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

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 褐色和米色脂肪细胞在调节适应性产热和控制能量中起关键作用 新陈代谢.冷暴露后，皮下白色脂肪组织内形成米色脂肪细胞 （sWAT），并与棕色脂肪组织（BAT）一起，有助于产热反应。因此，在本发明中， 人们对阐明冷诱导"褐变"或进一步的"布朗宁"的机制有着强烈的兴趣， 脂肪细胞，并确定推定的下游目标，介导产热和能量消耗 作为解决代谢疾病如肥胖症和糖尿病的治疗策略。初步 研究中，我们发现，小鼠暴露于寒冷（4 ℃）诱导ROCK 2的快速激活; 的sWAT;和上调产热基因程序组成的Pgc-1 β，Ucp1，和Prdm16。突变体 在脂肪细胞中缺乏Rock2的小鼠（脂肪Rock2-/-小鼠）不能发展冷诱导的sWAT褐变; 表现出减少的产热活动;并且不耐寒。有趣的是，我们还发现， 脂肪-Rock2-/-小鼠具有较少的线粒体含量。这一机制的部分原因可能是， 通过ROCK 2诱导的肌细胞增强因子（MEF）-2A丢失的线粒体生物发生 磷酸化和活化。因此，本提案的总体目标是确定脂肪细胞 Rock2可以通过诱导beiging和产热来调节能量代谢和肥胖。 具体目标1将检验脂肪细胞Rock2通过以下途径介导产热的假设： 在sWAT中诱导beiging和产热基因程序。单细胞转录组和蛋白质组 将对来自sWAT的脂肪细胞进行分析，以帮助描述Rock2介导的信号通路 可以调节冷诱导的褐变和产热。 具体目标2将检验Rock2通过MEF2A介导BAT产热的假设。 依赖线粒体的生物合成使用重构的Mef2a-/-小鼠胚胎成纤维细胞（MEF）或BA， 我们将确定MEF2A在Pgc-1 β基因转录中的作用。我们还将进行ATAC/ChIP/RNA-seq 为了评估磷酸化MEF2A结合区在基因启动子中的潜在富集， 参与线粒体的生物合成 具体目标3将检验脂肪细胞Rock2-Mef2a途径对能量至关重要的假设。 代谢和饮食诱导的肥胖（DIO）。我们将分析脂肪-Rock2-/-（丢失- 功能）和高脂肪饮食（HFD）的脂肪-caRock（功能获得）小鼠。我们也会用 使用CRISPR-Cas9基因编辑或Cre/loxP在MEF2A上的Rock2依赖性磷酸化位点中的突变 技术.这些Mef2a突变小鼠，如果存活，将被研究以确定它们对寒冷和HFD的反应 关于beiging，线粒体生物发生和产热。