Adipose Tissue Angiogenesis and Metabolic Disease

该项目的长期目标是了解区别人类产热脂肪细胞代谢和促进代谢健康的机制。在过去的资助期间，我们在以下三个方面取得了成功：a，我们开发了一种在小鼠体内询问人类脂肪组织生理功能的模型。B，我们发现了至少4种不同于人类间充质祖细胞的脂肪细胞亚型，它们与特定的脂肪组织功能相关，并在人类脂肪组织中存在差异；c，我们发现了LINC00473，这是一种灵长类动物特有的非编码RNA，在生热脂肪细胞中特异表达，参与脂解和线粒体氧化代谢。LINC00473水平在肥胖症和2型糖尿病患者中降低，一致 具有重要的生理作用。我们现在将进一步阐明LINC00473调节这些途径的分子机制及其对全身能量动态平衡的贡献。目的1.阐明LINC00473与脂滴和线粒体蛋白相互作用的机制及其对脂肪酸和线粒体氧化代谢的影响。利用亲和分离和质谱仪，我们已经鉴定了与LINC00473相互作用的脂滴和线粒体蛋白。我们现在将：1a。确定LINC00473和候选蛋白质之间的直接和间接相互作用，使用包括Chirp和APEX2邻近标记在内的分子方法。1B.定义LINC00473-蛋白质复合体的组装动力学及其与刺激的脂肪分解和呼吸作用的关系，以及1C。通过表达LINC00473突变结构来确定特定相互作用的作用。目的2.验证LINC00473介导反馈机制控制脂解的假设。我们将：2a。确定PLIN1耗竭时LINC00473表达的抑制是否是由于基础脂解作用增强所致。2B。确定PLIN1耗竭细胞脂解刺激受损是否是由于LINC00473抑制所致。2C。确定PLIN1缺失是否通过转录减少、增强降解或两者兼而有之来抑制LINC00473。目的3.检测LINC00473在生热脂肪组织发育和全身糖代谢中的作用。使用在小鼠身上产生人类脂肪组织的技术，我们将：3A。评估过表达或敲除LINC00473对脂肪组织生长、血管形成和神经支配的影响。3B。确定从表达高或低水平LINC00473的脂肪细胞发育而来的脂肪组织对寒冷等生理刺激的反应。3C。确定由表达高水平或低水平LINC00473的脂肪细胞形成的脂肪组织是否会对全身葡萄糖代谢产生不同的影响。这项工作将揭示定义人类产热脂肪细胞功能的分子机制，以及这些机制如何系统地运作并可能影响代谢性疾病。

The long-term objective of this project is to understand the mechanisms that distinguish human thermogenic adipocyte metabolism and contribute to metabolic health. During the past funding periods, we have been successful in three significant lines of research, as follows: a, We have developed a model to interrogate human adipose tissue physiological function in vivo in mice. b, We have discovered at least 4 distinct adipocyte subtypes differentiating from human mesenchymal progenitors, associated with specific adipose tissue functions and differentially enriched in human adipose tissue depots, and c, We have discovered LINC00473, a primate-specific long non-coding RNA specifically expressed in thermogenic adipocytes and involved in lipolysis and mitochondrial oxidative metabolism. LINC00473 levels are decreased in obesity and Type 2 Diabetes, consistent with an important physiological role. We will now further elucidate molecular mechanisms by which LINC00473 regulates these pathways and its contribution to systemic energy homeostasis. Aim 1. To elucidate the mechanism by which LINC00473 interacts with lipid droplet and mitochondrial proteins and the effect of these interactions on fatty acid and mitochondrial oxidative metabolism. Using affinity isolation and mass spectrometry, we have identified lipid droplet and mitochondrial proteins that interact with LINC00473. We will now: 1A. Define direct and indirect interactions between LINC00473 and candidate proteins, using molecular approaches including ChIRP and APEX2 proximity labeling. 1B. Define the kinetics of assembly of LINC00473-protein complexes and their relationship with stimulated lipolysis and respiration, and 1C. Define the role of specific interactions though expression of LINC00473 mutant constructs. Aim 2. To test the hypothesis that LINC00473 mediates a feedback mechanism to control lipolysis. We shall: 2A. Determine whether inhibition of LINC00473 expression upon PLIN1 depletion is due to enhanced basal lipolysis. 2B. Determine whether impaired stimulation of lipolysis in PLIN1 depleted cells is due LINC00473 supprssion. 2C. Determine whether PLIN1 depletion suppresses LINC00473 through decreased transcription, enhanced degradation or both. Aim 3. We will test the role of LINC00473 on thermogenic adipose tissue development and systemic glucose metabolism. Using techniques to generate human adipose tissue in mice we shall: 3A. Assess the effects of overexpression or knockdown of LINC00473 on dynamics of adipose tissue growth, vascularization, and innervation. 3B. Determine how adipose tissue developed from adipocytes expressing high or low levels of LINC00473 responds to physiological stimuli such as cold. 3C. Determine whether adipose tissue developed from adipocytes expressing high or low levels of LINC00473 differentially affects systemic glucose metabolism. This work will reveal molecular mechanisms that define human thermogenic adipocyte function, and how these mechanisms operate systemically and could impact metabolic disease.