Isolating and Characterizing the Fat-Regulating Hormone Leptin in the Fence Lizard

栅栏蜥蜴中脂肪调节激素瘦素的分离和表征

• 批准号: 0099303
• 负责人: Peter Niewiarowski
• 金额: $ 8万
• 依托单位: University of Akron
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0099303&HistoricalAwards=false
• 关键词: Isolating; Characterizing; Fat; Regulating; Hormone

Fat stores are an important source of energy for reproduction, maintenance, and growth in vertebrates. Recently, a protein hormone (leptin) that is central to regulation of lipid stores was discovered in mammals. Leptin also affects metabolic rate, age-at-maturity, and appetite (perhaps through regulation of lipid stores). Although leptin's biology has been vigorously studied in mammalian systems (3000 publications since 1994), its study in ectotherms is limited to a handful of papers. This research team initiated studies of leptin function in Sceloporus undulatus, the eastern fence lizard, and have demonstrated that injection of murine leptin in fence lizards induces the same short-term effects seen when leptin is injected into mammals. If long-term effects of leptin injection in fence lizards are consistent with mammalian studies, then leptin could be used to manipulate age-at-maturity, yielding a powerful new approach to test the fitness consequences of variation in this central life history trait. To reach that goal, leptin will be isolated and characterized from fence lizards. Initial results indicate that fence lizards express a protein of appropriate size that is recognized by an antibody against murine leptin. Taking advantage of this assay, the investigators will use three approaches to cloning and characterizing leptin from fence lizards. First, a cDNA expression library will be prepared from S. undulatus. This library will be screened with the mouse leptin antibody, to identify putative lizard leptin clones. Identity of clones will be verified via DNA sequencing and BLAST comparisons with GenBank sequences. Second, two-dimensional immunoblots of brain tissue will be screened with the mouse leptin antibody. Putative leptin spots will be partially sequenced by Edman degradation, and this primary sequence information will be used to make degenerate primers to use in amplifying lizard leptin by PCR. Lastly, the mouse leptin antibody will be immobilized to a chromatography matrix, and reacted with lizard brain tissue. Captured antigens will then be eluted and characterized by partial Edman degradation. These approaches will run concurrently, thus maximizing the chance of success. Although this research is focused on characterizing leptin in lizards in order to study life history variation, the isolation and characterization of leptin from fence lizards will significantly improve understanding of structure/function relationships in homologous leptins. The success of this project will yield the only leptin sequence from an ectotherm. By comparative sequence analysis with mammalian leptins, regions of functional importance will likely be revealed, which may be useful information for researchers seeking to use leptin as a natural chemical to ameliorate human obesity.

脂肪存储是脊椎动物繁殖，维持和生长的重要能源。 最近，在哺乳动物中发现了对脂质储存调节的核心激素（瘦素）。瘦素还会影响代谢率，成熟年龄和食欲（也许是通过脂质储存的调节）。 尽管瘦素的生物学在哺乳动物的系统中进行了大力研究（自1994年以来有3000个出版物），但其在热疗法中的研究仅限于少数论文。 该研究小组对东部栅栏蜥蜴sceloporus undulatus进行了瘦素功能的研究，并证明在栅栏蜥蜴中注射鼠瘦素会引起瘦素注射到哺乳动物中时看到的短期作用。 如果在栅栏蜥蜴中注射瘦素的长期作用与哺乳动物的研究一致，则可以使用瘦素来操纵成熟年龄，从而产生一种强大的新方法来测试这种中央生活历史特征中变异的适应性后果。为了实现这一目标，将瘦素与栅栏蜥蜴隔离。最初的结果表明，围栏蜥蜴表达​​了一种适当大小的蛋白质，该蛋白质由抗鼠瘦素抗体识别。 利用该测定法，研究人员将使用三种方法来克隆和表征栅栏蜥蜴的瘦素。 首先，将从S. undulatus制备cDNA表达式文库。 该库将使用小鼠瘦素抗体进行筛选，以识别推定的蜥蜴瘦素克隆。 克隆的身份将通过DNA测序和与GenBank序列进行爆炸的比较来验证。 其次，将用小鼠瘦素抗体筛选脑组织的二维免疫印迹。 推定的瘦素斑点将通过Edman降解部分测序，并且该主要序列信息将用于使PCR放大蜥蜴瘦素的变性引物。 最后，小鼠瘦素抗体将固定在色谱基质中，并与蜥蜴脑组织反应。 然后，捕获的抗原将洗脱并以部分Edman降解为特征。 这些方法将同时运行，从而最大程度地发挥成功的机会。 尽管这项研究的重点是表征蜥蜴中的瘦素以研究生活史的变化，但瘦素与栅栏蜥蜴的隔离和表征将显着提高对同源瘦素的结构/功能关系的理解。该项目的成功将产生唯一的瘦素序列。通过对哺乳动物瘦素的比较序列分析，可能会发现功能重要性的区域，这对于寻求使用瘦素作为天然化学物质来改善人类肥胖症的研究人员可能是有用的信息。