Long-term feeding of ethanol alone causes minimal pancreatic tissue injury in animal models [55
]. To account for this lack of injurious effect, we hypothesized that ethanol feeding causes ER stress and that a physiologic adaptive UPR responds to the ER stress preventing pathobiologic pancreatitis responses such as inflammation and cell death. As indicated above, previous studies [58
] demonstrated that all three ER stress/UPR transducers (i.e. IRE-1, ATF6 and PERK) and their downstream pathways are activated in experimental pancreatitis. However, there was no information on the nature of the ER stressors activating the UPR responses in the models of experimental pancreatitis. More importantly, there was no information on how the pancreas responds physiologically to ER stressors generated by alcohol abuse or other potentially toxic factors such as smoking.
To test our hypothesis, we designed a series of studies to characterize the effect of ethanol feeding on development of ER stress and the UPR, and determine the importance of the UPR in physiologic adaptation and prevention of pathologic consequences [66
]. We used rats and mice fed control diets or ethanol-containing diets for 4–6 weeks using the Tsukamoto-French intragastric model [67
] which provides continuous feeding of the diets to the animals. With this feeding protocol we found no obvious pancreatic damage using light microscopic examination of the tissue. However, a careful examination by electron microscopy demonstrated extensive dilation of the ER of acinar cells, an indicator of ER stress. In addition, measurements of the redox status of the ER found that there was decreased reduced glutathione and increased oxidized glutathione in the ethanol-fed rats indicating that the alcohol feeding generates ROS in the ER of the pancreatic acinar cell. Thus, ROS represent at least one set of ER stressors.
In an evaluation of the sensors and signals of the UPR, we found that ethanol feeding increased the expression of IRE-1 and XBP1-S along with a small increase in PERK activation [66
]. In addition, alcohol feeding significantly upregulated a key oxidoreductase, PDI. Finally, ethanol feeding altered PDI structure so that a greater proportion of the PDI was in its oxidized state in the ethanol-fed animals compared to the control-fed.
PDI is an oxoreductase that is critical for protein folding by catalyzing disulfide bond formation, a key step for maturation of proteins in the secretory pathway [68
]. This function is often referred to as ‘oxidative folding’ because PDI catalytic action requires reduced glutathione to sustain its ability to regenerate and sustain its ability to repeatedly form disulfide bridges [69
The combination of findings listed above indicates that ethanol feeding leads to an oxidative state in the ER of the acinar cell. The oxidative state could come from one or more biochemical mechanisms. For example, oxidative ethanol metabolism in the ER could directly lead to the oxidative states. On the other hand, ethanol and/or its metabolites could lead to modifications of nascent proteins in the ER that frustrate oxidative folding so that PDI is ineffective in catalytic redox cycles leading to more utilization and depletion of reduced glutathione. The end result in either situation would be a net generation of ROS in the ER, decreased reduced glutathione and increased oxidized glutathione and PDI itself.
Considering the results described above, we hypothesized that the effect of alcohol feeding to increase expression of IRE-1 and XBP1-S is necessary for the acinar cell to adapt to ER stress resulting from alcohol abuse. We tested this hypothesis using animals with heterozygous deficiency for XBP1. We chose this approach because homozygous depletion of XBP1 results in lethality, and because we hoped for a situation where we could specifically prevent the increase in XBP1 that we observed with ethanol feeding.
In the heterozygous animals, the ethanol feeding led to levels of XBP1-S expression similar to that observed in wild-type animals receiving the control diet allowing us to specifically observe the response of the pancreatic tissue in the absence of the UPR to ethanol feeding. In contrast to the effect in wild-type animals, ethanol feeding resulted in marked morphological and biochemical effects in animals with heterozygous deletion of XBP1.
In the pancreatic tissue of the XBP1-deficient animals receiving alcohol there was a seriously disorganized ultrastructure with a decreased number of zymogen granules, and the remaining ones were inappropriately scattered throughout the cell and not localized to their normal apical position. There was extensive dilation of the ER with occasional dense luminal inclusions, hallmarks of ER stress, as well as significant accumulation of autophagic vacuoles. By electron microscope examination, these abnormalities could be found in more than 40% of total acinar cells. There was also a marked decrease in levels of the digestive enzyme amylase corresponding to the decreased number of digestive enzyme-storing zymogen granules. Finally, about 20% of the pancreas contained lesions representing severe injury. These areas showed acinar cell necrosis, apoptosis and inflammation with replacement by stromal cells and ductular-appearing regenerating cellular structures.
In ethanol-fed animals the XBP1 deficiency prevented the increase in PDI while markedly enhancing PERK and eIF2α phosphorylation and expression of ATF4, all features of prolonged, severe and unchecked ER stress [69
]. These signals mediate translational inhibition (PERK and eIF2α phosphorylation) and expression of CHOP (ATF4) accounting for the decrease in digestive enzyme and increased cell death that we observed. Ethanol feeding in the XBP1-deficient animals also led to a decrease in expression of EDEM1, a key participant in the ERAD pathway for degradation for unfolded and misfolded proteins. Such a decrease in ERAD may account for the marked increase in autophagy in the pancreas of these animals because previous studies [70
] have shown that EDAM1 deficiency enhances autophagy to dispose of misfolded proteins.