Title: Activation and Dysregulation of the Unfolded Protein Response in Nonalcoholic Fatty Liver Disease
Abstract: Background & Aims: Nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) are associated with known triggers of the unfolded protein response (UPR). The aims were to (1) evaluate the activity of UPR in NAFL and NASH and (2) correlate expression of UPR pathways with liver histology. Methods: Messenger RNA (mRNA) and protein expression were measured by quantitative real-time PCR and Western blot, respectively. Apoptosis was assessed by TUNEL assay. Liver histology was scored using the NASH clinical research network criteria. Results: Compared with subjects with the metabolic syndrome and normal liver histology (n = 17), both NAFL (n = 21) and NASH (n = 21) were associated with increased eukaryotic initiation factor-2α (eIF-2α) phosphorylation. Activating transcription factor 4 (ATF4) mRNA and protein, C/EBP homologous protein (CHOP), and growth arrest, DNA damage-34 (GADD34) mRNA were not increased in NAFL or NASH. Whereas immunoglobulin heavy chain binding protein mRNA was significantly increased in NASH, unspliced X-box protein-1 (XBP-1) protein did not increase. Also, endoplasmic reticulum degradation-enhancing α-mannosidase-like protein mRNA levels were inversely related to spliced XBP-1 mRNA in NASH. NASH was specifically associated with low sXBP-1 protein and increased JNK phosphorylation. This correlated with increased TUNEL activity in NASH. The histologic severity correlated with sXBP-1 mRNA and JNK phosphorylation. Conclusions: There is a variable degree of UPR activation in NAFL and NASH. Although both NAFL and NASH are associated with eIF-2α phosphorylation, there is a failure to activate downstream recovery pathways, ie, ATF4-CHOP-GADD34. NASH is specifically associated with (1) failure to generate sXBP-1 protein and (2) activation of JNK. Background & Aims: Nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) are associated with known triggers of the unfolded protein response (UPR). The aims were to (1) evaluate the activity of UPR in NAFL and NASH and (2) correlate expression of UPR pathways with liver histology. Methods: Messenger RNA (mRNA) and protein expression were measured by quantitative real-time PCR and Western blot, respectively. Apoptosis was assessed by TUNEL assay. Liver histology was scored using the NASH clinical research network criteria. Results: Compared with subjects with the metabolic syndrome and normal liver histology (n = 17), both NAFL (n = 21) and NASH (n = 21) were associated with increased eukaryotic initiation factor-2α (eIF-2α) phosphorylation. Activating transcription factor 4 (ATF4) mRNA and protein, C/EBP homologous protein (CHOP), and growth arrest, DNA damage-34 (GADD34) mRNA were not increased in NAFL or NASH. Whereas immunoglobulin heavy chain binding protein mRNA was significantly increased in NASH, unspliced X-box protein-1 (XBP-1) protein did not increase. Also, endoplasmic reticulum degradation-enhancing α-mannosidase-like protein mRNA levels were inversely related to spliced XBP-1 mRNA in NASH. NASH was specifically associated with low sXBP-1 protein and increased JNK phosphorylation. This correlated with increased TUNEL activity in NASH. The histologic severity correlated with sXBP-1 mRNA and JNK phosphorylation. Conclusions: There is a variable degree of UPR activation in NAFL and NASH. Although both NAFL and NASH are associated with eIF-2α phosphorylation, there is a failure to activate downstream recovery pathways, ie, ATF4-CHOP-GADD34. NASH is specifically associated with (1) failure to generate sXBP-1 protein and (2) activation of JNK. Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease in North America.1Browning J.D. Szczepaniak L.S. Dobbins R. et al.Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity.Hepatology. 2004; 40: 1387-1395Crossref PubMed Scopus (2880) Google Scholar The clinical-histologic spectrum of NAFLD extends from a nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH).2Contos M.J. Choudhury J. Mills A.S. et al.The histologic spectrum of nonalcoholic fatty liver disease.Clin Liver Dis. 2004; 8: 481-500Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar NASH can progress to cirrhosis in up to 15% of subjects.3Ekstedt M. Franzen L.E. Mathiesen U.L. et al.Long-term follow-up of patients with NAFLD and elevated liver enzymes.Hepatology. 