Circadian regulation of liver energy metabolism: translational studies in diabetes and obesity

Obesity is a very common health issue, with a major impact on liver function. When fat builds up in the liver, this can lead to abnormal function, including insulin resistance, and type II diabetes. In addition, liver inflammation pushes the liver from non-alcoholic fatty liver disease (NAFLD) through non-alcoholic steatohepatitis (NASH - a form of hepatitis), and to cirrhosis and liver cancer. Every person has an internal body clock, or "circadian rhythm", which is built into every cell and helps the body function. There is new evidence of a circadian clock in the liver that controls fat metabolism. This offers a new perspective on how the progression of the above illnesses may be interrupted.

Here, I will test the hypothesis that the circadian clock, and circadian metabolic circuits in the liver can be affected by nutrient supply, and I will further test the role of signal mediators (chemicals) linked to feeding, and energy metabolism, including ceramides (sphingolipids derived from the intestinal epithelium), and short-chain fatty acids (products of the intestinal microbiome - bacteria colony - with proposed liver regulating activity). I will use primary human liver cells to create liver "organoids" (microlivers) for these studies. In parallel I will use primary mouse liver cells, as these also offer the opportunity to use cells from genetically modified mice carrying circadian clock reporter genes, which I can track in real-time through multiple circadian cycles, and in addition I can selectively delete particular circadian clock components including the genes BMAL1 and REVERBa.

I will go on to use this organoid system to investigate how targeting the circadian clock in the liver can regulate nutrient sensing, and handling of metabolites involved in energy conversion. In this way I can use small, drug-like molecules to regulate the clock (e.g. crytochrome activators, and REVERB ligands) along with the selective deletion of genes like BMAL1 (which stops the circadian clock), or REVERBa (which breaks the connection between the clock and the liver fat-production programme).

I will identify how signals derived from feeding, including glucose, fatty acids, as well as signalling mediators including short chain fatty acids and ceramides, impact on the core liver clock, and affect the circadian clock controlled metabolic network. I will go on to discover how the operation of the clock impacts on handling of metabolic changes whereby glucose and fatty acids are subject to oxidation, or in the case of glucose to lipogenesis. I will have the opportunity to further test the integrity of this system in response to inflammatory signals such as those experienced by people with chronic inflammatory disease such as rheumatoid arthritis, or the chronic low-grade inflammation that is a consistent feature of obesity.

Evidence suggests that circadian control mechanisms in the liver are essential for energy homeostasis, but how bioenergetic substrates communicate with the clock, and how liver circadian metabolism acts on the substrates is unknown.

I will model human liver using 3D spheroid culture systems, with primary human hepatocytes (PHH), as immortalized cell lines poorly replicate human liver. These human liver spheroids maintain insulin sensitivity in long term culture and this phenotype can be modulated by different intervention (i.e. glucose/insulin concentration). This technology offers me a perfect research training opportunity to investigate the interplay between energy metabolism, especially lipids, and the hepatic circadian clock, as explained below.

The circadian metabolic system in the liver is plastic, and responds to changes in state, such as presence of inflammation. This project capitalises on recent innovations which permit human liver organoids to be used as a translational model for diabetes and metabolic dysfunction. The microlivers will be challenged with lipogenic, high-energy culture medium, to drive lipid accumulation within the hepatocytes. The impact of this challenge, the mechanism underlying human hepatosteatosis, on the core circadian clock will be assessed by tracking the PER2-luc output, and by measuring gene expression profiles.

As 90% of the lipogenic programme in the liver lies under circadian control, I will target the core clock components BMAL1 (which stops the clock), and REVERBa, (major link between the clock and lipogenic gene expression). I have in house BMAL1, and REVERBa floxed mice crossed to albumin cre-ERT, which allows post-natal deletion of the clock genes. I will recover the livers, and use these to make microlivers, as above. My primary outcomes will include metabolic gene expression profiles through time, measured using nanostring technology, and this will be supplemented with stable isotope studies.

Obesity, type 2 diabetes, and other metabolic diseases are still highly stigmatised, and often misconstrued as "self-inflicted". This is despite the fact that we cannot explain the three-fold increase in the rates of obesity and overweight over the last 40 years. Globally, 39% of people are now overweight, and the obesity crisis shows no signs of stopping. Stigma acts as a barrier for patients to seek and receive appropriate care, and for their care to be funded. In my managerial role as part of the committee to set up Tier 4 bariatric services at the Buckinghamshire Healthcare NHS Trust, I have seen first-hand how difficult it is for patients with obesity to access the care they need.

It is my hope that this research will provide some answers on the pathophysiology of obesity and metabolic disease in the context of the circadian rhythm. Clinical research and population studies have shown that shift work and disrupted circadian rhythms have an adverse effect on the body's metabolism, with higher rates of conditions such as obesity. There is currently a paucity of data on how this works on a molecular level. Demonstrating how external factors, such as shift work and circadian dysrhythmias, contribute to these conditions on a cellular level may help to reduce some of the stigma associated with these diseases.

As per our communication strategy, we hope to publish outcomes of the research in high-impact academic and clinical journals. I would use my experience of disseminating research with the British Journal of Surgery and my large social media following to ensure that my published work would have a high reach, by producing infographics, running online chats and tutorials on the subject, and communicating the findings in plain English. This would allow the general public to have access to this information, and hopefully go some way to improving the stigma of these conditions. With an appropriate social media campaign (including the use of hashtags) following the publication of results, I would estimate we could see a positive effect in as little as two years. Thus, my research has the potential to improve the quality of life of patients themselves, as well as to have a positive impact on the care they receive. This may be in the form of improving funding for managing obesity and metabolic disease, and streamlining access to interventions such as bariatric surgery.

Another way to achieve this would be by communicating the findings with patient organisations and charities, policy-makers, and commissioning groups which fund the care of patients with obesity, diabetes, and metabolic disease, improving public health. This would also help alleviate the significant economic burden incurred by these conditions, both in terms of lost hours of work, and the costs to the health services.

It is hoped that this research will also pave the way for developing pharmacotherapies that could manipulate the circadian clockwork, and prevent or treat these conditions. This sort of research may require collaboration with pharmaceutical companies and would foster the economic competitiveness of the UK pharmaceutical industry.

Finally, I would benefit enormously from this project, in the invaluable practical, academic and scientific training I would receive, and how this would allow me to communicate with and manage patients with these conditions for the duration of my clinical career.