Studies of The Reciprocal Relationship Between Leptin and Fat

描述（由申请人提供）：瘦素是一种脂肪细胞激素，在调节脂肪组织质量的负反馈回路中作为传入信号发挥作用。作为这个反馈回路的关键元素，瘦素既响应脂肪组织质量的变化，又反过来调节脂肪库的大小。本申请提出了一组实验，试图阐明负责瘦素和脂肪组织质量之间的这种相互关系的分子机制。在第一组实验中，我们建议阐明控制瘦素基因表达的分子机制。瘦素血浆和RNA水平可以在禁食和肥胖状态之间变化数百倍，并且负责其脂肪组织特异性表达的转录机制和这种定量调节尚不清楚。与体外相比，瘦素在体内以显著更高的水平表达，使得有必要使用转基因小鼠研究瘦素基因表达。我们已经使用体内荧光素酶成像系统将组织特异性和定量调节所必需的顺式元件定位在瘦素基因的-22 kB和+18 kB之间。我们建议进一步的实验，以测试一组启动子缺失映射的顺式元件和反式因子调节瘦素表达的前奏，以确定相关的信号转导途径。我们假设瘦素是由脂质传感系统调节的，如果是真的，这些实验可以使我们了解细胞内脂质含量是如何被瘦素基因感知和读出的。在第二组实验中，我们将探索瘦素水平变化控制脂肪组织质量的机制。虽然瘦素缺乏的肥胖小鼠显示脂肪细胞数量大量增加，但调节脂肪细胞产生的因素在很大程度上是未知的。我们最近发现了一种脂肪干细胞，它能够重建脂肪库，并纠正脂肪缺乏性脂肪营养不良小鼠的代谢异常。我们现在建议进一步表征这种细胞类型在体内和体外作为一个前奏，研究瘦素对这种新的细胞类型的生长和发育的影响。我们还建议研究脂肪组织的发展，采用胚胎干细胞互补的方法，我们已经开发。在该方法中，将野生型ES细胞注射到脂肪营养不良动物的胚泡中。在由此产生的嵌合体中，ES细胞是脂肪组织的唯一来源。这项技术将使我们能够滴定ES细胞的数量，以确定脂肪块的发展所需的最小克隆大小，结果将对我们理解脂肪组织前体重建脂肪组织的能力具有重要意义。该方法还提供了一种用于研究特定基因产物的作用的稳健且有效的手段，所述特定基因产物将在调节脂肪组织发育和功能的其他实验中被鉴定。公共卫生相关性：肥胖与II型糖尿病、高血压、高脂血症和肝脂肪变性相关，是一个主要的公共卫生问题（1）。瘦素，脂肪细胞激素，调节脂肪组织质量的反馈回路的一部分，更全面地了解这个系统的元素可能有重要意义的病理生理和治疗肥胖。在本申请中，我们提出解决两个悬而未决的问题，瘦素生理学。是什么控制着瘦与肥胖状态下产生的瘦素的量？瘦素浓度的变化如何反过来调节脂肪组织质量？

DESCRIPTION (provided by applicant): Leptin is an adipocyte hormone that functions as the afferent signal in a negative feedback loop that regulates adipose tissue mass. As a key element of this feedback loop, leptin both responds to changes in adipose tissue mass and in turn modulates the size of the adipose depot. This application proposes a set of experiments that seek to elucidate the molecular mechanisms that are responsible for this reciprocal relationship between leptin and adipose tissue mass. In the first set of experiments, we propose elucidate the molecular mechanisms that control leptin gene expression. Leptin plasma and RNA levels can vary several hundred fold between the fasted and obese states and the transcriptional mechanisms responsible for its adipose tissue specific expression and this quantitative regulation are not known. Leptin is expressed at significantly higher levels in vivo vs. in vitro making it necessary to study leptin gene expression using transgenic mice. We have used an in vivo luciferase imaging system to localize the cis elements necessary for tissue specific and quantitative regulation to between -22 kB and +18 kB of the leptin gene. We propose further experiments to test a set of promoter deletions to map the cis elements and trans factors regulating leptin expression as a prelude to defining the relevant signal transduction pathway. We hypothesize that leptin is regulated by a lipid sensing system and, if true, these experiments could lead us to understand how intracellular lipid content is sensed and read out by the leptin gene, In the second set of experiments, we will explore the mechanism by which changing leptin levels control adipose tissue mass. While leptin deficient obese mice show a massive increase in the number of fat cells, the factors regulating fat cell production are largely unknown. We have recently identified an adipocyte stem cell that is capable of reconstituting a fat depot and correcting the metabolic abnormalities of fat deficient lipodystrophic mice. We now propose to further characterize this cell type in vivo and in vitro as a prelude to studies of the effects of leptin on the growth and development of this novel cell type. We also propose to study adipose tissue development by employing an ES cell complementation method that we have developed. In this method, wild type ES cells are injected into blastocysts of lipodystrophic animals. In the resulting chimaeras, the ES cells are the sole source of adipose tissue. This technique will allow us to titrate the number of ES cells to define the minimal clone size required for development of the adipose mass, results which will have important implications for our understanding of the ability of adipose tissue precursors to reconstitute adipose tissue. This method also provides a robust and efficient means for studying the role of specific gene products that will be identified in the other experiments in regulating adipose tissue development and function. PUBLIC HEALTH RELEVANCE: Obesity is associated with Type II diabetes, hypertension, hyperlipidemia and hepatic steatosis and represents a major public health problem (1). Leptin, an adipocyte hormone, regulates adipose tissue mass as part of a feedback loop and a fuller understanding of the elements of this system could have important implications for the pathophysiology and treatment of obesity. In this application, we propose to address two unanswered questions in leptin physiology. What controls the amount of leptin that is produced in the lean vs. obese state? How do changes in leptin concentration in turn regulate adipose tissue mass?