PGC1alpha Pathway: Novel Intracellular and Extracellular Mediators

PGC1alpha 通路：新型细胞内和细胞外介质

• 批准号: 10732540
• 负责人: BRUCE M. SPIEGELMAN
• 金额: $ 67.97万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10732540
• 关键词: Ablation; Adipocytes; Adipose tissue; Affect; Architecture; Binding; Biochemical; Biogenesis; Biological; Biology; Body Composition; Brain region; Cell Respiration; Cells; Data; Development; Diabetes Mellitus; Disease; Distant; Energy Metabolism; Enterobacteria phage P1 Cre recombinase; Fatty acid glycerol esters; Fiber; Gene Expression; Genes; Genetic; Grant; Health; High Fat Diet; Homeostasis; Human; Individual; Inflammation; Injections; Insulin Resistance; Intercellular Fluid; Knock-out; Knockout Mice; Link; Liquid substance; Liver; Luciferases; Maps; Mass Spectrum Analysis; Mediating; Mediator; Messenger RNA; Metabolic; Metabolic Diseases; Methods; Mitochondria; Molecular; Mus; Muscle; Muscular Atrophy; Muscular Dystrophies; Mutation; Neuromuscular Diseases; Neuromuscular Junction; Nuclear Protein; Obesity; Oxidative Regulation; PGC1a Regulation Pathway; Parkinson Disease; Pathologic; Pathway Analysis; Pathway interactions; Peripheral; Pharmaceutical Preparations; Physical Exercise; Physical activity; Physiological; Physiology; Play; Polyribosomes; Process; Proteins; RNA; RNA Binding; RNA Sequences; RNA-Binding Proteins; Recombinants; Regulation; Reporter; Reporting; Research; Ribosomes; Rodent; Role; Site; Skeletal Muscle; Small Interfering RNA; Specificity; Spirometry; Structure; System; Tertiary Protein Structure; Therapeutic; Therapeutic Intervention; Thermogenesis; Tissues; Titrations; Transcription Coactivator; Transgenic Mice; Translations; Weight Gain; Work; adeno-associated viral vector; adipokines; adiponectin; angiogenesis; cold temperature; crosslink; drug development; experimental study; extracellular; gain of function; genome wide association study; glucose tolerance; insight; interest; loss of function; nerve supply; nervous system disorder; neurotrophic factor; novel; polypeptide; promoter; reduce symptoms; ribosome profiling; targeted treatment; transcription factor; transcriptome sequencing

Project Abstract The PGC1a pathway has emerged as the dominant pathway for the modulation of mitochondrial biogenesis and oxidative metabolism in most tissues. Deficiencies in this pathway have been associated with pathological conditions such as obesity, diabetes and various neurological and neuromuscular disorders. Studies of the regulatory mechanisms at work here is thus fundamental to our understanding of energy homeostasis and the development of drugs to treat these and other disorders. The previous cycle of this grant illustrated that PGC1a is subject to robust control at the level of translation of its mRNA. In this proposal we interrogate trans-acting RNA-binding factors that might participate in the regulation of PGC1a translation and identify a little-known RNA- binding factor, RBM43, that is dramatically regulated in opposition to PGC1a. It is expressed lower in thermogenic fat than in energy-storing white fat, and it is suppressed by cold temperatures. Importantly, experimental reduction of RBM43 by siRNA causes an increase in PGC1a translation and oxidative metbolism. Conversely, increased expression of RBM43 decreases ribosome occupancy of the PGC1a mRNA, consistent with an action on the transational machinery. Here we propose mechanistic, genetic and biological studies of RBM43. Mice with a global mutation in Rbm43 have already been made in our lab, and show increased PGC1a levels and activity in adipose tissues. The physiological effects of this mutation will be determined by challenging mice with cold exposure and high fat diets. Gene expression in adipose tissues will be determined by RNA-seq, while weight gain, body composition and glucose tolerance will be determined using standard methods, including the use of metabolic cages. Fat-selective knockouts will be made using a Cre recombinase driven by the adiponectin promoter. To understand the mechanism by which RBM43 acts, its direct RNA targets will be identified by photo-crosslinking and the RBM43 protein domains and RNA sequences responsible for these interactions will be mapped. The PGC1a pathway also affects cells and tissues distant from the site of PGC1a expression, potentially through myokine or adipokine secretion. To more thoroughly investigate polypeptides secreted under the influence of PGC1a, a new method for the isolation of interstitial fluids from muscle and fat was developed. These fluids provide an excellent substrate for the application of sensitive and quantitative mass spectrometry. Using this approach, we have identified dozens of potential new myokines and adipokines under the control of PGC1a. Of particular interest is prosaposin (PSAP), a CNS neurotrophic factor not known to be secreted by peripheral tissues. Recombinant PSAP has effects on iWAT cells to stimulate thermogenic gene expression. We will characterize these effects in greater detail using RNA-seq and respirometry; the Psap gene will also be ablated in an adipose-selective fashion to determine its roles in thermogenesis and adipose innervation. Together, these studies will give broader insights into the regulation of the PGC1a pathway and potentially provide “actionable” targets for therapeutic intervention in metabolic diseases and other disorders.

