ORIGINAL RESEARCH Ethanol Induced Disordering of Pancreatic Acinar Cell Endoplasmic Reticulum: An ER Stress/Defective Unfolded Protein Response Model Richard T. Waldron,1,2 Hsin-Yuan Su,1 Honit Piplani,1 Joseph Capri,3 Whitaker Cohn,3 Julian P. Whitelegge,3 Kym F. Faull,3 Sugunadevi Sakkiah,1 Ravinder Abrol,1 Wei Yang,1 Bo Zhou,1 Michael R. Freeman,1,2 Stephen J. Pandol,1,2 and Aurelia Lugea1,2 1Department of Medicine, Cedars Sinai Medical Center, Los Angeles, California; 2Department of Medicine, or 3Psychiatry and Biobehavioral Sciences, University of California Los Angeles David Geffen School of Medicine, Los Angeles, California Pancreatic acinar cells Pancreatic acinar cells - no pathology - - Pathology - Ethanol feeding ER sXBP1 Pdi, Grp78… (adaptive UPR) aggregates Proper folding and secretion • disordered ER of proteins processed in the • impaired redox folding endoplasmic reticulum (ER) • ER protein aggregation • secretory defects SUMMARY METHODS: Wild-type and Xbp1þ/- mice were fed control and ethanol diets, then tissues were homogenized and fraction- Heavy alcohol consumption is associated with pancreas ated. ER proteins were labeled with a cysteine-reactive probe, damage, but light drinking shows the opposite effects, isotope-coded affinity tag to obtain a novel pancreatic redox ER reinforcing proteostasis through the unfolded protein proteome. Specific labeling of active serine hydrolases in ER with response orchestrated by X-box binding protein 1. Here, fluorophosphonate desthiobiotin also was characterized pro- ethanol-induced changes in endoplasmic reticulum protein teomically. Protein structural perturbation by redox changes redox and structure/function emerge from an unfolded was evaluated further in molecular dynamic simulations. protein response–deficient genetic model. RESULTS: Ethanol feeding and Xbp1 genetic inhibition altered ER redox balance and destabilized key proteins. Proteomic data and molecular dynamic simulations of Carboxyl ester lipase BACKGROUND & AIMS: Heavy alcohol drinking is associated (Cel), a unique serine hydrolase active within ER, showed an with pancreatitis, whereas moderate intake lowers the uncoupled disulfide bond involving Cel Cys266, Cel dimeriza- risk. Mice fed ethanol long term show no pancreas damage tion, ER retention, and complex formation in ethanol-fed, XBP1- unless adaptive/protective responses mediating proteostasis deficient mice. are disrupted. Pancreatic acini synthesize digestive enzymes (largely serine hydrolases) in the endoplasmic reticulum CONCLUSIONS: Results documented in ethanol-fed mice lack- (ER), where perturbations (eg, alcohol consumption) acti- ing sufficient spliced XBP1 illustrate consequences of ER stress vate adaptive unfolded protein responses orchestrated by extended by preventing unfolded protein response from fully spliced X-box binding protein 1 (XBP1). Here, we examined restoring pancreatic acinar cell proteostasis during ethanol- ethanol-induced early structural changes in pancreatic induced redox challenge. In this model, orderly protein ER proteins. folding and transport to the secretory pathway were disrupted, 480 Waldron et al Cellular and Molecular Gastroenterology and Hepatology Vol. 5, No. 4 and abundant molecules including Cel with perturbed sulfhydryls with isotope-coded affinity tags (ICAT). To structures were retained in ER, promoting ER stress- complement this approach, we also searched for peptides related pancreas pathology. (Cell Mol Gastroenterol Hepatol with cysteines modified by other biochemical residues. 2018;5:479–497; https://doi.org/10.1016/j.jcmgh.2018.01.001) Many abundant secretory proteins synthesized in the ER of the acinar cell belong to the serine hydrolase enzyme 17 Keywords: Alcohol Pancreatitis; Carboxyl Ester Lipase; Disulfide family. Although most serine hydrolases (eg, trypsin) are Bond; Unfolded Protein Response. produced as inactive proenzymes, aberrant activation within the cell has been mechanistically linked to pancreas pathology. Altered cysteine oxidation and disulfide bond lthough heavy drinking is a risk factor for both acute formation might overtly change serine hydrolase activity by A and chronic pancreatitis,1 only a small minority of altering protein structure. We therefore also investigated alcohol abusers acquire pancreatic diseases.2 Pancreatitis is the activity of serine hydrolases in the ER fraction to show initiated in the pancreatic acinar cell upon acinar cell their potential to act as causal links between ethanol dysfunction.3 The acinar cells are specialized secretory cells feeding, deficient UPR, and pancreatic pathology. with a prominent role as biosynthetic factories producing and secreting digestive enzymes. An extensive ribosome- Materials and Methods studded endoplasmic reticulum (ER) network in these cells 4 Experimental Animal Model of Impaired Oxidative enables high rates of enzyme production. Several studies have indicated that disorders in ER function are associated Refolding in Pancreas – fi þ þ þ with acute and chronic pancreatitis,5 10 but the precise al- XBP1-de cient (Xbp1 /-) and wild-type ([WT] Xbp1 / ) terations in ER components and ER protein processing mice were used for the study. Detailed information about þ during pancreatitis are poorly defined. Xbp1 /- mouse genotype and phenotype, diet composition, Protein folding in the acinar cell ER requires balanced and intragastric feeding procedure is provided later. 