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

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Abstract

Pancreatic beta cells synthesize and secrete the neurotransmitter GABA (γ-aminobutyric acid) 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, 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 patients with type 1 and type 2 diabetes, suggesting that loss of GABA as a synchronizing signal for hormone output may correlate with diabetes pathogenesis.

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Fig. 1: Cytosolic pools of GABA are depleted in type 1 and type 2 diabetic islets.
Fig. 2: Subcellular localization suggests a nonvesicular GABA release mechanism in beta cells.
Fig. 3: Islet GABA secretion is pulsatile and depends on GABA content.
Fig. 4: VRAC and TauT transport cytosolic GABA across the plasma membrane in beta cells.
Fig. 5: Cytosolic GABA secretion synchronizes insulin secretion.
Fig. 6: Cytosolic GABA secretion is interrupted in human islets from type 2 diabetic donors.

Data availability

The unique biological materials used in the manuscript are available from the corresponding authors upon reasonable request with the exception of those materials that the authors obtained via a materials transfer agreement that prohibits transfer to third parties; these include the GABA biosensor cells (obtainable from K. Kaupmann, Novartis Institute for BioMedical Research, Basal, Switzerland), LRRC8A−/− MIN6 cells and LRRC8Afl/fl mice (obtainable from R. Sah, Washington University in St. Louis, MO, USA), and NPY-pHluorin (obtainable from H. Gaisano, University of Toronto, Toronto, ON, Canada). Other requests for materials should be addressed to corresponding author A.C. or E.A.P. Source data for Figs. 1–6 and Extended Data Figs. 1, 4, and 5 are provided with the paper. The data that support the findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

This work was funded by the Intramural Research Program of UF’s Wertheim College of Engineering and J. Crayton Pruitt Family Department of Biomedical Engineering (E.A.P.), the Intramural Research Program of EPFL’s School of Life Sciences (S.B.), the Diabetes Research Institute Foundation, NIH grant nos. R56DK084321 (A.C.), R01DK084321 (A.C.) and R01DK106009 (R.S.), the NIDDK-supported Human Islet Research Network (HIRN, RRID:SCR_014393; https://hirnetwork.org; grant no. UC4DK104208 (E.A.P)), a JDRF award (grant no. 31-2008-416) to the European Consortium for Islet Transplantation (ECIT) Islets for Basic Research Program, a JDRF Faculty Transition Award (grant no. 1-FAC-2017-367-A-N) (E.A.P.), a JDRF Advanced Postdoctoral Fellowship (grant no. 3-APF-2014-208-A-N) (E.A.P.), a Whitaker International Program Postdoctoral Scholarship (E.A.P.), The Shepard Broad Foundation (E.A.P.), the Swedish Research Council (P.-O.B.), the Novo Nordisk Foundation (P.-O.B.), the Family Erling-Persson Foundation (P.-O.B.), the Stichting af Jochnick Foundation (P.-O.B.), the American Diabetes Association (grant no. 1-18-IBS-229) (R.S.) and the Canadian Institutes for Health Research (grant nos. PJT-159741 and PJT-148652) (H.Y.G.).

Human pancreatic islets were provided by the NIDDK-funded Integrated Islet Distribution Program (IIDP) at City of Hope, NIH grant no. 2UC4DK098085, and the JDRF-funded IIDP Islet Award Initiative (E.A.P.). This research was performed with the support of the Network for Pancreatic Organ donors with Diabetes (nPOD; RRID:SCR_014641), a collaborative type 1 diabetes research project sponsored by JDRF (grant no. 5-SRA-2018-557-Q-R) and The Leona M. & Harry B. Helmsley Charitable Trust (grant no. 2018PG-T1D053). The content and views expressed are the responsibility of the authors and do not necessarily reflect the official view of nPOD. Organ Procurement Organizations (OPO) partnering with nPOD to provide research resources are listed at http://www.jdrfnpod.org/for-partners/npod-partners/. The work with human pancreatic sections and islets was also made possible by the Human Islet Cell Processing Facility at the Diabetes Research Institute (University of Miami) and ECIT.

