Exploitation of metadherin as a regulator of hepatic energy metabolism

代谢综合征是肥胖、肝脏脂肪变性和胰岛素抵抗的集合，是一种重大的公共健康危机，医疗需求尚未得到满足。由于依从性差，包括改变生活方式在内的非手术干预策略通常无效。因此，我们必须继续确定和发展新的战略和治疗目标。一个基本的起始特征是甘油三酯在肝脏中过度堆积，可通过炎症和纤维化进展为脂肪性肝炎、肝硬变和肝细胞癌。肝脏中的糖皮质激素(GC)信号与进展的所有阶段有关。在寻找解决GC作用的代谢并发症的机制时，我们发现GC受体(GR)结合在一种多功能的蛋白质支架上，与肝脏的脂肪代谢、炎症和癌症有关。我们发现，胃粘附素是GC反式激活所必需的，但不是反式抑制所必需的，而且这是通过调节GR招募来增强元件来实现的。在缺乏胃粘附素的小鼠中，肝脏中的GC作用发生了深刻的变化，GC反应的脂代谢基因发生了重大变化，同时也出现了显著的高血糖反应。在Metherin缺失的小鼠中，我们还观察到H3K27Ac的全基因组变化，H3K27Ac是活性增强子的标志，对急性GC挑战的反应；可能表明GR环路的潜在重新连接。我们建立了糖皮质激素过量的小鼠模型，发现肝脏甘油三酯显著诱导，并伴有脂滴聚集；从而优化了GC诱导的肝骨病模型。我们现在将使用这种模式和更传统的饮食诱导的肥胖，来定义GR作用的控制机制，以及Met附着素的作用。我们将定义Met附着素如何调节GR的功能。我们有证据表明，胃粘附素促进了与修饰GR所需的激酶的相互作用。我们现在将全面定义翻译后修饰，使用新的质谱学方法，并将受体修饰与功能联系起来。随着糖皮质激素受体运输的改变(这可能解释了未能招募到增强剂来应对胃肠粘附素的干扰)，我们将使用单分子分辨率活细胞成像来跟踪胃泌素受体的运动，并使用荧光相关光谱来实时测量与胃泌素的相互作用。我们假设胃粘附素是一种已知的支架蛋白，它促进蛋白质-蛋白质的相互作用，调节细胞对环境应激的反应，例如肝脏中的能量过剩。为了发现胃粘附素的效应机制，我们将定义胃粘附素的翻译后动力学，并使用蛋白质组学来鉴定正常和脂肪肝中的胃粘附素客户蛋白。我们将使用有针对性的、定量的质谱学方法，具体测量与36个肝脏表达的核受体的相互作用。最后，我们将在正常条件下以及饮食诱导的肥胖和慢性GC治疗期间，通过选择性基因靶向在小鼠肝细胞中检测美粘附素作用的生理后果。总之，这项工作计划将定义一种新的GC信号调节模式，并揭示一个潜在的新治疗靶点，它具有选择性地改变GR作用的代谢结果的耐人寻味的潜力。

The metabolic syndrome, a collection of obesity, hepatic steatosis, and insulin resistance is a major public health crisis, with unmet medical need. Non-surgical intervention strategies including lifestyle modification are generally ineffective due to poor compliance. We must therefore continue to identify and develop novel strategies and therapeutic targets. An essential initiating feature is excess triglyceride accumulation in the liver, which can progress through inflammation and fibrosis to steatohepatitis, cirrhosis, and hepatocellular carcinoma. Glucocorticoid (Gc) signalling in the liver has been implicated at all stages of progression. Moreover, metabolic complication is a persistent and limiting issue in long-term Gc treatment in inflammatory disease.In seeking mechanisms to address metabolic complications of Gc action we discovered the Gc receptor (GR) binds to metadherin, a multifunctional protein scaffold, implicated in liver lipid metabolism, inflammation and cancer. We discovered that metadherin is essential for Gc transactivation, but not transrepression, and that this is accomplished by regulating GR recruitment to enhancer elements. In mice lacking metadherin, Gc action in the liver was profoundly altered with a major shift in Gc-responsive lipid metabolic genes, and also a striking hyperglycaemic response. In the metadherin null mice, we also observed genome wide changes in H3K27Ac, a mark of active enhancers, in response to acute Gc challenge; likely indicating an underlying re-wiring of the GR cistrome. We have established mouse models of glucocorticoid excess, and found a marked induction of hepatic triglyceride, with lipid droplet accumulation; thus optimising a model of Gc-induced hepatosteatosis. We will now use this mode and more conventional diet-induced obesity, to define control mechanisms in GR action, and the role of metadherin.We will define how metadherin regulates GR function. We have evidence that metadherin promotes interaction with kinases required to modify the GR. We will now comprehensively define post-translational modification, using new mass spectrometry approaches, and link receptor modification to function. As altered GR trafficking (as may explain failure of recruitment to enhancers in response to metadherin disruption), we will employ single molecule resolution live cell imaging to track GR movement, and use fluorescence correlation spectroscopy to measure interaction with metadherin in real time.We hypothesise that metadherin, a known scaffold protein, promotes protein-protein interaction to regulate cellular responses to environmental stress, such as energy excess in the liver. To find metadherin effector mechanisms, we will define post-translational dynamics of metadherin, and use proteomics to identify metadherin client proteins, and in normal and fatty liver. We will specifically measure interactions with the 36 liver-expressed nuclear receptors, using a targeted, quantitative mass spectrometry approach.Finally, we will examine the physiological consequence of metadherin action in vivo with selective genetic targeting of metadherin in hepatocytes in mice under normal conditions and during diet-induced obesity and chronic Gc treatment. Together, this programme of work will define a new regulatory mode in Gc signaling and reveal a potential new therapeutic target ,which has the intriguing potential to selectively modify metabolic consequence of GR action.