REVing-down: targeting the circadian clock in metabolic disease

Obesity and diabetes are major threats to public health, and the incidence of both conditions continues to rise. Non-surgical intervention strategies including lifestyle modification are generally ineffective due to poor compliance, and current pharmacological options are plagued by poor efficacy and adverse side effects. Novel strategies and therapeutic targets are needed. Human and animal studies have shown that adipose tissue inflammation is driving factor in the progress of obesity to life-threatening conditions, such as diabetes and cardiovascular disease. White adipose tissue (WAT) is an essential energy buffer, balancing energy storage and release during natural cycles of fasting and feeding. Chronic positive energy balance leads to expansion (hypertrophy) of the tissue, and this state can result in adipocyte dysfunction. Consequential to WAT hypertrophy there is a major shift in the immune system within the adipose tissue; where immune cells which are normally beneficial to adipocyte function (eg iNKT and ILC2 cells) are replaced by pro-inflammatory immune cells, such as inflammatory monocytes and macrophages. It is this inflammatory state which perpetuates WAT dysfunction, and drives dyslipidaemia, insulin resistance and adipocyte cell death. The challenge is to identify events that signal the transition from normal 'healthy' fat storage to adipose dysfunction and inflammation.

We have recently demonstrated in mice that lack of the protein REVERBa predisposes to efficient adipocyte function and resulting WAT hypertrophy. Strikingly, although fat mass is increased they do not show the expected reduced insulin sensitivity or high levels of adipose tissue inflammation normally associated hypertrophic adipose tissue. The mice also show an elevated production of adiponectin - a hormone produced by healthy adipose tissue, and known to have many beneficial metabolic effects in humans. This indicates that REVERBa plays an important role in the adaption of WAT during obesity. In this project, we want to understand how REVERBa controls WAT function, and in particular elucidate how REVERB acting in adipocytes, regulates WAT immune function to attenuate obesity-related inflammation and insulin resistance.

To achieve this, we will use novel genetic targeting in mice to manipulate REVERBa expression and activity specifically in adipocytes. This will allow us to define how REVERBa drives adipocyte function under normal circumstances and during obesity to promote/attenuate the development of tissue inflammation and insulin-resistance. We will also define mechanisms though which REVERBa controls adiponectin production (a valuable therapeutic target).

Exploiting this unique model, we will next characterize how immune cell dynamics differ between mice that develop obesity related inflammation and insulin resistance, with those that remain insulin-sensitive in the face of obesity. Using state-of-the-art imaging, cell sorting and genomic techniques, we will not only identify and quantify immune populations, but also determine their relative state of activation. This is important as it goes beyond the immediate impact of REVERBa to identify cells/events that signal more broadly, the development of WAT dysfunction.

Alongside animal models, we will similarly define adipose-immune dynamics in visceral adipose collected from obese patients undergoing bariatric surgery and normal weight controls. This will determine which cells and events are common in the progression of obese WAT to an inflamed insulin-resistant state in both humans and mice. Importantly, we will determine whether target pathways are amenable to pharmacological manipulation in human WAT using drugs that increase or decrease REVERBa activity. The parallel nature of these studies will allow important pathways to be cross validated between human and mouse tissues, and greatly increase our ability to identify new strategies in the fight against obesity-related disease.

In obesity, white adipose tissue (WAT) hypertrophy is associated with a shift in local immune cell populations, with displacement of beneficial cells by pro-inflammatory immune cells (eg M1 macrophages). From human and animal studies, we know that this inflammatory state drives WAT dysfunction, dyslipidaemia, and insulin resistance. What remain unclear are the events that signal the transition from efficient fat storage to WAT dysfunction and inflammation.

REVERBa-/- mice are profoundly obese; yet do not exhibit loss of insulin sensitivity or exacerbation of WAT inflammation. A role for REVERBa in lipid metabolism and within the immune system is already well established. REVERB typically inhibits macrophage inflammatory response. This led us to hypothesise that loss of REVERBa in adipocytes circumvents inappropriate programs of lipolysis and adipocyte stress which normally accompany WAT hypertrophy, and signals a selective preservation of distinct immune cell populations in the tissue (M2 macrophages, iNKT and ILC2 cells) even amid pronounced obesity.

To test these hypotheses, we will:
- Selectively delete REVERBa, or its DNA-binding domain (thereby segregating REVERBa control over metabolic and inflammatory processes) in adipocytes, and define the consequences of obesity in the mice.
- Identify, isolate and phenotype WAT immune populations during the development of obesity in normal (inflamed/insulin resistant) and transgenic (insulin sensitive) mice.

We will similarly define immune populations in WAT of obese and normal weight patients, to reveal common cells and events that define the inflamed insulin-resistant WAT in humans and mice, and use pharmacological manipulation of REVERBa to validate candidate pathways in cultured WAT. These studies will bring new understanding of WAT function and identify new strategies in the fight against obesity-related diseases.

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, adipose tissue function, inflammation 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. 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. brain awareness week), as well as through mass media. For example, our recent article in Curr Biol was reported widely in national and international newspapers, on local radio, and on the internet.

The proposed research is of interest to PHARMACEUTICAL COMPANIES due to direct implications for human metabolic disease. Pharmaceutical industry investment into circadian biology is rapidly growing due to the fact that circadian dysfunction has been linked to sleep disorders, mental health disorders, cancer, inflammation, and aging. In the context of "building partnerships to enhance take-up and impact, thereby contributing to the economic competitiveness of the United Kingdom", our laboratories are currently involved in collaborations with Pfizer and GSK on circadian-related projects, and regular communication with these companies will ensure research findings are taken-up by and impact upon industrial beneficiaries. The REV-ERB compounds to be used are provided through collaboration with GSK, and thus demonstrate directly stakeholder interest. 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. adiponectin production, macrophage polarisation, WAT inflammation). 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.