Inflammatory signals regulate neuroendocrine control of growth and energy balance through re-modelling of mammalian hypothalamus

Lead Research Organisation: University of Aberdeen
Department Name: Rowett Institute of Nutrition and Health

Abstract

Obesity and overweight are major problems in the developed world, which are due to the overconsumption of calories. Increasingly it is becoming a significant problem for countries in economic transition, such as China and India. In countries such as the UK obesity affects about 30% of the population and it is a serious problem that dramatically increases the risk of clinical disease such as type-2 diabetes, cardiovascular disease and cancer. At the other end of the spectrum, growth retardation or stunting is a major problem for the poorer parts of the developing world as this reduces healthy life expectancy. Interventions designed to reverse either obesity or stunting have proved difficult and while there may be many reasons for this, one possibility is that both obesity and stunting involve long-term changes in the mechanisms controlling energy balance and growth, which make them recalcitrant to reversal.
Both these physiological axes are controlled through the neuroendocrine system. In this study we aim to further our understanding of the mechanisms controlling long-term energy balance and growth by studying an animal model which is able to reversibly and naturally vary its energy balance and growth trajectory. For seasonal animals a simple change in environmental photoperiod induces dramatic and robust changes in food intake, body weight and growth. We will use the photoperiodically sensitive F344 rat in these studies. It is anticipated that the insights gained will contribute to improvements in our ability to control energy balance and growth in humans.
In our work to date we have identified two types of signalling molecules and two types of cellular changes that are potentially involved in the long-term neuroendocrine control of energy balance. The signalling molecules are retinoic acid and thyroid hormone, and the two processes they control are inflammation and the birth of new neurons. All show robust and marked changes in response to switch in photoperiod and are each associated with change in body weight in the F344 rat. Retinoic acid, which is nutritionally derived from Vitamin A, has been strongly associated with growth and development. Similarly thyroid hormone has a well-known association with energy metabolism. Independently, each has recently been proposed as a neuroendocrine regulator of energy balance and growth. Completely novel to this proposal is the explanation of how retinoic acid and thyroid hormone control the balance of inflammatory signaling and cell proliferation in the hypothalamus to regulate energy balance and growth. We postulate that these pathways drive a process of cellular re-modelling of the hypothalamus, which in turn modulates neuroendocrine control of energy balance and growth.
In this project we will test these ideas by blocking inflammatory signalling or cell proliferation to determine whether they are, as we predict, the long-sought for key to the mechanism that brings about long-term change in energy balance and growth - the very changes that, when abnormal, lead to obesity or metabolic disorders. We will also examine some of the details of the inflammatory signals and cell types involved. From these studies we hope to provide direct evidence for the role of inflammation and cell proliferation in the brain in the physiological control of energy balance. It is anticipated that this will provide new insights to help us understand how inflammation, which is a pathological consequence of obesity, may disrupt the control of energy balance in the obese state and more importantly how it may be reversed. Similarly the insights gained will help us understand how Vitamin A, thyroid hormone and inflammatory signalling influence growth and thus may also help us understanding why stunting can be resistant to reversal.

Technical Summary

Long-term and reversible changes in food intake, growth and energy balance are characteristic of seasonal animals. Photoperiod triggers these changes and recent work indicates that early events in the response involve altered thyroid hormone (TH) and retinoic acid (RA) signalling within the ependymal/tanycyte cells of the hypothalamus. This proposal will examine signalling downstream of TH and RA and how it links to the control of energy metabolism and growth in the photoperiodically sensitive F344 rat. The proposal focuses on the role of inflammatory signalling and cell proliferation, since gene expression of chemokines (chemerin and CCL22) and cytokines (CD40 L) and Ki67 staining in the hypothalamus are strongly regulated by photoperiod, RA or TH. In situ hybridization and immunocytochemical techniques will be used to examine the location of receptors for these signals (CMLKR1, CCR2 and CD40) as well as the nature and sites of signal transduction through pathways such as ERK, Akt and NF-kappaB. The phenotype of the cells involved in the downstream response will be studied using co-localisation techniques. Of particular interest will be the role of microglia (macrophage cells of the brain), which we have already established are radically changed in expression in response to photoperiod. To examine the importance of inflammatory signalling as a key mediator of the physiological effects of photoperiodic on growth and energy balance, lentiviral constructs, expressing either dominant negative or constitutively active constructs for IKK will be administered into the hypothalamus to either attenuate or mimic NF-kappa B signaling in the hypothalamus. To examine the role of cell proliferation in the RA and TH responses, BrdU labelling combined with immunocytochemical staining will be used to identify the cell types involved. The mitotic inhibitor, Ara C, will be injected into the brain to test if cell proliferation is critical to photoperiodic control of energy balance.

