Chronic GLP-1 receptor activation by exendin-4 induces expansion of pancreatic duct glands in rats and accelerates formation of dysplastic lesions and chronic pancreatitis in the Kras(G12D) mouse model

Abstract

Pancreatic duct glands (PDGs) have been hypothesized to give rise to pancreatic intraepithelial neoplasia (PanIN). Treatment with the glucagon-like peptide (GLP)-1 analog, exendin-4, for 12 weeks induced the expansion of PDGs with mucinous metaplasia and columnar cell atypia resembling low-grade PanIN in rats. In the pancreata of Pdx1-Cre; LSL-Kras(G12D) mice, exendin-4 led to acceleration of the disruption of exocrine architecture and chronic pancreatitis with mucinous metaplasia and increased formation of murine PanIN lesions. PDGs and PanIN lesions in rodent and human pancreata express the GLP-1 receptor. Exendin-4 induced proproliferative signaling pathways in human pancreatic duct cells, cAMP-protein kinase A and mitogen-activated protein kinase phosphorylation of cAMP-responsive element-binding protein, and increased cyclin D1 expression. These GLP-1 effects were more pronounced in the presence of an activating mutation of Kras and were inhibited by metformin. These data reveal that GLP-1 mimetic therapy may induce focal proliferation in the exocrine pancreas and, in the context of exocrine dysplasia, may accelerate formation of neoplastic PanIN lesions and exacerbate chronic pancreatitis.

FIG. 1.

The extent and frequency of PDGs surrounding the main pancreatic duct are increased by exendin-4 treatment in rats. Sections from the head of the pancreas from an untreated control rat (A) and after 12 weeks of daily exendin-4 injections (E), in which PDG clusters were identified surrounding the main pancreatic duct. PDGs were confined to the mesenchyme surrounding the main duct in controls but, after exendin-4, expanded to the extent that they projected into the lumen of the pancreatic duct as complex villous-like structures. A and E, insets: PDG cells were columnar in comparison with the cuboidal ductal cells and included goblet-like cells (arrowheads). B and C: PDGs contained mucin confirmed by Alcian blue and PAS staining. D: In contrast to duct cells, PDG cells also expressed Pdx-1 (red; combined staining with the duct cell marker cytokeratin [CK] in green). E: PDGs were more common in exendin-4–treated rats (Table 1). F–H: In addition, the epithelium often showed pseudostratification and pseudopapillary features, which are features characteristic for PanIN-like lesions. Scale bars = 200 μm (A and E) and 100 μm (B–D), and magnification ×20 (F–H). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

PDG cell replication is increased by exendin-4 treatment in rats. The frequency of replication ascertained by Ki-67 immunostaining (red; colabeled with cytokeratin [CK] in green) was increased in PDGs compared with adjacent duct cells (*lumen of the large duct) in both control (A) and exendin-4–treated (B) rats. Replication frequency showed variation within the PDGs in control (C) as well as exendin-4–treated (D) animals. E: However, both the abundance of PDGs and the frequency of replication were increased by exendin-4 treatment. Exendin-4 also increased replication in main duct cells but not in the duct cells in the tail of the pancreas. □, control (Ctrl); ■, exendin-4 (Ex). *P < 0.05, scale bars = 100 μm. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

