A glucose-responsive network

It is estimated that 4.5 million people in the UK suffer with diabetes and many of these patients regulate their blood-sugar levels by taking insulin. A major problem with this treatment is that is can lead to repeated bouts of low glucose or "hypos." This is when there is too little glucose in the bloodstream (hypoglycaemia), which can cause serious problems. For example, a patient might start feeling agitated or dizzy, and may even collapse unconscious. These feelings stimulate the body to release natural, protective hormones and alert the patient to eat something sugary. Unfortunately, diabetic patients taking insulin may suffer from bouts of hypoglycaemia so regularly that they start to become unaware of the warning signs, increasing the probability that they may collapse and even fall into a coma. If we are to help these patients in the future, it is critical that we understand how the brain normally detects and responds to hypoglycaemia. We have made the important discovery of a specific type of brain cell, which contains a chemical signal called PACAP. These cells can sense low sugar levels and initiate a series of physiological responses. We have mapped the other parts of the brain with which PACAP cells connect and, importantly, each of these areas has previously been implicated in the brain's response to hypoglycaemia. Thus, together they initiate the release of the protective hormones or produce a change in behaviour (e.g. cause hunger and sugar seeking). Critically, our discovery has provided us with a key to "open up" the brain and understand the complex electrical circuits which sense and respond to hypoglycaemia. Our experiments allow us to make PACAP cells shine under fluorescent light. This means we can cut slices of mouse brain from dead animals and record specifically the electrical activity of PACAP cells. This shows us exactly how PACAP cells respond to low sugar levels and how they then send this information to the other brain areas. Similar techniques also allow us to activate PACAP neurones in living mice, either by shining a light into their brains through an optic fibre or by giving the mice a special "designer" drug by an injection under their skin. The mice do not feel anything unusual, but by activating PACAP cells or other specific cell types in the brain (as happens naturally with hypoglycaemia), we will map the circuits which control the release of protective hormones and cause awareness of hypoglycaemia. By carefully profiling PACAP and other brain cells, we should be able to identify additional key cell types and fit them into our glucose-sensing network. Thus, we will be able to build up a picture of exactly how the brain senses and responds to hypoglycaemia. By understanding the complex circuits in this network, we should begin to understand why patients lose their awareness of hypoglycaemia after long-term insulin treatment. We hope to be able to suggest new ways of detecting and preventing impaired awareness of hypoglycaemia and, thus, improve the safety of diabetic patients.

The brain protects itself from low glucose levels by initiating a hierarchy of endocrine and behavioural counter-regulatory responses (CRR). This is particularly important for diabetic patients on insulin, who often experience hypoglycaemic events. Repeated "hypos" can lead to an impaired CRR and patients becoming unaware of their symptoms. Impaired awareness of hypoglycaemia is the major barrier preventing successful glycaemic control in diabetic patients and, therefore, it is critical that we understand how the brain normally senses and responds to hypoglycaemia. As with other systems regulating homeostatic function, the discovery of a distinct, primary-sensing neurone in a key brain region is often the catalyst to unravelling complex circuits (viz Agrp neurones and appetite or orexin neurones and sleep/wakefulness). We have discovered such a neurone: the PACAP-containing cell in the ventromedial hypothalamic nucleus, which is intrinsically sensitive to reduced glucose. We found that PACAPVMH neurones are "glucose-inhibited", respond to other relevant inputs and project to areas of the brain, each of which has been implicated in different CRR. We will confirm functional projections using channel-rhodopsin-assisted circuit mapping (CRACM) and cre-sensitive designer receptors (opto- and chemo-genetics). By mapping efferent outputs, as well as afferent inputs from other glucose-sensitive brain regions, we will demonstrate that PACAPVMH neurones are part of a robust and redundant glucose-responsive network. We will also use transcriptomic analysis of these and other VMH cells (based on their projections and physiological responses) to identify separate sensing and integratory neurones (such as "glucose-excited" or "presynaptically-excited" neurones). The network will be analysed by CRACM to dissect specific core pathways controlling different endocrine and behavioural CRR to hypoglycaemia.

Diabetic patients who take insulin can suffer from repeated bouts of low glucose, so eventually they may no longer recognise when they are experiencing a hypoglycaemic event. Impaired awareness of hypoglycaemia (IAH) can be extremely dangerous if undiagnosed and is seen as being the major barrier preventing successful glycaemic control. IAH affects approximately 25% of patients with T1DM, increases the risk of severe hypoglycaemia six-fold, and contributes to serious morbidity. Furthermore, T2DM is increasing rapidly with the burgeoning obesity epidemic and, since primary interventions of lifestyle changes or insulin-sensitising drugs can have limited success, patients with this condition often proceed onto insulin replacement. Thus, IAH is becoming more prevalent, so it is critical that we understand how patients normally respond to low glucose and why this regulation loses efficacy with repeat iatrogenic hypoglycaemia. Our laboratories are well placed to make a major impact on understanding the brain network senses and responds to hypoglycaemia, as we have available the necessary models, tools and expertise. Our findings will be disseminated to our academic and clinical colleagues at international conferences and by publication in high-impact journals during the grant's duration.
In 2015, over three million people in the UK were diagnosed with T2DM and a further 350,000 with T1DM. Currently about 10% of the National Health Service budget is spent on diabetes. The potential global market for diabetes drugs is over $100 billion. This project will provide underpinning knowledge which, in the future, more lead to the development of new interventions to prevent IAH. The PI has been involved previously in successful collaborative projects with a number of industrial partners, providing evidence for several novel targets for drug development that has underpinned programmes by AstraZeneca, Eli Lilly, Servier and Novo Nordisk. For example, the PI has previously been led on three Industrial Partnership Awards. Any intellectual property derived from this project will belong to the applicant and The University of Manchester. Any future contract negotiations will be carried out through the University's Intellectual Property company, UMI3 Ltd.
The PI will continue to provide consultations to different bodies regarding metabolic research (in the past with commercial companies, funding agencies, The Royal Society and with The Department for Business, Innovation and Skills), which will affect future funding policy. During the lifetime of the grant, the basic research will be discussed at meetings organised by the Child Health Research Network, the Diabetes and Obesity Research Network and the Association for the Study of Obesity. These annual meetings are forums for basic researchers, psychologists, clinicians, community nurses and other health professionals, patient group representatives and policy makers.
This project will provide strong training in in vivo skills and specialist techniques in electrophysiology, chemogenetics, optogenetics, behaviour and molecular profiling. In the last twelve years, the applicant has supervised 12 PhD students, 21 Masters students and 10 PDRAs, the majority of whom have remained in science (some have their own independent research groups and others have moved into the commercial sector). The applicant is external examiner on another University's integrative Masters course and regularly examines PhD theses. He directs a cross-University Integrative Mammalian Biology initiative to promote and expand research and training in in vivo biology. This problematic area is crucial to the UK economy and to the ambitions of Manchester to be a world-leading university.