Recruitment of skeletal muscle based on non-shivering thermogenesis in health and disease

描述（由申请人提供）：越来越多的证据表明，除了棕色脂肪组织（BAT）外，骨骼肌也是非寒颤产热（NST）的重要部位。虽然一些研究表明SR Ca2+循环可能在肌肉产热中起作用，但分子细节尚不清楚。我们实验室和其他人的研究表明，Sarcolipin （SLN）是SR Ca2+ ATP酶（SERCA）的调节剂，可以在Ca2+存在的情况下与SERCA结合，促进SERCA的解偶联并增加ATP水解。在SLN的存在下，SERCA变得低效；每摩尔ATP运输少于2摩尔Ca2+，从而增加ATP水解和产热。这些研究提示SLN可能在肌肉产热中起作用。为了确定SLN在肌肉中的相关性，我们采用了遗传学方法，最近研究表明，SLN的缺失使小鼠在急性冷暴露期间容易发生低体温，但在SLN -/-背景下重新引入SLN完全恢复了肌肉的产热作用。此外，饲喂高脂肪饮食（HFD）的Sln-/-小鼠体重明显高于WT对照组，而饲喂高脂肪饮食（HFD）的WT小鼠肥胖程度较轻，但Sln显著上调（3-4倍），表明Sln在饮食诱导的生热作用中被招募。这些新发现首次表明，SLN是通过解除泵耦合来增强SERCA依赖性热量产生的缺失环节，并且该机制被用于增加饮食过载期间的能量消耗。然而，关于肌肉NST募集的机制细节，SLN介导的SERCA解偶联，以及SLN如何增加肌肉代谢和能量学，人们知之甚少。在本应用中提出的研究挑战了目前认为BAT单独负责非颤抖产热的想法。它试图确定肌肉是NST的一个重要部位，并且可以在BAT缺失或无功能的动物中取代/替代产热。修订后的提案更加强调理解肌肉NST的机制基础。目前的建议旨在确定导致基于SERCA/SLN的产热激活的亚细胞机制。在Aim 1中，我们将研究寒冷暴露时肌肉NST激活背后的机制，并确定SLN是否可以替代哺乳动物BAT （UCP1）功能的丧失。在Aim 2中，我们将测试SLN的治疗相关性，并确定SLN的过表达是否可以通过增加能量消耗来预防饮食引起的肥胖。这个目的的另一个重要目标是发现SLN如何增加肌肉代谢，如果这涉及Ca2+依赖的信号通路。在Aim 3中，我们将确定SLN/SERCA相互作用的结构特征（结合位点和残基），并确定SLN相互作用如何导致解偶联和增加ATP水解。本文提出的实验将共同确定SLN/SERCA相互作用是基于骨骼肌的NST的机制，以及它们与Tc和全身能量代谢的相关性。最重要的是，它们将提供一个机制基础，表明SLN单独而不是磷蛋白与SERCA结合导致SERCA泵解偶联。我们认为，更好地理解肌肉产热对我们对包括人类在内的哺乳动物的肌肉代谢、能量消耗和明显肥胖的整体理解具有更广泛的意义。确定基于肌肉的NST背后的分子机制对人类至关重要，因为这种策略可以用来增加肌肉的能量消耗，从而为肥胖治疗提供新的靶点。

DESCRIPTION (provided by applicant): Increasing evidence suggests that in addition to brown adipose tissue (BAT), skeletal muscle is an important site for Nonshivering thermogenesis (NST). Although several studies have suggested that SR Ca2+ cycling may play a role in muscle thermogenesis, the molecular details were not known. Studies from our laboratory and others have shown that Sarcolipin (SLN), a regulator of SR Ca2+ ATPase (SERCA), can bind to SERCA in the presence of Ca2+ and promote uncoupling of SERCA and increase ATP hydrolysis. In the presence of SLN, SERCA becomes inefficient; transporting less than 2 mol Ca2+ per mol ATP, thereby increasing ATP hydrolysis and heat production. These studies hinted that SLN could play a role in muscle thermogenesis .To define the relevance of SLN in muscle, we took a genetic approach and recently showed that loss of SLN predisposes mice to develop hypothermia during acute cold exposure but reintroduction of SLN in the Sln-/- background fully restored muscle-based thermogenesis. In addition, when Sln-/- mice when fed on high fat diet (HFD) gained significantly more weight than WT controls, whereas WT mice fed on HFD were less obese but showed significant upregulation of SLN (3-4 fold) suggesting that SLN is recruited in diet induced thermogenesis. These novel findings, for the first time, suggest SLN is the missing link for enhancing SERCA dependent heat generation by uncoupling the pump and this mechanism is recruited to increase energy expenditure during diet overload. However the mechanistic details with regard to recruitment of muscle NST, SLN-mediated uncoupling of SERCA, and how SLN increases muscle metabolism and energetics are poorly understood. The research proposed in this application challenges current thinking that BAT alone is responsible for Nonshivering thermogenesis. It seeks to establish that muscle is an important site of NST and can replace/substitute for thermogenesis in animals where BAT is absent or nonfunctional. The revised proposal places a greater emphasis on understanding the mechanistic basis of muscle NST. The current proposal seeks to identify sub cellular mechanisms that lead to activation of SERCA/SLN based thermogenesis. In Aim 1, we will investigate the mechanism behind activation of muscle-based NST during cold exposure and determine if SLN can replace/substitute for loss of BAT (UCP1) function in mammals. In Aim 2, we will test the therapeutic relevance of SLN and determine if overexpression of SLN can protect against diet- induced obesity by increasing energy expenditure. Another important goal of this aim is to discover how SLN increases muscle metabolism, if this involves Ca2+ dependant signaling pathways. In Aim 3, we will identify the structural features (binding sites and residues) of SLN/SERCA interaction and determine how SLN interaction leads to uncoupling and increased ATP hydrolysis. The experiments proposed here will collectively establish that SLN/SERCA interaction is the mechanism for skeletal muscle based NST and their relevance to Tc and whole body energy metabolism. Most importantly they will provide a mechanistic basis to show that SLN alone but not Phospholamban binding to SERCA causes uncoupling of the SERCA pump. We suggest that a better understanding of muscle thermogenesis has broader implications to our overall understanding of muscle metabolism, energy expenditure and evidently obesity in mammals including humans. Identification of the molecular mechanisms behind muscle based NST is of paramount importance to humans, since this strategy could be exploited to increase energy expenditure in muscle, thereby providing newer targets for obesity treatment.