bioRxiv preprint doi: https://doi.org/10.1101/2020.01.30.927426; this version posted August 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Arginine-vasopressin mediates counter- 2 regulatory glucagon release and is 3 diminished in type 1 diabetes 4 Angela Kim1,2, Jakob G. Knudsen3,4, Joseph C. Madara1, Anna Benrick5, Thomas Hill3, 5 Lina Abdul Kadir3, Joely A. Kellard3, Lisa Mellander5, Caroline Miranda5, Haopeng 6 Lin6, Timothy James7, Kinga Suba8, Aliya F. Spigelman6, Yanling Wu5, Patrick E. 7 MacDonald6, Ingrid Wernstedt Asterholm5, Tore Magnussen9, Mikkel Christensen9,10,11, 8 Tina Visboll9,10,12, Victoria Salem8, Filip K. Knop9,10,12,13, Patrik Rorsman3,5, Bradford 9 B. Lowell1,2, Linford J.B. Briant3,14,* 10 11 Affiliations 12 1Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical 13 Center, Boston, MA 02215, USA. 2Program in Neuroscience, Harvard Medical School, 14 Boston, MA 02115, USA. 3Oxford Centre for Diabetes, Endocrinology and Metabolism, 15 Radcliffe Department of Medicine, University of Oxford, Oxford, OX4 7LE, UK. 4Section 16 for Cell Biology and Physiology, Department of Biology, University of Copenhagen. 17 5Institute of Neuroscience and Physiology, Metabolic Research Unit, University of Göteborg, 18 405 30, Göteborg, Sweden. 6Alberta Diabetes Institute, 6-126 Li Ka Shing Centre for Health 19 Research Innovation, Edmonton, Alberta, T6G 2E1, Canada. 7Department of Clinical 20 Biochemistry, John Radcliffe, Oxford NHS Trust, OX3 9DU, Oxford, UK. 8Section of Cell 21 Biology and Functional Genomics, Department of Metabolism, Digestion and Reproduction, 22 Imperial College London, W12 0NN, UK. 9Center for Clinical Metabolic Research, Gentofte 23 Hospital, Kildegårdsvej 28, DK-2900 Hellerup, Denmark. 10Department of Clinical pg. 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.30.927426; this version posted August 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 24 Pharmacology, Bispebjerg Hospital, University of Copenhagen, DK-2400 Copenhagen, 25 11Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of 26 Copenhagen, Copenhagen, Denmark. 12Steno Diabetes Center Copenhagen, DK-2820 27 Gentofte, Copenhagen, Denmark. 13Novo Nordisk Foundation Center for Basic Metabolic 28 Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, 29 Denmark.14Department of Computer Science, University of Oxford, Oxford, OX1 3QD, UK. 30 *Corresponding author: Linford J.B. Briant, [email protected] pg. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.30.927426; this version posted August 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 31 Abstract 32 Insulin-induced hypoglycemia is a major barrier to the treatment of type-1 diabetes (T1D). 33 Accordingly, it is important that we understand the mechanisms regulating the circulating 34 levels of glucagon – the body’s principal blood glucose-elevating hormone which is secreted 35 from alpha-cells of the pancreatic islets. Varying glucose over the range of concentrations 36 that occur physiologically between the fed and fuel-deprived states (from 8 to 4 mM) has no 37 significant effect on glucagon secretion in isolated islets (in vitro) and yet associates with 38 dramatic changes in plasma glucagon in vivo. The identity of the systemic factor(s) that 39 stimulates glucagon secretion remains unknown. Here, we show that arginine-vasopressin 40 (AVP), secreted from the posterior pituitary, stimulates glucagon secretion. Glucagon- 41 secreting alpha-cells express high levels of the vasopressin 1b receptor gene (Avpr1b). 42 Activation of AVP neurons in vivo increased circulating copeptin (the C-terminal segment of 43 the AVP precursor peptide, a stable surrogate marker of AVP) and increased blood glucose; 44 effects blocked by pharmacological antagonism of either the glucagon receptor or 45 vasopressin 1b receptor. AVP also mediates the stimulatory effects of hypoglycemia 46 produced by exogenous insulin and 2-deoxy-D-glucose on glucagon secretion. We show that 47 the A1/C1 neurons of the medulla oblongata drive AVP neuron activation in response to 48 insulin-induced hypoglycemia. Exogenous injection of AVP in vivo increased cytoplasmic 49 Ca2+ in alpha-cells (implanted into the anterior chamber of the eye) and glucagon release. 