Cellular Determinants of Adipocyte Phenotype and Function

项目总结 棕色和米色脂肪细胞在调节适应性产热和控制能量方面起着关键作用。 新陈代谢。冷暴露时，米色脂肪细胞在皮下白色脂肪组织内发育。 (SWAT)，并与棕色脂肪组织(BAT)一起参与生热反应。结果, 人们对阐明寒冷引起黄化或进一步“褐化”的机制非常感兴趣。 脂肪细胞，并识别可能的下游靶点，介导产热和能量消耗 作为解决肥胖症和糖尿病等代谢性疾病的治疗策略。在预赛中 研究发现，暴露在寒冷(4摄氏度)下的小鼠可诱导ROCK2的快速激活；增加褐变 并上调由pGC-1、UCP1和Prdm16组成的致热基因程序。突变型 脂肪细胞中缺乏ROCK2的小鼠(ADIPO-ROCK2-/-小鼠)未能形成冷诱导的SWAT褐变； 表现出生热活性降低；并有耐寒能力。有趣的是，我们还发现了蝙蝠 ADIPO-ROCK2-/-小鼠的线粒体含量较少。这种机制可能在一定程度上是由于 ROCK2诱导的心肌细胞增强因子(MEF)-2A缺失引起的线粒体生物发生 磷酸化和激活。因此，这项提议的总体目标是确定脂肪细胞如何 ROCK2可能通过诱导褐变和生热来调节能量代谢和肥胖。 特定目标1将检验脂肪细胞ROCK2通过 褐变的诱导和SWAT中的生热基因程序。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，以帮助描绘ROCK2介导的信号通路 这可以调节冷诱导的褐变和产热。 特定目标2将检验ROCK2通过MEF2A-2介导BAT产热的假设。 依赖线粒体的生物发生。使用重组的MEF2a/小鼠胚胎成纤维细胞(MEF)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/CHIP/RNA-SEQ 评估在以下基因的启动子中磷酸化的MEF2A结合区域的潜在富集性 参与线粒体的生物发生。 特殊目标3将验证脂肪细胞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)。我们将描述ADIPO-ROCK2-/-的代谢变化。 高脂饮食(HFD)和脂肪功能增强(ADI-O-CAROCK)小鼠。我们还将用 CRISPR-Cas9基因编辑或Cre/loxP对MEF2A上依赖于ROCK2的磷酸化位点的突变 技术这些mef2a突变小鼠，如果可行，将进行研究，以确定它们对寒冷和HFD的反应。 关于褐变、线粒体生物发生和产热。

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.