Molecular regulation of the brown-like ("beige") cells of white fat depots

描述（由申请人提供）：本提案描述了一个五年计划，用于培训Jun Wu，以实现她成为代谢研究独立研究者的目标。培训和职业发展计划包括具有潜在临床应用的引人注目的研究项目，实验室技术培训以及教学科学和职业发展研讨会和课程。申请人在分子和细胞生物学、小鼠遗传学和生理学方面有十多年的工作经验。她以前的重要发现已发表在许多高影响力的期刊上，并在她的同行随后的作品中被引用了2,000多次。布鲁斯Spiegelman博士，在糖尿病和肥胖领域的公认的领导者将指导申请人的科学和职业发展。Spiegelman博士培养了许多博士后研究员，他们现在在学术机构担任教职。此外，一个由分子代谢领域，特别是脂肪细胞生物学领域的知名专家组成的咨询委员会将为申请人提供科学和职业建议。该项目的总体目标是研究新分离的米色脂肪细胞的分子表型，并描绘负责米色细胞承诺和调节的机制。肥胖与糖尿病、高血压、血脂异常和心血管疾病有关。肥胖本质上是一种能量平衡紊乱，摄入超过支出。在哺乳动物中有两种主要的脂肪组织，白色和棕色。白色脂肪组织（WAT）以甘油三酯的形式储存多余的能量，而棕色脂肪组织（BAT）可以通过自适应产热安全地燃烧化学能来对抗肥胖。除了“经典的”棕色脂肪库，UCP 1阳性，棕色脂肪样细胞也被观察到散布在WAT中，以响应冷暴露。表达Myf 5的胚胎祖细胞产生BAT的经典仓库，但WAT中的棕色样细胞（所谓的米色细胞）并不来自该细胞谱系。米色细胞的细胞和发育起源目前仍不清楚。最近的研究表明，成年人有功能性的“BAT”，这就提出了关于这些代谢活性细胞的分子身份的问题。该提案试图阐明关于控制米色细胞发育和活化的基本机制，这将使人类“棕色”脂肪的分子定义成为可能，并可能为预防和治疗肥胖症及相关疾病提出新的方法。我们从腹股沟WAT、肩胛间BAT和附睾WAT库克隆衍生出多个永生化前脂肪细胞系。初步分析表明，腹股沟线的一个子集有一个基因表达模式类似的棕色线和可比的Ucp 1表达响应cAMP刺激。这些数据有力地支持了我们的假设，即在腹股沟贮库中有一个独特的祖细胞库，产生了米色细胞。目的#1是比较米色脂肪细胞的生理功能，特别是适应性产热潜力，真正的棕色脂肪细胞。将在培养的米色细胞中检测线粒体含量和呼吸速率，棕色细胞作为对照。将在免疫缺陷小鼠中由米色细胞衍生的移植脂肪垫中测定产热基因表达。米色脂肪细胞的作用 在提高能量消耗和防止小鼠肥胖方面的作用将被测试。目的#2是确定米色脂肪被提交和调节的分子机制。将通过计算生物学方法鉴定富含“米色脂肪基因特征”的米色细胞列表，并在多个体外和体内系统中进行验证。作为米色细胞调节因子的转录因子将通过三种互补的方法进行鉴定，它们的功能将通过我们的永生化细胞中的逆转录病毒功能获得/丧失进行体外测试，并在转基因小鼠模型中进行体内测试。目的#3是应用从这项研究中获得的见解来设计预防和治疗肥胖的新策略。为了确定人“BAT”的分子鉴定，将在PET阳性人“棕色”脂肪组织中研究“米色脂肪基因标签”和“棕色脂肪基因标签”的表达水平。我们将测试和验证目标2中确定的米色脂肪特异性表面标记，并设计米色细胞分选策略以从啮齿动物和人类脂肪组织中纯化米色细胞群。Spiegelman实验室和哈佛医学院朗伍德研究社区为培训未来的独立调查人员提供了理想的环境。该项目还将汇集世界领先的实验室， 咨询委员会相互补充的专业知识。这些优秀的资源将最大限度地提高申请人成功过渡到独立调查员的潜力。

DESCRIPTION (provided by applicant): This proposal describes a five-year plan for training Jun Wu to achieve her goal to become an independent investigator in metabolic research. The training and career development plan includes a compelling research project with potential clinical applications, training in laboratory techniques and didactic scientific and career development seminars and courses. The applicant has more than a decade of experiences working in molecular and cellular biology, mouse genetics and physiology. Her previous important findings have been published in many high-impact journals, and have been then cited more than 2,000 times in the subsequent works of her peers. Dr. Bruce Spiegelman, a well-recognized leader in the field of diabetes and obesity will mentor the applicant's scientific and career development. Dr. Spiegelman has trained numerous postdoctoral fellows who now have faculty positions in academic institutions. In addition, an advisory committee of highly- regarded experts in the field of molecular metabolism, adipocyte biology in particular, will provide the applicant scientific and career advices. The overall goal of the project is to study molecular phenotype of the newly isolated beige fat cells and delineate mechanisms responsible for beige cell commitment and regulation. Obesity is associated with diabetes, hypertension, dyslipidemia and cardiovascular disease. Obesity is essentially a disorder of energy balance, in which intake exceeds expenditure. There are two major types of adipose tissues in mammals, white and brown. White adipose tissue (WAT) stores excess energy in the form of triglycerides, whereas brown adipose tissue (BAT) can counteract obesity by safely burning off chemical energy