Stable isotope-based fate mapping to quantify adipogenesis in obesity

描述（由申请人提供）：最近来自人类研究的证据表明，脂肪细胞在整个成人生活中都有更新。虽然人们一直认为成年脂肪细胞是由干细胞群产生的，但最近在小鼠身上进行的谱系定位实验提供了强有力的证据，证明脂肪细胞前体存在于脂肪组织中。关于脂肪细胞转换在正常体内平衡和肥胖中的作用的许多问题仍未完全阐明，这在很大程度上是因为还没有明确的方法来定量体内脂肪生成。这个项目的中心假设是热量过剩导致依赖前体的白色脂肪周转的动态变化。解构脂肪形成在肥胖和糖尿病中的作用不仅会对这些疾病过程产生重要的机制见解，而且可能为直接针对脂肪形成的药物治疗提供理论依据。为了解决中心假设，将使用稳定同位素标记和两个基于质谱的平台来定量体内脂肪形成的新方法。稀有的稳定同位素在质量上不同于更常见的同位素形式的元素，但它们没有放射性，因此是完全安全的和生物惰性的。初步数据表明，稳定的同位素富集胸腺嘧啶是检测DNA合成和细胞分裂的有效示踪剂。利用脉冲追踪策略和质谱，包括多同位素质谱（MIMS），一种令人兴奋的新型显微镜方法，可以以亚细胞分辨率检测组织内稳定同位素掺入的区域，在生物相关干预后，可以在体内量化脂肪形成。目的1：验证高脂肪喂养会导致白色脂肪生成动态增加的假设。成年小鼠将被随机分配到正常饮食或高脂肪饮食，并使用两种基于稳定同位素的方法来确定肥胖中发生的脂肪扩张是否与新脂肪细胞的生成增加有关。目的2：验证长时间高脂肪喂养与肥胖相关的胰岛素抵抗导致皮下脂肪生成相对不足而有利于内脏脂肪生成增加的假设。研究人员将比较肥胖小鼠和瘦小鼠在高脂肪饮食下的脂肪形成率，目的是回答肥胖是否会导致脂肪形成中的储存特异性缺陷，这可能会导致共存的胰岛素抵抗。目的3：在体内验证ppar - γ激活会刺激白色脂肪生成的假设。稳定同位素方法将用于定量肥胖和瘦小鼠在噻唑烷二酮刺激ppar - γ后的脂肪生成。

DESCRIPTION (provided by applicant): Recent evidence from human studies suggests that adipocytes turnover throughout adult life. While it has long been thought that adult adipocytes are generated from a stem cell population, recent lineage mapping experiments in mice provide strong evidence for an adipocyte precursor residing within adipose tissue. Many questions regarding the role of adipocyte turnover during normal homeostasis and in obesity remain incompletely elucidated, in large part because there have not been definitive methods to quantitate adipogenesis in vivo. The central hypothesis of this project is that caloric excess results in dynamic changes in precursor-dependent white adipose turnover. Deconstructing the role of adipogenesis in obesity and diabetes will not only yield important mechanistic insight into these disease processes, but may provide the rationale to directly target adipogenesis with pharmacotherapies. To address the central hypothesis, novel approaches to quantitate adipogenesis in vivo using stable isotope labeling and two mass spectrometry based platforms will be utilized. Rare stable isotopes differ in mass from more common isotopic forms of elements, but they are not radioactive, and thus are entirely safe and biologically inert. Preliminary data suggests that stable isotope-enriched thymidine is an effective tracer to detect DNA synthesis and cellular division. Using pulse-chase strategies and mass spectrometry, including multi-isotope mass spectrometry (MIMS) an exciting new microscopy methodology that can detect areas of stable isotope incorporation within tissues with sub-cellular resolution, adipogenesis can be quantified after biologically relevant interventions, in vivo. Aim 1: To test the hypothesis that high fat feeding will result in a dynamic increase in white adipose adipogenesis. Adult mice will be randomized to a normal diet or an adipogenic (high-fat) diet, and two stable isotope based approaches will be used to determine whether adipose expansion as occurs in obesity is associated with increased generation of new adipocytes. Aim 2: To test the hypothesis that prolonged high fat feeding with resultant obesity-related insulin resistance results in a relative deficiency in subcutaneous adipogenesis in favor of increased visceral adipogenesis. The rates of adipogenesis will be compared between obese and lean mice in response to a high fat diet, with the aim to answer the question of whether obesity results in depot specific defects in adipogenesis, which may contribute to coexistent insulin resistance. Aim 3: To test the hypothesis that PPAR-gamma activation will stimulate white adipose adipogenesis, in vivo. Stable isotope methods will be used to quantitate adipogenesis after pharmacologic stimulation of PPAR-gamma with a thiazolidinedione in obese and lean mice.