Control of uncoupling protein-1 in brown adipose tissue

棕色脂肪组织中解偶联蛋白-1 的控制

• 批准号: RGPIN-2014-04973
• 负责人: Harper, MaryEllen
• 金额: $ 3.57万
• 依托单位: University of Ottawa
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=630628
• 关键词: Control; uncoupling; protein; brown; adipose

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.

棕色脂肪组织（BAT）是一种高度代谢的组织，存在于大多数小型哺乳动物、冬眠哺乳动物、新生儿和成年人中。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（UCP 1）含量高，约占线粒体蛋白的10%。当BAT被激活时，UCP 1作为质子泄漏蛋白;质子动力降低;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加倍。尽管最近发现BAT存在于成年人中令人兴奋，但我们对打开和关闭BAT的机制只有初步的了解。本研究的目的是阐明这些机制，并专注于BAT中这种独特蛋白UCP 1的控制。自1995年以来，我们的NSERC资助的研究一直专注于BAT代谢。总体而言，拟议的研究旨在阐明激活UCP 1蛋白的新生化机制，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，活性氧（ROS）的排放通常是高的，谷胱甘肽的氧化还原比是低的BAT与骨骼肌线粒体。然而，氧化损伤的标志物在BAT中很低。我们的初步研究结果使用质谱和研究在冬眠瘤细胞证明了UCP 1的脱乙酰化激活的作用。Sirtuin-3（SIRT 3）是线粒体基质中的NAD依赖性脱乙酰酶。SIRT 3在富含线粒体的组织中高度表达，包括BAT。在脂肪酸氧化的条件下，即，在冷暴露期间。SIRT 3去乙酰化并激活MnSOD，这是一种在淬灭超氧化物中重要的基质酶;它还增加MnSOD的转录。关于UCP 1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。本研究的目的是：1）表征细胞和小鼠模型中UCP 1和其他BAT蛋白的SIRT 3脱乙酰化; 2）确定SIRT 3介导的UCP 1控制对BAT产热的贡献程度; 3）确定线粒体氧化还原和ROS对SIRT 3依赖和非依赖BAT产热的作用。方法将包括研究BAT线粒体，细胞和蛋白质从各种类型的小鼠。例如，在一个示例中，SIRT 3敲除、SIRT 3过表达和UCP 1敲除，在不同的生理条件下。结果测定包括全身能量学（间接量热法）;蛋白质乙酰化水平（质谱法）;线粒体含量;耗氧量;氧化还原比; ROS排放;和氧化损伤。将探测UCP 1和其他蛋白质的翻译后修饰。体外细胞模型（例如，HIB-1B细胞）将用于研究突变关键SIRT 3残基对代谢参数的影响。据推测，UCP 1是通过脱乙酰化激活的;其他线粒体蛋白质是通过脱乙酰化协同激活的（SDH，ICD ACADL和几种OXPHOS蛋白质），这些过程是通过冷暴露激活的。进一步假设，虽然BAT中的解偶联呼吸期间ROS水平高，但MnSOD和其他过程的SIRT 3活化使氧化损伤保持在低水平，即使谷胱甘肽氧化还原比相对高度氧化。预计拟议的研究将导致对控制BAT产热和哺乳动物体温调节过程的深入了解。拟议的工作还将为学员提供代谢研究方面的尖端培训。