Abstract
Biphasic glucose-stimulated insulin secretion (GSIS) is essential for blood glucose regulation, but a mechanistic model incorporating the recently identified islet β cell heterogeneity remains elusive. Here, we show that insulin secretion is spatially and dynamically heterogeneous across the islet. Using a zinc-based fluorophore with spinning-disc confocal microscopy, we reveal that approximately 40% of islet cells, which we call readily releasable β cells (RRβs), are responsible for 80% of insulin exocytosis events. Although glucose up to 18.2 mM fully mobilized RRβs to release insulin synchronously (first phase), even higher glucose concentrations enhanced the sustained secretion from these cells (second phase). Release-incompetent β cells show similarities to RRβs in glucose-evoked Ca2+ transients but exhibit Ca2+–exocytosis coupling deficiency. A decreased number of RRβs and their altered secretory ability are associated with impaired GSIS progression in ob/ob mice. Our data reveal functional heterogeneity at the level of exocytosis among β cells and identify RRβs as a subpopulation of β cells that make a disproportionally large contribution to biphasic GSIS from mouse islets.
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Data availability
Data supporting the findings of this study are available in the figures and extended data figures. Other related data are available from the authors on reasonable request. As the original images contain other information that needs to be analysed, we cannot upload all of them to a publicly accessible repository presently. Instead, typical videos showing our signals are provided as supplementary data. However, if anyone is interested in them, original images may be requested from the corresponding authors. Source data are provided with this paper.
Code availability
Custom code and algorithm are available in Methods, publications from our laboratory (cited in the main text) or from the authors on request.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (81925022, 92054301, 92150301, T2288102, 91750203, 61827825, 12090053, 32088101, 32227802 and 32271235), the National Key Research and Development Program of China (2022YFC3400600, 2021YFA1101304, 2018YFA0900700, 2021YFF1200500 and 2020YFA0908200), Beijing Natural Science Foundation Key Research Topics (Z20J00059), the Fundamental Research Funds for the Central Universities (PKU2023XGK005, PKU2023LCXQ025) and the High-Performance Computing Platform of Peking University. L.C. was supported by New Cornerstone Science Foundation. X.P. was supported by the Postdoctoral Fellowship of Peking-Tsinghua Center for Life Sciences. H.R. was supported by the Boya Postdoctoral Fellowship of Peking University. We thank X. Yu (Shandong University) for the gift of the Stdt mouse. We thank K. Ouyang for advice on the experiments and the manuscript. We thank W. Ji for his help with the microscopy operation. We thank Z. Luo for help with data analysis.
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L.C. and H. Liu conceived and supervised the study. X.P. designed and performed experiments, image processing, and data analysis and interpretation. H.R. performed mathematical analyses and interpretation with the guidance of C.T. L.Y. performed experiments, image processing and data analysis. S.T. participated in experiments related to disease research and in image processing. R.Z. designed the perfusion apparatus. Yunxiang Wu, Z.C. and J.Z. developed the red zinc probe. L.W. participated in developing the ‘zinc flicker’ method. H. Long, Yongdeng Zhang, Yi Wu, J.S., G.Q and J.W. developed the software platform for fusion event identification and image processing. T.X. contributed to developing the zinc flicker method and the semi-automatic software platform. C.H., Yulin Zhang, M.Z. and Yiwen Zhao participated in mouse management and physiological experiments. L.C. and X.P. wrote the paper, with contributions from all authors.
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L.C., X.P., L.Y. and L.W. have authorized a patent application based on the zinc flicker method used in this work.
