Molecular Genetics of Thermogenesis

生热作用的分子遗传学

基本信息

批准号：
8067076
负责人：
Randall Lee Mynatt
金额：
$ 39万
依托单位：
LSU PENNINGTON BIOMEDICAL RESEARCH CTR
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-01 至 2014-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8067076
关键词：
Adipose tissue Adult Alleles Animals Binding Binding Proteins Biopsy Body Temperature Brown Fat Burn injury Calories Cardiovascular Diseases Cell Culture System Cell Culture Techniques Cells Characteristics Cytoplasm Development Diet Down-Regulation EF Hand Motifs EF-Hand Domain Ear Eating Embryo Energy Intake Energy Metabolism Energy Metabolism Pathway Fatty acid glycerol esters Fiber Fibroblasts Gene Expression Gene Expression Profile Generations Genes Genetic Models Genetically Engineered Mouse Genus Hippocampus Glycerol Glycerol-3-Phosphate Dehydrogenase Grant Heart Heating Homeostasis Human Image In Vitro Indirect Calorimetry Inner mitochondrial membrane Lead Location Maintenance Measures Mediating Mesenchymal Stem Cells Metabolic Metabolism Mitochondria Molecular Genetics Mus Muscle Muscle Cells Muscle Contraction Muscle Fatigue Muscle function Mutant Strains Mice Mutation Myocardial Myocardium Obesity Oxidoreductase Pathway interactions Phenotype Physical Endurance Physical activity Physiologic Thermoregulation Physiological Physiological Processes Physiology Play Predisposition Production Property Proteins Regulation Resistance Respiration Role SV40 T Antigens Sarcoplasmic Reticulum Skeletal Muscle Stress Structure System Testing Thermogenesis Tissues Up-Regulation Work base design diabetic diabetic patient energy balance extracellular in vivo inorganic phosphate insulin sensitivity mitochondrial uncoupling protein mutant novel null mutation nutrition oxidation protein expression public health relevance research study response subcutaneous

项目摘要

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.

描述（由申请人提供）：当能量摄入超过能量消耗时出现的正能量平衡是发展性肥胖的重要生理条件。虽然人们对控制食物摄入的基本机制了解很多，但除了体力活动之外，对生热机制的身份几乎一无所知，这些生热机制可以被激活以燃烧掉多余的卡路里。我们强迫线粒体解偶联蛋白 1 (UCP1) 被消除的小鼠通过逐渐暴露在寒冷环境中来激活生热的替代机制。使用 UCP1 缺陷的几种遗传模型，我们发现在冷应激和饮食诱导肥胖 (DIO) 的条件下，骨骼肌和腹股沟脂肪中持续诱导出一种新基因 Slc25a25。由于 Slc25a25 编码假定的 ATP-Mg2+/Pi 转运蛋白，该转运蛋白位于线粒体内膜上，被认为参与线粒体中 ATP 水平的调节，因此我们进行了实验来检验 Slc25a25 参与的假设能量稳态。 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下降。基于SLC25A25具有Ca2+结合EF-hand结构域并且其ATP-Mg2+/Pi活性受Ca2+调节的事实，我们设计了实验来检验SLC25A25在调节肌肉兴奋的最佳能量水平中发挥关键作用的假设- 收缩。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中和拥有 Ca2+ 结合 EF-hand 结构域。测试其在能量稳态中的作用。三个具体目标将检验以下假设：SLC25A25 和 GDM 通过维持支持 Ca2+ 穿过肌浆网循环所需的 ATP 水平，作为骨骼肌和心脏能量稳态的重要组成部分发挥作用。第一个目标是使用 Cre-LoxP 系统选择性地灭活心脏和骨骼肌中的 Slc25a25。将测定 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 Slc25a25 失活对参与底物氧化和能量代谢的基因表达的影响。第二个目标是对灌注的心脏和骨骼肌进行离体分析，以确定失活的 Slc25a25 和 Gdm 在营养改变的条件下对肌肉力特性的影响。第三个目标是利用由 Slc25a25 和 Gdm 失活的小鼠制备的小鼠胚胎成纤维细胞，使用 Seahorse XF 细胞外通量分析仪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。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。公共健康相关性：能量代谢的效率，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）非常重要。因此，糖尿病患者肌肉中 ATP 生成效率低下会导致心血管疾病。通过对突变小鼠的研究，我们发现了一种名为 SLC25A25 的蛋白质，它对于实现最大代谢效率非常重要，当它失活时会导致极度肌肉疲劳。我们发现肥胖和 2 型糖尿病患者的骨骼肌活检中 Slc25a25 基因表达显着降低，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 SLC25A25 通过支持肌肉功能和体温调节的能量需求来发挥作用的假设。