Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-cell loss

Abstract

Pancreatic insulin-producing beta-cells have a long lifespan, such that in healthy conditions they replicate little during a lifetime. Nevertheless, they show increased self-duplication after increased metabolic demand or after injury (that is, beta-cell loss). It is not known whether adult mammals can differentiate (regenerate) new beta-cells after extreme, total beta-cell loss, as in diabetes. This would indicate differentiation from precursors or another heterologous (non-beta-cell) source. Here we show beta-cell regeneration in a transgenic model of diphtheria-toxin-induced acute selective near-total beta-cell ablation. If given insulin, the mice survived and showed beta-cell mass augmentation with time. Lineage-tracing to label the glucagon-producing alpha-cells before beta-cell ablation tracked large fractions of regenerated beta-cells as deriving from alpha-cells, revealing a previously disregarded degree of pancreatic cell plasticity. Such inter-endocrine spontaneous adult cell conversion could be harnessed towards methods of producing beta-cells for diabetes therapies, either in differentiation settings in vitro or in induced regeneration.

Figure 1. β-cell ablation and regeneration

a, RIP-DTR mice express DTR on 100% or 50% of β-cells (blue cells in the cartoon: DTR-bearing β-cells). b-c, Measurement of β-cell mass after ablation and regeneration. Ablation of 99.6% of the β-cell mass is followed by a 3-fold increase between 15 and 30 days (from 5.9μg to 18.5μg), and up to 10-44-fold 10 months later. One-way ANOVA (p=0.0009) and Mann-Whitney tests (* p<0.05). d, Representative islets at various moments after DT. Bars: 20μm.

Figure 2. Conditional β-cell lineage tracing

a, Transgenes. b, Proportion of YFP+ β-cells. Controls: 95.4±0.5% (n=4; 159-499 β-cells scored/mouse; 5-12 islets/individual). Two weeks and 1 month after DT, 80.6±2.9% and 7.6±1.8% β-cells were labeled, respectively (15 days: n=3 mice, 80-174 β-cells from 15-25 islets/mouse; 1 month: n=3 mice, 54-73 β-cells from 15-24 islets/mouse). *P<0.01. One-way ANOVA (p=0.0181) and Dunn's multiple comparison test (* p<0.05). c-e, Most β-cells express YFP in controls. f-k, Few β-cells are YFP+ after one month (arrowhead) (f-h). In i-k, 2 β-cells are shown (arrow: YFP-negative β-cell). l, glucagon+/insulin+ cells are YFP-negative (arrows); YFP+/insulin+ cells are glucagon-negative (arrowhead). Bars: 20 μm (c-h); 10μm (i-l).

Figure 3. α-to-β reprogramming

a, Transgenes used for the conditional α-cell lineage tracing. b-e, DOX-treated mice, without DT. f, Most α-cells are YFP+ in controls (88.1±4.42%; n=3 mice; 2,258 α-cells scored, 108 islets). g-j, One month after DT, islets are mostly composed of YFP+ α-cells. k, Proportion of YFP+/insulin+ cells in DOX-treated mice. DT-treated group: 63.6±8.6% (511 β-cells from 239 islets; 5 mice). l-o, YFP+/insulin+/glucagon+ cell (arrowhead; 89.87±3.04% of insulin+ cells). p-s, YFP+/insulin+ cells, not expressing glucagon (arrowheads). Bars: 20 μm (b-j); 10 μm (l-s).

Figure 4. β-cell marker expression

a, Control mice: Nkx6.1 is expressed in β-cells. Bars: 20 μm. b, One month post-ablation, 5.05±0.8% of glucagon-expressing cells are Nkx6.1+ (2,855 cells in 277 islets, 5 mice, vs. 0.37±0.3% in controls; 674 α-cells in 39 islets, 3 mice; P=0.035). c, Nkx6.1 expression in glucagon+ cells (top). Some cells also express insulin (middle). YFP+/Insulin+/Nkx6.1+/Glucagon-negative cell (bottom). Bars: 10 μm. d, Proposed reprogramming sequence.