Figure 1.
Representative micrographs showing the range of exocrine pancreatic injury associated with drug doses and time points in HFD mice.
(A) Saline, 3 weeks; normal acinar cell morphology representative of all control time points. (B) 30 µg/mg exenatide, 3 weeks, acute focal acinar cell necrosis (black arrows), interstitial edema with hemorrhage (black arrowhead). (C) 30 µg/mg exenatide, 6 weeks, periductal acinar cell autophagy (white arrow), early ductal hyperplasia (black arrowhead), capillary dilatation (white arrowhead), and increase in early fibrosis (black arrow). (D) 30 µg/mg exenatide, 6 weeks, acinar cell autophagy, apoptosis (black arrows), ductal dilatation (black arrowhead) and early ductal hyperplasia (white arrowheads) with deposit of protein-rich materials in the interstitium (white arrow). (E) 3 µg/mg exenatide, 12 weeks, acinar cell hypertrophy (increased ratio of zymogen granules; white arrows) and autophagy (white arrowheads). (F) 3 µg/mg exenatide, 12 weeks, severe acinar cell necrosis (white arrowhead), interstitial inflammation (white arrows), and fat necrosis with calcification (black arrows). (G) 3 µg/mg exenatide, 12 weeks, severe, complex pancreatic injury including acinar cell loss, interstitial edema, and early ductal hyperplasia and fibrosis. Note adjacent non-affected (white arrows) acini. (H) 3 µg/mg exenatide, 12 weeks, severe acinar cell loss, atrophy, ductal hyperplasia (black arrows), fibroblast proliferation (black arrowheads), and deposit of protein-rich materials in the interstitium (white arrows). A–H, X630, H&E stain.
Figure 2.
Representative micrographs showing range of exocrine pancreatic injury associated with drug doses and time points in HFD mice.
(A) 10 µg/mg exenatide, 12 weeks, acinar cell hypertrophy (white arrows) in association with autophagy (black arrows), and interacinar edema (white arrowheads). (B) 10 µg/mg exenatide, 12 weeks, several mitoses (white arrows) indicative of cell proliferation (hyperplasia). (C) 10 µg/mg exenatide, 12 weeks, acinar cell apoptosis (white arrowheads), necrosis (black arrowheads) and inflammatory edema (black arrows) in the interstitium. (D) 10 µg/mg exenatide, 12 weeks, vasculitis (white arrows) in association with interstitial inflammation and edema (black arrows). (E) 30 µg/mg exenatide, 12 weeks, severe interstitial inflammation and edema (black arrows) in association with acinar cell hypertrophy (white arrows) and ductal hyperplasia (black arrowheads). (F) 30 µg/mg exenatide, 12 weeks, the most complex pancreatic injury: mixture of a variety of acinar cell injuries (black arrows), ductal hyperplasia (white arrows), interacinar fibrosis (white arrowheads). (G) 30 µg/mg exenatide, 12 weeks, deposit of a protein-rich fluid (exudate) surrounding injured arterioles (black arrows) and ductal hyperplasia (white arrows). (H) 30 µg/mg exenatide, 12 weeks, increase in fibroblast proliferation (interstitial fibrosis; white arrows) and ductal hyperplasia (black arrows). A–H, X630, H&E stain.
Table 1.
Histopathology evaluations link time exposure and dose concentration dependent increases in exocrine pancreatic injury to exenatide treatments.
Figure 3.
Representative micrographs comparing Reg3γ immunoreactivity to morphologic change in HFD mice.
(A & B) Saline, 12 weeks, weak Reg3γ immunoreactivity (A) characterized by discrete fine punctate and sparse coarse particles in the cytoplasm of acinar cells observed in areas of normal acinar cell morphology (B). (C & D) 30 µg/mg exenatide, 12 weeks, increased Reg3γ immunoreactivity (C) characterized by strongly intensive staining occupying almost all cytoplasm of acinar cells seen in areas demonstrating severe acinar cell injuries (D) including autophagy, apoptosis, necrosis. (E & F) 30 µg/mg exenatide, 12 weeks, increased Reg3γ immunoreactivity (E) characterized by intensive homogenous staining predominantly located in the zymogen granular zones of acinar cells. The staining pattern matched areas of acinar cell hypertrophy (F). Note normal acinar cells of separate lobule in top right corner. (G & H) 30 µg/mg exenatide, 12 weeks, Reg3γ showed a staining pattern (G) with distinct coarse particles in non-zymogen granular zones in areas of inflammation and necrosis (H). A–H, X600, A, C, E, G (IHC for Reg 3γ); B, D, F, H (H&E stain).
Figure 4.
Representative micrographs comparing proliferative (Ki-67) and apoptosis (TUNEL) immunoreactivity to morphologic changes in HFD mice.
(A & B) Saline, 12 weeks, Ki-67 staining (A) was occasionally identified in nuclei in areas of normal acinar and centroacinar cells (B). (C & D) 30 µg/mg exenatide, 12 weeks, increased Ki-67 immunoreactivity (C) characterized by more frequently stained nuclei in acinar, centroacinar, ductal, and interstitial cells observed in areas of severe acinar cell injury (D) including autophagy, apoptosis, and necrosis and ductal metaplasia. Two nuclei placed side by side with basophilic stained cytoplasm are suggestive of cell proliferation (hyperplasia). (E & F) 30 µg/mg exenatide, 12 weeks, increased Ki-67 immunoreactivity (E) characterized by nuclear staining in epithelial cells of the main duct observed in areas of pseudostratified columnar epithelium with increased number of goblet cells (F). Inset in F showing main pancreatic ductal cell proliferation. (G) Saline, 12 weeks, TUNEL staining revealed an occasional acinar cell undergoing apoptosis. (H) 30 µg/mg exenatide, 12 weeks, revealed many apoptotic cells in areas associated with acinar cell injury. A–H, X600, A, C, E, (IHC for Ki-67); B, D, F (H&E stain); G & H (TUNEL). Inset in F, X200.
Table 2.
Group mean of digital analysis of immunohistochemical staining reveals increased apoptosis, proliferation, and acinar cell stress in areas of exenatide injury.
Table 3.
Immunoreactivity scores of Reg 3γ, Ki-67, and apoptosis in pancreas of mice treated with saline or exenatide (30 µg/mg, s.c. injection) for 12 weeks.
Table 4.
Cell death/survival/proliferation/differentation related gene expression in mice treated with EXE for 12 weeks indicates increased cell survival compared to controls.
Table 5.
Oxidation/β-oxidation of fatty acid related gene expression in mice treated with EXE for 12 weeks indicates altered lipid metabolism compared to controls.