Discordant transcriptional regulation of gluconeogenic and lipogenic gene expression

摘要 糖脂代谢紊乱是胰岛素抵抗和2型糖尿病的特征。在 肝脏，表现为胰岛素不能抑制肝脏的葡萄糖输出，同时新生脂肪生成。 即使在禁食状态下仍保持升高，但仍能显示胰岛素刺激的增加。这个 胰岛素在激活新生脂肪生成的同时不能抑制肝脏葡萄糖输出已被提及 作为选择性胰岛素抵抗。糖异生和从头造脂的急性调节为主 然而，由于变构调节的快速变化，在慢性状态下，如胰岛素抵抗和 2型糖尿病患者糖异生和生脂基因表达有明显变化。我们已经获得了 初步数据表明，典型的生脂基因(即FASN、SCD1)主要受 转录启动，而糖异生基因(即：PCK1，G6Pc)主要在 转录延伸。在饮食诱导的肝脏胰岛素抵抗过程中，延长因子Spt5是 功能失调，从而增加糖异生基因在饲料状态下的表达。此外，使用 单细胞RNAseq我们已经确定了不同肝细胞亚群在 在肝细胞胰岛素抵抗的发展过程中，禁食/喂养周期也会改变。基于 这些数据，我们提出了两个具体的目标来检查几个新的分子和细胞机制， 是正常生理性肝细胞生脂和生糖基因的重要调节成分 在饮食诱导的胰岛素抵抗过程中，这些表达随后被失调。具体来说，我们使用1) ChIP-Seq法测定DNA占有率中各种起始和延伸因子的变化 结合，2)ATAC-Seq确定染色质组织，3)ChRO-Seq直接测量 跨基因体的转录速率。这些对总肝细胞功能的分析将是 辅以单细胞分子分析，包括1)单细胞RNA-Seq直接确定 肝细胞亚群的功能差异；2)单个肝细胞的单细胞ATAC-SEQ 染色质组织的变化，以及3)单分子FISH评估单个细胞 转录活性及其解剖区带的鉴定。这些方法将使我们能够 确定转录起始和延伸激活的分子机制 生脂和生糖基因表达之间的关系，并将扩大我们对个体的理解 在正常禁食/进食状态下和在 饮食诱导的肝脏胰岛素抵抗。

Abstract Dysregulated glucose and lipid metabolism are hallmarks of insulin resistance and type 2 diabetes. In the liver, this is manifested as the inability of insulin to suppress hepatic glucose output, while de novo lipogenesis remains elevated even in the fasted state but is still capable of displaying an insulin-stimulated increase. The inability of insulin to suppress hepatic glucose output while activating de novo lipogenesis has been referred to as selective insulin resistance. The acute regulation of both gluconeogenesis and de novo lipogenesis primarily results from rapid changes in allosteric regulation, however, in chronic states such as insulin resistance and type 2 diabetes there are marked changes in gluconeogenic and lipogenic gene expression. We have obtained preliminary data indicating that the prototypical lipogenic genes (i.e. FASN, SCD1) are primarily regulated by transcription initiation whereas gluconeogenic genes (i.e.: PCK1, G6Pc) are primarily regulated at the level of transcriptional elongation. During diet induced hepatic insulin resistance, the elongation factor Spt5 is functionally dysregulated thereby increasing gluconeogenic gene expression in the fed state. In addition, using single cell RNAseq we have identified the differential engagement of different hepatocyte subsets during the fasting/feeding cycle that are also altered during the development of hepatocyte insulin resistance. Based upon these data, we propose two specific aims to examine several novel molecular and cellular mechanisms that are important regulatory components of normal physiologic hepatocyte lipogenic and gluconeogenic gene expression that are subsequently dysregulated during diet-induced insulin resistance. Specifically, we use 1) ChIP-Seq to determine the changes in various initiation and elongation factors in their DNA occupancy binding, 2) ATAC-Seq to determine chromatin organization, and 3) ChRO-Seq to directly measure the rates of transcription across gene bodies. These analyses of total hepatocyte function will be complemented with single cell molecular analyses that include 1) single cell RNA-Seq to directly determine differential function of hepatocyte subsets, 2) single cell ATAC-Seq for individual hepatocyte chromatin organization changes, and 3) single molecule FISH to assess individual cellular transcriptional activity and identification of their anatomical zonation. These approaches will allow us to determine the molecular mechanisms responsible for the activation of transcriptional initiation and elongation between lipogenic and gluconeogenic gene expression, and will expand our understanding of the individual hepatocyte cellular responses that account for these changes in under normal fasting/feeding states and in diet-induced liver insulin resistance.