2006; 44: 865-873Crossref PubMed Scopus (1756) Google Scholar Although oxidative stress and cytokines have been implicated, the mechanisms of cell injury and progression of liver disease in NASH are not fully understood.4Sanyal A.J. Campbell-Sargent C. Mirshahi F. et al.Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities.Gastroenterology. 2001; 120: 1183-1192Abstract Full Text Full Text PDF PubMed Scopus (1716) Google Scholar, 5Diehl A.M. Li Z.P. Lin H.Z. et al.Cytokines and the pathogenesis of non-alcoholic steatohepatitis.Gut. 2005; 54: 303-306Crossref PubMed Scopus (185) Google ScholarThe endoplasmic reticulum (ER) plays a central role in the synthesis, folding, and trafficking of proteins. ER dysfunction is characterized by accumulation of unfolded proteins within the ER, which triggers the unfolded protein response (UPR). The UPR is initially characterized by translational arrest of protein synthesis, increased ER-associated degradation of proteins via a proteosomal pathway, and activation of genes that allow the cell to adapt to the trigger for ER dysfunction.6Kaufman R.J. Orchestrating the unfolded protein response in health and disease.J Clin Invest. 2002; 110: 1389-1398Crossref PubMed Scopus (1077) Google Scholar If the cell fails to adapt, alarm pathways are activated including c-jun-N-terminal kinase (JNK), which results in apoptosis and inflammation.7Schwabe R.F. Uchinami H. Qian T. et al.Differential requirement for c-Jun NH2-terminal kinase in TNF-α- and Fas-mediated apoptosis in hepatocytes.FASEB J. 2004; 18: 720-722Crossref PubMed Scopus (135) Google Scholar Activation of UPR has been implicated in the pathogenesis of insulin resistance, diabetes, and alcohol-induced liver disease.8Ozcan U. Cao Q. Yilmaz E. et al.Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes.Science. 2004; 306: 457-461Crossref PubMed Scopus (2932) Google Scholar, 9Hotamisligil G.S. Role of endoplasmic reticulum stress and c-Jun NH2-terminal kinase pathways in inflammation and origin of obesity and diabetes.Diabetes. 2005; 54: S73-S78Crossref PubMed Scopus (296) Google Scholar, 10Ji C. Mehrian-Shai R. Chan C. et al.Role of CHOP in hepatic apoptosis in the murine model of intragastric ethanol feeding.Alcohol Clin Exp Res. 2005; 29: 1496-1503Crossref PubMed Scopus (143) Google Scholar, 11Kaplowitz N. Ji C. Unfolding new mechanisms of alcoholic liver disease in the endoplasmic reticulum.J Gastroenterol Hepatol. 2006; 21: S7-S9Crossref PubMed Scopus (88) Google Scholar Saturated fat feeding, which is known to induce insulin resistance, activates UPR in the liver in mice.12Wang D. Wei Y. Pagliassotti M.J. Saturated fatty acids promote endoplasmic reticulum stress and liver injury in rats with hepatic steatosis.Endocrinology. 2006; 147: 943-951Crossref PubMed Scopus (427) Google Scholar Hyperhomocyteniemia, commonly present in the insulin-resistant state, induces UPR in cultured hepatocytes.13Werstuck G.H. Lentz S.R. Dayal S. et al.Homocysteine-induced endoplasmic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways.J Clin Invest. 2001; 107: 1263-1273Crossref PubMed Scopus (590) Google Scholar Also, C/EBP homologous protein (CHOP) and JNK activation has been noted in animal models of steatohepatitis.14Yang L. Jhaveri R. Huang J. et al.Endoplasmic reticulum stress, hepatocyte CD1d and NKT cell abnormalities in murine fatty livers.Lab Invest. 2007; 87: 927-937Crossref PubMed Scopus (81) Google Scholar, 15Schattenberg J.M. Singh R. Wang Y. et al.JNK1 but not JNK2 promotes the development of steatohepatitis in mice.Hepatology. 2006; 43: 163-172Crossref PubMed Scopus (316) Google Scholar, 16Rahman S.M. Schroeder-Gloeckler J.M. Janssen R.C. et al.CCAAT/enhancing binding protein β deletion in mice attenuates inflammation, endoplasmic reticulum stress, and lipid accumulation in diet-induced nonalcoholic steatohepatitis.Hepatology. 