Planned Impact

Who will benefit from this research and how?
This research project will provide basic underpinning knowledge, which in the longer term has the potential to influence a range of issues of policy relevance and industrial importance.

Government policy makers involved in health:
Chronic inflammation is now recognized as both a cause and consequence of ill health. Obesity is a major issue for Governments worldwide and it is well known to be associated with low-level inflammation.
While controlling obesity is a number one goal for policy makers, the solutions are not straightforward. A complementary approach is to enhance the quality of life by minimizing the metabolic side effects of obesity (inflammation). At present controlling inflammation is not on the radar of policy makers, as they are focused on controlling the obesity problem through calorie control. Ultimately it is anticipated that this research will inform those involved in public health of the importance of controlling inflammation in the brain (e.g. through diet) and this will re-shape current policy thinking on diet and health.

Members of the Bioscience industries:
To date therapeutics designed to control food intake have been generally unsuccessful. There are a variety of reasons for this, including unacceptable side effects and low efficacy. It is also true that the pharmaceutical industry have focused their attention on drugs that influence energy compensatory pathways within the brain. The work in this project offers potentially new insights into the control of energy balance, by revealing novel and unexplored routes to controlling long-term body weight, through inflammatory signalling. Given that the model being used in our studies exhibits reversible control of body weight, then it offers a new approach to exploring novel pathways involved in the control of energy balance pathways and thus is likely to identify new therapeutic targets for the future.

Policy makers and NGOs involved in developing solutions to Food Security issues:
A quite different issue, where this project could have impact is vitamin A deficiency, which remains one of the most significant nutritional deficiencies. It has been estimated that over 200 million people (mainly pregnant women and pre-school children) are at risk of vitamin A deficiency. Night-blindness is the clinical consequence most commonly associated with vitamin A deficiency, although there are many other clinical manifestations including poor immune function, stunting and increased risk of mortality. When vitamin A supplementation has been used to attempt to reverse the effects of vitamin A deficiency on growth, it has been met with variable success. This may be because stunting is not the consequence of a single micronutrient deficiency. However, we also know relatively little about how vitamin A influences the neuroendocrine control of growth. Based on our existing BBSRC funded work, we have identified novel retinoic acid signalling in the brain and its relationship to regulation of the growth axis. In our planned work, we will develop a more detailed understanding of how vitamin A/retinoic acid regulates long-term body weight and growth. This information is essential if we are to understand how to devise the best public health strategies for reversing the effects of vitamin A deficiencies in terms of growth.

Engagement with the public:
We are committed to the public understanding of science (see Pathways to Impact), and will continue to disseminate our research through public engagement activities, such as public science festivals, general lectures and though the media. This will not only have benefit for the public, but also for the post-doctoral scientists employed on the grant, who will gain experience of knowledge exchange activity and public engagement.

Publications

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Ransom J (2014) The rhythm of retinoids in the brain. in Journal of neurochemistry

