Exploitation of metadherin as a regulator of hepatic energy metabolism

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.

In the metabolic syndrome, an early event is triglyceride accumulation in the liver, which then leads to inflammation, fibrosis, disordered organ function, cirrhosis, and finally liver failure, or hepatocellular carcinoma. Glucocorticoid signalling lies at the heart of many of these processes, but how the glucocorticoid receptor (GR) engages effector mechanisms remains unclear. We discovered metadherin is required for some, but not all actions of the GR, and that this interaction is pronounced within the liver, with a target gene ontology of lipid metabolism. To define metadherin action and modification of GR function we will:

1. Use mass spectrometry and fluorescence correlation spectroscopy to define post-translational state, trafficking dynamics of GR, and GR:metadherin interaction under normal conditions and following metadherin disruption. We will validate GR modification using CRISPR engineered cells, and ascribe function using target gene expression measurements.

2. Metadherin serves as a protein scaffold, and in addition to our discovery of its interaction with GR, other metadherin client proteins include kinases and LXR. To reveal mechanisms of MTDH-mediated GR modification, we will determine the metadherin interactome, including potential effector kinases in livers from control and obese animals.

3. As metadherin regulates GR recruitment to the genome in vitro, and enhancer activation in response to glucocorticoid in vivo, we will map the GR cistrome in the liver in response to energy excess, and determine how metadherin regulates this action. Emerging mechanisms will be tested in primary hepatocytes recovered from wild type and metadherin null livers.

4. The physiological consequences of metadherin disruption on liver energy metabolism will be measured using hepatocyte metadherin null mice. We will use chronic glucocorticoid or high fat challenges, and measure lipid synthesis, oxidation, and export.

The research questions posed within this proposal are of major interest to ACADEMIC GROUPINGS in Biological, BioMedical, and Clinical Sciences. The academic community will benefit from elucidation of mechanisms involved in energy homeostasis, glucocorticoid action, and sensitivity, and obesity-related pathology. Understanding these pathways and identifying potential targets for intervention in obesity presents clear implication to human health and welfare. As such, research findings will impact greatly on the HEALTH CARE COMMUNITY. We will disseminate findings by publishing primary papers and reviews in high impact journals, and presenting work at national and international meetings. We anticipate that the proposed work will produce 2-4 high-quality primary research papers.

Our findings will be of interest to the GENERAL PUBLIC due to the prevalence of obesity and diabetes, and the very widespread usage of therapeutic glucocorticoids. At its most basic, the work will engage sections of the populous who wish to learn about their health and human physiology. This work also has potential to inform the general public about the pathogenesis of obesity and diabetes. Research findings will be delivered to the general public through public engagement activities (e.g.cafe scientific), as well as through mass media. For example, Ray is regularly featured on BBC television, radio, and on the BBC website.

The proposed research is of interest to PHARMACEUTICAL COMPANIES due to direct implications for human metabolic disease. Pharmaceutical industry investment into the problems resulting from obesity is rapidly growing due to the fact that the prevalence is increasing, and therapeutic options, or mitigating factors remain elusive. Furthermore, even in the non-obese population hepatic fatty infiltration, "fatty liver" is highly prevalent, and a recognised side-effect of therapeutic glucocorticoid treatment. In the context of "building partnerships to enhance take-up and impact, thereby contributing to the economic competitiveness of the United Kingdom", our laboratories have a successful track record of engagement with GSK on inflammation, and glucocorticoid projects, and regular communication with these companies will ensure research findings are taken-up by and impact upon industrial beneficiaries. UoM has taken a strong proactive role in developing links with major pharmaceutical companies, as well as identification and development of commercialisation opportunities.

Benefits of this research to the UK ECONOMY are not guaranteed. However, metabolic disorders (obesity, cardiovascular disease, diabetes etc) are, and will continue to be, a massive burden on the national health care service. This will only increase with the aging population, in which circadian and metabolic disturbance is common. Thus, future economic benefits may be substantial.

This proposal also offers a unique and significant opportunity for high-level in vivo training of the associated post-doctoral scientist, and any PhD students joining for related work. This is a significant benefit as a lack of in vivo research training has been highlighted as a weakness in UK bioscience. Numerous undergraduate and Master's degree students will be exposed to my research and gain valuable research skills through lab-based projects.

Although beyond the limits of this grant, our ultimate goal is to deliver novel therapies, which have real benefit for patients. Patients suffering with the associated co-morbidities of obesity, including type II diabetes urgently require new thinking to inform treatment development. Within the proposal, there are numerous pathways where therapeutic interest is already clear (e.g.modified GR ligands). Nevertheless, by incorporating non-biased and targeted approaches, mouse and human models, and adipose in different pathological states, this work is almost certain to identify novel and robust targets.