1. Biochemistry and Chemical Biology
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Blockade of glucagon signaling prevents or reverses diabetes onset only if residual β-cells persist

  1. Nicolas Damond
  2. Fabrizio Thorel
  3. Julie S Moyers
  4. Maureen J Charron
  5. Patricia M Vuguin
  6. Alvin C Powers
  7. Pedro L Herrera  Is a corresponding author
  1. University of Geneva, Switzerland
  2. Eli Lilly and Company, United States
  3. Albert Einstein College of Medicine, United States
  4. Columbia University, United States
  5. Vanderbilt University, United States
  6. VA Tennessee Valley Healthcare System, United States
Research Article
Cite this article as: eLife 2016;5:e13828 doi: 10.7554/eLife.13828
6 figures and 1 additional file

Figures

Figure 1 with 1 supplement
Gcgr-/- mice become diabetic after massive β-cell ablation.

(A) Random-fed glycemia (left) and area under the glycemia curve (AUC) between days 0 and 7 after DT (right) in untreated (Untr.) and DT-treated RIP-DTR;Gcgr+/- and RIP-DTR;Gcgr-/- females. (B) Body weight (left) and AUC body weight (days 0–7 after DT; right). , all mice of the group were dead at this time point (see Figure 1C). *p<0.05; **p<0.01; Mann-Whitney U test. C: Survival curve of RIP-DTR;Gcgr+/- and RIP-DTR;Gcgr-/- mice after DT treatment (N=5–6). Survival analysis of DT-treated animals (Gcgr+/- versus Gcgr-/-): p=0.044; Log-rank test.

https://doi.org/10.7554/eLife.13828.003
Figure 1—figure supplement 1
Insulin administration stabilizes body weight and allows survival of DT-treated Gcgr-/-mice.

Glycemia (left) and body weight (right) of RIP-DTR;Gcgr+/+ (blue triangles, N=7), RIP-DTR;Gcgr+/- (black squares, N=9), and RIP-DTR;Gcgr-/- (red circles, N=9) males following DT-mediated β-cell ablation and exogenous insulin treatment. Grey areas indicate the period during which mice were treated with insulin detemir (5 U/kg/day between days 6 and 25).

https://doi.org/10.7554/eLife.13828.004
Figure 2 with 2 supplements
Anti-GCGR mAb-treated mice become diabetic after massive β-cell ablation.

(A) Experimental design. (B-C) Random-fed glycemia (B) and body weight (C) after DT in C57BL/6 males pre-treated with vehicle or mAb (N=3).

https://doi.org/10.7554/eLife.13828.005
Figure 2—figure supplement 1
Anti-GCGR mAb administration recapitulates the metabolic and cellular phenotypes of Gcgr-/- mice.

(A) Experimental design. 9 mg/kg anti-GCGR mAb was injected i.p. 3 times per week for 3 weeks in C57BL/6 animals. (B) Left: Random fed glycemia of vehicle- (black squares) or mAb-treated males (red triangles). The grey area indicates the period of antibody treatment. Right: Area under the glycemia curves. *p<0.05; Mann-Whitney U test. C and D. Confocal images of pancreatic islet sections from vehicle- (C) and mAb-treated (D) males. α-cell hyperplasia and hypertrophy (compare C’ and D’, the dashed lines represent the cell perimeters) are observed in islets from mAb-treated mice. Scale bars: 20 μm. E and F. Intraperitoneal glucose tolerance test (E) and insulin tolerance test (F) performed in Gcgr+/+ (black squares, N=9), Gcgr-/- (grey circles, N=10), and mAb pre-treated Gcgr+/+ (red triangles, N=10) males. *p<0.05; **p<0.01; ***p<0.001; Gcgr+/+ versus mAb-treated Gcgr+/+ mice. †, p<0.05; ††, p<0.01; †††, p<0.001; Gcgr+/+ versus Gcgr-/- mice; two-way ANOVA. The difference between Gcgr-/- and mAb-treated Gcgr+/+ mice is not significant.

https://doi.org/10.7554/eLife.13828.006
Figure 2—figure supplement 2
Insulin administration is required to stabilize body weight and allow survival of anti-GCGR-treated mice after DT.

