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 PGC 1a途径已经成为调节线粒体生物发生的主要途径， 大多数组织的氧化代谢。这一通路的缺陷与病理性 例如肥胖症、糖尿病和各种神经和神经肌肉疾病。的研究 因此，在这里工作的调节机制是我们理解能量稳态和能量平衡的基础。 开发治疗这些和其他疾病的药物。上一个赠款周期表明，PGC 1a 在其mRNA的翻译水平上受到强有力的控制。在这个建议中，我们询问trans-acting RNA结合因子可能参与PGC 1a翻译的调节，并识别一种鲜为人知的RNA- 结合因子，RBM 43，这是显着调节反对PGC 1a。它的表达较低， 产热脂肪比储存能量的白色脂肪中的产热脂肪更少，并且它受到低温的抑制。重要的是， 实验性的通过siRNA减少RBM 43引起PGC 1a翻译和氧化代谢的增加。 相反，RBM 43的表达增加会降低PGC 1a mRNA的核糖体占有率，这与 在transational机器上采取行动。在这里，我们提出了机械，遗传和生物学研究， RBM43.我们的实验室已经制造出了Rbm 43全局突变的小鼠，并显示出增加的PGC 1a 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 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测定， 而体重增加、身体组成和葡萄糖耐量将使用标准方法测定，包括 代谢笼的使用脂肪选择性敲除将使用Cre重组酶进行，所述Cre重组酶由 脂联素启动子为了了解RBM 43的作用机制，将研究其直接的RNA靶点。 通过光交联和RBM 43蛋白结构域和负责这些的RNA序列鉴定 互动将被映射。PGC 1a通路也影响远离PGC 1a位点的细胞和组织 表达，可能通过肌因子或脂肪因子分泌。为了更彻底地研究多肽 在PGC 1a的影响下分泌，这是一种从肌肉和脂肪中分离间质液的新方法 开发了这些流体为应用灵敏和定量质量提供了极好的基质 光谱法使用这种方法，我们已经确定了几十个潜在的新的肌因子和脂肪因子， PGC 1a的控制。特别令人感兴趣的是鞘脂激活蛋白原（PSAP），一种CNS神经营养因子， 由外周组织分泌。重组PSAP对iWAT细胞产热基因的影响 表情我们将使用RNA-seq和呼吸测定法更详细地描述这些效应; Psap基因 也将以脂肪选择性方式消融，以确定其在产热和脂肪代谢中的作用。 神经支配总之，这些研究将为PGC 1a通路的调节提供更广泛的见解， 潜在地为代谢疾病和其它病症的治疗干预提供“可操作的”靶标。