Stable isotope-based fate mapping to quantify adipogenesis in obesity

描述（由申请人提供）：最近的人体研究证据表明，脂肪细胞在整个成年期的更替。虽然长期以来一直认为成体脂肪细胞是由干细胞群产生的，但最近在小鼠中进行的谱系作图实验为脂肪组织内存在脂肪细胞前体提供了强有力的证据。关于脂肪细胞周转在正常稳态和肥胖中的作用的许多问题仍然没有完全阐明，在很大程度上是因为还没有确定的方法来定量体内脂肪形成。该项目的中心假设是，热量过剩的结果，在动态变化的依赖性白色脂肪的周转。解构脂肪生成在肥胖和糖尿病中的作用不仅会产生对这些疾病过程的重要机制见解，而且可能提供直接靶向药物治疗脂肪生成的理论基础。为了解决中心假设，将使用稳定同位素标记和两种基于质谱的平台来定量体内脂肪生成的新方法。稀有稳定同位素的质量不同于更常见的同位素形式的元素，但它们没有放射性，因此是完全安全和生物惰性的。初步数据表明，稳定的同位素富集的胸苷是一种有效的示踪剂，以检测DNA合成和细胞分裂。使用脉冲追踪策略和质谱法，包括多同位素质谱法（MIMS），这是一种令人兴奋的新显微镜方法，可以用亚细胞分辨率检测组织内稳定同位素掺入的区域，可以在体内生物相关干预后定量脂肪形成。目的1：检验高脂喂养将导致白色脂肪生成动态增加的假设。将成年小鼠随机分配至正常饮食或脂肪形成（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症中发生的脂肪扩张是否与新脂肪细胞生成增加相关。目标二：为了验证这一假设，即长期高脂肪喂养导致肥胖相关的胰岛素抵抗导致皮下脂肪生成相对不足，有利于内脏脂肪生成增加。将比较肥胖和瘦小鼠对高脂饮食的反应的脂肪形成速率，目的是回答肥胖是否导致脂肪形成中的储库特异性缺陷的问题，这可能导致共存的胰岛素抵抗。目的3：检验PPAR-gamma激活将在体内刺激白色脂肪脂肪形成的假设。在肥胖和瘦小鼠中，使用噻唑烷二酮对PPAR-γ进行药理学刺激后，将使用稳定同位素方法定量脂肪生成。

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.