(A-C) Exogenous insulin, but not anti-GCGR mAb treatment, stabilizes body weight and improves survival after extreme β-cell loss. (A) Experimental design. (B) Evolution of body weight following DT administration in RIP-DTR males treated with anti-GCGR mAb and/or exogenous insulin (N=5–12). (C) Survival curves. Survival analyses are indicated next to the legend: n.s., not significant; **p<0.01; ***p<0.001; Log-rank test. Insulin was administered as subcutaneous implants (antibody-untreated mice), or as injections of long-acting insulin (antibody-treated mice) because insulin implants lead to hypoglycemia and death in mice with deficient glucagon signaling.

https://doi.org/10.7554/eLife.13828.007
Figure 3 with 2 supplements
DT administration leads to a more complete β-cell ablation than STZ.

(A) Islet sections stained for insulin (red) and glucagon (green) from untreated, STZ-, or DT-treated RIP-DTR;Gcgr-/- females, 6 days after the last STZ or DT injection. Scale bars: 20 μm. (B-D) β-cell mass (B), pancreatic insulin content (C) and fed plasma insulin levels (D) in untreated (Untr.), STZ-, or DT-treated RIP-DTR;Gcgr-/- males and females, 6 days after the last injection. STZ administration: two injections (200 and 150 mg/kg). *p<0.05; **p<0.01; Mann-Whitney U test.

https://doi.org/10.7554/eLife.13828.008
Figure 3—figure supplement 1
RIP-DTR;Gcgr-/- mice remain hyperglucagonemic and α-cell mass is not affected after STZ- or DT-treatment.

(A-B) fed plasma glucagon levels (A) and α-cell mass (B) in untreated (Untr.), STZ-, or DT-treated RIP-DTR;Gcgr+/- and RIP-DTR;Gcgr-/- males and females, measured 6 days after the last injection. STZ administration: two injections (200 and 150 mg/kg). **p<0.01; Mann-Whitney U test.

https://doi.org/10.7554/eLife.13828.009
Figure 3—figure supplement 2
Higher efficiency of β-cell ablation after DT- than after STZ-treatment in mice with normal glucagon signaling.

(A-B) β-cell mass (A) and pancreatic insulin content (B) in untreated (Untr.), STZ-, or DT-treated RIP-DTR;Gcgr+/- females, measured 6 days after the last injection. STZ administration: two injections (200 and 150 mg/kg). **p<0.01; Mann-Whitney U test.

https://doi.org/10.7554/eLife.13828.010
Figure 4 with 3 supplements
Inhibition of insulin action triggers hyperglycemia in STZ-treated Gcgr-/-mice.

(A) Random-fed glycemia after STZ and/or S961 administration in Gcgr+/- and Gcgr-/- females (left), and area under the glycemia curve (AUC) during S961 treatment (right). (B-D) Hepatic Pepck (top) and Glucokinase (bottom) mRNA levels relative to those of untreated Gcgr+/- (control) mice (N=4–6). (B) Glucagon deficiency: Gcgr-/- background. (C) Insulin deficiency: β-cell ablation or insulin signaling inhibition. (D) Combined deficiency: β-cell ablation and/or insulin signaling inhibition in a Gcgr-/- background. (E-G) FoxO1 mRNA levels in skeletal muscle, relative to those of untreated Gcgr+/- mice (N=4–6). STZ administration: 200 mg/kg at day 0 and 150 mg/kg at day 7. S961 treatment: osmotic pump (days 15 to 21). *p<0.05; **p<0.01; Mann-Whitney U test. Only groups that exhibited a > twofold regulation as compared to controls (dashed lines) were tested.

https://doi.org/10.7554/eLife.13828.011
Figure 4—figure supplement 1
Higher hepatic PEPCK protein expression after DT in both Gcgr+/- and Gcgr-/- mice.

Western blot analysis showing PEPCK and Tubulin expression in the liver of untreated (untr.) and DT-treated RIP-DTR-Gcgr+/- and RIP-DTR-Gcgr-/- females (left). Quantification of PEPCK band intensities relative to Tubulin is shown on the right.

https://doi.org/10.7554/eLife.13828.012
Figure 4—figure supplement 2
Liver glycogen concentration is reduced after DT-treatment in both RIP-DTR-Gcgr+/- and RIP-DTR-Gcgr-/- mice.

