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: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=588130
• 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) 是大多数小型哺乳动物中发现的高度代谢组织；冬眠的哺乳动物；新生儿和成年人中水平较低。其生理作用是生热作用，以在环境寒冷时进行体温调节。棕色脂肪细胞富含线粒体，其中解偶联蛋白-1 (UCP1) 含量很高，约占线粒体蛋白的 10%。 当BAT被激活时，UCP1充当质子泄漏蛋白；质子动势减小；并且呼吸急剧增加。事实上，在没有颤抖的情况下，小鼠 BAT 的激活使全身能量消耗增加了一倍。 尽管最近发现成年人体内存在 BAT 令人兴奋，但我们对开启和关闭 BAT 的机制只有初步的了解。 本研究的目的是阐明这些机制，并重点关注 BAT 中这种独特蛋白 UCP1 的控制。 自 1995 年以来，我们的 NSERC 资助的研究一直集中在 BAT 代谢上。总体而言，本研究旨在阐明激活 UCP1 蛋白的新生化机制，并探索谷胱甘肽氧化还原状态在控制 BAT 生热作用中的作用。我们发表的研究结果表明，与骨骼肌线粒体相比，BAT 中的活性氧 (ROS) 排放通常较高，而谷胱甘肽氧化还原比较低。然而，BAT 中氧化损伤的标志物较低。我们使用质谱法和冬眠瘤细胞研究的初步结果证明了 UCP1 去乙酰化激活的作用。 Sirtuin-3 (SIRT3) 是线粒体基质中的 NAD 依赖性脱乙酰酶。 SIRT3 在富含线粒体的组织（包括 BAT）中高度表达。在脂肪酸氧化条件下，即在冷暴露期间，其表达较高。 SIRT3 去乙酰化并激活 MnSOD，这是一种对于淬灭超氧化物很重要的基质酶；它还会增加 MnSOD 转录。关于 UCP1 的控制和 BAT 独特氧化还原特性的作用仍然存在许多问题。 研究目的是 1) 在细胞和小鼠模型中表征 SIRT3 对 UCP1 和其他 BAT 蛋白的脱乙酰作用； 2) 确定SIRT3介导的UCP1控制对BAT产热作用的程度； 3) 确定线粒体氧化还原和 ROS 对 SIRT3 依赖性和非依赖性 BAT 产热作用的作用。 方法将包括对来自不同类型小鼠的 BAT 线粒体、细胞和蛋白质的研究。 例如，不同生理条件下的 SIRT3 敲除、SIRT3 过表达和 UCP1 敲除。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；活性氧排放；和氧化损伤。 将探讨 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）将用于研究关键 SIRT3 残基突变对代谢参数的影响。假设 UCP1 通过脱乙酰化被激活；其他线粒体蛋白通过脱乙酰化协同激活（SDH、ICD ACADL 和几种 OXPHOS 蛋白），并且这些过程通过冷暴露激活。进一步假设，当 BAT 中的非偶联呼吸过程中 ROS 水平较高时，MnSOD 的 SIRT3 激活和其他过程使氧化损伤保持在较低水平，即使谷胱甘肽氧化还原比相对较高氧化。 预计拟议的研究将有助于加深对控制 BAT 产热和哺乳动物体温调节过程的理解。拟议的工作还将为学员提供代谢研究方面的前沿培训。