Extended Data Figure 1: Maintenance of α-cell plasticity in diabetic aged mice.

a, Evolution of glycaemia in β-cell-ablated adults (middle-aged) and aged mice. The area under the curve (AuC) in middle-aged (2-month-old, n = 4) and aged (1- and 1.5-year-old, n = 5 and n = 3) mice before and after stopping insulin administration revealed no statistical difference between groups (Welch’s test, P_{0–4.5 mpa =} 0.1029, 0.3321; P_4.5–7 mpa = 0.1748, 0.5007; one-way analysis of variance (ANOVA), P = 0.1161, P = 0.2681; and Mann–Whitney, P = 0.1640, 0.4519). b, Evolution of glycaemia in 14 aged mice over a period of 14 months post-ablation (mpa). Mice were treated with insulin for 4.5 months; most of them (5/7 in each group) subsequently recovered from diabetes. c–e, Pancreatic islets before (c) and after (d, e) β-cell ablation in 1.5-year-old mice; β-cell mass increases 3.5-fold between 0.5 and 1 mpa, 12-fold at 7 mpa and 32-fold at 14 mpa, in all age groups. Percentages (0.3% and 4.4%) indicate β-cell mass relative to unablated controls (Supplementary Table 1). Two-month-old: n_0.5 mpa = 4; n_1 mpa = 4; n_7 mpa = 4; 1-year-old: n_0.5 mpa = 5, n_1 mpa = 5, n_7 mpa = 5, n_14 mpa = 8; 1.5-year-old: n_0.5 mpa = 3; n_1 mpa = 3; n_7 mpa = 3, n_14 mpa = 8. f, β-Cell proliferation is very low in aged mice, whether control (1.5%; n = 8; 39,790 insulin⁺-cells scored) or ablated (0.2%; n = 6; 938 insulin⁺-cells scored) (Supplementary Table 2). g, Proportion of insulin⁺ cells also containing glucagon after DT is not different between groups (Supplementary Table 3). Control: n_2-month-old = 3; n_1-year-old = 3; n_1.5-year-old = 3; 0.5 mpa: n_2-month-old = 5; n_1-year-old = 5; n_1.5-year-old = 6; 1 mpa: n_2-month-old = 4; n_1-year-old = 6; n_1.5-year-old = 4; 7 mpa: n_2-month-old = 5; n_1-year-old = 5; n_1.5-year-old = 6. One-way ANOVA (P = 0.6796, 0.4297, 0.9266, 0.2411); note that 40% of the cells containing insulin at 1 mpa also contained glucagon. The proportion of glucagon⁺/insulin⁺ cells remains constant between 0.5 and 7 mpa, while the number of insulin⁺ cells increases with time (e; Supplementary Table 1), suggesting that there is a cumulative recruitment of α-cells into insulin production. h, Islet with YFP⁺/glucagon⁺/insulin⁺ cells in 1-year-old glucagon-rtTA; TetO-Cre; R26-YFP; RIP-DTR mice, 7 mpa; rtTA expression allows the selective irreversible YFP labelling of adult α-cells upon administration of doxycycline (DOX) before β-cell ablation. i, Proportion of YFP-labelled insulin-expressing cells in DOX-treated mice. Eighty per cent of insulin⁺ cells are YFP⁺ after 7 mpa, in all age groups (Supplementary Table 4). Control: n_2-month-old = 3; n_1-year-old = 3; n_1.5-year-old = 3; 1 mpa: n_2-month-old = 5; n_1-year-old = 3; n_1.5-year-old = 3; 7 mpa: n_2-month-old = 5; n_1-year-old = 5; n_1.5-year-old = 5. One-way ANOVA (P = 0.9417, 0.8910, 0.9641). j, k, YFP⁺/glucagon⁺/insulin⁺ cells at 7 mpa, following DOX pulse-labelling at 5.5 months after β-cell loss (Supplementary Table 5). Control: n_1-year-old = 5; n_1.5-year-old = 5; 7 mpa: n_1-year-old = 5; n_1.5-year-old = 5; Welch’s correction (P = 0.8272, 0.8926), Mann–Whitney (P = 0.9444). On average, 15% of the insulin⁺ cells found were YFP labelled, some of which no longer contained glucagon as in j, bottom row. Note the decreased proportion of YFP-labelled insulin⁺ cells when α-cells are tagged late after ablation (from 80% to 15%; compare i and k), and the presence of YFP-labelled insulin⁺/glucagon-negative cells in the latter situation (j), suggesting that bihormonal α-cells slowly but gradually lose glucagon gene activity. Scale bars, 20 µm. Error bars show s.d.