Cellular Determinants of Adipocyte Phenotype and Function

脂肪细胞表型和功能的细胞决定因素

• 批准号: 10410997
• 负责人: JAMES Kuang-Jan LIAO
• 金额: $ 16.4万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-19 至 2023-08-18
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10410997
• 关键词: ATAC-seq; Address; Adipocytes; Adipose tissue; Affect; Animals; Antibodies; Binding; Biogenesis; Body Weight; Brown Fat; CRISPR/Cas technology; Cells; ChIP-seq; Chromatin; Circadian Rhythms; Consumption; Cre-LoxP; DNA Binding; Data; Dependence; Diabetes Mellitus; Dual-Energy X-Ray Absorptiometry; Embryo; Energy Metabolism; Enhancers; Exhibits; Expenditure; FOXO1A gene; Fibroblasts; Genes; Genetic Transcription; Glucose; Heart; High Fat Diet; Human; Impairment; Insulin; Insulin Resistance; Lipoproteins; Location; Mediating; Metabolic; Metabolic Diseases; Metabolism; Mitochondria; Monitor; Morphology; Motor Activity; Mus; Muscle; Muscle Cells; Mutant Strains Mice; Mutate; Mutation; Nuclear; Obesity; Oxygen; Oxygen Consumption; Pathway Analysis; Pathway interactions; Phenotype; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Protein Isoforms; Proteome; ROCK1 gene; Reporting; Research; Role; Serine; Signal Pathway; Skeletal Muscle; System; Technology; Testing; Therapeutic; Thermogenesis; Transcript; Up-Regulation; XCL1 gene; adipocyte differentiation; brain tissue; cell motility; diet-induced obesity; gain of function; improved; interest; lipid biosynthesis; loss of function; metabolic profile; metabolic rate; muscle enhancer factor-2A; mutant; postnatal; programs; promoter; reconstitution; response; rho; rho GTP-Binding Proteins; subcutaneous; transcription factor; transcriptome; transcriptome sequencing; ATAC-seq; Address; Adipocytes; Adipose tissue; Affect; Animals; Antibodies; Binding; Biogenesis; Body Weight; Brown Fat; CRISPR/Cas technology; Cells; ChIP-seq; Chromatin; Circadian Rhythms; Consumption; Cre-LoxP; DNA Binding; Data; Dependence; Diabetes Mellitus; Dual-Energy X-Ray Absorptiometry; Embryo; Energy Metabolism; Enhancers; Exhibits; Expenditure; FOXO1A gene; Fibroblasts; Genes; Genetic Transcription; Glucose; Heart; High Fat Diet; Human; Impairment; Insulin; Insulin Resistance; Lipoproteins; Location; Mediating; Metabolic; Metabolic Diseases; Metabolism; Mitochondria; Monitor; Morphology; Motor Activity; Mus; Muscle; Muscle Cells; Mutant Strains Mice; Mutate; Mutation; Nuclear; Obesity; Oxygen; Oxygen Consumption; Pathway Analysis; Pathway interactions; Phenotype; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Protein Isoforms; Proteome; ROCK1 gene; Reporting; Research; Role; Serine; Signal Pathway; Skeletal Muscle; System; Technology; Testing; Therapeutic; Thermogenesis; Transcript; Up-Regulation; XCL1 gene; adipocyte differentiation; brain tissue; cell motility; diet-induced obesity; gain of function; improved; interest; lipid biosynthesis; loss of function; metabolic profile; metabolic rate; muscle enhancer factor-2A; mutant; postnatal; programs; promoter; reconstitution; response; rho; rho GTP-Binding Proteins; subcutaneous; transcription factor; transcriptome; transcriptome sequencing