11 Pancreas and pancreatic acinar cell pathology in this model redox conditions. We previously found adverse effects of 12,14 chronic ethanol feeding in pancreas ER, such as an increased was reported previously. oxidized-to-reduced-glutathione ratio.12,13 This perturbs protein folding and induces an unfolded protein response XBP1-Deficient Mice (UPR) involving up-regulation of the UPR transcription fac- We used mice with a null mutation in 1 Xbp1 allele tor, spliced X box-binding protein 1 (sXBP1) that limits (Xbp1þ/-) and littermate WT controls (Xbp1þ/þ, BALB/c ethanol-mediated damage.10,12 In this respect, genetic dele- background). Xbp1þ/- mice were generated by Ozcan tion of XBP1 in mice prevented increases in sXBP1, associ- et al,18 and have been described previously.12 We used Xbp1 ated with severe ER stress, disordered autophagy, decreased heterozygous mice because complete Xbp1 deletion results secretion of digestive enzymes from acinar cells, acinar cell in embryonic lethality, and pancreas-specific deletion death, and pancreas damage.12,14 More recently, we also results in rapid, extensive acinar cell death.19 In regular found that cigarette smoke extracts reduced sXBP1 levels, housing conditions and compared with WT mice, protein and increased ethanol-induced ER stress and cell death in levels of sXBP1 and unspliced (u) XBP1 in pancreas are acinar cells.10 Moreover, rats exposed to cigarette smoke reduced w40% and w30%, respectively, in Xbp1þ/- showed lower pancreatic sXBP1 levels and more severe mice.12 Xbp1þ/- mice are fertile and healthy, and grow at a alcoholic pancreatitis than control animals.10 These studies rate similar than their WT littermates when maintained on led us to propose that sXBP1 plays an essential role to pre- standard chow diets. However, compared with WT, Xbp1þ/- vent ethanol-induced ER dysfunction. Here, our goal was to mice show increased hepatic ER stress and insulin resis- identify novel ethanol feeding–induced changes in protein tance when placed on a high-fat diet for 16 weeks.18 processing in the ER that precede and sensitize the pancreas Moreover, Xbp1þ/- mice fed intragastrically with ethanol- to frank pathologic damage. containing diets show in pancreatic acinar cells signs of Because ethanol altered ER redox, and XBP1 genetic ER stress, decreased numbers of zymogen granules, deficiency (in Xbp1þ/- mice) increased damage in the increased autophagy, and 10%–15% loss of acinar cells.12 context of ethanol feeding, we focused on the role of ethanol-induced protein oxidation in pancreatic pathology. Abbreviations used in this paper: ATPase, adenosine triphosphatase; We hypothesized that the ethanol feeding-induced increase Cel, carboxyl ester lipase; DTT, dithiothreitol; ER, endoplasmic retic- in sXBP1 in the acinar cell supports normal protein pro- ulum; ERAD, endoplasmic reticulum–associated degradation; FAEE, fi fatty acid ethyl esters; FP, fluorophosphonate; ICAT, isotope-coded cessing and traf cking in wild-type mice by correcting affinity tags; LC-MS/MS, liquid chromatography-tandem mass spec- redox-induced changes in ER proteins. We further predicted trometry; MW, molecular weight; %-ox, percentage oxidized; RER, rough ER; sXBP1, spliced X box-binding protein 1; UPR, unfolded that ethanol would lead to accumulation of redox-driven protein response; WT, wild type. misfolding or other alterations in ER proteins in Xbp1þ/- Most current article mice (with a deficient UPR). We used liquid chromatography–tandem mass spectrometry (LC-MS/MS) © 2018 The Authors. Published by Elsevier Inc. on behalf of the AGA Institute. This is an open access article under the CC BY-NC-ND to characterize effects of ethanol feeding and XBP1 license (http://creativecommons.org/licenses/by-nc-nd/4.0/). deficiency on pancreatic ER proteins, and OXICAT15,16 to 2352-345X https://doi.org/10.1016/j.jcmgh.2018.01.001 quantify protein disulfides after reaction of cysteine 2018 Ethanol and the Pancreatic Acinar ER Proteome 481 Intragastric Ethanol Feeding in Mice expressed as a percentage of body weight at death vs initial Chronic ethanol feeding was performed