Functional characterization of brown adipose tissue in phylogenetically ancient eutherian mammals, Afrotheria

本研究旨在进一步探讨棕色脂肪组织(BAT)及其产热蛋白解偶联蛋白-1(UCP1)在真热哺乳动物进化中最基本的类群--非颤抖产热中的作用及其可塑性。Myurus、Elephantulus edwardii、AmblySomus hottenottus Longicep、Echinoptelairi)。我们提出的项目的中心目标是揭示非泡沫生热棕色脂肪是否已经传递了目前已知的所有关键特征，这些特征是推断经典的非颤抖生热所必需的。我们的方法是从自由放养动物在不同季节的体内代谢反应(麻木行为，非颤抖产热能力)扩展到产热的分子机制。通过体外和体内的线粒体质子泄漏动力学来获取产热蛋白的先进技术，将研究分离的蝙蝠线粒体中的分子参数和生物能量学。对于后者，我们将克隆并稳定地将其导入HEK293(人胚胎肾)细胞。在相同的遗传、细胞和线粒体背景下测量蛋白质功能将有助于确定UCP1活性和调控在非生物中的差异以及与现代真生物的比较，以及识别保守的结构-功能关系。

This study aims to further investigate the role of brown adipose tissue (BAT), the function of its thermogenic protein uncoupling protein-1 (UCP1) and the plasticity of adaptive non-shivering thermogenesis in the evolutionary most basal group of the eutherian mammals, the Afrotheria (Elephantulus. myurus, Elephantulus edwardii, Amblysomus hottentottus longiceps, Echinops telfairi). The central goal of our proposed project is to reveal whether or not Afrotherian thermogenic brown adipose already conveys all currently known key features necessary to infer classical non-shivering thermogenesis. Our approach is scaling from in vivo metabolic responses (torpor behaviour, capacity for nonshivering heat production) in free ranging animals, during different seasons, to the molecular mechanism of heat production. Molecular parameters and bioenergetics in isolated BAT mitochondria of E. edwardii and E. telfairi will be investigated with respect to sophisticated technologies accessing thermogenic proteins by mitochondrial proton leak kinetics in vitro and in vivo. For the latter, UCP1 cDNAs from E. myurus, E. edwardii, A. h. longiceps, and E. telfairi will be cloned and stably transfected into in HEK293 (human embryonic kidney) cells. Measuring protein function in front of an identical genetical, cellular and mitochondrial background will allow the determination of differences in UCP1 activity and regulation within Afrotherians and in comparison to modern eutherians, as well as the identification of conserved structure-function relationships.