Aging, Calorie Restriction and Insulin Sensitivity

胰岛素抵抗与许多最普遍和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。骨骼肌占到了 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 葡萄糖代谢热量限制（CR）增强老年大鼠骨骼肌胰岛素介导的GU 和人类，但细胞机制知之甚少。长远目标是推进 了解提高胰岛素敏感性的机制，从而实现健康的老龄化。具体目标是：1） 为了确定负责AS 160在CR诱导的改善中的作用的具体机制， 胰岛素介导的骨骼肌GU; 2）发现新的，CR-反应性磷蛋白， 3）研究CR对骨骼肌AMP激活蛋白的影响 在骨骼肌中测定AMPK异源三聚体特异性活性，并确定AMPK 激活化合物增强CR对胰岛素介导的肌肉GU的作用。新创建的AS 160-null大鼠 将使用腺相关病毒递送的野生型或磷酸突变的AS 160表达来揭示 AS 160位点选择性磷酸化对胰岛素介导的GU伴CR的增加至关重要。虽然AKT- 依赖性AS 160磷酸化对于胰岛素对GU的充分作用是重要的，很可能其他Akt- 底物也有助于CR对胰岛素敏感性的影响。然而，没有一个被确定。 因此，我们将使用基于定量质谱的磷酸蛋白质组学来分析肌肉（来自 老年AL与CR大鼠）治疗±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。基于磷酸化蛋白质组学数据，我们将创造转基因的 L 6肌细胞来测试这些磷蛋白是否控制胰岛素介导的GU。AMPK是细胞内 能量传感器一些研究报道了CR动物肌肉中AMPK的激活程度更高，但其他研究也报道了这一点。 没有。这些差异可能部分是因为先前的研究仅评估了总AMPK。AMPK是 由一个催化亚基（α1或α2亚型）和2个调节亚基组成的异源三聚体蛋白复合物 （β1或β2;以及γ1、γ2或γ3）。由于AMPK的多种生物效应依赖于特定的异源三聚体，我们将 如果CR效应是AMPK异源三聚体选择性的，则解决。典型啮齿动物CR方案（进食）中的CR水平 AL摄入量的60%）转化为人类是不现实的。我们将评估典型的CR方案（进食 60%的AL摄入量）和一个更可行的方案（吃85%的AL摄入量）。因为不太严重的CR可能是 不太有效，并且用AICAR（AMPK激活剂）预先治疗老年AL大鼠的肌肉提高了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的功效。 这些独特的方法将为CR的基本机制提供突破性的见解， 改善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.