Volume 140, Issue 3, Pages 987-997.e8 (March 2011) Adaptive Unfolded Protein Response Attenuates Alcohol-Induced Pancreatic Damage  Aurelia Lugea, David Tischler, Janie Nguyen, Jun Gong, Ilya Gukovsky, Samuel W. French, Fred S. Gorelick, Stephen J. Pandol  Gastroenterology  Volume 140, Issue 3, Pages 987-997.e8 (March 2011) DOI: 10.1053/j.gastro.2010.11.038 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Ethanol feeding activates UPR and XBP1 in rat pancreas. Rats were fed control (C) or ethanol (E) diets for 6 weeks. (A) Electron micrographs showing ultrastructure of pancreatic acinar cells. Ethanol-fed rats exhibit well-preserved acinar architecture (right panel) compared with controls (left panel), but ER is dilated (arrows). Bars = 2 μm. (B) RT-PCR showing higher XBP1 mRNA splicing (sXBP1) in pancreas from ethanol-fed than control-fed rats. (C) Western blot analysis of sXBP1, unspliced XBP1, Grp78, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; loading control). In B and C, each lane represents an individual rat. Graph shows densitometry for XBP1 and Grp78 (means ± SEM; n = 5).*P < .05 vs C diet (Student t test). Gastroenterology 2011 140, 987-997.e8DOI: (10.1053/j.gastro.2010.11.038) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Pancreatic acini isolated from Xbp1+/− mice are prone to ER stress. (A and B) Pancreatic acini isolated from wild-type and Xbp1+/− mice were incubated with and without CCK-8 for 30 minutes. (A) Immunoblot shows protein levels of sXBP1 and unspliced XBP1, phospho-PERK, phospho-eIF2α, and total eIF2α after stimulation with 0.1 nmol/L or 100 nmol/L CCK-8. (B) Graph shows amylase secretion by acini stimulated with CCK-8 (mean ± SEM, 4 independent studies). Immunoblot shows cellular amylase content. *P < .05 vs wild-type (2-way analysis of variance and Tukey posttests: CCK, P < .001; genotype, P = .002; interaction, P = .624). (C) Immunoblots show amylase content in media and cellular levels of sXBP1, amylase, PDI, and ERK1/2 (loading control) in unstimulated acini cultured for 24 hours (n = 2 independent experiments). (D) Graph shows densitometry for immunoblots depicted in C (mean ± SEM, n = 3). *P < .05 vs wild-type (Student t test). Gastroenterology 2011 140, 987-997.e8DOI: (10.1053/j.gastro.2010.11.038) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Ethanol feeding causes significant pancreatic damage in Xbp1+/− mice. Wild-type and Xbp1+/− mice were fed either control (C) or ethanol (E) diet. (A) Representative pancreatic H&E staining from ethanol-fed wild-type (panels i and ii) and Xbp1+/− mice (panels iii and iv) after 4 weeks on diets. Pancreas of ethanol-fed wild-type mice appeared normal. Pancreas of ethanol-fed Xbp1+/− mice displayed patchy areas of acinar cell necrosis (iii, white arrow) and areas with abundant stroma cells and tubular complexes (iii, black arrows). In panel iv, the typical eosin staining in apical areas of acini is decreased, suggesting loss of zymogen granules. Pancreatic sections from all control-fed mice appeared normal (not shown). Bars = 20 μm. (B) Electron micrographs from ethanol-fed wild-type (panel i) and Xbp1+/− mice (panels ii–iv) show features of ER stress in Xbp1+/− mice. Acinar cells in wild-type mice show regular ultrastructure, with a slightly dilated ER (panel i, arrows). In Xbp1+/− mice, acinar cells often displayed extensively dilated ER (panel ii; higher magnification in panel iii) with occasional accumulation of dense materials (panel iv). AV, autophagic vacuoles (arrowheads); L, acinar lumen; ZG, zymogen granules (arrows). Bars = 1 μm. (C) Graph shows percentage of acinar cell necrosis per total pancreatic area evaluated in H&E tissue sections (median ± 25th and 75th percentiles; n = 4–6 mice). *P < .05 vs wild-type mice (Kruskal–Wallis test and Dunn post hoc test). (D) Graph shows number of zymogen granules per cell section measured in electron microscopy pancreatic sections (mean ± SEM, n = 4–6 mice). *P < .05 vs wild-type mice (2-way analysis of variance and Tukey posttests). Criteria for quantification in C and D are explained in Supplementary Materials and Methods. (E) Immunoblots showing pancreatic levels of