SIRT1 and muscle insulin sensitivity

描述（由申请人提供）：胰岛素抵抗是衰老肌肉中常见的代谢并发症，是2型糖尿病（T2D）病因的主要缺陷。然而，肌肉中将年龄与胰岛素抵抗联系起来的信号机制尚不清楚。考虑到2010年，美国65岁及以上人群中约有27%的人患有T2D，而在未来20年，65岁及以上人口预计将翻一番，达到约7000万，解决这一根本性的知识差距势在必行。因此，本研究的长期目标是确定衰老过程中肌肉胰岛素抵抗的机制。热量限制（CR；定义为随意[AL]食物摄入的60%）强有力地逆转肌肉胰岛素抵抗，并延缓其在衰老过程中的发生。NAD+依赖性蛋白去乙酰化酶sirtuin 1 （SIRT1）被CR激活，被认为是连接年龄和CR与肌肉胰岛素作用的推定信号节点。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，将衰老和CR与肌肉胰岛素作用联系起来，主要通过调节主要信号节点的乙酰化，如信号转换器和转录激活器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型小鼠模型研究肌肉胰岛素信号传导和对年龄和CR的敏感性，在这种模型中，我们特别控制了肌肉中的SIRT1活性。我们的模型预测，衰老过程中SIRT1活性的变化，以及对CR的反应，导致SIRT1靶蛋白乙酰化状态和随后活性的变化，从而直接或间接调节肌肉胰岛素信号传导和敏感性。具体来说，Aim #1将阐明SIRT1和STAT3在衰老小鼠肌肉胰岛素抵抗发病机制中的作用，而Aim #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，我们将在体内使用高胰岛素-正糖钳测量肌肉胰岛素信号和胰岛素敏感性，在体外使用2-去氧葡萄糖摄取测定，在年轻（4个月），中年（12个月）和老年（20个月）饲喂AL饮食的小鼠中，并将其与饲喂短期（30天）或长期（9或17个月）CR饮食的小鼠进行比较。研究将在4种不同的转基因小鼠中进行，其中SIRT1活性的肌肉特异性增加，SIRT1去乙酰化酶活性的敲除（KO）， STAT3的KO或SIRT1和STAT3的KO。在Aim #3中，我们将在肌肉细胞中使用质谱技术和基于腺病毒的研究来鉴定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在肌肉生物学中的作用的理解，并可能对治疗肌肉胰岛素抵抗以及其他骨骼肌衰老疾病的治疗方法的发展产生广泛的影响。

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.! PUBLIC HEALTH RELEVANCE: Insulin resistance is a common metabolic defect in aged muscle that increases the risk for developing type 2 diabetes. These experiments aim to define why aging muscle becomes insulin resistant, and to define how caloric restriction reverses this insulin resistance, with particular focus on the "anti-aging" protein, SIRT1. Knowledge gathered by such studies will allow the design of improved therapies to treat insulin resistance.!