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Extended data
Extended Data Fig. 1 Visualization of zinc flickers by spinning-disc confocal microscopy.
a, Schematic diagram of imaging Zn2+/insulin corelease (‘Zinc flicker’) with spinning-disc confocal microscope. The islet was seeded on a glass coverslip, and only a thin region above the glass coverslip (∼1 μm) was imaged. b, Typical images of fusion events in the islet (from a male mouse) before and after stimulation with 18.2 mM glucose for 3 min. White puncta represented fused insulin granules labeled with fluorescent FluoZin-1. c, Dynamic insulin secretion evoked by 20 mM glucose with or without FluoZin-1 dye as determined by ELISA. Data were quantified from four independent experiments from male mice. d, Representative images of secretion (all fusion events in 15 minutes) in islets (from male mice) exposed to 11 mM glucose, either in the absence (Control) or presence of 250 µM diazoxide (Diazoxide). e, Averaged fusion events stimulated by 11 mM glucose (Control, n = 6 islets from 3 male mice) or with 250 μM diazoxide (Diazoxide, n = 5 islets from 3 male mice). f, Fusion events before (Diazoxide) and after (glucose) removal of diazoxide treatment on the same islet. n = 3 islets from 2 male mice. Significance was evaluated by ratio paired t test. g, Averaged fusion events detected in α-cells (n = 45 cells from 10 male islets) and δ−cells (n = 45 cells from 11 islets, mixed from male and female mice). h-i, Representative images of secretion (15 minutes) in male Glu-GCaMP (h) and Stdt islets (i) exposed to 18.2 mM glucose. White and cyan circles represented α and δ cells respectively. Cell membrane was coded by mpl-inferno color. Data was expressed as mean ± s.e.m. in c, e, g, and analyzed by two-sided unpaired Student t-test, * p < 0.05, ** p < 0.01. Scale bar = 10 μm.
Extended Data Fig. 2 The complementary cumulative distribution function of responsive cells under variable glucose stimulation.
a, Frequency histograms of secretory capacity in responsive cells under different glucose stimulation. b-c, Cumulative distribution function (CDF, b) and complementary cumulative distribution function (CCDF, c) of secretory capacity under different glucose stimulation. For datasets, 7 mM, 45 cells; 9.8 mM, 204 cells; 18.2 mM, 261 cells and 29.2 mM, 97 cells, respectively.
Extended Data Fig. 3 The synchronization of RRβ cells under variable glucose stimulation.
a–c, Exocytosis frequency histograms of fusion events from the top six cells stimulated by 9.8 mM (a, male), 18.2 mM (b, male) and 29.2 mM (c, male) glucose.
Extended Data Fig. 4 Repeated perfusion-stimulated exocytosis in mouse islets.
a, A schematic illustration of the perfusion apparatus and corresponding experimental procedure. Created with BioRender.com. b, Representative illustrations of insulin secretion in male and female islets under continuous perfusion stimulation. c, Heatmaps showcasing the β-cell Ca2+ dynamics within male and female islets undergoing intermittent 18.2 mM glucose stimulations. The color-bar indicates the fluorescent intensity within the islets. Each row represents individual β-cells within a singular islet. The x-axis represents the timeframe subsequent to glucose stimulation. d. Time-dependent fusion events in male and female islets triggered by recurring stimulation with 18.2 mM glucose. Male, n = 6 islets from 4 mice; female, n = 5 islets from 4 mice. Data was expressed as mean ± s.e.m. e. Correlations between the secretory capacity observed during two separate glucose stimulations. Scale bar = 10 μm.
Extended Data Fig. 5 Comparison of the secretory capacity of specific cell types with RRβ cells.
a, The proportion of various cell types classified based on their Ca2+ dynamics in relation to RRβ cells. b, The definition of 1st responder cells and 2nd follower cells, along with hub cells and leader cells. The left box presents the 1st responders and second responders; the right box portrays hub cells and leader cells. c, A representative functional connectivity map in a single male islet, with red dots symbolizing the hub cells. d, The distribution of connection numbers for different cells within an islet; the right panel depicts the log-log plot shown on the left panel. The current plot indicates that the Ca2+ connectivity map does not follow the power law distribution, which would be represented as an inverse linear relationship within the log-log plot.
