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.

脂肪储存是脊椎动物繁殖、维持和生长的重要能量来源。 最近，在哺乳动物中发现了一种蛋白质激素（瘦素），它是调节脂质储存的核心。瘦素还影响代谢率、成熟年龄和食欲（可能通过调节脂质储存）。 尽管瘦素的生物学在哺乳动物系统中得到了积极的研究（自1994年以来发表了3000篇论文），但其在外温动物中的研究仅限于少数论文。 该研究小组启动了对东部栅栏蜥蜴Sceloporus undulatus中瘦素功能的研究，并证明了在栅栏蜥蜴中注射鼠瘦素会诱导与将瘦素注射到哺乳动物中时相同的短期效应。 如果围栏蜥蜴注射瘦素的长期影响与哺乳动物研究一致，那么瘦素可以用来操纵成熟年龄，产生一个强大的新方法来测试这种中心生活史特征的变化的适应性后果。为了实现这一目标，将从栅栏蜥蜴中分离和表征瘦素。初步结果表明，栅栏蜥蜴表达的蛋白质的适当大小，是由抗体识别鼠瘦素。 利用这种检测方法，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，从S.波状的 将用小鼠瘦素抗体筛选该文库，以鉴定推定的蜥蜴瘦素克隆。 将通过DNA测序和与GenBank序列的BLAST比较验证克隆的同一性。 其次，将使用小鼠瘦素抗体对脑组织进行二维免疫印迹筛查。 通过Edman降解法对推定的瘦素点进行部分测序，并将该一级序列信息用于制备简并引物，以用于通过PCR扩增蜥蜴瘦素。 最后，将小鼠瘦素抗体固定到层析基质上，并与蜥蜴脑组织反应。 然后洗脱捕获的抗原，并通过部分Edman降解进行表征。 这些方法将同时运行，从而最大限度地提高成功的机会。 虽然这项研究的重点是表征瘦素在蜥蜴，以研究生活史的变化，从栅栏蜥蜴的瘦素的分离和表征将显着提高同源瘦素的结构/功能关系的理解。这个项目的成功将产生唯一的瘦素序列从外温动物。通过与哺乳动物leptins的比较序列分析，功能重要性的区域可能会被揭示，这可能是有用的信息，研究人员寻求使用瘦素作为一种天然化学物质，以改善人类肥胖。