1128 Diabetes Volume 67, June 2018 Adrenaline Stimulates Glucagon Secretion by Tpc2- Dependent Ca2+ Mobilization From Acidic Stores in Pancreatic a-Cells Alexander Hamilton,1 Quan Zhang,1 Albert Salehi,2 Mara Willems,1 Jakob G. Knudsen,1 Anna K. Ringgaard,3,4 Caroline E. Chapman,1 Alejandro Gonzalez-Alvarez,1 Nicoletta C. Surdo,5 Manuela Zaccolo,5 Davide Basco,6 Paul R.V. Johnson,1,7 Reshma Ramracheya,1 Guy A. Rutter,8 Antony Galione,9 Patrik Rorsman,1,2,7 and Andrei I. Tarasov1,7 Diabetes 2018;67:1128–1139 | https://doi.org/10.2337/db17-1102 Adrenaline is a powerful stimulus of glucagon secretion. It The ability of the “fight-or-flight” hormone adrenaline to acts by activation of b-adrenergic receptors, but the down- increase plasma glucose levels by stimulating liver gluconeo- stream mechanisms have only been partially elucidated. genesis is in part mediated by glucagon, the body’sprincipal Here, we have examined the effects of adrenaline in mouse hyperglycemic hormone (1). Glucagon is secreted by the and human a-cells by a combination of electrophysiology, a-cells of the pancreas (2). Reduced autonomic stimulation 2+ imaging of Ca and PKA activity, and hormone release of glucagon secretion may result in hypoglycemia, a serious measurements. We found that stimulation of glucagon and potentially fatal complication of diabetes (3). It has been secretion correlated with a PKA- and EPAC2-dependent estimated that up to 10% of insulin-treated patients die of (inhibited by PKI and ESI-05, respectively) elevation of hypoglycemia (4). Understanding the mechanism by which [Ca2+] in a-cells, which occurred without stimulation of i adrenaline stimulates glucagon secretion and how it becomes ISLET STUDIES electrical activity and persisted in the absence of extracel- perturbed in patients with diabetes is therefore essential. lular Ca2+ but was sensitive to ryanodine, bafilomycin, and The mechanism by which adrenaline stimulates glucagon thapsigargin. Adrenaline also increased [Ca2+] in a-cells in i secretion has only partially been elucidated. It is clear, how- human islets. Genetic or pharmacological inhibition of the b Tpc2 channel (that mediates Ca2+ release from acidic intra- ever, that it involves activation of -adrenergic receptors – cellular stores) abolished the stimulatory effect of adren- (5 7), leading to elevated intracellular cAMP ([cAMP]i), and 2+ aline on glucagon secretion and reduced the elevation of culminates in elevation of the cytoplasmic free Ca concen- 2+ 2+ 2+ tration ([Ca ] )withresultantCa -dependent stimulation [Ca ]i. Furthermore, in Tpc2-deficient islets, ryanodine i exerted no additive inhibitory effect. These data suggest of glucagon secretion (6,8,9). However, our understanding that b-adrenergic stimulation of glucagon secretion is con- of the spatiotemporal interrelationship between different 2+ 2+ a trolled by a hierarchy of [Ca ]i signaling in the a-cell that is intracellular Ca stores involved in -cell adrenaline signal- initiated by cAMP-induced Tpc2-dependent Ca2+ release ing remains patchy. from the acidic stores and further amplified by Ca2+-induced Here, we have explored how adrenaline triggers glucagon Ca2+ release from the sarco/endoplasmic reticulum. release by a combination of hormone secretion measurements, 1Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, 8Section of Cell Biology and Functional Genomics, Department of Medicine, University of Oxford, Headington, U.K. Imperial College London, London, U.K. 2Institute of Neuroscience of Physiology, Department of Physiology, Metabolic 9Department of Pharmacology, University of Oxford, Oxford, U.K. Research Unit, University of Göteborg, Göteborg, Sweden Corresponding author: Andrei I. Tarasov, [email protected], or 3 Diabetes Research, Department of Stem Cell Biology, Novo Nordisk A/S, Måløv, Patrik Rorsman, [email protected]. Denmark Received 15 September 2017 and accepted 14 March 2018. 4Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark This article contains Supplementary Data online at http://diabetes 5Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, U.K. .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-1102/-/DC1. 6Center for Integrative Genomics, Université de Lausanne, Lausanne, Switzerland © 2018 by the American Diabetes Association. Readers may use this article as 7Oxford National Institute for Health Research, Biomedical Research Centre, Oxford, long as the work is properly cited, the use is educational and not for profit, and the U.K. work is not altered. More information is available at http://www.diabetesjournals .org/content/license. diabetes.diabetesjournals.org Hamilton and Associates 1129 electrophysiology, and imaging of cytoplasmic Ca2+,cAMP, Pancreatic islets were isolated from mice by injecting and PKA activity in a-cells within intact mouse pancreatic collagenase solution into the bile duct, with subsequent islets. We have extended the measurements to human islets digestion of the connective and exocrine pancreatic tissue. and also tested