Hypothalamic glucose-sensitive transcriptomes.

Lead Research Organisation: University of Bristol


When blood sugar levels drop significantly, for example after a long fast, brain function becomes significantly impaired. This is due to the brain using sugar (e.g. glucose) to fuel all of its activities. However, because it cannot generate glucose itself, it depends on being provided with this fuel by other tissues in the body. The brain's dependence on glucose - it's cells, neurons, don't function properly without it - brings with it the need for a sophisticated machinery to sense glucose concentrations and to adjust whole body glucose levels accordingly. Although we understand that neurons in a brain site called the hypothalamus are very important in these glucose-sensing and -adjusting mechanisms, we lack fundamental knowledge of these processes. Furthermore, these mechanisms fail in response to recurrent bouts of very low glucose levels or chronic high levels of glucose, however we do not understand why neurons de-sensitize. Our proposal therefore aims to identify the basic machinery underlying hypothalamic glucose-sensing and corresponding adjustments to whole body glucose levels. On a wider level this research makes an important contribution to a key biological question: How do neurons adjust their function to response to varying fluctuating signals? We have recently identified a hypothalamic protein, CRTC2 (CREB regulated transcription co-activator), that links glucose-sensing with appropriate activation of specific genes. We also found that the genes CRTC2 controls are very important in the regulation of food intake. Our aim is now to demonstrate CRTC2's key role in the hypothalamic mechanisms adjusting whole body glucose levels. We hypothesize that glucose-dependent CRTC2-mediated changes in hypothalamic gene expression underpin the neuronal response to adjust glycemic state. We will work on several different experimental levels to investigate the function of this protein. On a cellular level, we will assess in which subset of neurons CRTC2 is found; physically hypothalamic neurons all look alike, but they express different proteins and perform very different functions. Furthermore, we can assess exactly which genes CRTC2 controls; by comparing the activation of CRTC2-dependent genes at different glucose levels, we can identify which glucose-dependent genetic 'program' CRTC2 controls. By performing these experiments with recurrent bouts of low glucose or chronic high levels of glucose, we can identify which aspects of this genetic program fail when neurons de-sensitize to glucose changes. Together with identifying the cell-type CRTC2 is found in, these experiments identify the neuronal processes that are activated by varying glucose levels. Finally, we will assess the physiological functions these CRTC2-dependent genetic programs bestow on particular hypothalamic areas. By rendering CRTC2 unable to be regulated by glucose changes in genetically modified mice, the genetic programs run by CRTC2 cannot be controlled properly and allowing us to study the consequences on whole-body glucose levels in these mice. Our work will thus create significant insight into the hypothalamic processes engaged to maintain glucose homeostasis in the body.

Technical Summary

The brain is dependent on peripheral supply of glucose as it's main metabolic fuel. Exquisite ability to sense and adjust whole body glycemic state is thus of crucial importance to neuronal function. While recent data demonstrate that the hypothalamus is a key CNS site sensing, integrating and adjusting glycemic state, there is considerable lack of information on the fundamental mechanisms translating hypothalamic glucose-sensing into changes in gene expression and ultimately amendment of neuronal function and glucose homeostasis. Using a multi-scale approach ranging from cellular to integrated physiological techniques, we aim to identify the molecular regulation and physiological roles of hypothalamic transcriptomes elicited by changes in glycemic state. In other words, we aim to gain insight into a wider, key biological question: how neurons adapt their functional phenotypes in response to changing extrinsic signals. We have uncovered a critical role for the CREB co-activator CRTC2 in the hypothalamic mechanisms linking glucose-sensing with appropriate gene expression. We hypothesize that glucose-dependent CRTC2-mediated changes in hypothalamic gene expression underpin the neuronal response to adjust glycemic state. We thus aim to identify CRTC2's molecular role in regulating glucose-sensitive transcriptomes using immunohistochemical, live cell imaging and promoter-occupancy bioinformatic approaches in mice harboring a fluorescently tagged CRTC2. These experiments will give us vital clues about the mechanisms neurons employ to adapt their functional phenotypes to changes in glucose. We will then link these glucose-sensitive transcriptomes to the physiological roles they bestow on hypothalamic areas. By generating hypothalamic subpopulation-specific genetically modified mice, expressing a constitutively active CRTC2, we will determine physiological consequences of glucose-insensitive transcriptome dysregulation on glucose homeostasis.

