Biological resonance: matching internal timing to environmental fluctuations

In virtually all organisms, inherent timing systems (circadian clocks) orchestrate rhythmic patterns of physiology and behaviour across the day. These timers are also responsive to external environmental cues, such as cycles in light and food availability. In mammals, the circadian clockwork regulates our daily patterns of behaviour (when and how long we sleep, when we eat), and also the underlying physiologies that support such behaviour (e.g. rhythms in body temperature, hormone release and liver function). Unfortunately, it now seems clear that disruption of our bodies' 'natural' rhythms by modern societies' 24h lifestyle (shift work, sleep restriction, disrupted eating patterns) is associated with metabolic disease (obesity, diabetes). Therefore, it is critical that we understand the basic biology behind how internal timers align our physiology to the environment and what the consequences are when that alignment is disrupted.

Until recently, it was believed that circadian timing in mammals was determined wholly by a small area of the brain, the suprachiasmatic nucleus (SCN). We now know this not to be true, and that clocks reside in many areas of the brain and virtually all peripheral organs. Within each tissue, many functions are controlled by the local clockwork, such as daily rhythms in liver glucose production. These peripheral tissue clocks are strongly influenced by rhythmic feeding activity (normally governed by the SCN) and hence rhythmic energy flux. As a consequence, many behavioural and physiological processes can be decoupled from the SCN when feeding does not adhere to an SCN driven rhythm. This can be modelled easily in laboratory mice using restricted feeding schedules, which force these nocturnal animals to eat in the day.

To understand how our physiology, and especially our ability to maintain energy balance, is affected by disrupted activity or feeding patterns, we will examine what happens when the external environment is put in opposition to internal clocks in the brain and liver, or when different clocks in the body are not able to keep in time with one another. This is especially important in tissues such as the liver, which must cope with large fluctuations in energy supply (due to feeding/fasting cycles). We will use genetically modified mice in which we have changed the speed of the clock (20h vs 24h per cycle) in all tissues or selectively in either the SCN or the liver. Our aim is not to stop or remove the clock within the mice (nor in any of their tissue systems), but reveal the consequences to energy metabolism when we run these clocks at different rates or phases to the external environment (light and meal times). This is much closer to the real world. We will also challenge these mice will high fat diet to determine their ability to cope with vastly altered energy intake. This may be particularly relevant to the real world, where large high calorie meals are often eaten at inappropriate times (such as before bed).

To date, it has been very hard to measure rhythmic responses in a particular tissue. However, we will use an exciting new method to track liver oscillations in free-moving mice. This is achieved by injecting a virus containing a light-emitting gene, which oscillates in response to activation of a specific metabolic pathway. Thus, we can track how the core clockwork of the liver responds to altered feeding schedules, or even of how metabolic genes regulating glucose production, fatty acid synthesis or protein metabolism change their expression. We are confident that this remarkable new technology will greatly increase understanding of how tissues such as liver respond to environmental cues, and be of wide-spread application, and also potentially lead to reduced animal usage as greatly more information can now be obtained from one animal than before.

This proposal examines the impact of physiological timing on energy balance in mammals. Our studies will determine the degree to which metabolic homeostasis is compromised when feeding and activity patterns oppose our internal clock, a common feature of modern society. We will test this hypothesis by modulating environmental input (light and food) into the clockwork, by weakening internal coordination of different body clocks, and by genetically altering clock speed in a tissue selective way to drive internal de-synchrony.

As we disrupt the alignment of the internal circadian clockwork with environmental cycles, and progressively drive internal desynchrony (between liver and SCN), mice will undergo comprehensive longitudinal assessment of behavioural (locomotor activity) and physiological rhythms (Tb, metabolic gas echange) and determination of metabolic efficiency based upon gross anatomical measures (BW, adiposity), nutrient profiling in the liver (TG, glycogen content) and serum (inc. glucose, TG, FFA, cholesterol, insulin, glucagon), and glucose tolerance testing. Genetic manipulations will employ established and validated mouse lines (Ck1etau, Albcre), as well as a new SCN-selective cre-driver line recently created by our collaborator Michael Hastings (LMB Cambridge).

Critically, we employ and further refine a novel technology to longitudinally monitor liver gene expression in vivo in free moving mice. This will allow for the first time, assessment of transcriptional changes in response to altered feeding schedules in real time. By this means, we will identify how feeding cycles drive not only the local hepatic clock-work genes (ie BMAL1), but crucially we will measure its impact on specific hepatic metabolic-regulating genes.

To our knowledge, this is the first study to use resonance protocols and genetic alteration of clock speed to determine the true impact on circadian timing on mammalian physiology.

The research questions posed within this proposal are of major interest to ACADEMIC GROUPINGS in Biological and BioMedical Sciences. The academic community will benefit from elucidation of novel mechanisms whereby metabolic factors interact with the circadian clockwork on a molecular and anatomical level. Examination of the adverse effects of disrupted clock function on metabolism 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.

All findings will be of high interest to the GENERAL PUBLIC due to the prevalence of obesity and 24hr lifestyles in our modern society. 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 realistic potential to inform the general public about how dietary habits (what you eat and when you eat it) may detrimentally affect health. 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 PNAS (re-entrainment of disrupted clocks) was reported widely in national and international newspapers, on local radio, and on the intranet following press releases issued by the University of Manchester and BBSRC.

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 have taken a major lead within the extensive community of researchers at the University of Manchester by developing significant interactions and links with major pharmaceutical companies. We 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 Faculty of Life Science at Manchester has taken a strong proactive role in developing links with major pharmaceutical companies, enhancing public communication of science, as well as identification and development of commercialisation opportunities. There are dedicated members of staff employed within the Faculty to assist in these areas.

Benefits of this research to the UK ECONOMY are neither immediate nor guaranteed. However, obesity and related disorders (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.