Discordant transcriptional regulation of gluconeogenic and lipogenic gene expression

摘要 葡萄糖和脂质代谢失调是胰岛素抵抗和2型糖尿病的标志。在 肝脏，这表现为胰岛素不能抑制肝脏葡萄糖输出，而从头脂肪生成 即使在禁食状态下也保持升高，但仍然能够显示胰岛素刺激的增加。的 胰岛素不能抑制肝葡萄糖输出，同时激活从头脂肪生成 选择性胰岛素抵抗对新生脂肪和新生脂肪的急性调节主要是 然而，在慢性状态下，如胰岛素抵抗， 在2型糖尿病中，促血管生成和脂肪生成基因表达有显著变化。我们所获得 初步数据表明，原型脂肪生成基因（即FIGH 3，SCD 1）主要受 转录起始，而致突变基因（即：PCK 1，G6 Pc）主要在以下水平进行监管： 转录延伸在饮食诱导的肝胰岛素抵抗期间，延伸因子Spt 5是 功能失调，从而增加进食状态下的致瘤基因表达。此外，使用 单细胞RNAseq，我们已经确定了不同的肝细胞亚群的差异参与过程中， 在肝细胞胰岛素抵抗的发展过程中，禁食/进食周期也会改变。基于 这些数据，我们提出了两个具体的目标，以检查几个新的分子和细胞机制， 是正常生理肝细胞脂肪生成和脂肪生成基因的重要调控成分 在饮食诱导的胰岛素抵抗过程中随后失调的表达。具体来说，我们使用1） ChIP-Seq以确定各种起始和延伸因子在其DNA占有率中的变化 结合，2）ATAC-Seq以确定染色质组织，和3）ChRO-Seq以直接测量染色质的结构。 跨基因体的转录速率。这些总肝细胞功能的分析将 补充单细胞分子分析，包括1）单细胞RNA-Seq，以直接确定 肝细胞亚群的差异功能，2）单个肝细胞的单细胞ATAC-Seq 染色质组织变化，以及3）单分子FISH，以评估个体细胞 转录活性和它们的解剖学分区的鉴定。这些方法将使我们能够 确定负责转录起始和延伸激活的分子机制 之间的脂肪和脂肪的基因表达，并将扩大我们对个人的理解， 在正常禁食/进食状态下以及在正常进食/进食状态下， 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.