Exendin-4 treatment increased chronic pancreatitis and the frequency of mPanIN lesions in Pdx1-Kras mice. Pancreata from Pdx1-Kras mice treated for 12 weeks with either vehicle (A) or exendin-4 (B) (20× objective). The pancreas from the exendin-4–treated animal demonstrates only scant residual intact acini (white arrow) with more extensive inflammation and fibrosis (stars) and more frequent mPanIN (black arrows). C and D: Low-grade mPanIN1a and mPanIN1b lesions with abundant apical mucin and basally oriented nuclei without significant nuclear pleomorphism or mitotic activity. E and F: Higher-grade mPanIN2 and mPanIN3 lesions with increased nuclear pleomorphism and focal loss of polarity. G: Quantitative analysis of mPanINs showing the percentage of pancreatic ducts with no dysplasia (□, normal [nl]); light-grey box, mPanIN1 (1); medium-grey box, mPanIN2 (2); or ■, mPanIN3 (3) lesions in control (Ctrl) and exendin-4 (Ex)-treated mice. H: Combined amylase (red) and cytokeratin (CK; green) immunofluorescent staining of the pancreas of a control Pdx1-Kras mouse. I: Intact acinar tissue (red) is replaced by cytokeratin-positive (green) ducts, and amylase-positive cells are rarely found in exendin-4–treated animals. Alcian blue staining (blue; counterstained with Nuclear Fast red) reveals mucin-containing lesions in control mice (J) and a higher frequency in treated mice (K). *P < 0.05; **P < 0.01 vs. control. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Duct cell replication frequency is increased in the pancreas of exendin-4–treated Pdx1-Kras mice. Immunohistochemical labeling of Ki-67–positive cells (brown; counterstained with hematoxylin) in benign ducts in areas of intact acinar tissue in control mice (A) and exendin-4–treated mice (B). An area of ductal proliferation embedded in fibrotic tissue shows an increase in Ki-67–positive cells in the exendin-4–treated group (D) compared with controls (C). Note the presence of proliferative ducts and mPanIN1a lesion in the exendin-4–treated animal. E: Analysis of duct cell proliferation by Ki-67 reveals an increase in the replication frequency in Pdx1-Kras mice treated with exendin-4 (Ex; ■) compared with vehicle control (Ctrl; □). *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

GLP-1R expression is present in PDGs in rats and humans. A: In the PDGs (shown here for an exendin-4–treated rat), GLP-1R expression (red) was detected by immunofluorescence with combined labeling for the duct cell marker cytokeratin (CK) in green and DAPI to mark the nuclei in blue. B: Colocalization of GLP-1R and cytokeratin is indicated in the merged images by the color orange. GLP-1R expression was similarly apparent in PDGs in duct cells in the human pancreas. Scale bars = 100 μm. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 6.

GLP-1R expression is present in PanIN lesions in Pdx1-Kras mice and humans. GLP-1R (red; shown with combined cytokeratin [CK] labeling in green) was detected in areas of acinar-to-ductal metaplasia (ADM) (A) and mPanIN lesion (B) in the pancreas of Pdx1-Kras mice. Colocalization of GLP-1R and cytokeratin is indicated in the merged images by the color orange. C: In human pancreas, GLP-1R expression was more apparent in the columnar cells (arrowheads) in regular ducts compared with adjacent normal cuboidal duct cells shown away from the arrowhead. D: Where duct cells adopt the columnar phenotype (PanIN1a lesion shown), GLP-1R expression becomes more apparent. E: In more advanced PanIN3 lesions, GLP-1R immunoreactivity also was clearly present. Scale bars = 100 μm. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 7.

Exendin-4 actions on human pancreatic duct cells. A and B: Time-course experiments of CREB (A) and ERK1/2 (B) phosphorylation in HPDE cells treated with exendin-4 (10 nmol/L) for 0–30 min as indicated. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Representative examples of Western blot experiments are shown in the top panels and the corresponding analysis in the bottom panels. C and D: Effect of long-term (0–9 h) stimulation on cyclin D1 (C) and cyclin A (D) protein levels. Data are expressed as the mean ± SD density ratio of total CREB, ERK1/2 (A and B), as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (C and D) from 3 to 5 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 vs. untreated control value.

FIG. 8.

Oncogenic Kras increases the effects of exendin-4 on human pancreatic duct cells, an effect that is counteracted by metformin. A: Representative Western blot of extracts from HPDE cells stably transfected with control vector (pBP) or oncogenic Kras showing CREB phosphorylation at Ser133. Cells were pretreated with metformin (Metf; 100 μmol/L) for 30 min as indicated, prior to a 15-min stimulation with exendin-4 (Ex; 10 nmol/L). Forskolin (Forsk; 10 μmol/L) was used as the positive control. B: Statistical analysis shows that phosphorylation of CREB by exendin-4 is higher in HPDE-Kras when compared with HPDE-pBP cells (P < 0.05). Metformin treatment abrogated the effect of exendin-4 in HPDE-Kras cells (P < 0.01) but not in HPDE-pBP cells. Data are expressed as the mean ± SD density ratio of total CREB from five independent experiments. *P < 0.05; **P < 0.01. Ctrl, control; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.