amylase and GAPDH (loading control). Each lane represents an individual mouse. Graph shows amylase quantification relative to control-fed wild-type mice (mean ± SEM, n = 5). Gastroenterology 2011 140, 987-997.e8DOI: (10.1053/j.gastro.2010.11.038) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Ethanol-induced XBP1 activation is blunted in pancreas of Xbp1+/− mice. Wild-type and Xbp1+/− mice were fed either control (C) or ethanol (E) diet for 4 weeks. (A) Pancreatic XBP1 splicing was assessed by RT-PCR. GAPDH was examined as a housekeeping gene. (B) Immunoblot analyses of sXBP1, unspliced XBP1, IRE1α, and ERK1/2 (loading control) in pancreas from wild-type and Xbp1+/− mice. In A and B, each lane represents an individual mouse. (C) Graphs show quantification of sXBP1 and IRE1α protein expression relative to control-fed wild-type mice (mean ± SEM, n = 5 mice).*P < .05 vs wild-type mice; #P < .05 vs C diet (2-way analysis of variance and Tukey posttests). Gastroenterology 2011 140, 987-997.e8DOI: (10.1053/j.gastro.2010.11.038) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 XBP1 deficiency augments pancreatic UPR in ethanol-fed mice. (A and B) Immunoblot analyses of key UPR regulators in pancreas from wild-type and Xbp1+/− mice fed control (C) or ethanol (E) diet for 4 weeks. Shown are expression levels of phospho-Thr980-PERK, total and phospho-Ser51-eIF2α, Grp78, and ATF4 (B). ERK1/2 was used as loading control. Each lane represents an individual mouse. (C) Densitometric quantification of phospho-PERK, phospho-eIF2α, and ATF4 levels. The graph shows increased protein content in ethanol-fed Xbp1+/− mice relative to ethanol-fed wild-type mice. *P < .05 vs wild-type mice (Student t test). Gastroenterology 2011 140, 987-997.e8DOI: (10.1053/j.gastro.2010.11.038) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 XBP1 deficiency diminishes ER components involved in protein folding and ERAD. Pancreatic expression of XBP1 targets was determined in wild-type and Xbp1+/− mice fed either ethanol (E) or control (C) diet. (A) Quantitative RT-PCR analyses of the chaperones ERdj4 and EDEM1 (mean ± SEM; n = 3). mRNA values were standardized to those of mouse acidic ribosomal phosphoprotein P0 and expressed relative to control-fed wild-type mice. *P < .05 vs wild-type mice (2-way analysis of variance). Immunoblots show pancreatic protein content of (B) EDEM1 and (C) the ER oxidoreductases PDI and ERp57. ERK1/2 and GAPDH were used as loading controls. (D) Quantitation of PDI and ERp57 expression in immunoblots relative to C diet, normalized to those of GAPDH. *P < .05 vs wild-type mice; #P < .05 vs C diet (2-way analysis of variance and Tukey posttests). (E) Immunoblot shows selective changes in PDI electrophoretic mobility associated with reduction/alkylation of thiol groups by Tris-2-carboxyethyl-phosphine and AMS treatment. AMS increases molecular weight about 500 daltons per thiol group. As judged by the relative mobility of the bands, alkylation was less in samples from ethanol-fed mice, suggesting oxidation of fewer PDI-free thiol groups. In B, C, and E, each lane represents an individual mouse. Gastroenterology 2011 140, 987-997.e8DOI: (10.1053/j.gastro.2010.11.038) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 XBP1 deficiency and ethanol feeding induce up-regulation of CHOP and apoptosis in mouse pancreas. (A) CHOP mRNA expression was assessed in pancreas from control (C)- and ethanol (E)-fed wild-type and Xbp1+/− mice by RT-PCR. GAPDH was used as a housekeeping gene. (B) Immunoblots show pancreatic levels of CHOP and the antiapoptotic proteins Bcl-2 and Bcl-xL. In A and B, each lane represents an individual mouse. (C) Apoptotic cells were assessed by TUNEL staining of pancreatic sections. Ethanol-fed wild-type mice lacked apoptotic nuclei (panel i). Similar results were found for all mice fed C diet (not shown). In Xbp1+/− mice fed ethanol, TUNEL-positive nuclei (brown staining) were found mainly in damaged areas of parenchyma displaying extensive loss of acinar cells (panel ii). Apoptotic cells comprise acinar and stroma cells. Bars = 20 μm. Gastroenterology 2011 140, 987-997.e8DOI: (10.1053/j.gastro.2010.11.038) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Long-term ethanol feeding in rats is associated with decreased levels of reduced GSH in pancreas. Pancreatic content of GSH and GSSG was measured in pancreas tissue samples obtained from rats fed control (C) or ethanol (E) diet for 6 weeks using the Tsukamoto–French intragastric infusion model. (A) The graph shows GSH content in whole tissue homogenates. GSH levels were measured by high-performance liquid chromatography as described in the Supplementary Materials and Methods. (B) GSH and GSSG were measured in pancreatic ER-enriched fractions obtained by differential centrifugation (see Supplementary Materials and Methods). Ratios of GSSH/GSH measurements obtained by these assays are shown in the graph. Graphs illustrate mean ± SEM of 3 to 5 rats per group; *P < .05 vs C diet (unpaired Student t test). (C) Representative immunoblots to characterize purity of ER-enriched factions obtained by differential centrifugation from rat pancreatic tissue homogenates. PDI, COX IV, and GAPDH were used as markers of ER, mitochondria (mit.), and cytosolic fractions, respectively. Gastroenterology 2011 140, 987-997.e8DOI: (10.1053/j.gastro.2010.11.038) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 The transcription factor XBP1 (sXBP1) is expressed in adult rat pancreas and in pancreatic acini. (A) Pancreatic expression levels of sXBP1 and unspliced XBP1 (uXBP1) proteins were assessed by Western blotting in pancreas homogenates from (A) 12-hour fasted and nonfasted rats fed chow diet and (B) pancreatic acini isolated from nonfasted rats. (A) Pancreatic levels of active XBP1 (sXBP1) were prominent in both nonfasted and fasted animals, likely related to the high rates of ER protein synthesis and processing in this organ. Immunoblots are representative of the results from 4 animals per group. (B) Freshly isolated rat pancreatic acini were stimulated with physiologic (0.1 nmol/L) or supraphysiological (100 nmol/L) CCK-8 for the indicated times, and cell lysates were processed for immunoblot analysis using antibodies to XBP1 and GAPDH (loading control). Immunoblots are representative of 3 independent studies. Gastroenterology 2011 140, 987-997.e8DOI: (10.1053/j.gastro.2010.11.038) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 Xbp1+/− mice have reduced pancreatic protein levels of sXBP1. The figure shows pancreas morphology and pancreatic expression levels of XBP1 proteins and other ER regulators in Xbp1+/− and wild-type mice fed standard chow diet. (A) Representative H&E staining in pancreatic sections from wild-type and Xbp1+/− mice. There was no detectable abnormality in the Xbp1+/− exocrine or endocrine (not shown) pancreas, and no difference was evident between the 2 genotypes. Bars = 20 μm. (B) Representative immunoblots showing levels of ER stress regulators and digestive enzymes (amylase and trypsin) in pancreatic homogenates. Each lane represents an individual mouse; results from 2 mice are shown. The graphs in C and D show densitometry of immunoblots illustrated in B (mean ± SEM of 2 mice per group); *P < .05 vs wild-type mice (unpaired Student t test). Compared with wild-type mice, protein levels of sXBP1 and unspliced XBP1 (uXBP1) were reduced 40% and 30%, respectively, in Xbp1+/− mice. sXBP1/uXBP1 ratios were comparable between genotypes, indicating that basal XBP1 splicing process is intact in pancreas of Xbp1+/− mice. In these mice, protein levels of the XBP1 target PDI were significantly reduced by 18%, consistent with a partial Xbp1 deletion. No differences were found in levels of other ER stress regulators such as IRE1α and Grp78. Pancreatic content of amylase and trypsin was similar between wild-type and Xbp1+/− mice. Taken together, data illustrated here and in Supplementary Table 2 indicate that the magnitude of decrease in sXBP1 levels found in Xbp1+/− mice has minor effects on pancreatic function in standard conditions. However, these data do not rule out involvement of sXBP1 in pancreatic function during long-term exposure to stressors such as ethanol or other environmental and dietary factors. Gastroenterology 2011 140, 987-997.e8DOI: (10.1053/j.gastro.2010.11.038) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 4 Short-term ethanol treatment markedly increases expression levels of uXBP1 mRNA in mouse pancreatic acini. Freshly isolated pancreatic acini obtained from untreated wild-type mice were cultured for 24 hours with and without 75 mmol/L ethanol. The image in the upper panel depicts expression of unspliced (uXBP1) and sXBP1 mRNA assessed by conventional RT-PCR. GAPDH was analyzed as housekeeping gene. Each lane shows data from an independent cell preparation; results from 2 independent cell preparations are shown. As illustrated in the picture, 24-hour ethanol treatment significantly increased mRNA levels of uXBP1 but had little effect on XBP1 splicing. Similarly, ethanol did not affect protein levels of sXBP1 (not shown). Gastroenterology 2011 140, 987-997.e8DOI: (10.1053/j.gastro.2010.11.038) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 5 Clinical parameters in wild-type and Xbp1+/− mice fed control or ethanol-containing diets. Three-month-old Xbp1+/− and littermate wild-type mice were fed either an ethanol (E) diet or a control (C) diet for 4 weeks using the Tsukamoto–French model. Graphs show clinical values and blood alcohol values at the time the mice were killed (mean ± SEM; n = 4–9 mice per group). (A) The graph shows average body weight gain during the 4-week feeding period. Mice in the 4 treatment groups had similar body weight at the beginning of the ethanol infusion. Wild-type mice on C or E diets steadily gained weight during the feeding period. However, average body weight of ethanol-fed Xbp1+/− mice slowly declined after the third week and at the time the mice were killed was significantly less than wild-type controls. *P < .05 vs wild-type (2-way analysis of variance and Tukey posttests: genotype, P = .045; diet, P = .200; interaction, P = .397). Compared with wild-type control, (B) blood amylase and (C) alanine aminotransferase (ALT) levels were elevated in XBP1-deficient mice fed ethanol, suggesting pancreas and liver damage in these mice. *P < .05 vs wild-type (2-way analysis of variance and Tukey posttests: for blood alanine aminotransferase levels, genotype, P = .038; diet, P = .002; interaction, P = .030). (D) Blood ethanol levels at the time the mice were killed were comparable between wild-type and Xbp1+/− mice, suggesting that XBP1 deficiency does not alter ethanol metabolism. Gastroenterology 2011 140, 987-997.e8DOI: (10.1053/j.gastro.2010.11.038) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 6 Evaluation of pancreatic LC3B levels in wild-type and Xbp1+/− mice fed control (C) or ethanol (E) diets. To assess accumulation of autophagic vacuoles in pancreas of mice fed C or E diets, we examined protein levels of the autophagosomal marker LC3B. (A) Immunofluorescence images of LC3B staining (red) in control and ethanol-fed wild-type and Xbp1+/− mice. Nuclei were counterstained with Hoechst 33342 (blue staining). Arrowheads indicate LC3B-stained autophagic compartments. Controls using only secondary antibody were negative (not shown). Images are representative of 3 mice analyzed per group; original magnification, 400×. (B) The graph shows the number and size of LC3B dots estimated by morphometric analysis of the total area of LC3B staining relative to total nuclei area (mean ± SEM; n = 3 mice per group). *P < .05 compared with wild-type mice and ethanol-fed Xbp1+/− mice (2-way analysis of variance and Tukey posttests: genotype, P = .014; diet, P = .040; genotype × diet, P = .106). (C) LC3B-I conversion into its lipidated form LC3B-II was analyzed by Western blotting. GAPDH expression was used as internal loading control. A representative immunoblot is shown, illustrating the results from 2 animals per group. (D) Data shown in the graph represent quantification of the optical density of LC3-II relative to LC3-I in mice fed E diet. LC3-I and LC3-II levels were normalized to those of GAPDH (mean ± SEM; n = 4 mice per group). *P < .05 compared with wild-type mice (unpaired Student t test). Gastroenterology 2011 140, 987-997.e8DOI: (10.1053/j.gastro.2010.11.038) Copyright © 2011 AGA Institute Terms and Conditions