Planned Impact

Whilst North America and Europe have experienced an increase in obesity for many years, expanded waistlines are now spreading across the globe. Due to associated co-morbidities, including type 2 diabetes and cardiovascular disease, obesity impacts negatively on quality of life and creates an impossible financial health service burden. However, our understanding of the genetic basis paving the path to an imbalance in energy intake and expenditure is incomplete. One of the challenges at the frontiers is to improve our understanding of the biology underlying the regulation of metabolic balance. The brain is a key player and this proposal outlines a program that will make important contributions to our understanding of how the brain senses nutrient state and integrates metabolic information, thereby guiding improved, future therapeutic design with significant socio-economic impact. In addition, this application investigates neuronal pathways engaged in the sensing of hypoglycemia and the correction thereof. These mechanisms can become impaired as a result of repeated hypoglycemic episodes, a potentially life-threatening condition. The glycemic threshold for counter-regulator responses shifts to lower glucose levels and iatrogenic hypoglycemia remains one of the most serious complications of insulin therapy in type 1 diabetes. An understanding of the patho-physiological molecular mechanisms through which falling glucose levels are detected and regulated is therefore key to developing therapies designed to minimize the impact of severe hypoglycemia in type 1 diabetes. The main beneficiary from our research is thus in the very long run the increasingly obese and overweight population, as well as patients with type 1 diabetes; defective neuronal nutrient-sensing being the common denominator. However incremental the novel piece of information may be, ultimately these pieces of a very large puzzle will generate an understanding of the neuronal mechanisms engaged in sensing and adjusting nutrient state. Novel therapeutic targets addressing impaired neuronal glucose sensing in recurrent hypoglycemia will significantly enhance quality of life for type 1 diabetic patients. Clearly, time scales from identification of interesting target genes to therapeutic approaches can be many decades. On a shorter time scale, the pharmaceutical industry will certainly benefit from our studies. Understanding the neuronal pathways sensing nutrient state is paramount interest to any body weight control drug hunting team. We already have ongoing collaborations with industry and will continue to foster these. On an even shorter time scale, research staff working on this project will gain significant experience in a wide range of techniques, including the sophisticated modification of gene expression in mice to model human disease, live imaging of cellular processes and in vivo physiological skills. The project would thus generate a highly skilled scientist, who can further apply their knowledge and expertise in future research projects, as well as research council and/or charity management positions. Engagement with the beneficiaries will be promoted by the PI and the post doc by continuing to work with industrial collaborators and the public. Dr. Balthasar already works with the University of Bristol Public Engagement Department and both her and the post doc will continue to actively take part in activities, such as Brain Awareness Week and the Science Festival. In addition, Dr. Balthasar is a volunteer for 'Understanding Animal Research' and regularly visits schools to talk about her research and the necessity of animals in medical research. In association with the SouthWest Science Learning Centre, she has also generated, prepared and delivered an RCUK Continuing Professional Development Course for teachers: Lifestyles and Health; Bringing Cutting-Edge Science to the Classroom. This course will continue to run in the foreseeable future.
Description ***Please note that I have been on maternity leave from 01/03/2014 until 27/10/2014***

Our programme assesses the basic machinery underlying hypothalamic glucose-sensing and corresponding adjustments to whole body glucose homeostasis in health and disease.

