Role of Forkhead Proteins and Sirtuins in Metabolism

描述（由申请人提供）：诱导胚胎发生是对禁食的重要适应。然而，过量的肝葡萄糖产生（HGP）也有助于糖尿病的病理生理学。在健康个体中，餐后血糖波动受到胰岛释放胰岛素的限制。胰岛素通过直接刺激葡萄糖摄取和抑制HGP来降低血糖水平。包含叉头盒的亚家族O蛋白1（FoxO 1）是胚胎发生的转录调节的主要介质。在禁食条件下，FoxO 1被发现在细胞核中，而在响应胰岛素受体信号FoxO 1被磷酸化，并迅速易位到细胞质中，通过蛋白酶体降解失活。来自我们实验室和其他实验室的证据表明，FoxO 1的转录活性分别被蛋白乙酰化酶和脱乙酰化酶如CBP/p300和SirT 1进一步修饰。我们推测，代谢应激积累，虽然老化和肥胖可以激活FoxO 1通过去乙酰化，从而有助于糖尿病的发病机制。沉默信息调节因子2（Sir 2 alpha）是一种NAD+依赖性蛋白脱乙酰酶，以FoxO 1依赖性方式控制果蝇和线虫的寿命。Sirt 1，Sir 2的哺乳动物直系同源物，调节肝细胞中的FoxO 1和PGC-1 α，从而导致其调节葡萄糖产生的建议。因此，我们建议研究Sirtl对小鼠代谢的影响。在目标1中，我们将利用Sirt 1 BAG产生具有低水平过表达的转基因小鼠（SirT 1-Tg），类似于已显示影响C.优雅我们假设Foxol是介导SirT 1有益作用所必需的。为了验证这一理论，我们将研究SirT 1-Tg小鼠的代谢表型是否是由其对FoxO 1或PGC-1 α脱乙酰化的影响引起的。在目标2中，我们建议用编码乙酰化缺陷或组成型乙酰化突变体的等位基因替换野生型FoxO 1基因，以评估对胰岛素信号传导和胰岛素生成的影响。相关性：胰岛素对基因表达的作用机制是生物学中的一个关键问题，对代谢紊乱的治疗具有重要的影响。在低等真核生物中，存在强有力的证据支持胰岛素/IGF 1信号通路与代谢和寿命调节之间的关系。通过基因扩增SirT 1基因，并使其下游靶点FoxO 1具有组成性活性，我们希望改善小鼠的血糖控制和可能的寿命。

DESCRIPTION (provided by applicant): Induction of gluconeogenesis is a vital adaptation to fasting. However, excessive hepatic glucose production (HGP) also contributes to the pathophysiology of diabetes. In healthy individuals, post-prandial glycemic excursions are limited by release of insulin from pancreatic islets. Insulin lowers blood glucose levels by directly stimulating glucose uptake and also by inhibiting HGP. The Forkhead Box-containing, sub-family O protein 1 (FoxO1) is the primary mediator of the transcriptional regulation of gluconeogenesis. During fasting conditions FoxO1 is found in the nucleus, while in response to insulin receptor signaling FoxO1 is phosphorylated and rapidly translocates to the cytoplasm for inactivation by proteasomal degradation. Evidence from our lab and others has shown that the transcriptional activity of FoxO1 is further modified by protein acetylases and deacetylases such as CBP/p300 and SirT1, respectively. We hypothesize that metabolic stress accrued though ageing and obesity can activate FoxO1 through deacetylation thereby contributing to the pathogenesis of diabetes. The silent information regulator 2 (Sir2 alpha) is a NAD+ dependent protein deacetylase that controls longevity in fruit flies and nematodes in a FoxO1- dependent manner. Sirtl, Sir2's mammalian ortholog, regulates FoxO1 and PGC-1 alpha in hepatocytes, thus leading to the suggestion that it modulates glucose production. We therefore propose to examine the effects of Sirtl on metabolism in mice. In Aim 1, we will utilize a Sirtl BAG to generate transgenic mice (SirT1-Tg) with low levels of over-expression, similar to those that have been shown to affect life span in C. elegans. We hypothesize that Foxol is required to mediate the salutary effects of SirT1. To test this theory, we will examine whether the metabolic phenotype of SirT1-Tg mice is caused by its effects on FoxO1 or PGC-1 alpha deacetylation. In Aim 2, we propose to replace the wild-type FoxO1 gene with alleles encoding either acetylation-defective or constitutively acetylated mutants to asses the effects on insulin signaling and gluconeogenesis. Relevance: The mechanism of insulin action on gene expression is a key question in biology with important ramifications for the treatment of metabolic disorders. In lower eukaryotes, strong evidence exists to support the relationship between the insulin/IGF1 signaling pathway and regulation of metabolism and lifespan. By genetically amplifying the SirT1 gene, and rendering its downstream target, FoxO1 constitutively active we hope to improve glycemic control and possibly lifespan in mice.