how the responsiveness to adrenaline is Human pancreatic islets were isolated at the Oxford Di- affected when islets are cultured under hyperglycemic con- abetes Research & Wellness Foundation Human Islet Isola- ditions to establish whether and how this regulation becomes tion Facility according to published protocols (12,13). impaired in diabetes. Our data indicate that Ca2+ release from Unless otherwise stated, experiments were performed on acidic (lysosomal) stores plays a critical and unexpected role acutely isolated islets. For experiments aiming to emulate in adrenaline-induced glucagon secretion. chronic hyperglycemia, islets were cultured for 48 h in RPMI medium containing 30 mmol/L or the standard 11 mmol/L RESEARCH DESIGN AND METHODS glucose, supplemented with 10% FBS, 100 IU/mL penicillin, and 100 mg/mL streptomycin (all reagents from Life Tech- Chemicals nologies, Paisley, U.K.). Recombinant sensors were deliv- The following substances were used (source given in ered via adenoviral (GCaMP6f, AKAR3) or BacMam (cADDis) parentheses): the L-type Ca2+ channel blocker isradipine vectors at 105 infectious units per islet, followed by 24–36 h and the P/Q Ca2+ channel blocker v-agatoxin (Alomone culturing at 11 mmol/L glucose (as described above) to Laboratories, Jerusalem, Israel); the a -antagonist prazosin 1 express the sensors. (Abcam, Cambridge, U.K.); the membrane-permeable PKA inhibitor myr-PKI, the inositol triphosphate receptor in- 2+ Imaging of [Ca ]i, cAMP, and PKA Activity in Islet Cells hibitor Xestospongin C, the nicotinic acid adenine dinucle- 2+ Time-lapse imaging of [Ca ]i in intact freshly isolated mouse otide phosphate (NAADP) antagonist Ned-19, the V-ATPase islets was performed on an inverted Zeiss AxioVert 200 mi- fi inhibitor ba lomycin, the sarco/endoplasmic reticulum croscope equipped with the Zeiss 510-META laser confocal (sER) ATPase inhibitor thapsigargin, and noradrenaline scanning system. Prior to Ca2+ imaging, mouse islets were (Tocris Bioscience, Bristol, U.K.); the EPAC2 inhibitor ESI- loaded with 6 mmol/L Fluo-4 at room temperature or 05 (BioLog, Bremen, Germany); and the insulin receptor 10 mmol/L Fluo4FF (Molecular Probes) at 37°C for 90 min. antagonist (S961; Novo Nordisk, Måløv, Denmark). All other Both dyes were excited at 488 nm and emission was collected compounds were obtained from Sigma-Aldrich (Dorset, at 530 nm, using the 512 3 512 frame scanning mode with U.K.). the pixel dwell time of 6 ms (image frequency: 0.25 Hz). Time- 2+ Solutions lapse imaging of [Ca ]i in groups of mouse islets was done on The extracellular solution (EC1) used for imaging and an Axiozoom.V16 microscope (0.1 Hz) using GCaMP6f (14). current-clamp electrophysiology (Fig. 2C)experiments PKA activity was reported in pancreatic islet cells using fl contained (in millimoles per liter ) 140 NaCl, 4.6 KCl, 2.6 recombinant uorescence resonance energy transfer (FRET) probe AKAR3 (15). AKAR3 CFP fluorescence was excited at CaCl2,1.2MgCl2,1NaH2PO4,5NaHCO3,and10HEPES (pH 7.4 with NaOH). The pipette solution (IC1) consisted of 458 nm using an Axiozoom.V16 microscope, which has been shown previously to excite CFP selectively (16); the emitted 76 K2SO4, 10 NaCl, 10 KCl, 1 MgCl2, and 5 HEPES (pH 7.35 with KOH). The extracellular solution (EC2) for recordings of light was collected at 485 nm (CFP) and 515 nm (YFP) the depolarization-induced exocytosis (Fig. 3C and D) con- (image frequency: 0.0 Hz). Time-lapse imaging of [cAMP]i was performed using a recombinant Green Downward cADDis tained 118 NaCl, 5.6 KCl, 2.6 CaCl2,1.2MgCl2,5HEPES, 20 TEA-Cl, and 6 glucose (pH 7.4 with NaOH). In the latter sensor (Montana Molecular, Bozeman, MT). The cADDis fl experiments, the pipette-filling medium (IC2) consisted of uorescence was excited at 485 nm and the emission recorded at 515 nm using an Axiozoom.V16 microscope. 76 Cs2SO4, 10 NaCl, 10 KCl, 1 MgCl2, and 5 HEPES (pH 7.35 with CsOH). PKA and cAMP were imaged at a frequency of 16 mHz. All imaging at 34°C was performed using an open cham- Animals, Islet Isolation, and Culture ber. The bath solution containing various stimuli was peri- NMRI mice (Charles River) were used throughout the fused continuously at a rate of 200 mL/min. In experiments study except for the experiments involving the genetic involving PKI, ESI-05, ryanodine, Xestospongin C, Ned-19 2/2 manipulation of TPC1 and -2 (Fig. 5). Tpcn1 and thapsigargin, and the Ca2+ channel blockers v-agatoxin and 2/2 Tpcn2 mice were developed on C57Bl/6N background isradipine, cells were preincubated in the solution of the 2/2 as previously described (10). Cd38 mice were developed respective agent for 90 min. For the bafilomycin experiments, on C57Bl/6J background (11). Age- and sex-matched the preincubation time was 20 min. By preincubating the islets C57Bl/6N and C57Bl/6J wild-type animals were used as in the agonist/antagonist solutions, we aimed to reach the controls. Mice were kept in a conventional vivarium with saturation of the effect by the start of the recording.