Molecular Genetics of Thermogenesis

描述（由申请人提供）：当能量摄入超过能量消耗时发生的正能量平衡是肥胖症发展的基本生理条件。虽然控制食物摄入的基本机制已经知道很多，但除了身体活动之外，几乎没有人知道生热机制的身份，这些机制可以被激活以燃烧多余的卡路里。我们已经迫使小鼠，其中线粒体解偶联蛋白1（UCP 1）已被消融，以激活替代机制的产热逐渐暴露于寒冷。使用几种UCP 1缺陷的遗传模型，我们发现一种新的基因，Slc 25 a25，在冷应激和饮食诱导的肥胖（DIO）条件下，在骨骼肌和腹股沟脂肪中一直被诱导。由于Slc 25 a25编码假定的ATP-Mg 2 +/Pi转运蛋白，其位于线粒体内膜上，在那里它被认为参与线粒体中ATP水平的调节，因此我们进行了实验以测试Slc 25 a25参与能量稳态的假设。Slc 25 a25整体失活的小鼠对DIO具有抗性，并且在跑步机上测试时具有降低的身体耐力。基于SLC 25 A25具有Ca 2+结合EF-手结构域并且其ATP-Mg 2 +/Pi活性受Ca 2+调节的事实，我们设计了实验来检验SLC 25 A25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，它编码线粒体甘油3-磷酸脱氢酶，并具有许多相同的表型和性质的SLC 25 A25，包括其在线粒体内膜和拥有Ca 2+结合EF-手结构域的位置，将测试其在能量稳态的作用。三个具体的目标将测试的假设，即SLC 25 A25和GDM功能的重要组成部分，在骨骼肌和心脏的能量稳态，通过维持必要的ATP水平，以支持Ca 2+循环通过肌浆网。在第一个目标中，将使用Cre-LoxP系统在心脏和骨骼肌中选择性地失活Slc 25 a25。将确定DIO和身体耐力以及肌肉的纤维类型分析和转录组分析，以确定Slc 25 a25失活对参与底物氧化和能量代谢的基因表达的影响。第二个目的将使用离体分析灌注的心脏和骨骼肌，以确定在改变营养的条件下，失活的Slc 25 a25和Gdm对肌肉的力特性的影响。第三个目标将利用小鼠胚胎成纤维细胞，从小鼠与失活的Slc 25 a25和Gdm，研究突变基因的影响Ca 2+成像，呼吸和ATP的生产细胞和分离的线粒体使用Seahorse XF细胞外通量分析仪。这些研究将确定SCL 25 A25和GDM是否通过维持ATP水平来支持Ca 2+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。 公共卫生相关性：能量代谢的效率被定义为响应细胞需求快速产生能量的能力，对于许多生理过程（例如心脏和骨骼肌的兴奋-收缩）是非常重要的。因此，糖尿病患者肌肉中ATP的低效生产可能导致心血管疾病。通过对突变小鼠的研究，我们发现了一种名为SLC 25 A25的蛋白质，它对实现最大代谢效率非常重要，当失活时会导致极度肌肉疲劳。我们发现肥胖和2型糖尿病患者骨骼肌活检组织中Slc 25 a25基因表达显著降低，这与胰岛素敏感性相关。在这项使用基因工程小鼠的提案中，我们将测试SLC 25 A25通过支持肌肉功能和体温调节的能量需求而发挥作用的假设。

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. PUBLIC HEALTH RELEVANCE: The efficiency of energy metabolism, defined as the ability to rapidly produce energy in response to a cellular requirement, is profoundly important for many physiological processes, such as excitation-contraction of heart and skeletal muscle. Consequently, the inefficient production of ATP in the muscle of diabetic patients can lead to cardiovascular disease. Through our studies of mutant mice we have identified a protein called SLC25A25 that is important for the achieving maximal metabolic efficiency, which when inactivated causes extreme muscle fatigue. We found major reductions of Slc25a25 gene expression in skeletal muscle biopsies from obese and type 2 diabetic humans that correlate with insulin sensitivity. In this proposal using genetically engineered mice we will test the hypothesis that SLC25A25 functions by supporting the energy requirements for muscle function and body temperature regulation.