Hypothalamic glucose-sensitive transcriptomes.

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.

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.

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.