Dynamic integration of ingestive behaviours and homeostasis by hypothalamo-neurohypophysial system glucagon like peptide 1 receptors

Obesity and its co-morbidities, such as diabetes, hypertension and COVID-19, can significantly reduce a person's quality of life and place huge pressure on healthcare resources. When we eat a meal our gut and brain release hormones to control the amount of food and fluid we ingest to prevent overeating. One of these hormones is called glucagon-like peptide 1 (GLP-1) and is released from intestinal cells in response to food intake, but also produced and released in the brain. Drug analogues of GLP-1 are already in use in the clinic to treat both diabetes and obesity. The aim of this program of work is to obtain fundamental knowledge about gut peptide signalling in the brain to see how these drug treatments affect brain functions.

To understand the actions of GLP-1 in the brain and periphery it is imperative to know where its receptor (GLP-1R) is located and how it functions. The GLP-1R is abundantly expressed in a part of the brain called the hypothalamus, specifically a region called the supraoptic nucleus (SON), which makes and secretes the hormones arginine vasopressin (AVP) and oxytocin (OXT) locally and from the pituitary gland. The most recognised roles of AVP in the periphery are the regulation body fluid and cardiovascular homeostasis and recent evidence suggests the control of blood glucose. OXT is best known for its roles in lactation and parturition and in the media it is known as the "love hormone". OXT also regulates renal sodium excretion, glucose and insulin homeostasis, gastric motility, eating behaviours, lipid metabolism, and bone formation, and within the brain it promotes satiety. We found that water deprivation and lactation, stimuli that that promote AVP and OXT secretion into the bloodstream, dramatically increase the expression of the GLP-1 receptor in the SON.

To identify sources of GLP-1 that target receptors in the SON, we will inject viruses into the SON expressing fluorescent proteins to trace neuronal connections with other areas of the brain. We will use fluorescently labelled probes to identify neurones that make GLP-1 and connect with the SON. We will then deliver specific viral tools switch off these cells to alter the supply of GLP-1 reaching the SON. In a second series of experiments, we will reduce the number of GLP-1 receptors in neurones of the SON, again using viruses to deliver genetic tools to decrease production of GLP-1 receptors by SON neurones. We will then feed these rats meals at a specific time of day (schedule-feeding) to investigate roles in feeding. In a third series of experiments, we will investigate hormone release from isolated SONs and pituitary glands using state-of-the-art biosensors. In a fourth series of experiments, we will investigate the pharmacological actions of GLP-1 using a selective receptor agonist called liraglutide, a drug that is approved for diabetes and obesity treatment in humans. To fill a gap in our knowledge about the effect of liraglutide on AVP and OXT release in humans we aim to perform a human clinical trial. In all animal studies we will measure food and water intake, hormone release, and alterations to cell functions, to better understand the physiological and pharmacological roles mediated by this receptor in the SON in relation to feeding.

Understanding GLP-1 receptors is essential for our knowledge of current treatments of diabetes and obesity that use stable peptide analogues in humans. Our data will advise about the functions of SON GLP-1 receptors which may influence treatment regimens for patients with diabetes who are at serious risk of dehydration. This is a particular problem for the elderly. This data may inform about some of the many undesired side effects of GLP-1 receptor treatments and as a result, these findings may help with design and development of new drugs for more targeted methods of activating receptors at specific sites, and thus influence current therapeutic strategies.

This proposal aims to characterise the exact physiological roles of gut peptide receptor GLP-1R in the hypothalamo-neurohypophysial system (HNS) of female and male rats. We will use adeno-associated viruses (AAVs) delivered by stereotaxic surgery to the rat brain. The AAV retrograde capsid variant, AAV2-retro, enables retrograde access to projection neurones including nucleus tractus solitarius (NTS) to supraoptic nucleus (SON) projections. We will firstly determine the neuropeptide identities of retrograde labelled NTS neurones projecting to the SON by RNAscope. We will inject AAV2-retro-Cre into the SON which will retrogradely trace back to mark NTS connecting neurones. We will deliver a separate Cre-dependent AAV directly to the NTS in the same surgery, for example, AAV FLEx system shRNA targeting the preproglucagon mRNA. In all studies we will investigate, feeding and drinking, hormone release, and cell signalling pathways. We will knockdown SON Glprs using AAV-shRNAs and investigate postprandial responses in a fasting-refed rats as well as schedule-fed rats to identify effects on feeding circuits.

We will use state-of-the-art biosensor cells to precisely monitor the real-time release of vasopressin and oxytocin in SON and posterior pituitary explant cultures maintained in a dish. We will investigate the short-term and longer-term consequences of daily liraglutide treatment on the rat SON and pituitary gland by exploring proteome-wide phosphorylation changes by phosphoproteomics. We further plan to recruit healthy human volunteers (male and female) as part of a randomized crossover trial to determine the short-term actions of liraglutide on HNS hormone release.

This project will inform about how the HNS integrates responses from food and fluid intake to maintain homeostasis. These data may help with design and development of new drugs for more targeted methods of activating receptors at specific sites, and thus influence therapeutic strategies.