Discordant transcriptional regulation of gluconeogenic and lipogenic gene expression

糖异生和脂肪生成基因表达的转录调控不一致

• 批准号: 10316350
• 负责人: Carolina Ines Eliscovich
• 金额: $ 59.9万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-15 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10316350
• 关键词: ATAC-seq; Acute; Address; Allosteric Regulation; Anatomy; Bile Acids; Binding; Cells; ChIP-seq; Chromatin; Chronic; Complement; Complex; Cytochrome P450; DNA; DNA Binding; Data; Development; Diet; Distal; Elongation Factor; Epigenetic Process; Event; FASN gene; Fasting; Fluorescent in Situ Hybridization; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Gluconeogenesis; Glutamine; Hepatic; Hepatocyte; Hyperglycemia; Hypertriglyceridemia; Ii-Key; Immunofluorescence Immunologic; Individual; Insulin; Insulin Resistance; Liver; Lobular; Measurement; Measures; Mediator of activation protein; Messenger RNA; Metabolic; Metabolism; Molecular; Molecular Analysis; Mus; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Organizational Change; Pathogenesis; Pattern; Peptide Initiation Factors; Physiological; Play; Process; RNA Polymerase II; Regulation; Regulator Genes; Rodent Model; Role; Running; Site; Testing; Time; Tissues; Transcription Elongation; Transcription Initiation; Transcriptional Activation; Transcriptional Regulation; base; chromatin immunoprecipitation; deep sequencing; feeding; genomic platform; glucose metabolism; glucose output; glucose production; human model; lipid biosynthesis; lipid metabolism; liver function; negative elongation factor; novel; oxidation; response; single molecule; single-cell RNA sequencing; transcription factor

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.

抽象的 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，这表现为胰岛素无法抑制肝脏葡萄糖输出，而从头脂肪生成 即使在禁食状态下仍保持升高，但仍能够表现出胰岛素刺激的增加。这 胰岛素无法抑制肝脏葡萄糖输出同时激活从头脂肪生成已被提及 如选择性胰岛素抵抗。主要是糖异生和脂肪从头生成的急性调节 然而，在胰岛素抵抗和慢性状态下，变构调节的快速变化是其结果 2型糖尿病的糖异生和脂肪生成基因表达有显着变化。我们已经获得了 初步数据表明，原型脂肪生成基因（即 FASN、SCD1）主要受 转录起始，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期间，延伸因子 Spt5 为 功能失调，从而增加进食状态下的糖异生基因表达。此外，使用 单细胞 RNAseq 我们已经确定了不同肝细胞亚群在 禁食/进食周期在肝细胞胰岛素抵抗的发展过程中也会发生改变。基于 根据这些数据，我们提出了两个具体目标来检查几种新颖的分子和细胞机制 是正常生理肝细胞脂肪生成和糖异生基因的重要调节成分 随后在饮食诱导的胰岛素抵抗过程中表达失调。具体来说，我们使用 1) ChIP-Seq 用于确定各种起始和延伸因子在 DNA 占据中的变化 结合，2) ATAC-Seq 确定染色质组织，3) ChRO-Seq 直接测量 跨基因体的转录率。这些总肝细胞功能的分析将 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 评估个体细胞 转录活性及其解剖分区的识别。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 脂肪生成和糖异生基因表达之间的关系，并将扩大我们对个体的理解 肝细胞的细胞反应解释了在正常禁食/进食状态下和在 饮食引起的肝脏胰岛素抵抗。