Adipose Tissue Angiogenesis and Metabolic Disease

本项目的长期目标是了解区分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，我们在三个重要的研究领域取得了成功，如下：a，我们已经开发了一种模型，在小鼠体内研究人类脂肪组织的生理功能。B，我们已经发现了至少4种不同的脂肪细胞亚型，其从人间充质祖细胞分化而来，与特定的脂肪组织功能相关，并且在人脂肪组织库中差异富集，以及c，我们已经发现了LINC00473，一种灵长类特异性长非编码RNA，其在产热脂肪细胞中特异性表达，并且参与脂解和线粒体氧化代谢。LINC00473水平在肥胖和2型糖尿病中降低，与 具有重要的生理作用。我们现在将进一步阐明LINC00473调节这些通路的分子机制及其对系统能量稳态的贡献。目标1.阐明LINC00473与脂滴和线粒体蛋白相互作用的机制，以及这些相互作用对脂肪酸和线粒体氧化代谢的影响。使用亲和分离和质谱，我们已经确定了与LINC00473相互作用的脂滴和线粒体蛋白。现在我们将：1A。定义LINC00473和候选蛋白之间的直接和间接相互作用，使用分子方法，包括ChIRP和APEX2邻近标记。1b.定义LINC00473-蛋白质复合物的组装动力学及其与刺激的脂解和呼吸以及1C的关系。通过LINC00473突变体构建体的表达定义特异性相互作用的作用。目标2.检验LINC00473介导反馈机制以控制脂解的假设。我们将：2A。确定PLIN 1消耗后LINC00473表达的抑制是否是由于基础脂解增强所致。2b.确定PLIN 1耗竭细胞中脂解刺激受损是否是由于LINC00473抑制所致。2c.确定PLIN 1缺失是否通过降低转录、增强降解或两者来抑制LINC00473。目标3：我们将测试LINC00473对产热脂肪组织发育和全身葡萄糖代谢的作用。使用在小鼠中产生人脂肪组织的技术，我们将：评估LINC00473的过表达或敲低对脂肪组织生长、血管化和神经支配的动力学的影响。3b.确定从表达高或低水平LINC00473的脂肪细胞发育的脂肪组织如何响应生理刺激，如寒冷。3c.确定从表达高或低水平LINC00473的脂肪细胞发育的脂肪组织是否差异性地影响全身葡萄糖代谢。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.