Mechanism and effects of pulsatile GABA secretion from cytosolic pools in the human beta cell

Menegaz, Danusa; Hagan, D. Walker; Almaça, Joana; Cianciaruso, Chiara; Rodriguez-Diaz, Rayner; Molina, Judith; Dolan, Robert M.; Becker, Matthew W.; Schwalie, Petra C; Nano, Rita; Lebreton, Fanny; kang, Chen; Sah, Rajan; Gaisano, Herbert Y.; Berggren, Per-Olof; Baekkeskov, Steinunn; Caicedo, Alejandro; Phelps, Edward A.

doi:10.1038/s42255-019-0135-7

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Mechanism and effects of pulsatile GABA secretion from cytosolic pools in the human beta cell

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Author(s): Danusa Menegaz ¹ ^, ¹⁶ , D. Walker Hagan ² ^, ¹⁶ , Joana Almaça ¹ , Chiara Cianciaruso ³ , Rayner Rodriguez-Diaz ¹ , Judith Molina ¹ , Robert M. Dolan ² , Matthew W. Becker ² , Petra C. Schwalie ³ ^, ⁴ , Rita Nano ⁵ , Fanny Lebreton ⁶ , Chen Kang ⁷ ^, ⁸ , Rajan Sah ⁷ ^, ⁸ , Herbert Y. Gaisano ⁹ , Per-Olof Berggren ¹⁰ ^, ¹¹ ^, ¹² , Steinunn Baekkeskov ³ ^, ¹³ ^, ^* , Alejandro Caicedo ¹ ^, ¹⁰ ^, ¹⁴ ^, ¹⁵ ^, ^* , Edward A. Phelps ² ^, ³ ^, ^*

Publication date (Electronic): 15 November 2019

Journal: Nature metabolism

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Abstract

Pancreatic beta cells synthesize and secrete the neurotransmitter γ-aminobutyric acid (GABA) as a paracrine and autocrine signal to help regulate hormone secretion and islet homeostasis. Islet GABA release has classically been described as a secretory vesicle-mediated event. Yet, a limitation of the hypothesized vesicular GABA release from islets is the lack of expression of a vesicular GABA transporter in beta cells. Consequentially, GABA accumulates in the cytosol. Here we provide evidence that the human beta cell effluxes GABA from a cytosolic pool in a pulsatile manner, imposing a synchronizing rhythm on pulsatile insulin secretion. The volume regulatory anion channel (VRAC), functionally encoded by LRRC8A or Swell1, is critical for pulsatile GABA secretion. GABA content in beta cells is depleted and secretion is disrupted in islets from type 1 and type 2 diabetic patients, suggesting that loss of GABA as a synchronizing signal for hormone output may correlate with diabetes pathogenesis.

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Most cited references 103

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A Single-Cell Transcriptomic Map of the Human and Mouse Pancreas Reveals Inter- and Intra-cell Population Structure.

Maayan Baron, Adrian Veres, Samuel Wolock … (2016)

Although the function of the mammalian pancreas hinges on complex interactions of distinct cell types, gene expression profiles have primarily been described with bulk mixtures. Here we implemented a droplet-based, single-cell RNA-seq method to determine the transcriptomes of over 12,000 individual pancreatic cells from four human donors and two mouse strains. Cells could be divided into 15 clusters that matched previously characterized cell types: all endocrine cell types, including rare epsilon-cells; exocrine cell types; vascular cells; Schwann cells; quiescent and activated stellate cells; and four types of immune cells. We detected subpopulations of ductal cells with distinct expression profiles and validated their existence with immuno-histochemistry stains. Moreover, among human beta- cells, we detected heterogeneity in the regulation of genes relating to functional maturation and levels of ER stress. Finally, we deconvolved bulk gene expression samples using the single-cell data to detect disease-associated differential expression. Our dataset provides a resource for the discovery of novel cell type-specific transcription factors, signaling receptors, and medically relevant genes.

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SWELL1, a plasma membrane protein, is an essential component of volume-regulated anion channel.

Zhaozhu Qiu, Adrienne E. Dubin, Jayanti Mathur … (2014)

Maintenance of a constant cell volume in response to extracellular or intracellular osmotic changes is critical for cellular homeostasis. Activation of a ubiquitous volume-regulated anion channel (VRAC) plays a key role in this process; however, its molecular identity in vertebrates remains unknown. Here, we used a cell-based fluorescence assay and performed a genome-wide RNAi screen to find components of VRAC. We identified SWELL1 (LRRC8A), a member of a four-transmembrane protein family with unknown function, as essential for hypotonicity-induced iodide influx. SWELL1 is localized to the plasma membrane, and its knockdown dramatically reduces endogenous VRAC currents and regulatory cell volume decrease in various cell types. Furthermore, point mutations in SWELL1 cause a significant change in VRAC anion selectivity, demonstrating that SWELL1 is an essential VRAC component. These findings enable further molecular characterization of the VRAC channel complex and genetic studies for understanding the function of VRAC in normal physiology and disease. Copyright © 2014 Elsevier Inc. All rights reserved.

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Channel-mediated tonic GABA release from glia.

