Pancreatic islet-autonomous insulin and smoothened-mediated signalling modulate identity changes of glucagon+ α-cells

Abstract

The mechanisms that restrict regeneration and maintain cell identity following injury are poorly characterized in higher vertebrates. Following β-cell loss, 1-2% of the glucagon-producing α-cells spontaneously engage in insulin production in mice. Here we explore the mechanisms inhibiting α-cell plasticity. We show that adaptive α-cell identity changes are constrained by intra-islet insulin- and Smoothened-mediated signalling, among others. The combination of β-cell loss or insulin-signalling inhibition, with Smoothened inactivation in α- or δ-cells, stimulates insulin production in more α-cells. These findings suggest that the removal of constitutive 'brake signals' is crucial to neutralize the refractoriness to adaptive cell-fate changes. It appears that the maintenance of cell identity is an active process mediated by repressive signals, which are released by neighbouring cells and curb an intrinsic trend of differentiated cells to change.

Figure 1.. α-cells engage insulin production after β-cell ablation in islets transplanted under the kidney capsule.

(a) Experimental design of islet transplantation underneath the kidney capsule of immunocompromised host mice (SCID). Exp. #1. WT islets are transplanted into RIP-DTR hosts; upon DT administration, β-cell ablation occurs in pancreatic islets, while transplanted islets remain unaffected and maintain normoglycemia. Exps. #2 & #3. Islets isolated from RIP-DTR donors are transplanted into either WT or RIP-DTR hosts; DT only ablates β-cells in transplanted islets (in WT hosts) or in both transplanted and endogenous pancreatic islets (in RIP-DTR hosts). (b) Immunofluorescence staining of insulin (red) and glucagon (green) in Exps. #1 to #3. Arrows indicate bihormonal glucagon+ insulin+ cells. Scale bars: 20 μm. Experiment repeated independently 3 times. (c) Random-fed blood glycemia after DT-treatment in 3 experimental conditions tested. The vertical dash line indicates the administration of one insulin pellet to the hyperglycemic mice. Experiment performed once. (d) Proportion of glucagon+ cells coexpressing insulin. Red: RIP-DTR islets; black: WT islets. n=3 biologically independent animals per condition. Data shown as mean ± s.d.; see Supplementary Table 1 for source data.

Figure 2.. Pdx1 expression inhibits glucagon production in adult α-cells.

(a) Transgenes required for α-cell tracing and ectopic Pdx1 expression. (b) Experimental design. (c) α-cells are specifically and efficiently YFP-labeled upon DOX administration in controls (upper panel). Pdx1-expressing YFP⁺ α-cells cease glucagon expression (bottom panel). The experiment was performed once. (d) Percentage of YFP-traced α-cells expressing glucagon after Pdx1 expression. Two-tailed unpaired t-test P<0.0001. n=3 control mice and 3 α-PdxOE mice. (e) Pancreatic glucagon content is decreased in αPdx1OE mice. Two-tailed unpaired t-test P=0.0362. n=3 control mice and 3 α-PdxOE mice. (f) Experimental design for Pdx1 induction in α-cells, and DT- or STZ-triggered β-cell loss. (g) A vast fraction of YFP⁺ α-cells (i.e. expressing Pdx1) starts insulin production upon DT- or STZ-mediated β-cell loss. The experiment was performed once with mice treated asynchronously according to their availability. (h) Fraction of YFP⁺ α-cells expressing insulin in αPdx1OE mice upon β-cell loss. Two-tailed Mann Whitney test, P=0.0167 for STZ+Pdx1 vs Pdx1 and P=0.0006 for DT+Pdx1 vs Pdx. n=6 and 8 untreated and DT-treated control mice, respectively and n=7, 7, 3 untreated, DT-treated and STZ treated α-PdxOE mice. (i) Transgenes required for α-cell tracing, DT-mediated β-cell ablation and ectopic Nkx6.1 expression. (j) Experimental design. (k) Nkx6.1 expression (GFP⁺ cells) does not induce insulin production in α-cells in presence of a normal β-cell mass. Glucagon expression persists in Nkx6.1OE α-cells (upper lane). After DT-mediated β-cell loss, most Nkx6.1-expressing α-cells start insulin expression and stop glucagon production (bottom lane). The experiment was performed once with all animals treated asynchronously according to their availability. (l) Pdx1 is not expressed in Nkx6.1OE α-cells when the β-cell mass is normal (upper lane) but is induced after DT (bottom lane). All centers indicate the mean. Scale bars: 20 μm. See Supplementary Tables 1b,c,d as source data.

