Role of Forkhead Proteins and Sirtuins in Metabolism

描述(由申请人提供)：糖异生的诱导是对禁食的重要适应。然而，过多的肝脏葡萄糖产生(HGP)也参与了糖尿病的病理生理过程。在健康个体中，餐后血糖漂移受到胰岛胰岛素释放的限制。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。含有Forkhead Box的亚家族O蛋白1(FoxO1)是糖异生的转录调控的主要调节因子。在禁食条件下，FoxO1在细胞核中被发现，而作为对胰岛素受体信号的响应，FoxO1被磷酸化并迅速转移到细胞质中，通过蛋白酶体的降解而失活。来自我们实验室和其他实验室的证据表明，FoxO1的转录活性分别被CBP/p300和SirT1等蛋白质乙酰基酶和去乙酰基酶进一步修饰。我们假设，通过衰老和肥胖而产生的代谢应激可以通过去乙酰化激活FoxO1，从而促进糖尿病的发病。沉默信息调节因子2(Sir2α)是一种依赖NAD+的蛋白质去乙酰基酶，以FoxO1依赖的方式控制果蝇和线虫的寿命。Sirt1，Sir2‘S哺乳动物同源基因，调节肝细胞中的FoxO1和PGC-1α，从而导致推测它调节葡萄糖的产生。因此，我们建议研究Sirtl对小鼠新陈代谢的影响。在目标1中，我们将利用Sirtl包来产生低水平过表达的转基因小鼠(SirT1-TG)，类似于那些已被证明影响线虫寿命的转基因小鼠。我们假设需要Foxol来调节SirT1的有益作用。为了验证这一理论，我们将检查SirT1-TG小鼠的代谢表型是由其对FoxO1或PGC-1α脱乙酰基的影响引起的。在目标2中，我们建议用编码乙酰化缺陷或成分乙酰化突变的等位基因替换野生型FoxO1基因，以评估其对胰岛素信号转导和糖异生的影响。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，有强有力的证据支持胰岛素/IGF1信号通路与代谢调节和寿命的关系。通过从基因上扩增SirT1基因，并使其下游靶基因FoxO1具有结构性活性，我们希望改善小鼠的血糖控制，并可能延长其寿命。

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.