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

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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.