Figure 3.. DT-mediated β-cell loss facilitates β-cell gene expression and elicits dual responses in α-cells.

(a) Experimental design for RNA-Seq. Transgenic mice allowing α- and β-cell lineage tracing were used to sort by FACS the following cell populations: i) native control α-cells (“native α”, n=6 biologically independent samples), ii) α-cells 1 month after DT-induced β-cell ablation (“DT”, n=3 biologically independent samples), iii) α-cells overexpressing Pdx1 (“Pdx1OE”, n=3 biologically independent samples), iv) α-cells overexpressing Pdx1 combined with β-cell ablation (“Pdx1OE+DT”, n=5 biologically independent samples). Native β-cells were also collected (“native β”, n=5 biologically independent samples) and analyzed to identify differentially-expressed genes (DEGs) between native α- and β-cells as reference gene sets (α-/β-cell genes). Note that insulin protein was detected in only 1 % of α-cells in the DT group, and 2% in Pdx1OE group (immunofluorescence). Experiment (RNA-Seq) performed once. (b) Venn diagrams showing the regulation of α-/β-cells enriched genes upon Pdx1OE, DT and Pdx1OE+DT. DEGs were calculated for each condition (DT, Pdx1OE, Pdx1OE+DT) compared to native α-cells and intersected with α-/β-cell enriched genes to identify α-/β-cell signature changes. DEGs in left and middle panels are upregulated β-cell genes and downregulated α-cell genes, respectively, which could be considered as an increased β-cell signature in α-cells. Conversely, all genes in the right panel are up-regulated α-cell genes, which could represent a resistance to reprogramming. For every category, only representative genes with FDR < 0.05 are shown. The entire gene lists are reported in Table S2. (c) Heatmap showing scaled expression (blue, high; white, low) of representative α-/β-cell genes in Fig. 3b and Fig. S3c. Gene clustering shown by dendrogram indicates separated gene clusters with different modulation patterns in each condition analyzed, as seen in Fig. 3b and Fig. S4c. See Suppl. Table 2 for source data.

Figure 4.. Decreased insulin signaling predisposes α-cells to insulin production in islets with an intact β-cell mass.

(a) Transgenes for α-cell tracing and insulin (IR) and IGF-1 receptor (IGF1R) downregulation in adult α-cells. (b) Experimental design. (c) Immunofluorescence on islets and (d) RT-qPCR on purified α-cells from αIR/IGF1R KO mice. Impaired insulin/IGF1 signaling does not lead to insulin protein production but induces insulin, Pdx1 and Nkx6.1 gene expression. Data shown as mean ± s.e.m.; n=5 independent biological samples (i.e. one mouse or pool of mice/sample). Experiment performed once. Two-tailed Mann Whitney test (P=0.0079, IR; P=0.0159, IGF1R; P=0.0317, IRS2; P=0.0317 INS1; P=0.0079 PDX1; P=0.0079 NKX6.1) (e) Experimental design for α-cell tracing and insulin signaling blockade with S961 in mice with intact β-cell mass. Islets were analyzed either immediately after stopping S961 (Analyses #1) or 1 month later (Analyses #2). (f) Immunofluorescence of islets of S961 treated mice. YFP-traced α-cells expressing insulin are present only in islets of mice analyzed during S961 treatment (Analyses #1). IF were performed at on 3–5 consecutive sections per animal with similar results. The experiment was performed once, with mice treated asynchronously according to their availability. (g) % of YFP-traced α-cells expressing insulin after ± S961 treatment (analyses #1 and #2). Center indicates the mean. n=5, 6 and 4 animals for control (no treatment), S961 1month and S961 1 month + STOP 1 month, respectively. Two-tailed Mann Whitney test, P=0.0025. (h) Experimental design for α-cell tracing and STZ-induced β-cell ablation followed either by blockade of residual insulin signaling with Wortmannin or S961, or insulin signaling enhancement through insulin administration (subcutaneous pellets). (i) Fraction of converted α-cells after β-cell loss and inhibition or enhancement of residual insulin signaling. n=7 mice for STZ and STZ+WORT groups and n=6 mice for STZ+S961 and STZ+INS groups. Center indicates the mean. Two-tailed Mann Whitney test, P=0.0070, STZ+WORT; P=0.0350, STZ+S961; P=0.0047, STZ+INS. Scale bars: 10 μm. See Supplementary Table 1e for source data.

