Molecular Genetics of Thermogenesis

描述（由申请人提供）：当能量摄入超过能量消耗时，能量正平衡是发生肥胖的必要生理条件。虽然人们对控制食物摄入的基本机制了解很多，但除了身体活动之外，人们对产热机制的身份几乎一无所知，这些产热机制可以被激活以燃烧掉多余的卡路里。我们强迫小鼠，其中线粒体解偶联蛋白1 （UCP1）已被切除，通过逐渐暴露在寒冷中来激活另一种产热机制。利用几种UCP1缺失的遗传模型，我们发现在冷应激和饮食性肥胖（DIO）条件下，一个新的基因Slc25a25在骨骼肌和腹股沟脂肪中持续被诱导。由于Slc25a25编码一种假定的ATP- mg2 +/Pi转运蛋白，该转运蛋白位于线粒体内膜上，被认为参与线粒体ATP水平的调节，因此我们进行了实验来验证Slc25a25参与能量稳态的假设。Slc25a25整体失活的小鼠对DIO有抵抗力，并且在跑步机上测试时身体耐力降低。基于SLC25A25具有Ca2+结合EF-hand结构域以及其ATP-Mg2+/Pi活性受Ca2+调节的事实，我们设计了实验来验证SLC25A25在调节肌肉兴奋-收缩的最佳能量水平中起关键作用的假设。此外，第二个基因Gdm编码线粒体甘油3-磷酸脱氢酶，具有许多与SLC25A25相同的表型和特性，包括其位于线粒体内膜和拥有Ca2+结合EF-hand结构域，将被测试其在能量稳态中的作用。三个特定的目标将测试假设SLC25A25和GDM作为骨骼肌和心脏能量稳态的重要组成部分，通过维持ATP水平来支持肌浆网Ca2+循环。在第一个目标中，将使用Cre-LoxP系统选择性地使Slc25a25在心脏和骨骼肌中失活。我们将通过测定DIO和体力耐力，以及肌肉纤维类型分析和转录组分析来确定Slc25a25失活对底物氧化和能量代谢相关基因表达的影响。第二个目标是对灌注的心脏和骨骼肌进行离体分析，以确定在营养改变的情况下，灭活的Slc25a25和Gdm对肌肉力特性的影响。第三个目标将利用小鼠胚胎成纤维细胞，由灭活的Slc25a25和Gdm制备，研究突变基因对细胞和分离线粒体中Ca2+成像、呼吸和ATP产生的影响，使用Seahorse XF细胞外通量分析仪。这些研究将确定SCL25A25和GDM是否通过维持ATP水平来支持Ca2+循环，以及它们的失活是否会导致代谢效率低下，从而影响肌肉的身体耐力和肥胖。

DESCRIPTION (provided by applicant): A positive energy balance that occurs when energy intake exceeds energy expenditure is an essential physiological condition for the development obesity. While much is known of the basic mechanisms controlling food intake, almost nothing is known of the identity of thermogenic mechanisms, apart from physical activity, that could be activated to burn off excess calories. We have forced mice, in which the mitochondrial uncoupling protein1 (UCP1) has been ablated, to activate alternative mechanisms of thermogenesis by gradually exposing them to the cold. Using several genetic models of UCP1 deficiency we have found that a novel gene, Slc25a25, has been consistently induced in skeletal muscle and inguinal fat under conditions of cold stress and diet-induced obesity (DIO). Since Slc25a25 encodes a putative ATP-Mg2+/Pi transporter, which is located on the inner mitochondrial membrane where it is thought to be involved in the regulation of ATP levels in the mitochondria, we have pursued experiments to test the hypothesis that Slc25a25 is involved in energy homeostasis. Mice with a global inactivation of Slc25a25 are resistant to DIO and have reduced physical endurance when tested on a treadmill. Based upon the fact that SLC25A25 has Ca2+-binding EF-hand domains and its ATP-Mg2+/Pi activity is regulated by Ca2+, we have designed experiments to test the hypothesis that SLC25A25 plays a crucial role in regulation of optimal energy levels for muscle excitation- contraction. In addition, a second gene, Gdm, which encodes the mitochondrial glycerol 3-phosphate dehydrogenase and has many of the same phenotypes and properties as SLC25A25, including its location in the inner mitochondrial membrane and possession of Ca2+-binding EF-hand domains, will be tested for its role in energy homeostasis. Three specific aims will test the hypothesis that SLC25A25 and GDM function as important components of energy homeostasis in skeletal muscle and heart by maintaining the level of ATP necessary to support Ca2+ cycling across the sarcoplasmic reticulum. In the first aim Slc25a25 will be inactivated selectively in heart and skeletal muscle using the Cre-LoxP system. DIO and physical endurance will be determined as well as fiber type analysis and transcriptome analysis of muscle to determine the effects of Slc25a25 inactivation on expression of genes involved in substrate oxidation and energy metabolism. The second aim will use ex vivo analysis of the perfused heart and skeletal muscle to determine the effects of inactivated Slc25a25 and Gdm on force characteristics of muscle under conditions of altered nutrition. The third aim will utilize mouse embryonic fibroblasts, prepared from mice with inactivated Slc25a25 and Gdm, to investigate the effects of mutant genes on Ca2+ imaging, respiration and ATP production in cells and isolated mitochondria using the Seahorse XF Extracellular Flux analyzer. These studies will establish whether SCL25A25 and GDM support Ca2+ cycling through maintenance of ATP levels and that their inactivation leads to metabolic inefficiency with effects on both muscle physical endurance and adiposity.