The role of circulating Slit2 in adipose thermogenesis and diabetes

摘要 全球范围内肥胖症的增加导致医疗保健费用和慢性病患者数量的增加 患有肥胖相关疾病，如2型糖尿病。这项研究的首要目标是 建议是更好地了解棕色脂肪的激活，并开发新的治疗操作靶向 棕色脂肪用于治疗代谢疾病，如2型糖尿病。这项工作将测试的假设， 分泌蛋白Slit 2参与脂肪产热，如果给予该蛋白可以是安全的， 有效治疗糖尿病。我们以前利用无偏定量蛋白质组学来鉴定 参与布朗宁的新型分泌蛋白。使用这种方法，我已经确定了新的机制， 可在脂肪组织中激活产热。来自该方法的候选者之一将Slit 2识别为 产热脂肪细胞分泌的一种因子。Slit 2以前曾被描述为在大脑中的功能， 并且不被认为在血液中循环。此外，我还发现了一个C-末端切割片段， Slit 2，Slit 2-C，没有已知的外周功能，通过未知的细胞表面受体发挥作用。我 初步数据表明，增加肥胖、胰岛素抵抗小鼠的Slit 2-C循环水平， 全身葡萄糖稳态和能量消耗。这些小鼠在体内的耗氧量增加， 棕色脂肪组织以及产热转录程序的激活。至少在某种程度上，狭缝2-C 通过激活经典的PKA通路。在该奖项的指导阶段，目标1将测试 假设Slit 2是通过代谢调节体内脂肪组织产热所必需的 脂肪细胞特异性敲除Slit 2的表征。在指导阶段的目标2中，我将测试 假设向糖尿病啮齿动物施用Slit 2-C重组蛋白将改善饮食诱导 胰岛素抵抗目标3将在独立阶段进行，并将采用多种方法， 确定Slit 2-C的功能性受体和信号通路，进一步具有临床应用的潜力。 翻译.我将结合动物生理学和遗传学，生化蛋白质纯化， 质谱技术来解决提案中的问题。如果成功，我预计 这项提案中的发现有可能为2型糖尿病的新治疗做出贡献。 我目前和长期的职业目标是确定参与脂肪组织的途径 本发明的目的是开发新的蛋白质治疗剂，其调节葡萄糖稳态，并具有 改善糖尿病的可能性。我一直对研究配体-受体相互作用感兴趣， 大分子摄取和肿瘤发展中的细胞内信号传导机制。在这方面的发现 建议是直接建立在我的发现在我的博士后工作，并将完成在 确定奖励期限。因此，研究分泌型Slit 2-C因子作为糖尿病新的蛋白质治疗药物是一个重要的研究方向。 我研究的逻辑延伸在K99的指导阶段之后，我的职业轨迹是成为一名 一家领先的学术研究机构的助理教授。布鲁斯斯皮格曼博士，一个公认的领导者，在 棕色脂肪和糖尿病领域将指导我的科学和职业发展。斯皮格曼医生 成功地培养了许多博士后研究员，现在在学术机构担任教职。的 Spiegelman实验室和哈佛医学院研究社区为培训提供了理想的环境 未来的独立调查员我的职业活动计划包括一个持续的进展评估， 导师，生物化学和蛋白质治疗培训，监督和领导培训，教育 课程作业和求职指导。这些职业活动对于准备建立 我自己的研究实验室在独立阶段，我计划利用我的生化技能， 开发新的蛋白质生物制剂和信号通路，以科学地将我自己与我的导师分开。 我的长期科学目标是建立自己作为一个独立的研究人员，以推动代谢领域 糖尿病向前发展。我的重点将是研究循环激素的生物途径，并进一步 研究肥胖症背景下的产热激活的肾上腺素能受体非依赖性途径， 糖尿病再加上哈佛医学院的优秀资源，这将使我的潜力最大化 成功过渡到独立。NIH独立之路奖对我来说至关重要。 因为它将使我能够获得额外的训练，在小鼠模型的肥胖和蛋白质疗法 以及提高我的写作和沟通技巧所需的成功过渡。

ABSTRACT The increase in obesity worldwide has led to rising health care costs and the number of chronically ill people suffering from obesity-related disorders such as type 2 diabetes. The overarching goal of this research proposal is to better understand brown fat activation and to develop new therapeutic manipulations targeting brown fat to treat metabolic disease such as type 2 diabetes. This work will test the hypothesis that the secreted protein Slit2 is involved in adipose thermogenesis and if administration of the protein could be a safe and effective treatment for diabetes. We have previously utilized unbiased quantitative proteomics to identify novel secreted proteins involved in browning. Using this method, I have identified new mechanisms by which thermogenesis can be activated in adipose tissue. One of the candidates from this approach identified Slit2 as a secreted factor from thermogenic adipocytes. Slit2 had previously been described for its function in brain, and were not believed to be circulating in blood. In addition, I have found a C-terminal cleavage fragment of Slit2, Slit2-C, which has no known peripheral function and acts through an unknown cell-surface receptor. My preliminary data demonstrate that increasing Slit2-C circulating levels in obese, insulin resistant mice improves whole body glucose homeostasis and energy expenditure. These mice show increased oxygen consumption in the brown fat tissue as well as activation of a thermogenic transcriptional program. At least in part, Slit2-C acts through activation of the canonical PKA pathway. In the mentored phase of the award, Aim 1 will test the hypothesis that Slit2 is required for regulating adipose tissue thermogenesis in vivo