Table 1.
Sequences of target crRNAs flanking exons 4 to 13 of the Gcgr gene, and corresponding donor single-stranded oligodeoxynucleotides (ssODNs), including loxP.
Fig 1.
Deficiency of proteins encoded by the proglucagon gene induces PP-cell hyperplasia and the formation of multi-hormone-producing cells.
(A) Representative immunofluorescence staining of GCG or GFP (green), PP (red), and nuclei (DAPI; blue) in pancreas sections from 40-week-old GCG+/+ mice and Gcggfp/gfp mice. A magnified view of the boxed region is shown on the very right. (B) Representative immunofluorescence staining of PP (blue), GCG or GFP (green), and SST (red) in pancreas sections from 40-week-old Gcg+/+ mice and Gcggfp/gfp mice (B). Scale bars represent 30 μm or 50 μm.
Fig 2.
GCG signaling deficiency induces PP-cell hyperplasia and the appearance of GCG+ PP+ cells.
(A) Schematic diagram of the loxP target site. (B) Representative immunofluorescence staining of GCG (green), PP (red), and nuclei (DAPI; blue) in pancreas sections from 10-week-old Gcgr+/+ mice and GcgrΔ/Δ mice. Magnified images of the boxed region are shown at the bottom. Scale bar represents 30 μm. PP-cell mass (C) and the proportion of GCG⁺ PP⁺ cells among the GCG⁺ cells (D) in 10-week-old Gcgr+/+ mice and GcgrΔ/Δ mice (n = 4–7 mice). Data are shown as the mean ± SEM, and was analyzed by the two-tailed unpaired Student t-test. **p < 0.01, ***p < 0.001.
Fig 3.
PP-cell hyperplasia involves both the proliferation of PP cells and an increase in GCG+ PP+ cells.
(A) Representative immunofluorescence staining of GCG (green), PP (red), and nuclei (DAPI; blue) in pancreas sections from 4-week-old Gcgr+/+ mice and GcgrΔ/Δ mice. Scale bar represents 30 μm. (B) PP-cell mass in 4-week-old Gcgr+/+ mice and GcgrΔ/Δ mice (n = 6 mice each). (C) Representative immunofluorescence staining of GCG (green), PP (red), and nuclei (DAPI; blue) in pancreas sections from 4-week-old and 10-week-old Gcgr+/+ mice and GcgrΔ/Δ mice. Scale bar represents 30 μm. (D) Proportions of GCG⁺ PP⁺ cells among GCG⁺ cells in 4-week-old and 10-week-old Gcgr+/+ mice and GcgrΔ/Δ mice (n = 4 mice each). (E) Representative immunofluorescence staining of PP (blue), GCG (green), and Ki67 (red) in pancreas sections from 4-week-old Gcgr+/+ mice and GcgrΔ/Δ mice. Scale bar represents 30 μm. A magnified image of the boxed region is shown on the very right. (F) Proportions of Ki67⁺ PP⁺ GCG‒ cells among PP⁺ cells in 10-week-old Gcgr+/+ mice and GcgrΔ/Δ mice (n = 4 mice each). Data are shown as the mean ± SEM, and analyzed by the two-tailed unpaired Student t-test (B and F) or one-way ANOVA followed by the Tukey test (D). **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 4.
GCG dysfunction in the liver induces PP-cell hyperplasia and the appearance of GCG+ PP+ cells.
(A) Representative immunofluorescence staining of GCG (green), PP (red), and nuclei (DAPI; blue) in pancreas sections from 10-week-old Gcgrf/f mice and Alb-Cre;Gcgrf/f mice. Scale bar represents 30 μm. The PP-cell mass (B) and the proportion of GCG⁺ PP⁺ cells among GCG⁺ cells (C) in 10-week-old Gcgrf/f mice and Alb-Cre;Gcgrf/f mice (n = 5 mice each) are shown. (D) Representative immunofluorescence staining of GCG (green), PP (red), and nuclei (DAPI; blue) in pancreas sections from 40-week-old Gcgrf/f mice and Alb-Cre;Gcgrf/f mice. (E) Representative immunofluorescence staining of INS (green), PP (red), and nuclei (DAPI; blue) in pancreas sections from 40-week-old Gcgrf/f mice and Alb-Cre;Gcgrf/f mice. Scale bar represents 30 μm. In (B) and (C), data are shown as the mean ± SEM, and analyzed by the two-tailed unpaired Student t-test. *p < 0.05, **p < 0.01. In C, D, and E, magnified images of the boxed regions are shown in the bottom row.
Fig 5.
GCG+ PP+ cells are increased in isolated islets upon incubation in high concentrations of glutamine, and this increase is mediated by the mTOR pathway.
(A) Representative immunofluorescence staining of PP (red), GCG (white), and nuclei (DAPI; blue) in pancreatic islets from 10-week-old wild-type mice, either untreated (Ctrl, upper panels) or incubated with a high concentration glutamine (Gln, lower panels). Scale bar represents 30 μm. Magnified images of the boxed region are shown at the bottom. The proportion of GCG⁺ cells among islet cells (B) (n = 10 islets), the proportion of PP cells among islet cells (C) (n = 10 islets), and the proportion of GCG⁺ PP⁺ cells among GCG⁺ islet cells (D) (n = 10 islets) from 10-week-old wild-type mice, either untreated (Ctrl) or incubated in a high concentration of glutamine (Gln). (E) Representative immunofluorescence staining of PP (red) and GCG (white) in pancreatic islets isolated from 10-week-old wild-type mice. Untreated islets (Ctrl, left panel), islets incubated with a high concentration of glutamine (Gln, middle panel), and islets incubated with both a high concentration of glutamine and rapamycin (Gln+Rap, right panel) are shown. Scale bar represents 30 μm. (F) Proportions of GCG⁺ PP⁺ cells among GCG⁺ cells of the islets (n = 10 islets) from 10-week-old wild-type mice, which were either untreated (Ctrl), incubated with a high concentration of glutamine (Gln), or incubated with both a high concentration of glutamine and rapamycin (Gln+Rap). Data are shown as the mean ± SEM, and analyzed by the two-tailed unpaired Student t-test (B–D) or one-way ANOVA followed by the Tukey test (F). **p < 0.01, ***p < 0.001, ns, not significant.