Neural circuits of glucagon-like peptide-1 (GLP-1) action in health and disease

Obesity, diabetes and co-morbidities, such as hypertension, are a serious health burden for the patient and a strain on public resources. Promising drugs that might be beneficial for the treatment of these conditions are glucagon-like peptide-1 (GLP-1) analogues. GLP-1 is a hormone that is secreted from the gut after a meal. Its principal role is to improve the digestion of sugars, and to generate the sensation of fullness, or satiety. Besides their anti-diabetic actions, GLP-1 analogues have been shown to cause nausea, mild hypertension and tachycardia, possibly indicating action in the brain. In fact, microinjection of GLP-1 into the brain reduces food intake and lowers blood glucose. The aim of our project is to improve our knowledge of the relative importance of the brain and gut GLP-1 signalling in the control of blood sugar and food intake, and to clarify whether GLP-1 analogues, which are already in clinical use, activate the brain GLP-1 system.
To understand GLP-1 action in brain it is imperative to know the detailed expression pattern of the GLP-1 receptor (GLP-1R) in brain. In addition, manipulation of GLP-1R in the brain, by controlling the activity of GLP-1R expressing cells, would enable clarification of the role of these receptors in vivo. We will use a novel transgenic mouse (glp-1r-cre) that expresses both a red fluorescent label (RFP) and cre-recombinase in GLP-1R expressing neurons, and thus allows in vitro identification of these cells as well as their genetic targeting with 'flex-switch' virus constructs in vivo. We will use a gene therapy approach where new genes are delivered by a virus to GLP-1R cells in specific areas of the brain. These genes will produce proteins allowing cell-specific suppression of the activity of GLP-1R cells with unprecedented temporal and spatial resolution. By measuring the effects of inhibiting the activity of specific GLP-1R-expressing cells groups on blood glucose control, food intake and behaviour, we will be able to tease out the exact physiological function played by GLP-1R-expressing central neurons.
Using this mouse line we propose to define and validate the precise distribution of GLP-1R expressing cells in mouse brain. We will then determine the functional significance of these cells in different brain regions and characterise the GLP-1R expressing neuron populations in vitro by electrophysiological and optical Ca2+ recordings. Finally, we will assess the action of GLP-1 analogues in brain and whether it changes under high fat diet.
This project will clarify the physiological importance of brain GLP-1Rs and provide vital information regarding activation of central GLP-1Rs by stable GLP-1 analogues. This is especially important for understanding potential unwanted effects that may be observed during GLP-1 therapy. These data will provide further insight into the clinical benefit of GLP-1-based treatments, possibly not only for patients with diabetes, but more generally in metabolic disease. Furthermore, it will provide information on the cause of undesired side effects such as nausea, as well as clarifying whether there is a role for neuronal GLP-1Rs in cardiovascular disease and in olfactory signal processing. Since GLP-1 analogues are already on the market for the treatment of type 2 diabetes, we expect that the knowledge from this project will feed into drug development and formulation, and thus an influence on therapy could be hoped for in the near future.

This proposal aims at characterising the exact physiological role of GLP-1R-expressing neurons in the control of energy balance and autonomic functions. We will experimentally silence or ablate GLP-1R-expressing neuronal populations in vivo by using viral gene transfer. Specific targeting will be achieved by combining a transgenic mouse line that expresses Cre-recombinase selectively in cells that express GLP-1R with viral expression vectors that are only active in cells expressing Cre-recombinase (FLEX-switch technique). The adeno-associated virus (AAV)-based vectors are injected stereotaxically into individual brain regions containing GLP-1R-expressing neurons. 3-5 weeks after the infections the animals will be tested for effects of GLP-1R neuron modulation on food intake, blood glucose control, cardiovascular and circadian activities. AAV vectors produced for this purpose will include those that inactivate or ablate GLP-1R cells permanently (Tetanus toxin light chain; Diphteria toxin A subunit), and those that inactivate GLP-1 cells transiently (DREADD hM4D), when the appropriate agonist is applied (clozapine-N-oxide).
Food and water intake will be recorded, blood glucose control will be evaluated with intraperitoneal glucose tolerances tests, and cardiovascular parameters will be assessed using telemetry in awake animals. Our results will further define the physiological role of GLP-1R-expressing neurons in specific brain regions and provide crucial data on the usefulness of a GLP-1 based therapy for metabolic disease. These experiments will also specifically address the question whether GLP-1R expressed in autonomic neurons contributes to cardioprotection, and if GLP-1 in the olfactory system constitutes a novel target for the control of energy balance by GLP-1 analogues.

The basic research we propose here is likely to significantly increase understanding of how GLP-1 affects energy balance and cardiovascular control. We expect these results to be of considerable interest to the pharmaceutical industry. This is particularly likely because GLP-1 analogues are already in clinical use for the treatment of type 2 diabetes.
A benefit to the general population, in terms of improvements in healthcare, might be possible over a time-course of 10 years if our findings influence the availability of GLP-1 therapy for diabetic and for obese patients. Additionally, our research might identify new treatment strategies for metabolic disease, which could hopefully enter clinical practice over a 10-15 year time-course. This again would elicit interest from the pharmaceutical industry. Currently ~10% of the NHS budget (£9billion) is spent on the 2.6 million diabetics in the UK. Diabetes UK predicts this number to rise to 4 million by 2025, mainly due to an increased incidence of obesity, driven by increasingly sedentary lifestyles. In 2009 more than 60% of the adult UK population were overweight. The complications of these diseases include stroke, retinopathy, neuropathy, renal failure, cardiovascular disease and cancer. The increased prevalence of diabetes, obesity and co-morbidities was recently predicted to contribute to a lowered overall life expectancy in the UK (http://www.independent.co.uk/life-style/health-and-wellbeing/health-news/diabetes-may-cause-first-fall-in-life-expectancy-for-200-years-966914.html) for the first time in 200 years. GLP-1 analogues show great potential in the treatment of type 2 diabetics mainly due to their actions as insulin secretagogues and positive trophic effect on beta-cell mass. The anorexic effect of GLP-1 is mediated by central actions and so might be some of the insulin secretagogue effect. Since GLP-1 has various central effects (including induction of nausea and vomiting, in addition to satiety) some of which might be considered detrimental, a detailed understanding of GLP-1 action in brain down to a cellular level is urgently needed. Our study allows for the first time the precise functional targeting of GLP-1 receptor expressing cells in the brain. It will educate the scientific community about the different pathways involved and thus clarify the role of central GLP-1 in metabolic and cardiovascular control. In particular, this work will address roadblocks in diabetes research as identified recently by the European Commission's Support Action "DIAMAP: A Road Map for Diabetes in Europe" (http://www.diamap.eu/) including "Determine central nervous master switches that control food intake and energy expenditure", which is identified as lacking "available in vivo methodology and imaging techniques", which we are addressing with this proposal. Additionally, our proposal addresses several priority areas identified at the recent "MRC Neuroscience of Obesity Workshop", including 'the metabolic role of the autonomic nervous system' and 'capitalise on new techniques to discriminate between the action of peptides in the gut and the brain'.