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

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.

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.

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.