Fig. 5: Smo inactivation in α-cells facilitates their engagement in insulin production.

a, Transgenes required for simultaneous α-cell lineage tracing, Smo co-receptor downregulation and diphtheria toxin-induced β-cell ablation. b, Experimental design. c, Smo inactivation in α-cells leads to insulin production when combined with β-cell loss (upper panels) or insulin receptor antagonism (lower panels). Immunofluorescence was performed once on 3–5 consecutive sections per animal with similar results. The mice were treated asynchronously according to their availability. Scale bars, 10 µm. GSIS, glucose-stimulated insulin secretion. d, Percentage of YFP⁺ cells producing insulin following the inactivation of Smo in α-cells combined with diphtheria toxin or S961 treatment. Treatment groups with no diphtheria toxin, n = 4, 4 and 3 for Smo^WT/WT, Smo^fl/fl and Smo^fl/fl mice, respectively; diphtheria toxin treatment groups, n = 4, 10 and 5 for Smo^WT/WT, Smo^fl/fl and Smo^fl/fl mice, respectively; S961 treatment groups, n = 4, 5 and 6 for Smo^WT/WT, Smo^fl/fl and Smo^fl/fl mice, respectively. Horizontal bars indicate the mean; two-tailed Mann–Whitney test; Smo^fl/fl diphtheria toxin versus Smo^WT/WT diphtheria toxin treatment, P = 0.0079; Smo^fl/fl diphtheria toxin versus Smo^WT/WT S961 treatment, P = 0.0095; Smo^fl/fl S961 versus Smo^WT/WT S961 treatment, P = 0.0286. e, In vivo glucose challenge in α-Smo-KO mice. Two-tailed Wilcoxon test, P = 0.012; n = 18 mice. f, Pipeline for α-cell sorting, in vitro pseudoislet reconstruction and functional tests. g, Live imaging of seven-day-cultured pseudoislet reconstituted using α-cells from α-Smo-KO mice. Representative images from three independent experiments are shown. h, Immunofluorescence of α-Smo-KO pseudoislet at day seven of aggregation culture. Representative images from three independent experiments are shown. Scale bar, 25 µm. i, Percentage of YFP⁺ cells producing insulin in pseudoislets from control α-Smo-WT (no β-cell ablation) or α-Smo-KO mice after cell ablation (diphtheria toxin treatment). Three independent cohorts each from 18 α-Smo-WT and 18 α-Smo-KO mice. Horizontal bars indicate the mean; P = 0.049, two-tailed unpaired t-test. j, Glucose-stimulated C-peptide secretion. α-Smo-KO cells secrete C-peptide in response to glucose in vitro, whereas α-Smo-WT (no diphtheria toxin control) cells have no measurable secretion. Three independent cohorts each from 18 α-Smo-Wt and 18 α-Smo-KO mice. P = 0.048, one-tailed paired t-test. The dashed line indicates the detection threshold documented by the manufacturer (0.0127 nmol pseudoislet⁻¹ h⁻¹). Each data point represents one independent experiment using biologically different samples. *P < 0.5, **P < 0.01. See Supplementary Table 1f–l for the source data.