The Regulation of Hepatic Metabolic Zonation by the Diabetes Gene TCF7L2

摘要 哺乳动物的肝脏由称为小叶的功能单位组成，这些小叶在空间上被分成几个相对的区域 门静脉、三联体和中央静脉。每个区域都包含具有不同功能的肝细胞 影响新陈代谢过程。肝脏的这种空间分工几十年来一直受到重视，但 对代谢区划的分子和生理决定因素知之甚少。此外，是否会-- 有序的肝脏代谢结构在常见肝病中的病理生理作用 安全。作为我们长期努力的一部分，以剖析领先的2型糖尿病(T2D)候选患者的功能作用 基因，我们已经确定转录因子7-样2(TCF7L2)基因编码的蛋白质是一个关键的调节因子。 肝脏代谢性区带的激光。利用TCF7L2在肝脏和肝脏中失活的独特小鼠模型 结合细胞、基因组和体内生理学方法，我们将研究这种调节 TCF7L2的代谢区带，并将确定由TCF7L2调控的区带途径的意义 TCF7L2对非酒精性脂肪性肝炎(NASH)肝纤维化发展的影响在已发表的研究中，我们 揭示了TCF7L2在肝脏一系列代谢基因转录调控中的显著作用- 细胞，包括那些参与葡萄糖、脂肪和氨基酸代谢的带状通路的细胞。在预赛中 研究发现，TCF7L2基因的表达普遍存在于肝小叶，而TCF7L2 转录活性高度受限于成人中央静脉周围的一群肝细胞 老鼠的肝脏。这些数据，以及其他的初步研究，促使我们开发了一种独特的老鼠模型 只在肝脏表达转录失活的TCF7L2(HEP-TCF7L2ΔDBD)。使用这个模型，在这个 建议我们将检验TCF7L2是肝脏代谢分区的重要调节因子的中心假设， TCF7L2介导的中枢氨基酸代谢紊乱使HEP-TCF7L2ΔDBD小鼠易患 饮食诱导的纤维化和NASH。在特定的目标1中，我们将剖析时空转录功能 转座酶可及染色质测序技术应用于肝脏TCF7L2基因的研究 (SnATAC-Seq)和SnRNA-Seq在小鼠肝小叶的不同肝细胞群中。以特定的目标 2、我们将确定TCF7L2在维持肝脏代谢区带中的作用并测试其影响 TCF7L2灭活对HEP-1大鼠中心周围和门静脉周围肝细胞群小叶分布的影响 TCF7L2ΔDBD小鼠。最后，在具体目标3中，我们将把肝纤维化扰乱区域氨基酸代谢联系起来 在HEP-TCF7L2ΔDBD小鼠中，采用体内综合生理学方法。这些实验将剖析 TCF7L2影响肝脏代谢区带的分子和生理机制 记述肝脏组织紊乱对代谢性疾病发展的影响。

ABSTRACT The mammalian liver consists of functional units called lobules, which are spatially separated into zones relative to the portal triad and central vein. Each zone contains hepatocytes with distinct functions that differentially impact metabolic processes. This spatial division of labor in liver has been appreciated for decades, but the molecular and physiological determinants of metabolic zonation are poorly understood. Moreover, whether dis- ordered hepatic metabolic architecture plays a pathophysiological role in prevalent liver diseases remains un- clear. As part of our long-term effort to dissect the functional role of leading type 2 diabetes (T2D) candidate genes, we have identified the protein encoded by the transcription factor 7-like 2 (TCF7L2) gene as a key regu- lator of hepatic metabolic zonation. Leveraging a unique mouse model of TCF7L2 inactivation in liver and an integrative combination of cellular, genomic and in vivo physiological approaches, we will examine the regulation of metabolic zonation by TCF7L2, and will determine the significance of the zonated pathways regulated by TCF7L2 to the development of hepatic fibrosis in nonalcoholic steatohepatitis (NASH). In published studies, we unveiled a striking role for TCF7L2 in the transcriptional regulation of a diverse set of metabolic genes in hepato- cytes, including those involved in zonated pathways of glucose, lipid, and amino acid metabolism. In preliminary studies we discovered that the expression of the Tcf7l2 gene is ubiquitous across the liver lobule, but TCF7L2 transcriptional activity is highly restricted to a population of hepatocytes surrounding the central vein in adult mouse liver. These data, and additional preliminary studies, prompted us to develop a unique mouse model that expresses transcriptionally inactive TCF7L2 exclusively in the liver (Hep-TCF7L2ΔDBD). Using this model, in this proposal we will test the central hypothesis that TCF7L2 is an important regulator of hepatic metabolic zonation, and that TCF7L2-mediated disruption of pericentral amino acid metabolism predisposes Hep-TCF7L2ΔDBD mice to dietary induced fibrosis and NASH. In specific aim 1, we will dissect the spatiotemporal transcriptional function of TCF7L2 in liver using cutting-edge single-nuclei Assay for Transposase-Accessible Chromatin sequencing (snATAC-Seq) with snRNA-Seq in distinct hepatocyte populations across the mouse liver lobule. In Specific Aim 2, we will determine the role of TCF7L2 in the maintenance of hepatic metabolic zonation and will test the impact of TCF7L2 inactivation on the lobular distribution of pericentral and periportal hepatocyte populations in Hep- TCF7L2ΔDBD mice. Finally, in Specific Aim 3 we will link hepatic fibrosis disrupted zonal amino acid metabolism in Hep-TCF7L2ΔDBD mice using integrative in vivo physiological approaches. These experiments will dissect the molecular and physiological mechanisms by which TCF7L2 influences hepatic metabolic zonation, and will de- scribe the consequences of disordered hepatic organization on the development of metabolic diseases.