Marked expansion of exocrine and endocrine pancreas with incretin therapy in humans with increased exocrine pancreas dysplasia and the potential for glucagon-producing neuroendocrine tumors

Abstract

Controversy exists regarding the potential regenerative influences of incretin therapy on pancreatic β-cells versus possible adverse pancreatic proliferative effects. Examination of pancreata from age-matched organ donors with type 2 diabetes mellitus (DM) treated by incretin therapy (n = 8) or other therapy (n = 12) and nondiabetic control subjects (n = 14) reveals an ∼40% increased pancreatic mass in DM treated with incretin therapy, with both increased exocrine cell proliferation (P < 0.0001) and dysplasia (increased pancreatic intraepithelial neoplasia, P < 0.01). Pancreata in DM treated with incretin therapy were notable for α-cell hyperplasia and glucagon-expressing microadenomas (3 of 8) and a neuroendocrine tumor. β-Cell mass was reduced by ∼60% in those with DM, yet a sixfold increase was observed in incretin-treated subjects, although DM persisted. Endocrine cells costaining for insulin and glucagon were increased in DM compared with non-DM control subjects (P < 0.05) and markedly further increased by incretin therapy (P < 0.05). In conclusion, incretin therapy in humans resulted in a marked expansion of the exocrine and endocrine pancreatic compartments, the former being accompanied by increased proliferation and dysplasia and the latter by α-cell hyperplasia with the potential for evolution into neuroendocrine tumors.

FIG. 1.

Pancreas, β-cell, and α-cell mass. A: Mean pancreatic weight. *P < 0.05 DM-I vs. DM. B: Pancreatic fractional insulin area. ***P < 0.0001 DM-I vs. DM, **P < 0.001 DM vs. ND. C: β-Cell diameter. *P < 0.05 DM-I vs. DM. D: β-Cell mass. ^P < 0.01 DM-I vs. DM, *P < 0.05 DM-I vs. ND, *P < 0.05 DM vs. ND. E: Pancreatic fractional glucagon area. ***P < 0.0001 DM-I vs. DM and ND. F: α-Cell diameter. ^P < 0.01 DM-I vs. DM, #P < 0.005 DM-I vs. ND. G: α-Cell mass. ^P < 0.01 DM-I vs. DM. *P < 0.05 DM-I vs. ND. Pancreatic weight was 40% increased in DM-I compared with DM (P < 0.05). β-Cell mass was decreased in DM compared with ND but was approximately sixfold increased in DM-I compared with DM. α-Cell mass was comparable in DM and ND but was approximately fivefold increased in DM-I compared with DM. The increase in β-cell and α-cell mass with incretin treatment was predominantly due to endocrine hyperplasia rather than hypertrophy.

FIG. 2.

Glucagon immunohistochemistry in pancreas in DM after incretin therapy. A–E: Sections of pancreas from DM-I donors (cases 6185, 6186, 6206, and 6203 with sitagliptin) immunostained for glucagon (pink) with hematoxylin counterstain. Exuberant expansion of glucagon immunoreactive cells is seen as enlarged eccentrically shaped islets as well as nodular and linear aggregates of cells intimately associated with ducts and demonstrating variable extension into duct lumens (arrow). C–E: Higher-power images show glucagon immunoreactivity in cells lining ducts. F–I: Pancreas sections from DM-I donors (case 6199, sitagliptin; 6189, exenatide) show immunofluorescent costaining for cytokeratin (green), glucagon (red), and DAPI nuclear counterstain (blue). Glucagon-expressing cells are present within and adjacent to keratin-positive duct structures. G: One cell costaining for cytokeratin and glucagon is indicated by the arrow.

FIG. 3.

Distribution of α-cells and β-cells in relation to ducts in DM-I donors. Serial adjacent sections of pancreas from DM-I donors (case 6189, exenatide; 6185, sitagliptin) were immunostained for glucagon (A and C) or insulin (B and D) with hematoxylin counterstain. These serial sections indicate that the exuberant endocrine growth associated with ducts is predominantly comprised of glucagon immunoreactive cells. The percentage of glucagon-positive cells in ducts (E) and insulin-positive cells in ducts (F) is shown. The percentage of glucagon-positive cells in ducts was increased in the DM-I group compared with the DM and ND groups (*P < 0.05). In contrast, the percentage of insulin-positive cells in ducts was unchanged in the DM-I group.

FIG. 4.

Evidence for a direct role of GLP-1 mimetic action in α-cell hyperplasia. Images of pancreatic sections (A–F) are provided to illustrate a similar pattern of α-cell hyperplasia in the donor treated with exenatide (6189) as in the sitagliptin-treated donors, implying a role for GLP-1 action independent of DPP-4 inhibition in α-cell hyperplasia. Enlarged and often eccentrically shaped islets are apparent, as well as increased numbers of glucagon immunoreactive cells associated with and surrounding ductal structures.

FIG. 5.

Pancreatic glucagon expressing neuroendocrine tumor and microadenoma. Grossly visible lesion (A) and corresponding section stained with hematoxylin and eosin (B) of the clinically undetected glucagon-expressing neuroendocrine tumor in the pancreas of a DM-I donor (nPOD case 6185) after sitagliptin therapy. Gross specimen (C) and corresponding hematoxylin and eosin–stained section (D) of a glucagon-expressing microadenoma in nPOD case 6206, DM after sitagliptin therapy. The inset shows high-power view of representative cells stained for glucagon by immunohistochemistry. (See Supplementary Fig. 3 for an additional image of microadenoma in case 6206.)

FIG. 6.

Insulin and glucagon coexpression. Co-immunofluorescent images of islets from case 6185, sitagliptin (A–H) show endocrine cells coexpressing insulin (green) (A and E), glucagon (red) (B and F), and merged (D and H). The proportion of endocrine cells per islet that were thus detected as coexpressing insulin and glucagon was markedly increased in DM-I. Confocal images are shown of an islet from case 6185 stained for insulin (green) (I) and glucagon (red) (J), and a merged image (K) shows a mixture of cells, some expressing insulin or glucagon only (arrowheads) and some showing coexpression (arrows). DAPI nuclear counterstain (blue).

FIG. 7.

Pancreatic intraepithelial neoplasia, endocrine complexes, and cellular replication. Photomicrographs show sections with immunohistochemical staining for Ki67 (brown) and glucagon (pink) in PanIN lesions (A and C) with hematoxylin counterstain or Ki67 (brown) and insulin (pink) (B and D) with Alcian blue counterstain to highlight mucin. Glucagon-expressing endocrine cells are shown intimately associated with PanIN lesions to varying degrees. Foci of replication (arrows, Ki67 nuclei) are also apparent. (See Supplementary Fig. 4 for additional examples of foci of increased replication in incretin treated pancreas.) E: Pancreas cell replication is increased in DM-I (Ki67). ***P < 0.0001 DM-I vs. DM and ND. F: Frequency of PanIN1 and 2 (lesions/mm² ×10³ of pancreas) is increased in DM-I. ^P < 0.01 DM-I vs. DM, ***P < 0.0001 DM-I vs. ND.