The role of circulating Slit2 in adipose thermogenesis and diabetes

摘要 全球范围内肥胖人数的增加导致了医疗费用和慢性病患者数量的上升 患有与肥胖相关的疾病，如2型糖尿病。这项研究的首要目标是 建议更好地了解棕色脂肪的激活，并开发新的靶向治疗操作 棕色脂肪用于治疗代谢性疾病，如2型糖尿病。这项工作将检验这样一个假设，即 分泌蛋白Slit2参与脂肪产热，如果服用该蛋白可能是安全的 糖尿病的有效治疗。我们之前已经利用无偏见的定量蛋白质组学来识别 与褐变有关的新的分泌蛋白。使用这种方法，我已经确定了新的机制，通过它 生热作用可以在脂肪组织中被激活。这种方法的一个候选者将Slit2标识为 产热脂肪细胞的一种分泌因子。Slit2之前已经被描述为它在大脑中的功能， 而且据信不是在血液中循环。此外，我还发现了一个C-末端的裂解片段 Slit2，Slit2-C，它没有已知的外周功能，通过未知的细胞表面受体发挥作用。我的 初步数据显示，提高肥胖、胰岛素抵抗小鼠的SLIT2-C循环水平可以改善 全身葡萄糖动态平衡和能量消耗。这些小鼠在体内表现出更多的氧气消耗 棕色脂肪组织以及一个产热转录程序的激活。至少在一定程度上，Slit2-C起作用 通过激活规范的PKA途径。在奖项的指导阶段，Aim 1将测试 Slit2在体内通过代谢调节脂肪组织产热所需的假说 脂肪细胞特异性Slit2基因敲除的特征。在指导阶段的目标2中，我将测试 糖尿病啮齿动物给予Slit2-C重组蛋白可改善饮食诱导的假说 胰岛素抵抗。目标3将在独立阶段进行，并将使用多种方法 确定Slit2-C的功能受体和信号通路，具有进一步临床应用的潜力 翻译。我将使用动物生理学和遗传学的结合，生化蛋白质纯化，以及 质谱学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型糖尿病的新疗法做出贡献。 我目前和长期的职业目标是确定与脂肪组织有关的途径 新陈代谢，并开发新的蛋白质疗法，调节血糖稳态，并具有 改善糖尿病的可能性。长期以来，我一直对研究配体-受体相互作用感兴趣， 肿瘤发展中的大分子摄取和细胞内信号机制。这篇文章中的发现 提案直接基于我在博士后工作中的发现，并将在 确定的奖励期限。因此，分泌型因子Slit2-C作为治疗糖尿病的一种新的蛋白质疗法的研究具有重要意义。 我研究的合乎逻辑的延伸。在K99的指导阶段结束后，我的职业轨迹是成为一名 在一家领先的学术研究机构担任助理教授。布鲁斯·斯皮格曼博士，一位公认的 棕色脂肪和糖尿病领域将指导我的科学和职业发展。斯皮格尔曼博士已经 成功培养了众多博士后研究员，目前在学术机构担任教职。这个 斯皮格曼实验室和哈佛医学院研究社区为培训提供了理想的环境 未来的独立调查人员。我的职业活动计划包括与我的 导师，生物化学和蛋白质疗法培训，监督和领导力培训，教育 课程作业和有指导的求职。这些职业活动将是建立 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和 开发新的蛋白质生物制剂和信号通路，以科学地将我和我的导师分开。 我的长期科学目标是让自己成为一名推动新陈代谢领域的独立研究人员 糖尿病后遗症。我的重点将是研究荷尔蒙循环的生物学途径，并进一步 在肥胖和肥胖的背景下研究肾上腺素能受体非依赖性产热激活途径 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独立之路奖将是我的 过渡，因为它将使我能够在肥胖和蛋白质疗法的小鼠模型中获得额外的培训 以及提高我的写作和沟通技能，这是成功过渡所必需的。

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.