Extended Data Fig. 6 Both δ- and α- cell influence β-cell secretion with the islet.
a. A representative example of fusion events from islet 1 in c, during the initial round of glucose stimulation and the subsequent round with 10 nM MK4256. b, A representative example of fusion events from islet 5 in c, during the initial round of glucose stimulation and the second round with 200 nM CYN 154806. c, A tabular representation detailing fusion events from islets treated with either of the SSTR antagonists. Islet 3 isolated from male mouse, and other islets isolated from female mice. d, A typical example of insulin granule fusion evoked by 18.2 mM glucose (upper, male) or with 1 μM MK0893 and 1 μM Exendin9-39 (down, male). e, Total number of fusion events evoked by 18.2 mM glucose (Control, n = 5 islets from 3 male mice) or with 1 μM MK0893 and 1 μM Exendin9-39 (MK+Ex, n = 4 islets from 3 male mice). Data was expressed as mean ± s.e.m. and analyzed by two-sided unpaired Student t-test, ** p < 0.01.
Extended Data Fig. 7 Physiology phenotypes of 4-week and 8-week ob/ob mice.
a, b, The glucose tolerance test (a) and body weight (b) of 4w and 8w ob/ob mice and their littermates. n = 12 mice (6 male and 6 female) from 4w Ins-GCaMP ob/wt mice, 10 mice (5 male and 5 female) from 4w Ins-GCaMP ob/ob mice, 5 male mice from 8w wt/wt and 5 male mice from 8w ob/ob mice. Data was expressed as mean ± s.e.m., p values were analyzed by two-sided unpaired Student’s t-tests, *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Extended Data Fig. 8 The synchronization of RRβ cells within ob/ob islets.
a, Exocytosis frequency histograms of fusion events from the top six RRβ cells in 4-week Ins-GCaMP (left, male) and ob/ob islets (right, male). b, Exocytosis frequency histograms of fusion events from the top six RRβ cells in adult Ins-GCaMP ob/wt (left, male) and Ins-GCaMP ob/ob islets (right, female).
Supplementary information
Supplementary Table 1
Statistical tests and exact P values for all figures.
Supplementary Video 1
Glucose-stimulated Zn2+–insulin corelease under the spinning-disc microscope. The puncta (cyan hot) represent individual fusion events of insulin granules from β cells after 18.2 mM glucose stimulation.
Supplementary Video 2
Glucose-evoked Ca2+ dynamics and insulin secretion in the same Ins-GCaMP islets. The puncta (cyan hot) represent individual fusion events of insulin granules labelled with PKZnR-1. The amplitude of Ca2+ dynamics was coded by mpl-inferno colour.
Supplementary Video 3
Distribution of long-lasting fluorescence puncta within Stdt mouse islet in three dimensions (related to Fig. 5). The puncta represent individual fusion events of insulin granules labelled with FluoZin-1. The plasma membrane was labelled with FM 4-64, and δ cells were labelled with tdTomato. Both were coded by mpl-inferno colour.
Supplementary Video 4
Fusion events evoked by 18.2 mM glucose in the ob/ob mouse islet (related to Fig. 6). The puncta (cyan hot) represent individual fusion events of insulin granules labelled with PKZnR-1.
Supplementary Video 5
Glucose-evoked Ca2+ dynamics and insulin secretion in the same Ins-GCaMP ob/ob islets (related to Fig. 7). The puncta (cyan hot) represent individual fusion events of insulin granules labelled with PKZnR-5. The amplitude of Ca2+ dynamics was coded by mpl-inferno colour.
Supplementary Video 6
Glucose-evoked Ca2+ dynamics and insulin secretion in the same Ins-GCaMP ob/wt islets (related to Fig. 7). The puncta (cyan hot) represent individual fusion events of insulin granules labelled with PKZnR-5. The amplitude of Ca2+ dynamics was coded by mpl-inferno colour.
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Peng, X., Ren, H., Yang, L. et al. Readily releasable β cells with tight Ca2+–exocytosis coupling dictate biphasic glucose-stimulated insulin secretion. Nat Metab 6, 238–253 (2024). https://doi.org/10.1038/s42255-023-00962-0
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DOI: https://doi.org/10.1038/s42255-023-00962-0
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