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: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=611358
• 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；新生人类和成年人的较低水平。它的生理作用是为了在环境寒冷中进行温度调节目的的生热作用。棕色脂肪细胞富含线粒体，其中，解偶联蛋白-1（UCP1）高，占线粒体蛋白的约10％。激活BAT时，UCP1充当质子泄漏蛋白。质子力降低；呼吸急剧增加。实际上，在没有发抖的情况下，小鼠中蝙蝠的激活使全身能量消耗增加了一倍。尽管最近发现蝙蝠存在于成年人中，但我们对打开和关闭蝙蝠的机制只有基本的理解。这项研究的目的是阐明这些机制，并专注于对BAT中这种独特蛋白质的控制，UCP1。 自1995年以来，我们的NSERC资助研究集中在BAT代谢上。总体而言，拟议的研究旨在阐明激活UCP1蛋白的新型生化机制，并探讨谷胱甘肽氧化还原状态在控制蝙蝠热发生中的作用。我们发表的发现表明，反应性氧（ROS）发射通常很高，而谷胱甘肽氧化还原比在BAT与骨骼肌线粒体中的氧化还原比很低。然而，氧化损伤的标记在BAT中很低。我们使用质谱法的初步发现和在休眠瘤细胞中的研究表明了UCP1的启动性激活的作用。 Sirtuin-3（SIRT3）是线粒体基质中的NAD依赖性脱乙酰基酶。 SIRT3在富含线粒体的组织中高度表达，包括蝙蝠。在脂肪酸氧化条件下，即在冷暴露期间，其表达很高。 SIRT3脱乙酰酸盐并激活MNSOD，这是一种对淬氧化物淬氧化物很重要的基质酶；它还增加了MNSOD转录。关于UCP1的控制以及BAT独特的氧化还原特性的作用仍然存在许多问题。 研究目的是1）表征在细胞和小鼠模型中，UCP1和其他BAT蛋白的SIRT3脱乙酰化； 2）确定SIRT3介导的UCP1的控制有助于蝙蝠热发生的程度； 3）确定线粒体氧化还原和ROS在SIRT3依赖性和非依赖性BAT热发生上的作用。 方法将包括对各种类型小鼠的蝙蝠线粒体，细胞和蛋白质的研究。例如，在不同的生理条件下，SIRT3敲除，SIRT3过表达器和UCP1敲除。结果确定包括全身能量学（间接量热法）；蛋白质乙酰化水平（质谱法）；线粒体含量；消耗氧气；氧化还原比； ROS排放；和氧化损伤。将探测UCP1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SIRT3残基对代谢参数的影响。假设UCP1通过脱乙酰化激活。其他线粒体蛋白是通过脱乙酰基化（SDH，ICD Acadil和几种Oxphos蛋白）协调激活的，并且这些过程被冷暴露激活。它是进一步假设的，而在蝙蝠中未耦合呼吸期间，ROS水平较高，MNSOD的SIRT3激活和其他过程保持低氧化损伤，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热生成和哺乳动物温度调节的过程有深入的了解。拟议的工作还将为学员提供代谢研究的尖端培训。