Control of uncoupling protein-1 in brown adipose tissue

褐色脂肪组织（BAT）是一种在大多数小型哺乳动物中发现的高度代谢组织；冬眠哺乳动物;新生儿和较低水平的成年人。它的生理作用是产热，在环境寒冷中进行体温调节。棕色脂肪细胞富含线粒体，其中解偶联蛋白-1 （uncoupling protein-1, UCP1）含量高，约占线粒体蛋白的10%。当BAT被激活时，UCP1作为质子泄漏蛋白；质子力减小；呼吸急剧增加。事实上，在没有颤抖的情况下，小鼠体内BAT的激活使全身能量消耗增加了一倍。尽管最近发现BAT存在于成年人体内令人兴奋，但我们对BAT开启和关闭的机制只有初步的了解。本研究的目的是阐明这些机制，并重点研究BAT， UCP1中这一独特蛋白的控制。*自1995年以来，我们的NSERC资助的研究主要集中在BAT代谢。总的来说，本研究旨在阐明激活UCP1蛋白的新的生化机制，并探索谷胱甘肽氧化还原状态在BAT产热控制中的作用。我们发表的研究结果表明，BAT与骨骼肌线粒体的活性氧（ROS）排放通常较高，谷胱甘肽氧化还原比较低。然而，BAT的氧化损伤标志物较低。我们使用质谱法和冬眠瘤细胞研究的初步发现证明了UCP1的去乙酰化激活作用。Sirtuin-3 （SIRT3）是线粒体基质中nadd依赖的去乙酰化酶。SIRT3在富含线粒体的组织中高表达，包括BAT。在脂肪酸氧化条件下，即在冷暴露期间，其在其中的表达量很高。SIRT3脱乙酰并激活MnSOD，一种在淬灭超氧化物中起重要作用的基质酶；它也会增加MnSOD的转录。关于UCP1的控制和BAT独特的氧化还原特性的作用仍然存在许多问题。*研究目的：1)在细胞和小鼠模型中表征UCP1和其他BAT蛋白的SIRT3去乙酰化；2)确定sirt3介导的UCP1调控对BAT产热的贡献程度；3)确定线粒体氧化还原和ROS在sirt3依赖性和非依赖性BAT产热中的作用。*方法将包括研究BAT线粒体、细胞和来自不同类型小鼠的蛋白质。例如，在不同生理条件下SIRT3基因敲除、SIRT3过表达基因敲除、UCP1基因敲除。结果测定包括全身能量（间接量热法）；蛋白质乙酰化水平（质谱法）；线粒体的内容;耗氧量;氧化还原率;ROS排放;还有氧化损伤。探讨UCP1等蛋白的翻译后修饰。体外细胞模型（如HIB-1B细胞）将用于研究SIRT3关键残基突变对代谢参数的影响。假设UCP1是通过去乙酰化激活的；其他线粒体蛋白通过去乙酰化（SDH， ICD， ACADL和几种OXPHOS蛋白）协调激活，并且这些过程被冷暴露激活。进一步的假设是，尽管在BAT的解偶联呼吸过程中ROS水平很高，但即使谷胱甘肽氧化还原比相对较高，SIRT3激活MnSOD等过程也会使氧化损伤保持在较低水平。*预计拟议的研究将导致对BAT产热和哺乳动物体温调节控制过程的深入了解。这项工作还将为学员提供代谢研究方面的前沿培训。

Brown adipose tissue (BAT) is a highly metabolic tissue found in most small mammals; hibernating mammals; newborn humans and at lower levels in adult humans. Its physiological role is thermogenesis for the purpose of thermoregulation in environmental cold. Brown adipocytes are rich with mitochondria, and therein, uncoupling protein-1 (UCP1) is high comprising ~10% of mitochondrial protein. When BAT is activated, UCP1 acts as a proton leak protein; protonmotive force decreases; and respiration increases dramatically. Indeed, the activation of BAT in mice doubles whole body energy expenditure in the absence of shivering. Despite the excitement surrounding the recent discovery that BAT is present in adult humans, we only have a rudimentary understanding of the mechanisms that turn BAT on and off. The purpose of this research is to elucidate these mechanisms, and to focus on the control of this unique protein in BAT, UCP1. * Our NSERC funded research since 1995 has focused on BAT metabolism. Overall, the proposed research aims to elucidate novel biochemical mechanisms that activate UCP1 protein and to explore the role of glutathione redox status in the control of BAT thermogenesis. Our published findings show that reactive oxygen species (ROS) emission is normally high, and the glutathione redox ratio is low in BAT vs. skeletal muscle mitochondria. Markers of oxidative damage however are low in BAT. Our preliminary findings using mass spectrometry and studies in hibernoma cells demonstrate a role for deactylation activation of UCP1. Sirtuin-3 (SIRT3) is an NAD-dependent deacetylase in the mitochondrial matrix. SIRT3 is highly expressed in tissues enriched with mitochondria, including BAT. Its expression therein is high under conditions of fatty acid oxidation, i.e., during cold exposure. SIRT3 deacetylates and activates MnSOD, a matrix enzyme important in quenching superoxide; it also increases MnSOD transcription. Many questions remain regarding the control of UCP1 and the role of unique redox characteristics of BAT.* The research aims are 1) Characterize SIRT3 deacetylation of UCP1 and other BAT proteins in cellular and mouse models; 2) Determine the degree to which SIRT3-mediated control of UCP1 contributes to BAT thermogenesis; and 3) Determine the roles of mitochondrial redox and ROS on SIRT3-dependent and -independent BAT thermogenesis. * Approaches will include studies of BAT mitochondria, cells and proteins from various types of mice. E.g., SIRT3 knockouts, SIRT3 overexpressors and UCP1 knockouts, under different physiological conditions. Outcome determinations include whole body energetics (indirect calorimetry); levels of protein acetylation (mass spectrometry); mitochondrial content; oxygen consumption; redox ratios; ROS emission; and oxidative damage. Post-translational modification of UCP1 and other proteins will be probed. In vitro cellular models (e.g., HIB-1B cells) will be used to study the effects of mutating key SIRT3 residues on metabolic parameters. It is hypothesized that UCP1 is activated by deacetylation; that other mitochondrial proteins are coordinately activated by deacetylation (SDH, ICD ACADL and several OXPHOS proteins) and that these processes are activated by cold exposure. It is further hypothesized while ROS levels are high during uncoupled respiration in BAT, SIRT3 activation of MnSOD and other processes keep oxidative damage low, even as the glutathione redox ratio is relatively highly oxidized. * It is anticipated that the proposed research will lead to an advanced understanding of the processes controlling BAT thermogenesis and mammalian thermoregulation. The proposed work will also provide a cutting-edge training in metabolic research to trainees.