Soojung Lee, Bo-Eun Yoon, Ken Berglund … (2010)

Synaptic inhibition is based on both tonic and phasic release of the inhibitory transmitter γ-aminobutyric acid (GABA). Although phasic GABA release arises from Ca(2+)-dependent exocytosis from neurons, the mechanism of tonic GABA release is unclear. Here we report that tonic inhibition in the cerebellum is due to GABA being released from glial cells by permeation through the Bestrophin 1 (Best1) anion channel. We demonstrate that GABA directly permeates through Best1 to yield GABA release and that tonic inhibition is eliminated by silencing of Best1. Glial cells express both GABA and Best1, and selective expression of Best1 in glial cells, after preventing general expression of Best1, fully rescues tonic inhibition. Our results identify a molecular mechanism for tonic inhibition and establish a role for interactions between glia and neurons in mediating tonic inhibition.

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Author and article information

Journal

Journal ID (nlm-journal-id): 101736592

Journal ID (pubmed-jr-id): 48119

Journal ID (nlm-ta): Nat Metab

Journal ID (iso-abbrev): Nat Metab

Title: Nature metabolism

ISSN (Electronic): 2522-5812

Publication date Nihms-submitted: 18 October 2019

Publication date (Electronic): 15 November 2019

Publication date (Print): November 2019

Publication date PMC-release: 19 May 2020

Volume: 1

Issue: 11

Pages: 1110-1126

Affiliations

[1. ]Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA.

[2. ]J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL 32611, USA.

[3. ]Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.

[4. ]Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland.

[5. ]Pancreatic Islet Processing Facility, Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy

[6. ]Cell Isolation and Transplantation Center, Faculty of Medicine, Department of Surgery, Geneva University Hospitals and University of Geneva, CH-1205 Geneva, Switzerland

[7. ]Center for Cardiovascular Research and Division of Cardiology, Department of Internal Medicine, Washington University School of Medicine, St Louis, MO, 63110, USA

[8. ]Department of Internal Medicine, Division of Cardiovascular Medicine, University of Iowa, Carver College of Medicine, Iowa City, IA, 52242, USA.

[9. ]Department of Medicine, University of Toronto, Toronto, ON, Canada M5S 1A8.

[10. ]Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA.

[11. ]The Rolf Luft Research Center for Diabetes & Endocrinology, Karolinska Institutet, Stockholm, SE-17177, Sweden.

[12. ]Division of Integrative Biosciences and Biotechnology, WCU Program, University of Science and Technology, Pohang, 790-784 Korea.

[13. ]Departments of Medicine and Microbiology / Immunology, Diabetes Center, University of California San Francisco, San Francisco, CA, 94143, USA.

[14. ]Department of Physiology and Biophysics, Miller School of Medicine, University of Miami, Miami, FL 33136, USA.

[15. ]Program in Neuroscience, Miller School of Medicine, University of Miami, Miami, FL 33136, USA.

[16. ]These authors contributed equally

Author notes

AUTHOR CONTRIBUTIONS

E.A.P. and S.B. conceived and carried out subcellular studies of GABA-ergic components in islet cells. D.M. and A.C. conceived and identified GABA release from islets in pulses and pioneered the biosensor cell technique for analyzing the dynamics of islet GABA release. E.A.P. conceived and identified the role of VRAC and TauT in GABA release and uptake. D.W.H. and E.A.P. analyzed the genetic models for LRRC8A ^−/− MIN6 cells, βc-LRRC8A ^−/− murine islets, and knock-down LRRC8A-shRNA human islets; D.M., D.W.H., and E.A.P. performed experiments to detect GABA, taurine, and serotonin/insulin secretion; J.M. and J.A. performed hormone assay experiments and ELISAs; J.A. conducted NPY-pHluorin experiments to measure exocytosis; H.Y.G. generated adeno-NPY-pHluorin vectors; R.S. generated genetic models for LRRC8A knock-out MIN6 cells and LRRC8A ^fl/fl murine islets; C.K. isolated and shipped LRRC8A ^fl/fl murine islets; M.W.B. isolated rodent islets and performed Western blot analyses; C.C. prepared cultures of primary rat hippocampal neurons; P.C.S. performed bioinformatics analysis; R.N. and F.L. isolated human islets for research; E.A.P., D.M., D.W.H., J.A., C.C., R.M.D., and R.R.-D. collected, analyzed, and quantified immunohistochemical data; P.-O.B. provided critical equipment, reagents, expertise, and support. D.M., D.W.H., S.B., A.C., and E.A.P. designed the study, analyzed data, and wrote the paper. All authors discussed the results and commented on the manuscript.

[* ]Correspondence: ephelps@ 123456bme.ufl.edu (E.A.P.), sbaekkeskov@ 123456ucsf.edu (S.B.), and acaicedo@ 123456med.miami.edu (A.C.)

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Manuscript ID: NIHMS1541052

DOI: 10.1038/s42255-019-0135-7

PMC ID: 7236889

PubMed ID: 32432213

SO-VID: da5798a6-a17e-4cb5-8c4e-b1c4c2ff40cd

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Mechanism and effects of pulsatile GABA secretion from cytosolic pools in the human beta cell

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Most cited references 103

A Single-Cell Transcriptomic Map of the Human and Mouse Pancreas Reveals Inter- and Intra-cell Population Structure.

SWELL1, a plasma membrane protein, is an essential component of volume-regulated anion channel.

Channel-mediated tonic GABA release from glia.

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