Gastrin induces ductal cell dedifferentiation and β-cell neogenesis after 90% pancreatectomy

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   Figure 1

   Metabolic evolution of sham-operated and Px rats. Blood glucose concentrations of sham (S+V) and Px rats treated with vehicle (Px+V) or with gastrin (Px+G). Values are means±s.e.m. n≥5.
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   Figure 2

   Gastrin and CCKBR expression following 90%-Px. Gene expression of (A) gastrin and (B) CCKBR in pancreatic remnant-equivalents from sham-Px rats (S+V, white bars) and pancreatic remnants from 90%-Px rats (Px+V, hatched bars), 1 and 3 days after surgery. (C) Representative image of CCKBR (red) and KRT20 (green) double immunofluorescence of pancreatic remnants from Px+V rats. An area of regeneration is depicted. (D) Quantification of CCKBR expressing cells per mm² of pancreas in sham (S+V, white bars) and Px rats-treated with vehicle (Px+V, hatched bars) or with gastrin (Px+G, black bars). Values are means±s.e.m. ^#P<0.05 vs Px+V for Student's t-test. ANOVA, P<0.05; *P<0.05 vs all other groups; and ^†P<0.05 vs Px+V for Tukey's test. ND, not detected.
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   Figure 3

   Focal areas of regeneration and ductal-cell relative volume after pancreatectomy (Px). (A) Representative fluorescence microscopy image of keratin 20 (KRT20) (Alexa Fluor 488, green) and vimentin (Alexa Fluor 555, red) in focal areas of regeneration in pancreatic remnants of Px rats, 3 days after Px. (B) Relative volume of the focal areas of regeneration in pancreatic remnants from Px+V (hatched bars) and Px+G (black bars) rats, 1 and 3 days after Px. (C) KRT20+ cell relative volume in pancreatic remnant-equivalents from sham-Px (S+V, white bars), and pancreatic remnants from Px+V (hatched bars) and Px+G (black bars) rats, 3 days after surgery. Values are means±s.e.m. from five independent experiments. ^#P<0.05 vs 1 day after Px for Student's t-test. ANOVA, P<0.05; *P<0.05 vs other groups for Tukey's test.
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   Figure 4

   Ductal and epithelial cell markers expression after Px and gastrin treatment. (A) Gene expression profile of ductal markers in remnant-equivalents from sham-Px rats (S+V, white bars) and pancreatic remnants from Px+V (hatched bars) and Px+G (black bars) rats on days 1, 3, 5, and 7 after surgery. (B) Confocal microscopy images of keratin 20 (KRT20) immunofluorescence (Alexa Fluor 555, red) in areas of regeneration from pancreatic remnants of Px+V and Px+G rats, 3 days after Px. (C) KRT20 immunofluorescence intensity quantification. (D) Fluorescence microscopy images of β-catenin immunofluorescence (Alexa Fluor 555, red) in areas of regeneration from pancreatic remnants of Px+V and Px+G rats, 3 days after Px. (E) β-catenin immunofluorescence intensity quantification. (F) Ductal cell replication in the focal areas of regeneration of pancreatic remnants from Px+V and Px+G rats 3 days after Px. Px, pancreatectomy; RQ, relative quantity. Values are means±s.e.m. from five independent experiments. ANOVA, P<0.05; *P<0.05 vs Px+V and ^†P<0.05 vs Px+V for Tukey's test. ^#P<0.05 for Student's t-test.
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   Figure 5

   Effects of gastrin treatment on the expression of transcription factors involved in endocrine specification. (A) Gene expression profile of transcription factors involved in endocrine specification in pancreatic remnant equivalents from sham-Px rats (S+V; white bars) and pancreatic remnants from Px+V (hatched bars) and Px+G (black bars) rats. (B) Fluorescence microscopy images of neurogenin 3 (NEUROG3) immunofluorescence (fluorescein, green) in the focal areas of regeneration from pancreatic remnants on day 1 after Px. NEUROG3 positivity is found in epithelial ductal cells (asterisk) and in the surrounding mesenchymal cells. (C) Confocal microscopy images of keratin 20 (KRT20; Alexa Fluor 488, green), insulin (Alexa Fluor 647, blue) and NKX6-1 (Alexa Fluor 555, red) immunofluorescence in the focal areas of regeneration from pancreatic remnants 3 days following Px. Ductal structures with low expression of NKX6.1 in the nuclei (arrows) and insulin clusters with high expression of NKX6.1 in the nuclei (arrow head) are shown. (D) Quantification of KRT20 positive cells, within the focal areas of regeneration, expressing low levels of NKX6.1. Px, pancreatectomy. Values are means±s.e.m. ANOVA, P<0.05; *P<0.05 vs all other groups and ^†P<0.05 vs Px+V for Tukey's test. ^#P<0.05 for Student's t-test.
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   Figure 6

   Increased β-cell neogenesis and mass in gastrin-treated rats. (A) Representative confocal microscopy image of keratin 20 immunofluorescence (Alexa Fluor 647, red) and insulin (Alexa Fluor 488, green) of areas of regeneration from pancreatic remnants, 3 days after pancreatectomy (Px). (B) Number of small β-cell clusters per mm² of regenerative pancreas. (C) β-cell apoptosis and (D) β-cell replication in pancreatic remnant equivalents of sham-operated pancreases (S+V, white bars) and pancreatic remnants from Px+V (hatched bars) and Px+G (black bars) rats, 3 days after surgery. (E) Cell replication of β-cells located within the non-regenerative (white bars) or regenerative (hatched bars) pancreas, 3 days after pancreactectomy. (F) Evolution of the β-cell relative area after 90%-Px in pancreatic remnants from Px+V (black squares) and Px+G (open circles). (G) β-cell mass of pancreatic remnant equivalents of sham pancreas (S+V, white bars) and pancreatic remnants from Px+V (hatched bars) and Px+G (black bars) rats, the day of surgery, 3 and 14 days after Px. Values are means±s.e.m. Student's t-test, ^†P<0.05. ANOVA, P<0.05; ^#P<0.05 vs Px+V group for Student's t-test; *P<0.05 vs all other groups for Tukey's test.

• © 2014 Society for Endocrinology