PROJECT SUMMARY Brown and beige adipocytes play key roles in mediating adaptive thermogenesis and controlling energy metabolism. In response to cold exposure, beige adipocytes develop within subcutaneous white adipose tissue (sWAT), and together with brown adipose tissue (BAT), contribute to the thermogenic response. As a result, there is intense interest in elucidating the mechanism by which cold induces “beiging” or further “browning” of adipocytes, and identifying putative downstream targets that mediate thermogenesis and energy expenditure as a therapeutic strategy for addressing metabolic diseases such as obesity and diabetes. In preliminary studies, we found that exposure of mice to cold (4oC) induces the rapid activation of ROCK2; increases beiging of sWAT; and upregulates the thermogenic gene program consisting of Pgc-1, Ucp1, and Prdm16. Mutant mice lacking Rock2 in adipocytes (adipo-Rock2–/– mice) failed to develop cold-induced beiging of sWAT; exhibited reduced thermogenic activity; and have cold intolerance. Interestingly, we also found that BAT of adipo-Rock2–/– mice have less mitochondria content. The mechanism may be due, in part, to decreased mitochondrial biogenesis through the loss of ROCK2-induced myocyte enhancer factor (MEF)-2A phosphorylation and activation. The overall aim of this proposal, therefore, is to determine how adipocyte Rock2 could regulate energy metabolism and obesity through the induction of beiging and thermogenesis. Specific aim 1 will test the hypothesis that adipocyte Rock2 mediates thermogenesis through the induction of beiging and the thermogenic gene program in sWAT. Single-cell transcriptome and proteome analyses will be performed on adipocytes from sWAT to help delineate Rock2-mediated signaling pathways that could regulate cold-induced beiging and thermogenesis. Specific aim 2 will test the hypothesis that Rock2 mediates BAT thermogenesis through MEF2A- dependent mitochondrial biogenesis. Using reconstituted Mef2a–/– mouse embryonic fibroblasts (MEFs) or BA, we will determine the role of MEF2A in Pgc-1 gene transcription. We will also perform ATAC/ChIP/RNA-seq to assess for potential enrichment of phosphorylated MEF2A-binding regions in promoters of genes that are involved in mitochondrial biogenesis. Specific aim 3 will test the hypothesis that adipocyte Rock2-Mef2a pathway is critical for energy metabolism and diet-induced obesity (DIO). We will profile metabolic changes in adipo-Rock2–/– (loss-of- function) and adipo-caRock (gain-of-function) mice on high fat diet (HFD). We will also generate mice with mutations in Rock2-dependent phosphorylation sites on MEF2A using CRISPR-Cas9 gene editing or Cre/loxP technology. These Mef2a mutant mice, if viable, will be studied to determine their response to cold and HFD with regards to beiging, mitochondrial biogenesis, and thermogenesis.

项目摘要 棕色和米色脂肪细胞在介导适应性热和控制能量中起关键作用 代谢。为了响应冷暴露，米色脂肪细胞在皮下白色脂肪组织中发展 （SWAT）以及与棕色脂肪组织（BAT）一起有助于热反应。因此， 人们对阐明冷诱导“存在”或进一步“褐变”的机制非常感兴趣 脂肪细胞，并识别介导生热和能量消耗的推定的下游靶标 作为解决肥胖和糖尿病等代谢疾病的治疗策略。在初步 研究，我们发现小鼠暴露于寒冷（4oC）会诱导Rock2的快速激活。增加变得 特警；并上调由PGC-1，UCP1和PRDM16组成的热基因程序。突变体 在脂肪细胞（adipo-rock2 - / - 小鼠）中缺乏岩石2的小鼠未能发展出SWAT的冷引起的变为。 暴露的热活动降低；并具有冷肠。有趣的是，我们还发现 adipo-rock2 - / - 小鼠的线粒体含量较少。该机制可能部分归因于 线粒体生物发生因岩石2诱导的肌细胞增强因子（MEF）-2a而丧失。 磷酸化和激活。因此，该提案的总体目的是确定脂肪细胞如何 Rock2可以通过诱导变为和热生成来调节能量代谢和肥胖。 具体目标1将检验以下假设：脂肪细胞岩石2通过 SWAT中的诱导和热基因程序的诱导。单细胞转录组和蛋白质组 分析将在SWAT的脂肪细胞上进行，以帮助划定Rock2介导的信号通路 这可能调节冷诱导的变为和热生成。 特定的目标2将检验以下假设，即Rock2通过MEF2A-介导BAT的热发生 依赖性线粒体生物发生。使用重构的MEF2A - / - 鼠标胚胎成纤维细胞（MEFS）或BA， 我们将确定MEF2A在PGC-1基因转录中的作用。我们还将执行ATAC/芯片/RNA-seq 评估潜在的基因启动子中磷酸化的MEF2A结合区域的富集 参与线粒体生物发生。 特定目标3将检验以下假设：脂肪细胞rock2-mef2a途径对于能量至关重要 代谢和饮食引起的肥胖症（DIO）。我们将介绍adipo-rock2 - / - 的代谢变化（丧失 - 功能）和高脂饮食（HFD）的小鼠（功能获得）小鼠（HFD）。我们还将与 使用CRISPR-CAS9基因编辑或CRE/LOXP，依赖Rock2依赖性磷酸化位点的Rock2依赖性磷酸化位点的突变 技术。这些MEF2A突变小鼠（如果可行）将是研究它们对冷和HFD的反应 关于存在，线粒体生物发生和热生成。