akt调节。基于磷蛋白质组学数据，我们将创造转基因 L6肌肉细胞，以测试这些磷酸蛋白是否控制胰岛素介导的GU。AMPK是细胞内一种关键的 能量传感器。一些研究报道了CR动物肌肉中AMPK的更大激活，但另一些研究 还没有。这些差异可能部分是因为先前的研究只评估了AMPK的总和。AMPK是 由催化亚基(α1或α2亚基)和2个调节亚基组成的异三聚体蛋白质复合体 (β1或β2；以及γ1、γ2或γ3)。由于AMPK的不同生物效应取决于特定的杂三聚体，我们将 解决CR效应是否具有AMPK异源三聚体选择性。典型啮齿动物CR方案(进食)中的CR水平 60%的AL摄入量)对人类的翻译是不现实的。我们将评估两种典型的CR协议(饮食 60%的AL摄入量)和更可行的方案(食用85%的AL摄入量)。因为不那么严重的CR可能会 AICAR(一种AMPK激活剂)对老年AL大鼠肌肉的预先治疗效果较差，可提高GU， 我们将测试CR加AICAR最佳地增强老年大鼠肌肉中胰岛素介导的GU的效果。 这些独特的方法将提供对CR基础机制的开创性见解- 改善了老年人肌肉中的胰岛素敏感性。

Insulin resistance is linked to many of the most prevalent and devastating age-related pathologies, including Type 2 diabetes, cardiovascular disease and cognitive dysfunction. Skeletal muscle accounts for up to 85% of insulin-mediated blood glucose clearance, and glucose uptake (GU) is a rate-controlling step for muscle glucose metabolism. Calorie restriction (CR) enhances insulin-mediated GU in skeletal muscle from old rats and humans, but the cellular mechanisms are poorly understood. The broad, long-term goal is to advance understanding of mechanisms to improve insulin sensitivity, leading to healthy aging. The Specific Aims are: 1) To identify the specific mechanism that is responsible for AS160's role in the CR-induced improvement in insulin-mediated GU by skeletal muscle; 2) To discover novel, CR-responsive phosphoproteins that are insulin- regulated and Akt-dependent in skeletal muscle; 3) To determine the influence of CR on AMP-activated protein kinase (AMPK) heterotrimer-specific activity in skeletal muscle and to determine the extent to which an AMPK activating compound enhances CR's effect on insulin-mediated GU by muscle. Newly created AS160-null rats with adeno-associated virus-delivered wildtype or phosphomutated AS160 expression will be used to reveal if AS160 site-selective phosphorylation is essential for greater insulin-mediated GU with CR. Although Akt- dependent AS160 phosphorylation is important for insulin's full effect on GU, it is likely that other Akt- substrates also contribute to CR's effects on insulin sensitivity. However, none have been identified. Accordingly, we will use quantitative mass spectrometry-based phosphoproteomics to analyze muscles (from old AL vs. CR rats) treated ±insulin and ±selective Akt-inhibitor to discover novel protein phosphorylation sites regulated by CR, insulin and/or Akt. Based on the phosphoproteomics data, we will create genetically modified L6 muscle cells to test if these phosphoproteins control insulin-mediated GU. AMPK is a key intracellular energy sensor. Some studies have reported greater AMPK activation in muscles of CR animals, but others have not. These discrepancies may be in part because prior studies have only assessed total AMPK. AMPK is a heterotrimeric protein complex comprised of a catalytic subunit (α1 or α2 isoform) and 2 regulatory subunits (β1 or β2; and γ1, γ2 or γ3). Because AMPK’s diverse bioeffects depend on specific heterotrimers, we will resolve if CR effects are AMPK heterotrimer-selective. The level of CR in a typical rodent CR protocol (eating 60% of AL intake) is unrealistic for translation to humans. We will assess both a typical CR protocol (eating 60% of AL intake) and a more feasible protocol (eating 85% of AL intake). Because less severe CR may be less effective, and prior treatment of muscles from old AL rats with AICAR (an AMPK activator) elevates GU, we will test the efficacy of CR plus AICAR to optimally enhance insulin-mediated GU in muscles from old rats. These unique approaches will provide groundbreaking insights into fundamental mechanisms underlying CR- improved insulin sensitivity in muscle of older individuals.