2007; 45: 1108-1117Crossref PubMed Scopus (124) Google Scholar, 17Svegliati-Baroni G. Candelaresi C. Saccomanno S. et al.A model of insulin resistance and nonalcoholic steatohepatitis in rats: role of peroxisome proliferator-activated receptor-α and n-3 polyunsaturated fatty acid treatment on liver injury.Am J Pathol. 2006; 169: 846-860Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar NAFLD is strongly associated with insulin resistance and several known triggers of UPR, eg, ATP depletion.4Sanyal A.J. Campbell-Sargent C. Mirshahi F. et al.Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities.Gastroenterology. 2001; 120: 1183-1192Abstract Full Text Full Text PDF PubMed Scopus (1716) Google Scholar, 18Cortez-Pinto H. Chatham J. Chacko V.P. et al.Alterations in liver ATP homeostasis in human nonalcoholic steatohepatitis: a pilot study.JAMA. 1999; 282: 1659-1664Crossref PubMed Scopus (414) Google ScholarWe hypothesized that (1) NAFLD was associated with activation of UPR and that (2) increasing disease activity correlated with the severity of the UPR. This was tested by (1) evaluating the expression of the UPR in subjects with the metabolic syndrome with and without NAFL or NASH and (2) correlation of expression of specific pathways with liver histology.Materials and MethodsStudy CohortConsecutive subjects who were referred for either obesity management or suspected NAFLD were screened for this study. All subjects underwent routine clinical assessment and radiologic, hematologic, biochemical, and serologic testing. The metabolic syndrome was diagnosed using the Adult Treatment Panel III criteria.19Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III).JAMA. 2001; 285: 2486-2497Crossref PubMed Scopus (24024) Google Scholar Alcohol consumption was assessed clinically and considered to be significant when >20 g/day for females and >30 g/day for males. All subjects were tested for hepatitis B and C, autoimmune hepatitis, hemochromatosis, Wilson disease, α1-antitrypsin deficiency, and primary biliary cirrhosis. NAFLD was suspected in those with (1) either abnormal liver enzymes or radiologic evidence of a fatty liver along with negative studies for other common etiologies of liver disease and (2) absence of clinically significant alcohol consumption.Subjects with the metabolic syndrome with or without features suggestive of NAFLD were considered for this study. Consecutive subjects who gave informed consent underwent a core liver biopsy, using a 15-gauge Microvasive gun (Microinvasive, Quincy, MA), by a percutaneous route under ultrasound or laparoscopic guidance. One liver core was fixed in formalin for assessment of histology, and another was snap frozen in liquid nitrogen and stored at −80°C for future studies. Based on the liver histology, 3 groups were identified: (1) subjects with the metabolic syndrome and normal liver histology and liver enzymes, (2) subjects with the metabolic syndrome and NAFL, and (3) subjects with the metabolic syndrome and NASH. Subjects with bridging fibrosis or cirrhosis were excluded. The study was performed according to Virginia Commonwealth University regulations for the protection of human research subjects, and the protocol was reviewed and approved by the Institutional Review Board.Histologic AssessmentA true core biopsy of the liver was used for histologic assessment in all cases. A minimum of 1.7 cm of liver tissue was sent for histopathologic studies. Hepatic steatosis and other histologic parameters of fatty liver disease were scored separately using the NASH clinical research network criteria.20Kleiner D.E. Brunt E.M. Van Natta M. et al.Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology. 2005; 41: 1313-1321Crossref PubMed Scopus (6903) Google Scholar Only those with steatosis alone were classified as NAFL for this study. Steatohepatitis was diagnosed by the presence of steatosis, cytologic ballooning, and inflammation.21Ludwig J. Viggiano T.R. McGill D.B. et al.Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease.Mayo Clin Proc. 1980; 55: 434-438PubMed Google ScholarEvaluation of the Adaptive Pathways of the UPRRNA-activated protein kinase (PKR)-like ER kinase (PERK) activation was evaluated by measurement of phosphorylated eukaryotic initiation factor-2α (eIF-2α), activating transcription factor (AFT) 4 messenger RNA (mRNA) and protein, CHOP, and growth arrest and DNA damage-34 (GADD34) mRNA expression.22Marciniak S.J. Ron D. Endoplasmic reticulum stress signaling in disease.Physiol Rev. 2006; 86: 1133-1149Crossref PubMed Scopus (774) Google Scholar ATF6 activation was assessed from immunoglobulin heavy chain binding protein (BiP) and unspliced X-box protein-1 (uXBP-1) mRNA and uXBP-1 protein.23Okada T. Yoshida H. Akazawa R. et al.Distinct roles of activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) in transcription during the mammalian unfolded protein response.Biochem J. 2002; 366: 585-594Crossref PubMed Scopus (407) Google Scholar Inositol requiring enzyme-1 (IRE-1) activation splices uXBP-1 to form spliced XBP-1 (sXBP-1) mRNA; sXBP-1 protein transcriptionally activates ER degradation-enhancing α-mannosidase-like protein (EDEM), which promotes proteasomal protein degradation.24Yoshida H. Matsui T. Hosokawa N. et al.A time-dependent phase shift in the mammalian unfolded protein response.Dev Cell. 2003; 4: 265-271Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar IRE-1 activation was assessed from the EDEM mRNA, sXBP-1 mRNA, and protein levels.25Yoshida H. Matsui T. Yamamoto A. et al.XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor.Cell. 2001; 107: 881-891Abstract Full Text Full Text PDF PubMed Scopus (2904) Google Scholar, 26Lee A.H. Iwakoshi N.N. Glimcher L.H. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response.Mol Cell Biol. 2003; 23: 7448-7459Crossref PubMed Scopus (1584) Google ScholarEvaluation of the Alarm Pathways of the UPRAlarm signaling in the UPR is mediated by JNK and p38 mitogen-activated protein kinase (MAPK).27Urano F. Wang X. Bertolotti A. et al.Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1.Science. 2000; 287: 664-666Crossref PubMed Scopus (2281) Google Scholar, 28Takeda K. Matsuzawa A. Nishitoh H. et al.Roles of MAPKKK ASK1 in stress-induced cell death.Cell Struct Funct. 2003; 28: 23-29Crossref PubMed Scopus (197) Google Scholar The status of the alarm/death pathways was evaluated by measurement of total and phosphorylated JNK and p38 MAPK proteins and apoptotic activity, as measured by the terminal dUTP nick-end labeling (TUNEL) assay.29Zinszner H. Kuroda M. Wang X. et al.CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum.Genes Dev. 1998; 12: 982-995Crossref PubMed Scopus (1667) Google ScholarRNA Preparation and Real-Time Quantitative Polymerase Chain Reaction AnalysisTotal RNA was extracted from the frozen liver tissue using 1 mL TRIzol (Invitrogen Life Technologies, Carlsbad, CA) following the manufacturer’s protocol and quantified spectrophotometrically (Bio-Rad, Hercules, CA) from absorbance at 260 nm. Three measures for quality control were taken to ensure a high quality of the extracted RNA: 260 nm/280 nm ratio, NanoDrop ND-1000 UV-Vis Spectrophotometer to look for any additional genomic products and agarose gel electrophoresis for 18S and 28S components.Sequence-specific primers were designed to assess the mRNA expression of specific genes (Table 1). β-Actin was used as the normalizing gene. The design ensured that the polymerase chain reaction (PCR) product spanned an intron/exon boundary to minimize the possibility of coamplifying genomic DNA. The oligonucleotides were synthesized from Sigma Genosys (Sigma-Aldrich Co, St. Louis, MO).Table 1Oligonucleotide Primers Used in This StudyNameSequence (5′−3′)ATF4 FwTGGCTGGCTGTGGATGGATF4 RevTCCCGGAGAAGGCATCCTGADD34 FwGTGGAAGCAGTAAAAGGAGCAGGADD34 RevCAGCAACTCCCTCTTCCTCGBiP FwCGTGTTCAAGAACGGCCGBiP RevCGTAGACAGTACGACAGCAACTGTuXBP-1 FwAGTGAGCTGGAACAGCAAGTGGTAuXBP-1 RevTGCAGAGGTGCACGTAGTCTGAGTsXBP-1 FwAGTGAGCTGGAACAGCAAGTGGTAsXBP-1 RevACATGACTGGGTCCAAGTTGTCCAEDEM FwCCGTCCAAGTCTTTGAGGCCACGEDEM RevGTGCATGTCTCATTATTGGTGTCAGGAGGβ-actin FwCCAGAGCAAGAGAGGCATCCβ-actin RevCCGTGGTGGTGAAGCTGTAGFw, forward; Rev, reverse. Open table in a new tab Real-time quantitative polymerase chain reaction (qPCR) was performed in a 2-step reaction. Complementary DNA (cDNA) was synthesized with oligo-dT from 1.25 μg total RNA in a final volume of 20 μL using ThermoScript Reverse Transcriptase (Invitrogen Life Technologies) according