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

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（可能是通过调节脂质储存）。尽管瘦素的生物学在哺乳动物系统中得到了大力研究（自1994年以来发表了3000篇论文），但它在变温动物中的研究仅限于少数几篇论文。这个研究小组开始了对东部栅栏蜥蜴（Sceloporus undulatus）瘦素功能的研究，并证明在栅栏蜥蜴中注射小鼠瘦素会产生与在哺乳动物中注射瘦素相同的短期效果。如果瘦素注射对栅栏蜥蜴的长期影响与哺乳动物的研究一致，那么瘦素可以用来控制成熟年龄，从而产生一种强有力的新方法来测试这一核心生活史特征变异的适应性后果。为了达到这一目标，瘦素将从栅栏蜥蜴中分离出来并进行表征。初步结果表明，栅栏蜥蜴表达一种适当大小的蛋白质，这种蛋白质可以被一种抗小鼠瘦素的抗体识别。利用这一分析，研究人员将使用三种方法克隆和表征栅栏蜥蜴的瘦素。首先，从波曲菌中制备cDNA表达文库。该文库将与小鼠瘦素抗体进行筛选，以鉴定假定的蜥蜴瘦素克隆。克隆的身份将通过DNA测序和BLAST与GenBank序列的比较来验证。其次，用小鼠瘦素抗体筛选脑组织的二维免疫印迹。假设的瘦素点将通过Edman降解部分测序，这些初级序列信息将用于制作退化引物，用于用PCR扩增蜥蜴瘦素。最后，将小鼠瘦素抗体固定在色谱基质上，与蜥蜴脑组织反应。然后，捕获的抗原将被洗脱，并以部分埃德曼降解为特征。这些方法将同时运行，从而使成功的机会最大化。虽然本研究的重点是研究蜥蜴瘦素的生活史变化，但从栅栏蜥蜴中分离和鉴定瘦素将大大提高对同源瘦素结构/功能关系的理解。这个项目的成功将产生唯一的瘦素序列来自恒温动物。通过与哺乳动物瘦素的比较序列分析，可能会揭示功能重要的区域，这可能为研究人员寻求将瘦素作为一种天然化学物质来改善人类肥胖提供有用的信息。

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.