Figure 5.. Smoothened (Smo) inactivation in α-cells facilitates their engagement into insulin production.

(a) Transgenes required for simultaneous α-cell lineage tracing, Smo co-receptor downregulation and DT-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 (bottom panels). IF were performed at on 3–5 consecutive sections per animal with similar results. The experiment was performed once, with mice treated asynchronously according to their availability. (d) Fraction of YFP⁺ cells producing insulin upon inactivation of Smo in α-cells combined with DT or S961. n=4,4,3 mice for Smo^wt/wt, Smo^fl/fl and Smo^fl/fl noDT, respectively; n=4,10,5 mice for Smo^wt/wt, Smo^fl/fl and Smo^fl/fl DT, respectively; n=4,5,6 mice for Smo^wt/wt, Smo^fl/fl and Smo^fl/fl S961, respectively. Center indicates the mean. Two-tailed Mann Whitney test, P=0.0079 Smo^fl/fl DT vs Smo^wt/wt DT, P=0.0095 Smo^fl/fl DT vs Smo^wt/wt S961 and P=0.0286 Smo^wt/wt vs Smo^wt/wt S961. Scale bars: 10 μm. (e) In vivo glucose challenge in α-Smo-KO mice. n=18 mice, P=0.012, Wilcoxon test, two-tailed. (f) Pipeline for α-cell sorting, in vitro pseudoislet reconstruction and functional tests. (g) Live imaging of 7-day-cultured pseudoislet reconstituted using α-cells from α-Smo-KO mice. Representative images from 3 independent experiments. (h) α-Smo-KO pseudoislet at day 7 of aggregation culture (immunofluorescence). Representative images from 3 independent experiments. Scale bar: 25 μm. (i) Percentage of YFP+ cells producing insulin in pseudoislets from control α-Smo-WT animals (no β-cell ablation) or α-Smo-KO mice after cell ablation (DT). n = 3 independent cohorts from 18 α-Smo-WT mice, and 3 independent cohorts from 18 α-Smo-KO mice, P=0.049, unpaired t-test, two-tailed. Center indicates the mean. (j) Glucose-stimulated C-peptide secretion. α-Smo-KO cells secrete C-peptide in response to glucose in vitro, while α-Smo-WT (no DT control) cells have no measurable secretion. n = 3 independent cohorts obtained from 18 α-Smo-Wt mice, n=3 independent cohorts obtained from 18 α-Smo-KO mice, P=0.048, paired t test, one-tailed. The dash line indicates the detection threshold documented by manufacturer (0.0127 nmole/pseudoislet/hour). All values are means ± s.e.m. See Supplementary Tables 1f-l for source data.

Figure 6.. Smo inactivation in δ-cells leads to enhanced α-to-β cell conversion.

(a) Transgenes required for simultaneous δ-cell lineage tracing, Smo co-receptor downregulation and DT-induced β-cell ablation. (b) Experimental design (c) Immunofluorescence staining of islets from δ-Smo-KO mice 1,5 month after DT-induced β-cell loss. δ-Smo-KO mice display increased numbers of insulin⁺/glucagon⁺ coexpressing cells as compared to controls. (d) Percentage of cells coexpressing insulin and glucagon in δ-Smo-KO mice. n=3 mice wt/wt, wt/fl, fl/fl no DT; n =4, 6, 4 mice in wt/wt, wt/fl, fl/fl DT respectively. Two-tailed Mann Whitney test, P=0.0095 wt/fl vs wt/wt and P=0.0286 fl/fl vs wt/wt. Center indicates the mean. (e) Transgenes for simultaneous inactivation of Smo in α- and δ-cells along with their lineage tracing, and for DT-induced β-cell ablation. (f) Immunofluorescence staining of islets from α+δ-Smo-KO mice 1,5 month after DT. (g) Fraction of insulin⁺/glucagon⁺ coexpressing cells traced with YFP. n=5 wt/fl mice, n=4 fl/fl mice. Two-tailed Mann Whitney test, P=0.84. Center indicates the mean. Experiments in c-f were repeated two times independently with similar results. Scale bars: 10 μm. See Supplementary Tables 1n-p for source data.