Extended Data Fig. 4: Glucagon stimulates δ-cell Ca²⁺ activity via G_s and G_q pathways.

a, δ-cell [cAMP]_i (cADDis signal in Sst-tdRFP islets; ΔF/F₀) at 1 mM glucose in response to 1 μM glucagon and 10 μM forskolin (positive control). Data represents mean value (solid red trace) ± SEM (shaded ribbon) (41 cells from 15 islets, 2 mice). Timings and durations of the application of treatments are indicated with horizontal red bars and grey shaded areas. The inset summarizes δ-cell [cAMP]_i AUC in the absence or presence of 1 μM glucagon. ****p = 2.9×10⁻⁹ vs. control, two-sided unpaired t-test. b, Representative δ-cell [Ca²⁺]_i oscillations (ΔF/F₀) in Sst-GCaMP6 islets at 1 mM glucose in the absence or presence of 1 μM glucagon in response to pre-treatment and continuous presence of control medium, ESI-05, PKI 14-22 amide, ryanodine, YM-254890 or xestospongin C. Concentrations and treatments are as indicated. c, Percentage of active δ-cell at 1 mM glucose under indicated experimental conditions as shown in (b) (control, n = 3 islets; ESI-05, n = 7 islets; PKI, n = 5 islets; ryanodine, n = 5 islets; YM-254890, n = 5 islets; Xestospongin C, n = 5 islets; 4 mice). *p < 0.05, **p < 0.01 vs. control, one-sided unpaired t-test. d, Summary of δ-cell Ca²⁺ spike frequency under indicated experimental conditions as shown in (b) (control, n = 79 cells in 3 islets; ESI-05, n = 133 cells in 7 islets; PKI, n = 71 cells in 5 islets; ryanodine, n = 72 cells in 5 islets; YM-254890, n = 86 cells in 5 islets; Xestospongin C, n = 102 cells in 5 islets, 4 mice). For comparison, the responses with and without glucagon but in the absence of blockers are shown as the controls (dark grey, data taken from Fig. 3h). **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control of the respective groups, two-sided Mann-Whitney test; ^###p = 0.0001 vs. without glucagon, two-sided Wilcoxon matched-pairs signed rank test. e-f, SST secretion at 1 mM glucose with and without 1 μM glucagon in mouse (e) (n = 4 experiments for control, n = 3 experiments/group for all other groups, 4 mice) and human (f) islets (n = 11 experiments for control, n = 10 experiments/group for all other groups, 3 donors, different colours mark individual donors) pre-treated with conditions as indicated. *p < 0.05, **p < 0.01, ***p < 0.001, ****p = 7.4×10⁻⁵ vs. control of the respective groups; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 vs. without glucagon in the same pre-treatment group, two-sided unpaired t-test. In (b-f), glucagon was applied acutely and all the blockers were applied during preincubation and perfusion (Ca²⁺ imaging) or incubation (secretion experiments). In dot plots, rectangles and error bars behind data points represent mean values ± S.E.M. and violin plots the median with quartiles.

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