Molecular Mechanisms of Mitochondrial Uncoupling and Thermogenesis

项目摘要 通过底物氧化，线粒体在线粒体膜内产生高电势 (IMM)。这种势能的能量通常由线粒体三磷酸腺苷合成酶转化为三磷酸腺苷，但 由于跨IMM的氢泄漏的存在，馏分以热的形式散失。线粒体H的泄漏是 由IMM的特殊蛋白介导，如解偶联蛋白1(UCP1)，它具有重要的 生理功能。它控制着身体的新陈代谢效率，有助于支持核心身体 温度，并减少线粒体产生的活性氧物种，以保护免受氧化 损坏。线粒体H的泄漏被认为在肥胖的保护机制中很重要， 糖尿病和与年龄相关的退行性疾病，以及与 线粒体氧化应激，如缺血再灌流。尽管它的生理和病理生理学 重要的是，线粒体H的泄漏仍然知之甚少，主要是由于缺乏直接的方法 来研究它。我们最近开发了一种方法，消除了这一技术障碍，并首次允许 膜片钳直接记录整个IMM的H泄漏电流。这种方法帮助我们解决了长期- 关于依赖于UCP1的生热H泄漏穿过IMM的机制的悬而未决的问题 棕色脂肪。在这一应用中，我们建议使用膜片钳技术来进一步表征 线粒体H泄漏存在于多种组织中，这些组织在产热和能量代谢中发挥重要作用。 这项建议的具体目的是：1)确定导致线粒体H泄漏的蛋白质(S)。 非脂肪组织；2)表征脂肪酸激活的线粒体H泄漏的机制 腺嘌呤核苷酸转运体(ANT)；3)表征了线粒体H漏的机制 原生团DNP和FCCP。这些具体目标的实现将有助于我们阐明 调节新陈代谢效率和产热的机制，这些知识将有助于 开发治疗干预措施以控制肥胖、糖尿病和与年龄相关的退行性疾病。

Project Summary Through substrate oxidation, mitochondria generate a high potential across the inner mitochondrial membrane (IMM). The energy of this potential is usually converted into ATP by the mitochondrial ATP synthase, but a fraction is dissipated as heat due to the presence of a H+ leak across the IMM. This mitochondrial H+ leak is mediated by specialized proteins of the IMM, such as uncoupling protein 1 (UCP1), and it has important physiological functions. It controls the metabolic efficiency of the body, helps to support the core body temperature, and reduces mitochondrial production of reactive oxygen species to protect against oxidative damage. The mitochondrial H+ leak is considered to be important in protective mechanisms against obesity, diabetes, and age-related degenerative disorders as well as against pathological conditions involving mitochondrial oxidative stress such as ischemia-reperfusion. Despite its physiological and pathophysiological significance, the mitochondrial H+ leak remains poorly understood, primarily due to the lack of direct methods to study it. We recently developed a method that removes this technical barrier and for the first time allows direct patch-clamp recording of H+ leak currents from the whole IMM. This method helped us resolve long- standing questions about the mechanism of the UCP1-dependent thermogenic H+ leak across the IMM of brown fat. In this application, we propose to use the patch-clamp technique to further characterize the mitochondrial H+ leak in several tissues that play important roles in thermogenesis and energy metabolism. The specific aims of this proposal are to: 1) identify the protein(s) responsible for the mitochondrial H+ leak in non-adipose tissues; 2) characterize the mechanism of the fatty acid-activated mitochondrial H+ leak via the adenine nucleotide translocator (ANT); 3) characterize the mechanism of the mitochondrial H+ leak induced by protonophores DNP and FCCP. Accomplishment of these specific aims will help us elucidate the principal mechanism that regulates metabolic efficiency and thermogenesis, and such knowledge will aid the development of therapeutic interventions to control obesity, diabetes, and age-related degenerative disorders.