We wish to extend our thanks to the following individuals: D. Bosco and T. Berney, University of Geneva, and L. Piemonti, San Raffaele Scientific Institute, Milan, for human islets through ECIT; C. Mathews, University of Florida, and R. Bottino, Institute of Cellular Therapeutics, Allegheny Health Network, Pittsburgh, Pennsylvania, for human islets through the nPOD Islet Isolation Program; K. Kaupmann, Novartis Institutes for BioMedical Research, Switzerland, for providing the genetically modified GABA biosensor CHO cells; and S. D. Roper for conceptual input on the biosensor cell approach and for critically reading the manuscript; J. Hubbell and M. Swartz, EPFL and University of Chicago, for support; C. Rancourt, University of Florida, for mouse colony management; and M. Pasquier, K. Johnson, B. Benjamin, D. Garcia, P. Parente, A. Arzu, L. Barash, M. Formoso, R. Arrojo e Drigo and A. Tamayo for technical assistance.

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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−/− MIN6 cells and LRRC8Afl/fl murine islets. C.K. isolated and shipped LRRC8Afl/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 to Steinunn Baekkeskov or Alejandro Caicedo or Edward A. Phelps.

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Extended data

Extended Data Fig. 1 Delta cells in human islets contain GABA and express GAD65; taurine content is preserved in diabetic islets.

a-b. Islets in a non-diabetic human pancreas immunostained for GABA and somatostatin (a); or GAD65 and somatostatin (b). GABA and GAD65 are present in somatostatin producing human delta cells. Scale bar 50 µm. Right panels show higher magnification views of the boxed region showing channels for: (a) (1) GABA only, (2) somatostatin only, and (3) GABA and somatostatin; (b) (1) GAD65 only, (2) somatostatin only, and (3) GAD65 and somatostatin. Images are representative of data plotted in Figures 1b and 1j. Scale bar 20 µm. c. Quantification of taurine mean fluorescence intensity (MFI) per islet in confocal images of human pancreas sections from non-diabetic (n = 21 islets, 6 donors), type 2 diabetic (n = 24 islets, 8 donors), and type 1 diabetic donors (n = 24 islets, 8 donors). Background (BKGD) indicates average taurine MFI in acinar tissue outside of the islet. One-way ANOVA: ND vs. T2D (*P = 0.0430), ND vs. T1D (ns, P = 0.6667). Center line indicates the mean. d. Human pancreas sections immunostained for taurine, insulin, and glucagon from a non-diabetic, type 2 diabetic and, type 1 diabetic donor. Left panels show insulin and glucagon channels, while right panels show taurine channel from the same image. Images are representative of the dataset plotted in panel c. Scale bars 50 µm. e. Representative confocal image of a monolayer of human islet endocrine cells showing immunostaining for GAD65, insulin, and glucagon (left panel) and GAD65 alone (right panel). Images are representative of 3 human islet preparations. Scale bar 20 µm. Source Data

Extended Data Fig. 2 VGAT expression is concentrated in delta cells of human islets and alpha cells of rat islets; GABA colocalizes with GAD65 and VGAT in synaptic vesicles in neurons.

a-b. Islets in a non-diabetic human pancreas (a) and rat pancreas (b) immunostained for VGAT, insulin, glucagon, somatostatin, and pancreatic polypeptide. VGAT is absent in most beta cells but present in somatostatin producing human delta cells and glucagon producing rat alpha cells. Results are representative of the dataset plotted in Figure 1b. Scale bar 50 µm. c. Rat hippocampal neuron immunostained for GABA and the GABA biosynthesizing enzyme GAD65. Scale bar 10 µm. Right panels show higher magnification views of the boxed region showing channels for: (1) GABA only; (2) GAD65 only; (3) GABA and GAD65. GAD65 and GABA colocalize in vesicles. Results are representative of n = 3 rat neuron preparations. Scale bar 5 µm. d. Rat hippocampal neuron immunostained for GAD65 and the vesicular GABA transporter VGAT, which is present in synaptic vesicle membranes. Scale bar 10 µm. Right panels show higher magnification views of the boxed region showing channels for: (1) GAD65 only; (2) VGAT only; (3) GAD65 and VGAT. GAD65 and VGAT colocalize in synaptic vesicles. Results are representative of n = 3 rat neuron preparations. Scale bar 5 µm.

