Exploitation of metadherin as a regulator of hepatic energy metabolism

代谢综合征是肥胖症、肝脂肪变性和胰岛素抵抗的集合，是一个重大的公共卫生危机，医疗需求未得到满足。由于依从性差，包括生活方式改变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策略和治疗靶点。一个基本的初始特征是肝脏中过量的甘油三酯积累，可通过炎症和纤维化进展为脂肪性肝炎、肝硬化和肝细胞癌。肝中的糖皮质激素（Gc）信号在进展的所有阶段都有牵连。此外，代谢并发症是炎症性疾病长期Gc治疗的持续和限制性问题。在寻找解决Gc作用代谢并发症的机制时，我们发现Gc受体（GR）与metadherin结合，metadherin是一种多功能蛋白质支架，与肝脏脂质代谢、炎症和癌症有关。我们发现met粘附素对Gc反激活至关重要，但不是反抑制，这是通过调节GR募集到增强元件来实现的。在缺乏met粘附素的小鼠中，Gc在肝脏中的作用发生了深刻的改变，Gc反应性脂质代谢基因发生了重大变化，并且出现了显著的高血糖反应。在metadherin无效的小鼠中，我们还观察到H3K27Ac（活性增强子的标志）在急性Gc挑战下的全基因组变化；很可能表明GR池有潜在的重新布线。我们建立了糖皮质激素过量的小鼠模型，发现肝甘油三酯明显诱导，脂滴积聚；从而优化了gc诱导的肝骨附着症模型。我们现在将使用这种模式和更传统的饮食引起的肥胖，来定义GR作用的控制机制，以及metadherin的作用。我们将定义metadherin如何调节GR功能。我们有证据表明metadherin促进与修饰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，使用新的质谱方法，并将受体修饰与功能联系起来。由于GR运输的改变（这可能解释了metadherin中断时增强剂招募失败的原因），我们将采用单分子分辨率活细胞成像来跟踪GR的运动，并使用荧光相关光谱来实时测量与metadherin的相互作用。我们假设metadherin，一种已知的支架蛋白，促进蛋白-蛋白相互作用，调节细胞对环境压力的反应，如肝脏中的能量过剩。为了找到metadherin效应机制，我们将定义metadherin的翻译后动力学，并使用蛋白质组学来识别metadherin客户蛋白，以及正常和脂肪肝。我们将使用靶向定量质谱方法，专门测量与36种肝脏表达的核受体的相互作用。最后，我们将在正常情况下、饮食诱导的肥胖和慢性Gc治疗期间，通过在小鼠肝细胞中选择性地靶向metadherin，研究metadherin在体内作用的生理后果。总之，这项工作将定义一种新的Gc信号调节模式，并揭示一个潜在的新的治疗靶点，它有可能选择性地改变GR作用的代谢后果。

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.