Key findings include:

1) Neurons within the paraventricular hypothalamus are glucose-sensitive. Using neuronal sub-population-specific genetically modified mice and electrophysiological techniques, we have been able to demonstrate that a specific subpopulation of neurons within the paraventricular hypothalamus respond to changes in extracellular glucose levels by altering their firing activity.
2) Maternal diet programmes offspring glucose homeostasis. By feeding lean mouse dams a high-fat/high-sugar diet during pregnancy and lactation only, we have demonstrated that adult offspring glucose homeostasis dysregulation is programmed in utero by maternal diet. Female offspring is particularly affected, potentially by diet-mediated misprogramming of paraventricular hypothalamus genes involved in the maintenance of glucose homeostasis.
3) We have developed a technique for genome-wide analysis of diet-mediated epigenetic modifications within the paraventricular hypothalamus. This method to perform chromatin immunoprecipitation, followed by next generation sequencing and bioinformatic analysis will be invaluable to researchers wishing to analyze epigenetic modifications in small subareas of the CNS.
4) Diet-induced obesity activates a specific epigenetic programme to modify the synaptic landscape within the paraventricular hypothalamus. Using CHIP Seq methods described above we identified a specific epigenetic programme that modifies synaptic transmission within the paraventricular hypothalamus downstream of diet-induced obesity.
Exploitation Route We are in the process of uploading our raw ChIP Seq data on an open access database managed by the University of Bristol , from where it can be mined by other researchers.
In addition, identifying neuronal pathways that are dysregulated by maternal diet or long-term high-fat diet feeding has the potential to uncover novel therapeutic targets and guide improved, future therapeutic design in diabetes and may thus be of interest to the therapeutics industry.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

Description To ensure beneficiaries in the Scientific and Health Research Community have an opportunity to learn of our results we have : - published our findings in high profile, open access (where possible) scientific journals so that our findings are readily accessible and will continue to do so. - communicated our findings at the appropriate national and international meetings, that are open to basic researchers and clinicians alike (e.g. specialized Keystone Symposia, Society for Neuroscience Annual Meeting). - encouraged further collaborations with industry, clinicians and epidemiologists with the aim of translating our work into studies on humans. To ensure Public Audiences have an opportunity to learn of our results we have : - Participated in e.g. Brain Awareness Week and Festival of Science events. - Interacted closely with the University of Bristol Centre for Public Engagement. - Ensured that the impact of our research reaches as wide a public audience as possible by providing press releases on our published findings through the University of Bristol's Press Office.
First Year Of Impact 2013
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

Description EBI Clinical Primer
Amount £29,500 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Institutional Strategic Support Fund
Sector Charity/Non Profit
Country United Kingdom
Start 07/2015 
End 02/2016
Description Lister Institute of Preventive Medicine Summer Studentship
Amount £2,000 (GBP)
Organisation Lister Institute of Preventive Medicine 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2015 
End 09/2015
Description Pilot Project
Amount £45,434 (GBP)
Funding ID ARUK-PPG2013B-10 
Organisation Alzheimer's Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2013 
End 02/2015
Title ChIpSeq sample prep 
Description generation of good quality DNA libraries for ChIP Seq experiments from mouse CNS areas. 
Type Of Material Biological samples 
Year Produced 2013 
Provided To Others? Yes  
Impact reduction in the number of mice needed per experiment 
Title genetically modified mouse models 
Description genetically modified mice expressing a dominant negative or fluorescently tagged version of CRTC2 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact these mouse models allow the study of CRTC2 function in vivo and in vitro 
Title ChIP Seq DIO mouse PVH data 
Description genome-wide analysis of epigenetic alterations in paraventricular hypothalamus genes downstream of diet-induced obesity 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact we are in the process of making this data available to other researchers via UoB-managed open-access database. once this has been accomplished, this data will be mineable by all researchers with an interest in epigenetic programming and/or diet-induced obesity 
Description Bristol Neuroscience Festival 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Talk sparked questions and discussion from lay audience. Talk caused public to engage with other events of the festival and thereby informed about Bristol Neuroscience research.

After my talk the public were inspired to engage with other activities of the Bristol Neuroscience festival.
Year(s) Of Engagement Activity 2013
URL http://www.bristol.ac.uk/neuroscience/events/diary/2013/101365.html
Description South Western Obstetrical and Gynaecological Society Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Talk sparked questions and discussions over lunch.

Increased engagement of clinicians with our basic research.
Year(s) Of Engagement Activity 2014
URL http://swogs.org.uk/