Liver glycogen concentration in different conditions of insulin and/or glucagon deficiency (N=4). *p<0.05; Mann-Whitney U test.

https://doi.org/10.7554/eLife.13828.013
Figure 4—figure supplement 3
Expression of genes negatively regulated by insulin signaling in skeletal muscle.

mRNA levels of genes inhibited by insulin in skeletal muscle (gastrocnemius), relative to those of untreated Gcgr+/- females (normalized to Actb, Gapdh, and Gusb) (N=4–6). Irs2, Insulin receptor substrate 2; Fbxo32, F-box only protein 32 (Atrogin-1); Trim63, Tripartite motif-containing 63 (MuRF1); 4e-bp1, Eukaryotic translation initiation factor 4E binding protein 1 (Eif4ebp1); Gadd45a, Growth arrest and DNA-damage-inducible 45 alpha; p21, Cyclin-dependent kinase inhibitor 1A (Cdkn1a). p27, Cyclin-dependent kinase inhibitor 1B (Cdkn1b). *p<0.05; **p<0.01; Mann-Whitney U test. Only groups that exhibited a > twofold regulation as compared to controls (dashed lines) were tested.

https://doi.org/10.7554/eLife.13828.014
Figure 5 with 1 supplement
Anti-GCGR mAb treatment does not normalize hyperglycemia after efficient STZ-mediated β-cell ablation.

(A) Random-fed glycemia in C57BL/6 males treated with STZ (single injection at day 0: 175 or 225 mg/kg) and/or anti-GCGR mAb (osmotic pump, days 6 to 14; N=3–6). (B) Area under the glycemia curves during mAb treatment. (C) Pancreatic insulin content. *p<0.05; **p<0.01; Mann Whitney U test.

https://doi.org/10.7554/eLife.13828.015
Figure 5—figure supplement 1
Hepatic Pepck and Glucokinase expression after STZ and/or anti-GCGR mAb treatment.

Liver Pepck (left) and Glucokinase (right) mRNA levels in mice treated with STZ (single injection at day 0: 175 or 225 mg/kg) and/or anti-GCGR mAb (osmotic pump, days 6 to 14) relative to those of untreated Gcgr+/- mice (N=4–7). *p<0.05; **p<0.01; Mann-Whitney U test. Only groups that exhibited a > twofold regulation as compared to controls (dashed lines) were tested.

https://doi.org/10.7554/eLife.13828.016
Figure 6 with 1 supplement
Absence of glucagon signaling does not block the appearance of new glucagon-insulin bihormonal cells after β-cell ablation.

(A) Islet sections exhibiting glucagon-insulin co-expressing cells (arrowheads) from RIP-DTR;Gcgr+/+ and RIP-DTR;Gcgr-/- females (1 m after DT). Scale bars: 20 μm. (B-D) Percentage of glucagon+ cells that co-express insulin (B), bihormonal cells per islet section (C), and pancreatic insulin content (D) in RIP-DTR;Gcgr+/+ and RIP-DTR;Gcgr-/- females (1 m after DT, N=5–6). (E-F) Percentage of glucagon+ cells that co-express insulin (E), and bihormonal cells per islet section (F) in vehicle- or anti-GCGR mAb- treated RIP-DTR males (2 weeks after DT, N=3). *p<0.05; Mann-Whitney U test.

https://doi.org/10.7554/eLife.13828.017
Figure 6—figure supplement 1
Newly formed bihormonal cells in Gcgr-/- mice are reprogrammed α-cells.

(A) Transgenes required to irreversibly lineage-trace pancreatic α-cells with YFP before β-cell ablation. Inverted triangles represent loxP sites. (B) Experimental design. Upon DOX administration, the transgenic rtTA protein expressed in α-cells binds to the TetO promoter and activates Cre expression, which in turn recombines the STOP sequence in the R26-YFP transgene, leading to irreversible YFP expression. C and D: Example of YFP-traced cells that co-express insulin, as observed after β-cell ablation in a RIP-DTR;Gcg-rtTA;TeTO-Cre;R26-YFP;Gcgr-/- female (C) or in an anti-GCGR mAb-treated RIP-DTR;Gcg-rtTA;TetO-Cre;R26-YFP male (D). Higher magnification of the dotted areas is shown on the right side of panels C and D. YFP was detected using an anti-GFP antibody. Scale bars: 20 μm.

https://doi.org/10.7554/eLife.13828.018

Additional files

Supplementary file 1

Primer sequences used for RT-qPCR.

https://doi.org/10.7554/eLife.13828.019

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