50 Hypoglycemia also increases circulating levels of AVP in humans and this hormone 51 stimulates glucagon secretion from isolated human islets. In patients with T1D, 52 hypoglycemia failed to increase both plasma copeptin and glucagon levels. These findings 53 suggest that AVP is a physiological systemic regulator of glucagon secretion and that this 54 mechanism becomes impaired in T1D. pg. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.30.927426; this version posted August 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 55 Introduction 56 Glucagon is secreted from alpha-cells of the pancreatic islets and has many physiological 57 actions; most notably the potent stimulation of hepatic glucose production to restore 58 euglycemia when blood glucose has fallen below the normal range (a process referred to as 59 counter-regulation). The importance of glucagon for glucose homeostasis is well established 60 (1). In both type-1 diabetes (T1D) and type-2 diabetes (T2D), hyperglycemia results from a 61 combination of complete/partial loss of insulin secretion and over-secretion of glucagon (2). 62 In addition, counter-regulatory glucagon secretion becomes impaired in both forms of 63 diabetes (particularly T1D), which may result in life-threatening hypoglycemia (3). Despite 64 the centrality of glucagon to diabetes etiology, there remains considerable uncertainty about 65 the regulation of its release and the relative importance of intra-islet effects and systemic 66 factors (4, 5). Based on observations in isolated (ex vivo) islets, hypoglycemia has been 67 postulated to stimulate glucagon secretion via intrinsic (6-8) and/or paracrine mechanisms (9, 68 10). While it is indisputable that the islet is a critical component of the body’s ‘glucostat’ (11) 69 and has the ability to intrinsically modulate glucagon output, it is clear that such an ‘islet- 70 centric’ viewpoint is overly simplistic (12). Indeed, many studies have clearly demonstrated 71 that brain-directed interventions can profoundly alter islet alpha-cell function, with glucose- 72 sensing neurons in the hypothalamus being key mediators (13-16). This ability of the brain to 73 modulate glucagon secretion is commonly attributed to autonomic innervation of the 74 pancreas (14, 17, 18). However, glucagon secretion is not only restored in pancreas 75 transplantation patients but also insensitive to adrenergic blockade (19, 20), suggesting that 76 other (non-autonomic) central mechanisms may also regulate glucagon secretion in vivo. 77 78 Arginine-vasopressin (AVP) is a hormone synthesised in the hypothalamus (reviewed 79 in (21)). AVP neurons are divided into two classes: parvocellular AVP neurons (which either pg. 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.30.927426; this version posted August 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 80 project to the median eminence to stimulate ACTH and glucocorticoid release or project 81 centrally to other brain regions) and magnocellular AVP neurons (which are the main 82 contributors to circulating levels of AVP). The parvocellular neurons reside solely in the 83 paraventricular nucleus of the hypothalamus (PVH), whereas magnocellular neurons are 84 found in both the PVH and supraoptic nucleus (SON). Stimulation of the magnocellular AVP 85 neurons causes release of AVP into the posterior pituitary, where it enters the peripheral 86 circulation. 87 88 Under normal conditions, Avpr1b is one of the most enriched transcripts in alpha-cells 89 from both mice and humans (22, 23). This raises the possibility that AVP may be an 90 important regulator of glucagon secretion under physiological conditions. Indeed, the ability 91 of exogenous AVP and AVP analogues to potently stimulate glucagon secretion ex vivo has 92 been known for some time (24). However, whether circulating AVP contributes to 93 physiological counter-regulatory glucagon release and how this regulation is affected in 94 diabetes remains unknown. 95 96 Here, we have investigated the regulation of glucagon by circulating AVP. We first 97 explored the role of AVP during hypoglycemia, a potent stimulus of glucagon secretion. 98 Next, we explored the link between AVP and glucagon in humans and provide