Extended Data Fig. 3 Characterization of GABA biosensor cells for detecting GABA released from human islets.

a. Schematic and image of the GABA biosensor cell assay setup (left panel). Biosensor cells consist of CHO cells stably expressing the heteromeric GABAB receptor (GABAB R1b and GABAB R2) and the G-protein α subunit, Gαqo5 to allow for GABA detection by intracellular Ca2+ mobilization (Δ[Ca2+]i) (right panel). GABA biosensor cells are pre-loaded with the [Ca2+]i indicator Fura-2 and plated on poly-d-lysine coated cover slips in a perfusion chamber. Individual islets are placed on top of this layer of biosensor cells and connected to a closed bath small volume imaging chamber to ensure linear solution flow and fast exchange. b. Titration of exogenous GABA showing concentration-dependence of Ca2+ flux in GABA biosensor cells. The plot shows the average 340/380 Fura-2 ratio of n = 5 GABA biosensor cells in the same field of view. Mean ± SEM. c. Effect of the selective GABAB receptor antagonist CGP5584 on biosensor cell responses to exogenously applied GABA. n = 5 biosensor cells in the field of view. Mean ± SEM. d. Biosensor cell intracellular Ca2+ responses remain elevated during sustained (30 min) exposure to GABA (100 µM shown). n = 5 GABA biosensor cells in the field of view. Mean ± SEM. e. GABA release from a human islet maintained in 3 mM glucose. n = 5 GABA biosensor cells located under or immediately downstream of the islet. This is a representative trace of experiments performed on 40 human islet preparations. Mean ± SEM. f. Biosensor cells have tonic responses to continuously applied GABA and phasic responses to GABA pulses released from islets. Periods of pulsatile GABA release measured from n = 22 human islet preparations, ≥ 3 islets per preparation (black circles) as shown in panel e. Calculated periods for biosensor cell responses to continuously applied GABA (gray circles) at 0.1, 1, 10, and 100 µM GABA as shown in panel d. Center line indicates the mean. g. GABA release from a human islet maintained in 3 mM glucose without addition of inhibitors. n = 5 GABA biosensor cells located under or immediately downstream of the islet. This is a representative trace of experiments performed on 40 human islet preparations. Mean ± SEM. h. Effect of the selective GABAB receptor antagonist CGP5584 on biosensor cell detection of GABA released from a single human islet. n = 5 GABA biosensor cells located under or immediately downstream of the islet. Trace is representative of 3 independent experiments with different human islet preparations. Mean ± SEM.

Extended Data Fig. 4 GABA release does not depend on glucose but is activated by VRAC opening.

a-b. Titration of glucose concentrations from 0-25 mM has no effect on islet GABA release. HPLC quantification of GABA released from rat (a) and human (b) islets during 30 mins static incubation in KRBH of the indicated glucose concentrations. n = 4 samples of 100 islets. One-way ANOVA, P = 0.5563 rat (a), P = 0.2053 human (b), ns = not significant. Mean ± SEM. c. HPLC quantification of GABA released from human islets in 5.5 mM glucose (n = 4 samples of 100 human islets), 30 mM KCl (n = 3 samples of 100 human islets), or diazoxide (100 µM) (n = 4 samples of 100 human islets). One-way ANOVA, P = 0.1511, ns = not significant. Mean ± SEM. d. Effect of the GAT inhibitors SNAP5114 (50 µM), NNC05-2090 (50 µM), and NNC711 (10 µM) on biosensor detection of GABA secretion from a human islet. GAT inhibitors were present throughout the shaded portion of trace. Results are representative of the data plotted in panel e. e. Quantification of GABA release following treatment with GAT inhibitors. Box extends from 25th to 75th percentiles, center line represents the median, whiskers represent smallest to largest values. n = 3 islets, One-way ANOVA, 0-20 min vs. 20-40 min (*P = 0.002), 0-20 min vs. 40-60 min (P = 0.9194), 0-20 min vs. 60-80 min (*P = 0.0058). Source Data

