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途径已成为线粒体生物发生和调控的主要途径。 大多数组织中的氧化代谢。这一途径的缺陷与病理 肥胖、糖尿病以及各种神经和神经肌肉疾病。对中国传统文化的研究 因此，在这里发挥作用的调节机制对于我们理解能量动态平衡和 开发治疗这些疾病和其他疾病的药物。这笔赠款的上一个周期表明，PGC1a 在其mRNA的翻译水平上受到强有力的控制。在这项提议中，我们审问了变装 可能参与调节PGC1a翻译的RNA结合因子，并确定一个鲜为人知的RNA- 结合因子RBM43，与PGC1a相反，受到戏剧性的调控。它在中表达得较低 生热脂肪比储存能量的白色脂肪要好，而且它受到低温的抑制。重要的是 通过siRNA实验降低RBM43导致PGC1a翻译增加和氧化代谢增加。 相反，RBM43的表达增加减少了PGC1a mRNA的核糖体占有率，这与 在转运机上采取行动。在这里，我们建议从机制、遗传学和生物学角度研究 RBM43。我们实验室已经制造了Rbm43基因全球突变的小鼠，并显示出PGC1a增加 脂肪组织中的水平和活性。这种突变的生理效应将通过挑战来确定 冷暴露和高脂肪饮食的小鼠。脂肪组织中的基因表达将通过RNA-SEQ来确定， 虽然体重增加、身体成分和葡萄糖耐量将使用标准方法确定，包括 新陈代谢笼的使用。脂肪选择性敲除将使用Cre重组酶进行，该重组酶由 脂联素启动子。为了了解RBM43的作用机制，它的直接RNA靶标将是 通过光交联和与此相关的RBM43蛋白结构域和RNA序列进行鉴定 交互将被映射。PGC1a途径也影响远离PGC1a部位的细胞和组织 表达，可能通过肌肉细胞因子或脂肪细胞因子的分泌。更深入地研究多肽 在PGC1a影响下分泌的一种分离肌肉和脂肪间质液体的新方法 是发展起来的。这些流体为灵敏和定量质量的应用提供了极好的底物 光谱分析。使用这种方法，我们已经确定了数十种潜在的新的肌肉运动因子和脂肪运动因子。 前列环素1a的控制。特别令人感兴趣的是丙皂苷(PSAP)，一种未知的中枢神经营养因子 由外周组织分泌。重组PSAP对IWAT细胞致热基因的激活作用 表情。我们将使用rna-seq和呼吸测量法更详细地描述这些影响；PSAP基因。 也将以脂肪选择性的方式被消融，以确定其在产热和脂肪中的作用 神经支配。总之，这些研究将使人们对PGC1a途径的调控和 有可能为代谢性疾病和其他疾病的治疗干预提供“可操作的”靶标。