Control of uncoupling protein-1 in brown adipose tissue

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、新生儿和成年人类中，含量较低。它的生理作用是生热，以调节环境寒冷中的体温。棕色脂肪细胞富含线粒体，其中解偶联蛋白-1(UCP1)含量较高，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的激活会使全身的能量消耗增加一倍。尽管最近发现蝙蝠存在于成年人身上，人们对此感到兴奋，但我们对蝙蝠开启和关闭的机制只有一个初步的了解。本研究的目的是阐明这些机制，并重点控制这种独特的蛋白在蝙蝠，UCP1。 我们的NSERC资助的研究自1995年以来一直专注于蝙蝠的新陈代谢。总之，这项研究旨在阐明激活UCP1蛋白的新的生化机制，并探索谷胱甘肽氧化还原状态在控制蝙蝠产热中的作用。我们发表的研究结果表明，与骨骼肌相比，蝙蝠线粒体中的活性氧(ROS)释放通常很高，谷胱甘肽氧化还原比率很低。然而，蝙蝠体内氧化损伤的标记物较低。我们使用质谱学的初步发现和对冬眠瘤细胞的研究表明，UCP1的去乙酰化激活具有作用。Sirtuin-3(SIRT3)是线粒体基质中一种依赖NAD的脱乙酰酶。SIRT3在包括蝙蝠在内的富含线粒体的组织中高度表达。在脂肪酸氧化的条件下，即在冷暴露期间，它在其中的表达是高的。SIRT3去乙酰化并激活MnSOD，这是一种在猝灭超氧化物歧化过程中起重要作用的基质酶；它还可以增加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。 本研究的目的是：1)在细胞和小鼠模型中研究UCP1和其他BAT蛋白的SIRT3去乙酰化；2)确定SIRT3对UCP1的调控在多大程度上促进BAT的产热；3)确定线粒体氧化还原和ROS在SIRT3依赖和非依赖的BAT产热中的作用。 方法将包括对不同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基因敲除、SIRT3过表达和UCP1基因敲除。结果决定包括全身能量学(间接量热法)；蛋白质乙酰化水平(质谱学)；线粒体含量；氧气消耗；氧化还原比率；ROS释放；以及氧化损伤。将探讨UCP1和其他蛋白质的翻译后修饰。体外细胞模型(如HIB-1B细胞)将被用来研究突变的关键SIRT3残基对代谢参数的影响。它假设UCP1是通过脱乙酰基激活的；其他线粒体蛋白是通过脱乙酰基激活的(SDH、ICD ACADL和几个OXPHOS蛋白)，这些过程是通过冷暴露激活的。进一步的假设是，当BAT在非耦合呼吸过程中ROS水平较高时，MnSOD和其他过程的SIRT3激活保持低氧化损伤，即使谷胱甘肽氧化还原比率相对较高。 预计拟议的研究将导致对控制蝙蝠产热和哺乳动物体温调节的过程有更深入的了解。拟议的工作还将为受训人员提供新陈代谢研究方面的尖端培训。

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.