Extended Data Fig. 5 Allylglycine inhibition of beta cell GABA content and secretion.

a-b. Validation of GABA antibody via immunostaining of paraformaldehyde-fixed rat hippocampal neurons (a) or rat islet cell monolayers (b) for GAD65, GABA, and insulin (not shown) without or with addition of soluble GABA to the primary antibody incubation buffer; or without or with preincubation of cells with allylglycine (10 mM) to inhibit GABA biosynthesis. Images are representative of 3 experimental replicates. Scale bars 20 µm. c. Immunostaining of paraformaldehyde-fixed rat islets cell monolayers for GABA, insulin, and GAD65, following allylglycine (10 mM) addition and removal. Images are representative of the dataset plotted in panel d. d. Quantification of GABA mean fluorescence intensity (MFI) in rat islet cell monolayers in allylglycine timecourse experiments shown in panel c. n = 4 coverslips. Mean ± SEM. e. HPLC analysis of GABA release from human islets during a 30 min addition of allylglycine (no pre-incubation). n = 3 samples of ~100 islets each. Statistical analysis by two-tailed t-test, *P = 0.0104. Mean ± SEM. Source Data

Extended Data Fig. 6 Human islet single-cell RNA-seq for expression of genes of interest.

a. Expression of neurotransmitter transporter family genes (SLC6A). Mean ± SEM. b. Expression of genes of interest reported in the literature as related to GABA or putative GABA membrane transporters. Mean ± SEM. Data shown are from two datasets54,55, but results agree with and are representative of three different curated human single-cell RNA-seq datasets analyzed54,55,56 (see also Figure 4).

Extended Data Fig. 7 Kymographs of individual GABA biosensor cells.

a-b. Still image, kymographs, and average trace from timelapse videos of Fura-2 [Ca2+]i signals in GABA biosensor cells in a perfusion flow field in 3 mM glucose isotonic KRBH exposed to (a) 0.1, 1, and 10 μM GABA, (b) downstream from a wild type mouse islet, and (c) downstream from a βc-LRRC8A-/- mouse islet. GABA (1 μM) is added to (c) at 23 min. GABA-responsive cells were selected for analysis, while unresponsive cells were not analyzed. Data are representative of three independent experiments. See also Supplementary Videos 13.

Supplementary information

Supplementary Table 1

Data summary comparing the expression of GAD65, VGAT and GABA in human and rat islet endocrine cell subtypes.

Reporting Summary

Supplementary Tables 2 and 3

Average relative mRNA expression of all SLC genes in human islets from three different single-cell RNA-seq datasets. Buffer composition and osmolarity calculations.

Supplementary Video 1

GABA biosensor cell calibration. Timelapse video of Fura-2 [Ca2+]i signal in GABA biosensor cells exposed to 0.1, 1 and 10 μM GABA. Data are representative of five independent experiments. See also Extended Data Fig. 7.

Supplementary Video 2

GABA biosensor cell responses to a wild-type mouse islet. Timelapse video of Fura-2 [Ca2+]i signal in GABA biosensor cells in proximity to a wild-type mouse islet. Data are representative of four independent experiments. See also Extended Data Fig. 7.

Supplementary Video 3

GABA biosensor cell responses to a βc-LRRC8A−/− mouse islet. Timelapse video of Fura-2 [Ca2+]i signal in GABA biosensor cells in proximity to a βc-LRRC8A−/− mouse islet. GABA (1 μM) is added at 23 min. Data are representative of three independent experiments. See also Extended Data Fig. 7.

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Menegaz, D., Hagan, D.W., Almaça, J. et al. Mechanism and effects of pulsatile GABA secretion from cytosolic pools in the human beta cell. Nat Metab 1, 1110–1126 (2019) doi:10.1038/s42255-019-0135-7

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