FOXN3 Regulation of Fasting Glucose Metabolism

项目摘要 人类全基因组关联研究已将单核苷酸多态联系在一起 (或接近)近100个基因中的SNPs与2型糖尿病的发生有关。其中大多数都是 关联缺乏机制基础，因为风险等位基因大多是非编码的(偶尔会导致 微妙的错义突变)。阐明单个GWAs“命中”的功能后果是一个核心问题 后基因组学时代的挑战。在最近一次强大的全球气候变化研究中(Manning et al.2012)，一份“联合”分析报告 测定空腹血糖和空腹胰岛素。这一方法确定了近20部小说 与其中一个参数或两个参数的关联。SNP与空腹血糖有关 Rs8004664，位于FOXN3基因的第一内含子。这个基因还没有牵涉到BASIC或 先前对血糖稳态的临床研究。编码基因在人类进化过程中具有保守的作用 排除成人生活中功能丧失研究的早期颅面发育：全球缺失 编码基因会导致致命缺陷。 我们发现在原始人中FOXN3转录丰度和FOXN3蛋白丰度增加 肝细胞rs8004664风险等位基因纯合。此外，FOXN3蛋白迅速减少 人HepG2肝癌细胞(rs8004664保护等位基因纯合)的血清提取 在禁食的大鼠肝脏中降低。我们假设FOXN3表达的增加或过度是由 风险等位基因，促进肝脏代谢的基因调节变化，导致血糖升高。我们 观察到(1)转基因斑马鱼FOXN3和人FOXN3在斑马鱼肝脏中的过表达 增加整个幼虫的游离葡萄糖和成虫的空腹血糖；和(2)FOXN3推动这种增加 通过抑制葡萄糖利用转录程序的表达来控制血糖。具体来说，FOXN3 抑制MYC的表达，MYC是一种推动葡萄糖利用糖酵解表达的转录因子) 携带危险等位基因的原代人肝细胞中的酶和转基因斑马鱼肝脏。我们在一个 Foxn3基因转录和蛋白丰度在肝脏下调的小鼠胰岛素缺乏模型； 相反，下调小鼠肝脏中胰高血糖素受体的表达会增加Foxn3的表达。在一个 综合方式(生理和基因调控；使用斑马鱼、小鼠和细胞培养方法)， 我们将揭示FOXN3是如何调节肝脏葡萄糖代谢的。我们还将确定胰高血糖素信号如何 调节FOXN3的表达。这些研究将导致禁食的新的、潜在的“可下药”的方面 新陈代谢。

Project Summary Human genome-wide association studies (GWAS) have linked single nucleotide polymorphisms (SNPs) in (and near) nearly 100 genes to the development of type 2 diabetes mellitus. Most of these associations lack a mechanistic basis, in that the risk alleles are mostly non-coding (and occasionally cause subtle mis-sense mutations). Elucidating the functional consequences of individual GWAS “hits” is a central challenge of the post-genomics era. In a recent, well-powered GWAS (Manning et al. 2012), a “joint” analysis was conducted for fasting blood glucose and fasting insulin. This approach identified nearly 20 novel associations with either or both parameters. An association with fasting glucose was revealed for the SNP rs8004664, which resides in the first intron of the FOXN3 gene. This gene has not been implicated in basic or clinical studies in glucose homeostasis previously. The encoding gene has a conserved evolutionary role in early cranio-facial development that has precluded loss-of-function studies in adult life: global deletion of the encoding gene causes lethal defects. We found that FOXN3 transcript abundance and FOXN3 protein abundance are increased in primary human hepatocytes homozygous for the rs8004664 risk allele. Furthermore, FOXN3 protein is rapidly decreased by withdrawal of serum in human HepG2 hepatoma cells (homozygous for the protective rs8004664 allele), and is decreased in fasted rat livers. We hypothesize that increased or excessive FOXN3 expression, driven by the risk allele, promotes gene regulatory changes in liver metabolism that result in increased blood glucose. We observe that (1) transgenic over-expression of both zebrafish foxn3 and human FOXN3 in the liver of zebrafish increases whole larval free glucose and adult fasting blood glucose; and (2) FOXN3 drives this increase in blood glucose by suppressing expression of a glucose utilization transcriptional program. Specifically, FOXN3 suppresses the expression of MYC, a transcription factor that drives expression of glucose utilization glycolytic) enzymes in primary human hepatocytes carrying the risk allele and transgenic zebrafish livers. We find in a mouse insulin-deficiency model that Foxn3 transcript and protein abundance is down-regulated in the liver; conversely, knock down of Glucagon Receptor expression in mouse liver increases Foxn3 expression. In a comprehensive manner (physiological and gene-regulatory; using zebrafish, mice and cell culture approaches), we will reveal how FOXN3 regulates liver glucose metabolism. We will also determine how glucagon signaling regulates FOXN3 expression. These studies will lead to novel, potentially “drugable” aspects of fasting metabolism.