The Regulation of Hepatic Metabolic Zonation by the Diabetes Gene TCF7L2

摘要 哺乳动物肝脏由称为小叶的功能单位组成，小叶在空间上被分隔成相对的区域。 到门静脉三联体和中央静脉每个区域包含具有不同功能的肝细胞， 影响新陈代谢过程几十年来，肝脏的这种空间分工一直受到重视，但 代谢分区的分子和生理决定因素知之甚少。此外，无论dis- 有序的肝脏代谢结构在普遍的肝脏疾病中起着病理生理作用， 清楚作为我们长期努力剖析2型糖尿病（T2D）候选者的功能作用的一部分， 基因，我们已经确定了由转录因子7样2（TCF7L2）基因编码的蛋白质作为关键调节因子。 肝代谢分区的影响因素。利用TCF 7L2在肝脏中失活的独特小鼠模型， 细胞，基因组和体内生理方法的综合结合，我们将研究调控 TCF 7L2的代谢分区，并将确定由TCF 7L2调节的分区途径的意义。 TCF 7L2与非酒精性脂肪性肝炎（NASH）肝纤维化发展的关系在已发表的研究中，我们 揭示了TCF7L2在肝细胞中多种代谢基因的转录调控中的惊人作用， 细胞，包括参与葡萄糖、脂质和氨基酸代谢的带状途径的细胞。初步 我们的研究发现，Tcf7l2基因的表达在整个肝小叶中是普遍存在的，但Tcf7l2基因在肝小叶中的表达是不稳定的。 转录活性高度限制于成人中央静脉周围的肝细胞群 小鼠肝脏。这些数据和额外的初步研究促使我们开发了一种独特的小鼠模型， 仅在肝脏中表达转录失活的TCF7L2（Hep-TCF7L2 Δ DBD）。利用这种模式，在 建议我们将测试TCF 7L2是肝代谢分区的重要调节剂的中心假设， TCF7L2介导的中心周围氨基酸代谢的破坏易使Hep-TCF7L2 Δ DBD小鼠 饮食诱导的纤维化和NASH。在具体的目标1中，我们将剖析时空转录功能 使用先进的单核分析法进行转座酶可识别染色质测序， 在整个小鼠肝小叶的不同肝细胞群体中，使用snRNA-Seq进行snATAC-Seq的比较。具体目标 2、我们将确定TCF 7L2在维持肝脏代谢分区中的作用，并将测试TCF 7L2对肝脏代谢分区的影响。 TCF 7L2失活对Hep-3肝细胞中心周围和门静脉周围肝细胞群小叶分布的影响 TCF7L2 Δ DBD小鼠。最后，在具体目标3中，我们将肝纤维化破坏带状氨基酸代谢 在Hep-TCF7L2 Δ DBD小鼠中使用整合的体内生理学方法。这些实验将剖析 TCF 7L2影响肝脏代谢分区的分子和生理机制，并将使肝脏代谢分区的功能丧失。 阐明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.