Reduction in O-GlcNAcylation Mitigates the Severity of Inflammatory Response in Cerulein-Induced Acute Pancreatitis in a Mouse Model

Abstract

Acute pancreatitis (AP) involves premature trypsinogen activation, which mediates a cascade of pro-inflammatory signaling that causes early stages of pancreatic injury. Activation of the transcription factor κB (NF-κB) and secretion of pro-inflammatory mediators are major events in AP. O-GlcNAc transferase (OGT), a stress-sensitive enzyme, was recently implicated to regulate NF-κB activation and inflammation in AP in vitro. This study aims to determine whether a pancreas-specific transgenic reduction in OGT in a mouse model affects the severity of AP in vivo. Mice with reduced pancreatic OGT (OGT^Panc+/-) at 8 weeks of age were randomized to cerulein, which induces pancreatitis, or saline injections. AP was confirmed by elevated amylase levels and on histological analysis. The histological scoring demonstrated that OGT^Panc+/- mice had decreased severity of AP. Additionally, serum lipase, LDH, and TNF-α in OGT^Panc+/- did not significantly increase in response to cerulein treatment as compared to controls, suggesting attenuated AP induction in this model. Our study reveals the effect of reducing pancreatic OGT levels on the severity of pancreatitis, warranting further investigation on the role of OGT in the pathology of AP.

Keywords: O-GlcNAc transferase (OGT); O-GlcNAcylation; acute pancreatitis; cerulein; inflammation; pancreas; pancreatitis.

Figure 1

Body weight and glucose homeostasis did not differ between mice with reduced pancreatic OGT and controls. (A) Representative immunostaining image of a pancreas from an OGT^Panc+/− mouse with Cre reporter GFP, insulin (red), and DAPI (blue). Magnification 10×. (B) Western blotting for OGT and Vinculin (loading control) and quantification of OGT over Vinculin, n = 3–6. (C) Body weight, (D) non-fasted, and (E) fasted blood glucose of mice before induction of pancreatitis at 8–10 weeks (n = 26–33 for (C,D) and n = 4–7 for (E)). (F) IPGTT and calculated AUC (n = 4–7). (G) Pancreas to body weight ratio following treatment with saline or cerulein (n = 12–17). (B–E) were presented as mean ± SEM, two-tailed t-test, and (G) was two-way ANOVA, p values, * p < 0.05, *** p < 0.001.

Figure 2

Mice with reduced pancreatic OGT had a decrease in the histological severity of pancreatitis. (A) Representative H&E image for scoring severity of pancreatitis of a control mouse treated with cerulein. Edema disrupted and separated acini (solid arrows), inflammatory infiltrate in the parenchyma and between lobules (dashed arrows), focal necrosis (asterisks). Magnification 20×. Histological scoring of (B) edema, (C) inflammatory infiltrate, and (D) necrosis, respectively, on a scale of 0–3 (n = 9–12). (E) Cumulative histoscore for the severity of pancreatitis (n = 9–12 animals). (F) Representative H&E images for each genotype and treatment group. Magnification 20×, scale bar 100 µm. (B–E) were presented as mean ± SEM, one-way ANOVA. p values, * p < 0.05, ** p < 0.01.

Figure 3

Lipase, LDH, and TNF-α did not increase in OGT^Panc+/− mice treated with cerulean. (A) Serum amylase (n = 8), (B) lipase (n = 7–13), and (C) LDH (n = 10–13) as well as (D) serum cytokine levels of TNF-α, (E) IL-1β, and (F) IL-6 (n = 6–13) following treatment with saline or cerulein. (A–F) were presented as mean ± SEM, one-way ANOVA. p values, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Macrophage infiltrate following cerulein treatment was reduced when pancreatic OGT was reduced. (A) Macrophage infiltrate, as determined by CD68+ cells in the pancreatic tissue (n = 5–6). (B) Representative immunostaining images for each genotype and treatment group with CD68 (red) and DAPI (blue). Magnification 60×, scale bar 50 µm. (C) Quantification of TUNEL positive cells/high-power field (n = 6–8 animals). (D) Representative image from a control treated group with Amylase (green), TUNEL (red), and DAPI (blue). Magnification 60×, scale bar 50 µm. A was presented as mean ± SEM, one-way ANOVA, and C was presented as mean ± SEM, U-test. p values, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Pancreatic mRNA of the inflammatory and NFκB response. (A) Pancreatic mRNA levels of Ccl2, (B) TNF-α, (C) IL-1β, (D) IL-6, (E) RelA/p65, and (F) IκBα. Values are relative to the saline-treated control condition and normalized to the reference gene 36B4, n for experiments = 4–10 animals. (A–F) were presented as mean ± SEM, one-way ANOVA. p values, * p < 0.05, ** p < 0.01.