Normal glucagon signaling and β-cell function after near-total α-cell ablation in adult mice

Abstract

Objective: To evaluate whether healthy or diabetic adult mice can tolerate an extreme loss of pancreatic α-cells and how this sudden massive depletion affects β-cell function and blood glucose homeostasis.

Research design and methods: We generated a new transgenic model allowing near-total α-cell removal specifically in adult mice. Massive α-cell ablation was triggered in normally grown and healthy adult animals upon diphtheria toxin (DT) administration. The metabolic status of these mice was assessed in 1) physiologic conditions, 2) a situation requiring glucagon action, and 3) after β-cell loss.

Results: Adult transgenic mice enduring extreme (98%) α-cell removal remained healthy and did not display major defects in insulin counter-regulatory response. We observed that 2% of the normal α-cell mass produced enough glucagon to ensure near-normal glucagonemia. β-Cell function and blood glucose homeostasis remained unaltered after α-cell loss, indicating that direct local intraislet signaling between α- and β-cells is dispensable. Escaping α-cells increased their glucagon content during subsequent months, but there was no significant α-cell regeneration. Near-total α-cell ablation did not prevent hyperglycemia in mice having also undergone massive β-cell loss, indicating that a minimal amount of α-cells can still guarantee normal glucagon signaling in diabetic conditions.

Conclusions: An extremely low amount of α-cells is sufficient to prevent a major counter-regulatory deregulation, both under physiologic and diabetic conditions. We previously reported that α-cells reprogram to insulin production after extreme β-cell loss and now conjecture that the low α-cell requirement could be exploited in future diabetic therapies aimed at regenerating β-cells by reprogramming adult α-cells.

FIG. 1.

α-Cell ablation in Glucagon-DTR mice. A: DT injection triggers α-cell ablation in Glucagon-DTR transgenic mice but not in wild-type mice. Two days after DT injection, dying cells are detected by TUNEL, mainly at islet periphery in Glucagon-DTR animals (middle). At 1 week after DT injection, islets in transgenic mice are devoid of glucagon-expressing cells (right). The dashed lines delineate islets. Scale bars = 20 μm. B: At 1 week after DT treatment, 97.9% of glucagon-expressing cells were destroyed (*P = 0.05, one-tailed Mann-Whitney U test; values in Table 1). C: At 1 week after DT, pancreatic glucagon content was reduced to 0.86% of control (***P = 0.0001, one-tailed Mann-Whitney U test; values in Table 1). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

Extreme α-cell loss moderately decreases circulating glucagon and has no effect on the counter-regulatory response. Evolution of body weight (A) and glycemia (B) in fasted controls (n = 3; DT-untreated, black ♦) and DT-treated (n = 3; red ▲) Glucagon-DTR mice. C: Glycemia after insulin-induced hypoglycemia 1 week after α-cell ablation. Blood glucose increased in both DT-treated mice (red ▲) and controls (black ♦) after an insulin challenge (n = 3/group). D: Plasma glucagon 1 week and 6 months after DT (red □) injections and in control (black □) mice. Glucagonemia was significantly reduced by 35% 1 week after DT (***P = 0.001) but returned to normal levels 6 months later (values in Supplementary Table 2). Confocal images of pancreatic sections stained for insulin (green) and glucagon (red) in controls (E) and Glucagon-DTR mice (F). Very few pancreatic α-cells can be observed 1 week after DT. E’ and F’: higher magnification of the dotted areas depicted in E and F, respectively. Scale bars = 100 μm in E and F and 20 μm in E’ and F’. G: Arginine-induced glucagon secretion from perfused pancreas 1 week after DT or in controls. H: Quantification of arginine-induced glucagon secretion upon arginine stimulation (1,290.2 ± 281.9 for controls, and 423.3 ± 85.3 pg/mL for DT-treated Glucagon-DTR mice; *P = 0.014). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

β-Cell function is unaltered after α-cell ablation. A: Pancreatic insulin content measured 1 week after DT. Consistent with the specificity of α-cell ablation, pancreatic insulin content was unaffected after DT (control: 131.1 ± 5.8, n = 6; DT: 134.0 ± 11.0 ng/mg of pancreas, n = 7). B: Glucose tolerance test. DT-treated Glucagon-DTR mice (red ▲) were not intolerant to glucose 1 week after massive α-cell ablation (n = 6 for each group; control, black ♦). C: Insulin secretion measured from perfused pancreas after glucose stimulation. No significant change in insulin secretion was observed 1 week after α-cell ablation (n = 4 for each group).

FIG. 4.

Changes in pancreatic glucagon content and α-cell number after near-total α-cell loss. A: Pancreatic glucagon content was increased almost sevenfold between 1 week and 1 month (from 23.52 to 157.9 pg/mg; **P = 0.0048, one-tailed Mann-Whitney U test) and doubled between 1 and 6 months (157.9 to 331.4 pg/mg; *P = 0.0317, one-tailed Mann-Whitney U test) in DT-treated animals. B: Pancreatic α-cell number was not increased between 1 week and 1 month after DT but was doubled at 6 months. C: The total number of islets (defined as clusters of at least three β-cells) remained unchanged after α-cell ablation. D: The number of islet sections containing at least one α-cell was dramatically reduced after DT treatment, throughout the whole period of analysis. E: The number of α-cells in islet sections containing α-cells after DT treatment was almost doubled at 6 months. F: The number of α-cells located outside of islets was always lower in DT-treated mice than in controls and did not increase significantly with time after DT. A–F: Black ♦: control; red ▲: DT-treated mice.

FIG. 5.

Newly formed α-cells after ablation are not reprogrammed β-cells. A: Transgenes required for the inducible β-cell tracing and α-cell ablation. B: Experimental design for irreversible labeling of adult β-cells before α-cell ablation. C–F: Almost all β-cells were YFP-labeled after tamoxifen (TAM) administration in control animals (DT-untreated). G–J: At 6 months after DT, none of the very rare glucagon-expressing cells were YFP-positive (the arrowhead points to one α-cell, also shown in the top right inset at higher magnification). Scale bars = 20 μm. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 6.

α-Cell ablation does not prevent streptozotocin (STZ)-induced diabetes. A: Experimental design. One high dose of STZ (200 μg/g of mouse weight) was administered to Glucagon-DTR mice to ablate β-cells 1 week after massive DT-mediated α-cell removal. Animals were killed 2 weeks after STZ. B–G: Confocal images of pancreatic sections show DT-mediated α-cell ablation and STZ-mediated β-cell removal. White arrowheads show remaining α-cells after DT. Scale bars = 20 μm. H: Follow up of glycemia. All STZ-treated mice become hyperglycemic irrespective of DT administration. By contrast, animals that did not receive STZ remain normoglycemic (red ♦: Glucagon-DTR mice treated with both DT and STZ, n = 6; black ▼: Glucagon-DTR mice treated only with STZ, n = 6; black ■: untreated Glucagon-DTR mice, n = 3; red ▲: Glucagon-DTR mice treated only with DT). I: Body weight 15 days after STZ. All mice treated with STZ lose weight and develop typical diabetes symptoms. (A high-quality digital representation of this figure is available in the online issue.)