through adaptive thermogenesis. In addition to "classical" brown adipose depots, UCP1 positive, brown fat-like cells have also been observed interspersed in WAT in response to cold exposure. Myf5-expressing embryonic progenitors give rise to the classic depots of BAT, but the brown-like cells in WAT (so-called beige cells) do not arise from this cell lineage. The cellular and developmental origin of beige cells remains unknown at present. Recent studies that revealed adult humans have functional "BAT" have raised the question regarding the molecular identity of these metabolically active cells. This proposal attempts to clarify the basic mechanisms regarding the control of beige cell development and activation, which will enable molecular definition of human "brown" fat and may suggest new approaches for the prevention and treatment of obesity and associated medical conditions. We clonally derived multiple lines of immortalized preadipocytes from inguinal WAT, interscapular BAT and epididymal WAT depots. Preliminary analysis shown that a subset of inguinal lines have a gene expression pattern similar to the brown lines and comparable Ucp1 expression in response to cAMP stimulations. These data strongly support our hypothesis that there is a distinct pool of progenitors in the inguinal depot that gives rise to the beige cells. Aim #1 is to compare physiological functions of beige fat cells, adaptive thermogenesis potential in particular, to thos of the bona fide brown fat cells. Mitochondrial content and respiration rate will be tested in cultured beige cells with brown cells as controls. Thermogenic gene expression will be assayed in transplanted fat pads derived by beige cells in immunodeficient mice. A role for beige fat cells in raising energy expenditure and protecting mice against obesity will be tested. Aim #2 is to determine molecular mechanisms by which beige fat is committed and regulated. A list of beige cell enriched "beige fat gene signature" will be identified through computational biology approach and validated in multiple in vitro and in vivo systems. Transcription factors as beige cell regulators will be identified through three complementary approaches and their functions will be tested in vitro via retroviral gain/loss of function in our immortalized cells and in vivo in transgenic mouse models. Aim #3 is to apply insights gained from this study to design novel strategies of prevention and treatment of obesity. To define molecular identify of human "BAT", the expression levels of "beige fat gene signature" and "brown fat gene signature" will be investigated in PET positive human "brown" fat tissues. We will test and validate beige fat specific surface markers identified in aim 2 and design beige cell sorting strategies to purify beige cell populations from rodent and human adipose tissues. The Spiegelman laboratory and Harvard Medical School Longwood research community provide an ideal setting for training future independent investigators. This project will also bring together leading laboratories of the advisory committee that complement each other's expertise. These outstanding resources will maximize the potential for the applicant to successfully transition to an independent investigator.