 
Description The brain, and in particular a specialised region called the hypothalamus, is the master regulator of energy balance and growth. In this project we investigated how long-term changes (several days to weeks) in food intake, body weight and growth are regulated in the hypothalamus. Such long-term changes can be induced in photoperiodically sensitive species by changing the daylength (photoperiod), because long and short daylengths can stimulate markedly different rates of growth and body weight.
In this project, we studied the role of the hypothalamus in the regulation of food intake and body weight in growing F344 rats, which show a marked difference in these parameters in response to daylength. The hypothesis of the grant was that a cycle of cell proliferation/neurogenesis within a specific region of the hypothalamus (cells surrounding the ventricular region), together with associated morphological changes underpins the mechanism involved in body weight regulation in response to daylength. Amongst others, we hypothesised that this might be driven by inflammatory molecules.
The photoperiodic response involves local changes in thyroid hormone and retinoic acid synthesis in the hypothalamus. Both of these factors are elevated in long daylength, and reduced in short daylength, suggesting that they may be involved in suppressing cell proliferation in long daylength.
Retinoic acid is known to be involved in differentiation, and thus the high levels of retinoic acid under long daylength could also be important to the differentiation of newly generated cells. Vimentin is an intermediate filament protein that is characteristic of cells called tanyctyes, which are known to have progenitor cell characteristics. These are not neurons, but they project radially into the arcuate nucleus of the hypothalamus acting as important structural and supporting cell types. In meeting the award objectives, firstly we showed that vimentin is expressed more strongly under long days when retinoic acid levels are highest, consistent with increased differentiation of vimentin positive cell types.
Furthermore, we found that daylength markedly alters the rate of cell proliferation within the ventricular region of the hypothalamus consistent with our hypothesis. Under short daylength, cell proliferation is at least 5 times higher than under long daylength. The proliferating cell types include cells stained positively for neuronal markers, suggesting that neurogenesis is an essential part of the photoperiodic response. To test the importance of the cell proliferation response to photoperiod regulation of body weight, a mitotic inhibitor was used to block cell proliferation in the hypothalamus. This appears to partly block the normal daylength induced change in body weight, but was not sufficient to prevent neural progenitor cells from dividing.
Significant achievements from the award include that, in support of our hypothesis, we were able to provide evidence for the mechanism through which daylength stimulates increased differentiation, which is reflected by increased vimentin expression in long daylength. We found that retinoic acid stimulated by day length increases the expression of the inflammatory chemokine gene, chemerin in the ventricular region of the hypothalamus, and that chemerin protein infused into the hypothalamus stimulates increased expression of vimentin within the periventricular region and into the arcuate nucleus.
Further towards meeting the grant objectives, through in vitro studies we showed that thyroid hormone can increase the expression of the gene responsible for retinoic acid synthesis. Together with the above, this illuminates a pathway involved in the photoperiodic control of differentiation - namely: photoperiod thyroid hormone retinoic acid chemerin vimentin within the hypothalamus.
Another significant achievement was revealing the importance and relevance of the chemerin pathway to the long-term changes in body weight and growth seen in photoperiodic animals. This was shown by infusion of chemerin into the hypothalamus which resulted in significantly increased food intake after 2 weeks of treatment, a time-course consistent with effects of photoperiod. However, since the response was transient and not sustained, these results show that while chemerin is an important part of the pathway controlling energy balance in photoperiod-sensitive F344 rats, there must be other pathways that also contribute to the full response.
Overall the results from this study demonstrate that a cycle of cell proliferation, neurogenesis and differentiation underpins the photoperiodic response of animals to daylength, with increased proliferation occurring in short daylength, associated with a reduced thyroid hormone levels, and increased differentiation under long daylength when retinoic acid levels are at their peak. Interrupting the cycle of proliferation and differentiation appears to block the photoperiodic response underlining its importance to long-term seasonal body weight regulation. This together with the elucidation of the photoperiodic control of differentiation) through chemerin means that the hypothesis for the project was addressed and the objectives of the project were fully met.
Exploitation Route This study has shown the importance of cell proliferation and differentiation to the long-term regulation of energy balance in photoperiodic animals. It therefore makes an important step forward in terms of understanding of the neuroendocrine control of energy balance by photoperiod, but more widely it adds to the growing body of evidence that neurogenesis within the ventricular region of the hypothalamus in adult animals is a new and important dimension in neuroendocrine control. For example, high fat diets are recognised to influence neurogenesis in the hypothalamus, and through this influence the neuroendocrine control of appetite and energy balance. This may prove to be hugely important as a new target area of relevance in the worldwide efforts to combat obesity. Thus, this is an emerging area in neuroendocrinology where the importance and contribution of cell proliferation and neurogenesis from progenitor cells to the control of physiology in adult animals is really starting to be appreciated and picked up by neuroendocrinologists and the wider community in neurobiology.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description The results of our studies are too early to be have been taken up for economic benefit. Nevertheless, members of the research team have been active in engaging with the public and society through a variety of avenues, such as lay articles in the Conversation and public understanding of science lectures (e.g. Cafe Scientifique), science demonstrations to schools and in particular through Open door events which are held annually at the University, and where the local community can come and learn about the research we do. Our team have been instrumental in organising open door events with specific focus on the brain and it is through these types of engagement activities that we have been able to describe how our research on the brain is opening our understanding about how food (through inflammation) can potentially influence appetite and hence body weight.
First Year Of Impact 2016
Sector Other
Impact Types Societal