by performing metabolic characterization of the adipocyte-specific knockout of Slit2. In aim 2 of the mentored phase, I will test the hypothesis that administration of Slit2-C recombinant protein to diabetic rodents will improve diet-induced insulin resistance. Aim 3 will be conducted in the independent phase and will use multiple approaches to determine the functional receptor and signaling pathways for Slit2-C with further potential for clinical translation. I will use a combination of animal physiology and genetics, biochemical protein purification, and mass spectrometry techniques to address the questions in the proposal. If successful, I anticipate that the findings in this proposal has the potential to contribute with new treatments for type-2 diabetes. My current and long-term career objectives are to identify pathways involved in adipose tissue metabolism and to develop new protein therapeutics that regulates glucose homeostasis and has the possibility to improve diabetes. I have a longstanding interest in studying ligand-receptor interactions, macromolecular uptake and mechanisms of intracellular signaling in tumor development. The findings in this proposal are directly building upon my discovery in my postdoctoral work and would be completed in the defined award period. Therefore, study of secreted factor Slit2-C as a new protein therapy for diabetes is a logical extension of my research. My career trajectory after the mentored phase of the K99 is to become an assistant professor at a leading academic research institute. Dr. Bruce Spiegelman, a well-recognized leader in the field of brown fat and diabetes will mentor my scientific and career development. Dr. Spiegelman has successfully trained numerous postdoctoral fellows now holding faculty positions in academic institutions. The Spiegelman laboratory and Harvard Medical School research community provide an ideal setting for training future independent investigators. My plan for career activities includes a continous progress evaluation with my mentor, training in biochemistry and protein therapies, supervision and leadership training, educational coursework, and mentored job search. These career activities will be imperative in the preparation to establish my own research laboratory. In the independent phase, I plan to leverage my biochemical skills and development of new protein biologics and signaling pathways to scientifically separate myself from my mentor. My long-term scientific goal is to establish myself as an independent researcher to drive the field of metabolism and diabetes forward. My focus will be to study biological pathways of circulating hormones and to further study adrenergic receptor-independent pathways of thermogenesis activation in the context of obesity and diabetes. Together with the outstanding resources at Harvard Medical School, this will maximize my potential to successfully transition to independence. The NIH Pathway to Independence Award will be essential for my transition because it will enable me to gain additional training in mouse models of obesity and protein therapies as well as improving my writing and communication skills required for a successful transition.