项目摘要 PGC1A途径已成为调节线粒体生物发生和 大多数组织中的氧化代谢。该途径中的缺陷与病理有关 肥胖，糖尿病以及各种神经和神经肌肉疾病等疾病。研究 因此，这里工作的监管机制是我们对能量体内稳态和 开发用于治疗这些疾病和其他疾病的药物。该赠款的上一个周期说明了PGC1A 在其mRNA的翻译水平上受到强大的控制。在此提案中，我们询问跨性别 可能参与PGC1A翻译调节并确定鲜为人知的RNA-的RNA结合因子 结合因子RBM43，在与PGC1A相反的情况下受到了极大的调节。它的表达较低 热脂肪比能量储能的白脂肪在寒冷的温度下被抑制。重要的是， siRNA对RBM43的实验降低会导致PGC1A翻译和氧化元病的增加。 相反，RBM43的表达增加可降低PGC1A mRNA的核糖体占用率，一致 采用对翻译机械的动作。在这里，我们提出了机械，遗传和生物学研究 RBM43。在我们的实验室中已经制造了RBM43中全球突变的小鼠，并显示PGC1A增加 脂肪组织的水平和活性。该突变的物理影响将由挑战决定 感冒暴露和高脂饮食的小鼠。脂肪组织中的基因表达将由RNA-seq确定 尽管体重增加，但将使用标准方法（包括）确定身体成分和葡萄糖耐受性 代谢笼的使用。脂肪选择敲除将使用CRE重组酶驱动器进行 脂联素启动子。要了解RBM43的作用机制，其直接RNA靶标将是 通过照片 - 跨链接和RBM43蛋白结构域和RNA序列确定 互动将被映射。 PGC1A途径还会影响远离PGC1A部位的细胞和组织 表达，有可能通过肌动物或脂肪因子分泌。更彻底地研究多肽 在PGC1A的影响下分泌，这是一种从肌肉和脂肪中分离间质流体的新方法 这些烟道为应用敏感和定量质量提供了极好的底物 光谱法。使用这种方法，我们已经确定了数十种潜在的新肌动物和脂肪因子 PGC1A的控制。特别感兴趣 由周围组织分泌。重组PSAP对IWAT细胞具有刺激热基因的影响 表达。我们将使用RNA-seq和呼吸测定法更详细地表征这些效果。 PSAP基因 还将以脂肪选择性的方式消融，以确定其在热发生和脂肪中的作用 神经。这些研究一起将对PGC1A途径的调节和 有可能为代谢性疾病和其他疾病的热干预提供“可行的”靶标。