SIRT1 and muscle insulin sensitivity

描述(申请人提供)：胰岛素抵抗是老年肌肉中常见的代谢并发症，也是2型糖尿病(T2D)病因的主要缺陷。然而，肌肉年龄与胰岛素抵抗相关的信号机制尚不清楚。考虑到2010年，美国65岁及以上的人中约有27%患有T2D，预计在未来20年，65岁及以上的人口将翻一番，达到约7000万，解决这一根本的知识差距是当务之急。因此，这项研究的长期目标是确定衰老过程中肌肉胰岛素抵抗的潜在机制。卡路里限制(CR；定义为60%的随意[AL]食物摄入)有力地逆转了肌肉胰岛素抵抗，并在衰老过程中延缓了它的发生。NAD+依赖的脱乙酰基酶sirtuin1(SIRT1)被CR激活，被认为是连接AGE和CR与肌肉胰岛素作用的信号节点。然而，令人惊讶的是，关于SIRT1对肌肉胰岛素作用的调节作用的知识，特别是在体内，非常有限。我们的中心假设是SIRT1是一个关键的信号分子，它将衰老和CR与肌肉胰岛素的作用联系起来，主要是通过调节主要信号节点的乙酰化，如信号转导和转录激活因子3(STAT3)，然后影响许多信号通路，包括胰岛素信号通路。为了解决这一假设，我们的方法将是使用新的小鼠模型来研究肌肉胰岛素信号和对年龄和CR的敏感性，在这种模型中，我们操纵了SIRT1在肌肉中的活动。我们的模型预测，在衰老过程中SIRT1活性的变化，以及对CR的响应，会导致SIRT1靶蛋白的乙酰化状态和随后的活性变化，这直接或间接地调节肌肉胰岛素信号和敏感性。具体地说，目标1将阐明SIRT1和STAT3在老年小鼠肌肉胰岛素抵抗发病机制中的作用，而目标2将确定SIRT1-STAT3信号轴是否支持CR增强小鼠肌肉胰岛素敏感性的能力。在这些研究中，我们将使用高胰岛素-正常血糖钳在体内测量肌肉胰岛素信号和胰岛素敏感性，并在体外使用2-脱氧葡萄糖摄取试验，测量喂食AL饮食的年轻(4个月)、中年(12个月)和老年(20个月)小鼠的肌肉胰岛素信号和胰岛素敏感性，并将它们与短期(30天)或长期(9或17个月)CR饮食喂养的小鼠的测量结果进行比较。将在4种不同的转基因小鼠中进行研究，这些小鼠的肌肉特异性SIRT1活性增加、SIRT1脱乙酰酶活性敲除(KO)、STAT3的KO或SIRT1和STAT3的KO。在目标3中，我们将使用质谱学技术和腺病毒在肌肉细胞中的研究来确定SIRT1的新靶点及其乙酰化对胰岛素作用的功能影响。总之，这些研究将拓宽我们对SIRT1在肌肉生物学中作用的理解，并可能对不仅治疗肌肉胰岛素抵抗，而且还治疗其他骨骼肌衰老疾病的疗法的发展产生广泛影响。

DESCRIPTION (provided by applicant): Insulin resistance is a common metabolic complication in aged muscle, and is a primary defect underlying the etiology of type 2 diabetes (T2D). However, the signaling mechanisms in muscle linking age to insulin resistance are unknown. Considering that in 2010, ~27% of individuals 65 yrs and older in the United States were afflicted with T2D, and in the next 20 years the 65 and older population is anticipated to double to ~70 million, it is imperative that this fundamental gap in knowledge be resolved. Thus, the long-term objective of this research is to define the mechanisms underlying muscle insulin resistance in aging. Caloric restriction (CR; defined as 60% of ad libitum [AL] food intake) robustly reverses muscle insulin resistance, and delays its onset during aging. The NAD+-dependent protein deacetylase, sirtuin 1 (SIRT1) is activated by CR, and is thought to be the putative signaling node linking age and CR to muscle insulin action. Surprisingly, however, knowledge regarding the regulatory role of SIRT1 on muscle insulin action, particularly, in vivo, is remarkably limited. Our central hypothesis is that SIRT1 is a key signaling molecule that links aging and CR to muscle insulin action, primarily by modulating the acetylation of major signaling nodes, such as signal transducer and activator of transcription 3 (STAT3), that then influence many signaling pathways, including the insulin signaling pathway. To address this hypothesis, our approach will be to study muscle insulin signaling and sensitivity in response to age and CR using novel mouse models, in which we have manipulated SIRT1 activity specifically in muscle. Our model predicts that changes in SIRT1 activity during aging, and in response to CR, leads to changes in the acetylation status and subsequent activity of SIRT1 target proteins, which directly or indirectly regulates muscle insulin signaling and sensitivity. Specifically, Aim #1 wil elucidate the contribution of SIRT1 and STAT3 to the pathogenesis of muscle insulin resistance in aging mice, whilst Aim #2 will determine whether a SIRT1- STAT3 signaling axis underlies the ability of CR to enhance muscle insulin sensitivity in mice. For these studies, we will measure muscle insulin signaling and insulin sensitivity, in vivo, using hyperinsulinemic- euglycemic clamps, and ex vivo, using 2-deoxyglucose uptake assays, in young (4 months), mid-aged (12 months) and old (20 months) mice fed an AL diet, and compare them to measurements in mice fed a short- term (30 d) or long-term (9 or 17 months) CR diet. Studies will be conducted in 4 different transgenic mice with either a muscle-specific increase of SIRT1 activity, knockout (KO) of SIRT1 deacetylase activity, KO of STAT3 or KO of SIRT1 and STAT3. In Aim #3, we will use mass spectrometry techniques and adenoviral-based studies in muscle cells to identify novel targets of SIRT1 and the functional effects of their acetylation on insulin action. Altogether, these studies will broaden our understanding of the role of SIRT1 in muscle biology, and may have wide-reaching impact on the development of therapies to treat not only muscle insulin resistance, but also other diseases of aging in skeletal muscle.!