 
Description Evidence to Health and Sport Committee of Scottish Parliament
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Participation in a guidance/advisory committee
 
Description International collaboration 
Organisation Albert Einstein College of Medicine
Country United States 
Sector Academic/University 
PI Contribution The collaboration is on-going. The collaborators will benefit ultimately through publications arising from the work
Collaborator Contribution Interactions with the Albert Einstein College of Medicine, New York, USA on work with lentivirus for disruption of the NF-kB signalling pathway and have set up collaborative agreements for transfer of materials and future publications.
Impact None as yet. The work is still active.
Start Year 2013
 
Description 107th Annual Meeting of German Zoological Society 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Information exchange with other delegates with discussion about new collaborations

Discussion about new collaboration initiated with fellow scientists attending conference
Year(s) Of Engagement Activity 2014
 
Description 8th International Congress for Neuroendocrinology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Generated peer to peer discussion about data and interpretation of results

important feedback on talk helped to shape thinking about research
Year(s) Of Engagement Activity 2014
 
Description Alison Douglas Memorial Lecture at the International Congress of Neuroendocrinology in Toronto. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was a talk aimed at early career researchers, using my own career path and some history around the developments in neuroendocrinology to inspire the younger up and coming scientists.
Year(s) Of Engagement Activity 2018
 
Description Conference on translational medicine (Berlin) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Oral presentation: Retinoic Acid Homeostasis in the Adult CNS and its Role in Health and Disease

Engagement with fellow scientists in field of Neuroscience and translational medicine
Year(s) Of Engagement Activity 2014
 
Description Invited talk at the Neurex Workshop on the Arcuate Nucleus at the University of Strasbourg 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Participation in specialist workshop on metabolic function of the hypothalamus. Talk entitled: 'Photoperiodic Regulation of Tanycytes: Timing seasonal food intake and energy balance?' was given. The talk sparked considerable discussion afterwards with specialists in the area.
Year(s) Of Engagement Activity 2018
 
Description Neuroendocrinology conference (Manchester) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact Engagement with fellow scientists in the field of Neuroendocrinology across the UK

No notable impacts other than information exchange with fellow scientists in the field
Year(s) Of Engagement Activity 2013
 
Description Neuroscience Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact Engagement with members of scientific community in British Neuroscience

No notable impacts, other than exchange of information and ideas with fellow conference delegates
Year(s) Of Engagement Activity 2013
 
Description Science Week, March 2013 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact Science demonstrations to Primary 2 and 4/5 at Stoneywood Primary School, Bucksburn, Aberdeen

no actual impacts realised to date
Year(s) Of Engagement Activity 2013
 
Description Scottish Food and Innovation Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact The workshop was used to inform the future Innovation strategy for Scotland Food and Drink

The output of the workshop were taken forward for discussion with Scotland Food and Drink Federation onward discussion with funders and other stakeholders
Year(s) Of Engagement Activity 2014
 
Description Scottish Government workshop on changing behaviour 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Policymakers/politicians
Results and Impact Identified gaps in achieving behaviour change in relation to diet and health

Opportunity for future engagement identified
Year(s) Of Engagement Activity 2014
 
Description Scottish Neuroscience Group (Glasgow) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Participants in your research and patient groups
Results and Impact Engagement with fellow scientists in field of neuroscience in Scotland

No notable impacts other that information sharing with other scientists
Year(s) Of Engagement Activity 2014
 
Description Seminar Presentation to School of Agriculture, Food and Rural Development, University of Newcastle 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Participants in your research and patient groups
Results and Impact Oral presentation: , Retinoic Acid Homeostasis in the Adult CNS

No notable impact other that engagement with scientists at UK University
Year(s) Of Engagement Activity 2014
 
Description Seminar at the Netherlands Institute for Neuroscience 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk entitled: 'A unifying hypothesis for seasonal control of body weight and reproduction' was given to a general neuroscience audience, which sparked a diverse range of questions following the talk from students and researchers, plus more targeted advice to specialists about their own research outcomes and future study ideas.
Year(s) Of Engagement Activity 2018