FOXN3 Regulation of Fasting Glucose Metabolism

项目摘要 人类全基因组关联研究（GWAS）将单核苷酸多态性与 2型糖尿病的发生与近100个基因的单核苷酸多态性（SNPs）有关。大多数这些 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， 后基因组时代的挑战。在最近的一项强有力的GWAS（Manning et al. 2012）中，一项“联合”分析 空腹血糖和空腹胰岛素。这种方法确定了近20种新的 与其中一个或两个参数关联。SNP与空腹血糖相关 rs 8004664，其位于FOXN 3基因的第一内含子中。该基因与基础或 葡萄糖稳态的临床研究。编码基因在进化过程中具有保守的作用， 早期颅面发育排除了成年期功能丧失的研究： 编码基因导致致命缺陷。 我们发现，FOXN 3转录本丰度和FOXN 3蛋白丰度在原代人类中增加， rs 8004664风险等位基因纯合子肝细胞。此外，FOXN 3蛋白被快速减少， 在人HepG 2肝癌细胞（保护性rs 8004664等位基因纯合）中抽取血清， 在禁食大鼠肝脏中减少。我们假设FOXN 3表达的增加或过度表达，是由细胞凋亡驱动的。 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foxn 3和人FOXN 3在斑马鱼肝脏中的过表达 增加整个幼虫游离葡萄糖和成人空腹血糖;和（2）FOXN 3驱动这种增加， 通过抑制葡萄糖利用转录程序的表达来降低血糖。具体来说，FOXN 3 抑制MYC的表达，MYC是一种驱动葡萄糖利用糖酵解表达的转录因子） 携带风险等位基因的原代人肝细胞和转基因斑马鱼肝脏中的酶。我们发现在一个 小鼠胰岛素缺乏模型，Foxn 3转录物和蛋白质丰度在肝脏中下调; 相反，小鼠肝脏中胰高血糖素受体表达的敲低增加Foxn 3表达。中 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FOXN 3如何调节肝脏葡萄糖代谢。我们还将确定胰高血糖素信号 调节FOXN 3的表达。这些研究将导致新的，潜在的“药物”方面的禁食 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.