Ethanol feeding accelerates pancreatitis progression in CPA1 N256K mutant mice

Abstract

Alcoholic pancreatitis is a multifactorial, progressive, inflammatory disorder of the pancreas. Alcohol initiates pancreatitis and promotes its progression in the context of genetic susceptibility and/or other environmental risk factors such as smoking. Genetic mutations can cause digestive enzyme misfolding, which induces endoplasmic reticulum (ER) stress and elicits pancreatitis. Here, we tested the hypothesis that alcohol synergizes with misfolding in promoting ER stress and thereby accelerates chronic pancreatitis progression. To this end, we fed an ethanol-containing diet to CPA1 N256K mice, which carry the human p.N256K CPA1 mutation and develop spontaneous chronic pancreatitis. Inexplicably, CPA1 N256K mice suffered generalized seizures after 2-3 wk of ethanol feeding, which resulted in high mortality and the early termination of the study. Analysis of CPA1 N256K mice euthanized after 3-3.5 wk of ethanol feeding revealed more severe chronic pancreatitis associated with significantly increased Hspa5 [ER chaperone immunoglobulin heavy chain-binding protein (BiP)] mRNA levels when compared with CPA1 N256K mice on a control liquid diet. In contrast, ethanol feeding of C57BL/6N mice for 4 wk increased Hspa5 levels to a lesser degree and caused no pancreatitis. We conclude that ethanol feeding synergizes with the misfolding CPA1 mutant in promoting ER stress and thereby accelerates progression of chronic pancreatitis in CPA1 N256K mice.NEW & NOTEWORTHY Alcoholic pancreatitis is a multifactorial, progressive, inflammatory disorder of the pancreas. This study demonstrates that alcohol synergizes with digestive enzyme misfolding in promoting endoplasmic reticulum stress and thereby accelerates progression of chronic pancreatitis.

Keywords: chronic pancreatitis; endoplasmic reticulum stress; genetic mutation; mouse model.

Graphical abstract

Fig. 1.

Experimental design of ethanol feeding and effect on mortality. A: experimental protocol and actual experiments performed. B: Kaplan-Meier plot of survival of ethanol-fed mice. Note that 2 carboxypeptidase A1 (CPA1) N256K male mice were euthanized for malocclusion on days 9 and 12. From the 24 spontaneously deceased CPA1 N256K mice, we were able to obtain pancreas tissue for analysis in 13 cases.

Fig. 2.

Food consumption of C57BL/6N and carboxypeptidase A1 (CPA1) N256K mice. The average daily consumption (in mL units) of ethanol-fed and pair-fed mice from experiments 1, 2, and 4 was plotted as individual data points. The table indicates means ± SD values. Control, control liquid diet; EtOH, ethanol-containing liquid diet.

Fig. 3.

Effect of ethanol feeding on body weight. The average weekly body weights (in gram units) of C57BL/6N and carboxypeptidase A1 (CPA1) N256K mice are shown as individual data points. The table indicates means ± SD values. Control, control liquid diet; EtOH, ethanol-containing liquid diet.

Fig. 4.

Effect of ethanol feeding on pancreas protease zymogen content and plasma amylase levels. A: trypsinogen, chymotrypsinogen, and carboxypeptidase A (CPA) content were measured from pancreas homogenates of C57BL/6N mice, as detailed in methods. B: plasma amylase levels of C57BL/6N and carboxypeptidase A1 (CPA1) N256K mice were determined as described in methods. Individual values with means ± SD are shown. Note that a mOD/min substrate cleavage rate value corresponds to 23.8 U/L amylase activity. Control, control liquid diet; EtOH, ethanol-containing liquid diet.

Fig. 5.

Effect of ethanol feeding on pancreas weight. A: pancreas weight of C57BL/6N and carboxypeptidase A1 (CPA1) N256K mice in mg units. B: normalized pancreas weight expressed as % body weight. Individual values with means ± SD are shown. Control, control liquid diet; EtOH, ethanol-containing liquid diet. Gray squares indicate female mice.

Fig. 6.

Effect of ethanol feeding on pancreas histology. Pancreas sections from C57BL/6N and carboxypeptidase A1 (CPA1) N256K mice were stained with hematoxylin and eosin. The scale bars correspond to 50 µm and 20 µm [control liquid diet slides (control)] and 50 µm, 20 µm, and 20 µm [ethanol-containing liquid diet (EtOH) slides]. The extent of acinar cell loss in CPA1 N256K mice was quantitated by visual scoring and expressed as percent of tissue area. Gray squares indicate female mice.

Fig. 7.

Effect of ethanol feeding on macrophage infiltration in the pancreases of carboxypeptidase A1 (CPA1) N256K mice. Immunohistochemistry was performed on representative pancreas sections for the F4/80 macrophage marker. Scale bars correspond to 50 µm. Control, control liquid diet; EtOH, ethanol-containing liquid diet.

Fig. 8.

Effect of ethanol feeding on fibrosis of the pancreas. Representative pancreas sections from C57BL/6N and carboxypeptidase A1 (CPA1) N256K mice were stained with Masson’s trichrome staining, which highlights collagen in blue. The scale bars correspond to 50 µm and 20 µm [control liquid diet slides (control)] and 50 µm, 20 µm, and 20 µm [ethanol-containing liquid diet (EtOH) slides].

Fig. 9.

Effect of ethanol feeding on the hydroxyproline content of the pancreas. Hydroxyproline was measured from pancreas homogenates of C57BL/6N and carboxypeptidase A1 (CPA1) N256K mice, as described in methods. Individual values with means ± SD are shown. Gray squares indicate female mice. Control, control liquid diet; EtOH, ethanol-containing liquid diet.

Fig. 10.

Effect of ethanol feeding on the mRNA expression of endoplasmic reticulum stress markers Hspa5 (A) and Ddit3 (B). Quantitative PCR was performed on pancreatic cDNA samples from C57BL/6N and carboxypeptidase A1 (CPA1) N256K mice, and results were expressed as fold change relative to the average of the C57BL/6N results. Individual values with means ± SD are shown. Gray squares indicate female mice. Control, control liquid diet; EtOH, ethanol-containing liquid diet.