Mechanistic Connection between Interorganellar Communication and Obesity-associated Diseases

项目摘要 肥胖是2型糖尿病、非酒精性脂肪性肝病和心血管疾病的主要危险因素 疾病。肥胖相关疾病发病的一个中心驱动因素是脂肪储存不足。 脂肪细胞的能力和随后在脂肪外器官中的脂肪沉积。脂滴(LD)是 脂肪细胞中负责储存和动员脂肪的细胞器。目前尚不清楚是否 蛋白质和调节LD结构和功能的途径构成了控制脂肪储存的限制因素 脂肪细胞的能力，因此在决定一个人对肥胖相关的易感性方面起着至关重要的作用 精神错乱。我们观察到，缺乏哺乳动物脂肪细胞特异性蛋白CLSTN3B的小鼠 与体重匹配的野生型小鼠相比，更容易出现高脂饮食引起的代谢紊乱， 而脂肪特异性clstn3b转基因小鼠则表现出相反的表型。初步证据 显示CLSTN3B定位于内质网(ER)/LD接触部位和CLSTN3B的消融 导致磷脂和蛋白质对LDS的包膜造成损害。我们的总体目标是：(I)建立 白色脂肪细胞表达的CLSTN3B对代谢表型的意义；(Ii)揭示 CLSTN3B在ER/LD接触点作用的分子机制。中心假设是CLSTN3B 增强LDS的结构和功能的完整性，提高白色脂肪细胞的脂肪储存能力 有助于在肥胖条件下维持新陈代谢健康；从机制上讲，这是通过 补充LD表面磷脂，促进LD靶向蛋白的结合。我们将对此进行测试 通过追求三个具体目的提出假说：1)表明CLSTN3B在白色脂肪细胞中表达是主要的 对高脂饮食的代谢益处的贡献；2)表明CLSTN3B促进 磷脂在内质网和内质网之间的转移；3)探讨内质网C端管腔段在内质网中的作用 CLSTN3B参与了ER/LD接触的形成。对于第一个目标，我们将构建遗传模型，以 白色脂肪细胞来源的CLSTN3B的特异性评估。对于第二个目标，我们将在体外设计 重组磷脂转移测定及LD表面磷脂的功能意义 密度。对于第三个目标，我们将使用生化方法来确定ER的潜在结合伙伴 CLSTN3B的管腔C-末端片段，然后使用 细胞和动物模型。提出的研究具有创新性，因为它剖析了分子机制。 并从一个新的角度解释了肥胖相关疾病的易感性。这个 拟议的研究具有重要意义，因为它的目标是建立一个涵盖以下内容的综合理解 有机体水平上的细胞器间通讯和代谢生理学。我们的长期目标是利用 CLSTN3B作为一个分子句柄来深入了解ER/LD在特定的 研究脂肪细胞的背景并确定肥胖相关疾病的新治疗靶点。

Project Summary Obesity is a leading risk factor for type 2 diabetes, nonalcoholic fatty liver disease, and cardiovascular diseases. A central driver of pathogenesis in obesity-associated disorders is the insufficient lipid-storing capacity of adipocytes and subsequent lipid deposition in extra-adipose organs. The lipid droplet (LD) is the organelle responsible for lipid storage and mobilization in adipocytes. It remains to be elucidated whether proteins and pathways regulating LD structure and function constitute limiting factors governing the lipid-storing capacity of adipocytes, and thus play an essential role in determining one’s susceptibility to obesity-associated disorders. We observed that mice deficient in CLSTN3B, a mammalian adipocyte-specific protein, are more prone to high-fat diet-induced metabolic disorders compared with body weight-matched wild-type mice, whereas the adipose-specific clstn3b transgenic mice display the opposite phenotype. Preliminary evidence shows that CLSTN3B localizes to endoplasmic reticulum (ER)/LD contact sites and ablation of CLSTN3B results in an impaired coating of LDs by phospholipids and proteins. Our overall objectives are to (i) establish the significance of CLSTN3B expressed in white adipocytes to the metabolic phenotype; (ii) reveal the molecular mechanism of CLSTN3B action at the ER/LD contact sites. The central hypothesis is that CLSTN3B enhances the structural and functional integrity of LDs, improves white adipocyte lipid-storing capacity, and contributes to the maintenance of metabolic health under obese conditions; mechanistically, this is achieved by replenishing LD surface phospholipids and promoting the binding of LD-targeting proteins. We will test this hypothesis by pursuing three specific aims: 1) Show that CLSTN3B expressed in white adipocytes is the main contributor to the metabolic benefits upon high-fat diet feeding; 2) Show that CLSTN3B promotes phospholipids transfer between ER and LD; 3) Probe the role of the C-terminal ER luminal segment of CLSTN3B in the formation of ER/LD contacts. For the first aim, we will construct genetic models allowing specific assessment of white adipocyte-derived CLSTN3B. For the second aim, we will design in vitro reconstituted phospholipid transfer assays and examine the functional significance of LD surface phospholipid density. For the third aim, we will use biochemical approaches to identify potential binding partners of the ER luminal C-terminal fragment of CLSTN3B, followed by assessing the significance of such interactions using cellular and animal models. The proposed research is innovative because it dissects the molecular mechanism of a novel protein and explains susceptibility to obesity-associated disorders from a novel perspective. The proposed research is significant because it aims to establish an integrated understanding encompassing interorganelle communication and metabolic physiology at the organismal level. Our long-term goal is to use CLSTN3B as a molecular handle to derive a thorough understanding of ER/LD interactions in the specific context of adipocytes and identify novel therapeutic targets for obesity-associated diseases.