to the manufacturer’s instructions. The qPCR was performed in duplicate using the Stratagene Mx3000P QPCR system and 2X SYBR Green Master Mix (Bio-Rad). The cycling parameters for qPCR reaction included 45 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and elongation at 74°C for 60 seconds. After amplification, a final melting curve was recorded by heating to 95°C.The specificity of qPCR was established by incorporating no template and no reverse transcript controls. The amplification of specific transcripts was confirmed by melting curve profiles generated at the end of the PCR program. Total RNA extracted from a histologically normal liver of a lean normal individual obtained at the time of living donor liver transplantation was used as an internal calibrator across all experiments. Cycle threshold (Ct) values were normalized to β-actin, and comparative quantification of target mRNA was done by the ΔΔCt method using integrated software with the Stratagene Mx3000P QPCR system.Western Blot AnalysisLiver biopsy tissues were lysed using lysis buffer (catalogue No. C2978; Sigma Chemical Co, St. Louis, MO). Samples (10 μg protein/lane) were separated by 4%–12% Bis-Tris Nu-PAGE7 gel (Invitrogen Life Technologies) (phos-eIF-2α, ATF4, and XBP-1) or 10% SDS-PAGE (JNK and p38 MAPK). Western blots were performed with appropriate antibodies (ATF4 and XBP-1; Santa Cruz Biotechnology, Santa Cruz, CA) and (JNK and p38 MAPK; Signalchem, Richmond, BC, Canada). β-Actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as internal controls. Immunoblots were developed with horseradish peroxidase (HRP)-linked secondary antibodies (Amersham catalogue No. RPN210891; Cell Signaling catalogue No. 7074) and a chemiluminescence kit (ECL) and observed with Gel Doc 1000 system (Bio-Rad). Image density of blots was quantified by Scion image software (Scion Corp, Frederick, MD).Evaluation of ApoptosisApoptotic activity was evaluated by the TUNEL assay, which was performed using the in situ cell death detection kit (catalog no. 11684817910; Roche Diagnostics, Penzberg, Germany) according to the manufacturer’s instruction manual. Methyl green (5%) was used as a counterstain. For each subject, a total of 10 high-power fields (×40) from each of 2 sections were studied, and the number of brown-stained TUNEL-positive cells counted. Thus, approximately 20,000 cells from each group were evaluated. The mean number of TUNEL-positive cells per high-power field was compared across study groups.Statistical AnalysisThe RNA levels for a given gene were compared across groups using Kruskal–Wallis analysis of variance (ANOVA), a distribution-free test. A Dunn posttest was used for multiple comparisons as indicated. Linear regression was used to analyze the relationship between the levels of expression of multiple genes along a given pathway. ANOVA was used to compare the slopes of 2 or more linear regressions. Ordinal logistic regression was used to evaluate the relationship between expression of genes in specific UPR pathways and the severity of histologic abnormalities. Significance was set at a P value of .05.ResultsA total of 89 subjects were screened: 59 subjects were enrolled (controls [normal histology + normal ALT] = 17, NAFL = 21, and NASH = 21). The reasons for exclusion included refusal to consent (n = 17), insufficient amount of tissue for experimental studies (n = 9), and alanine aminotransferse (ALT) elevation in those with normal histology (n = 4). The baseline characteristics of the study population are shown in Table 2. The 3 groups were comparable with respect to gender, ethnicity, and features of the metabolic syndrome. However, subjects with NASH were older and, surprisingly, less obese. As expected, subjects with NASH had higher aspartate aminotransferase (AST), ALT, and alkaline phosphatase levels. The liver biopsy specimen length used for histologic studies varied from 1.7 to 3.5 cm (mean, 2.3 cm). Subjects with NAFL had a mean steatosis grade of 2.1 ± 0.6 and had no other features of NAFLD. Subjects with NASH all had steatosis, inflammation, cytologic ballooning, and pericellular fibrosis. The mean NAFLD activity score was significantly higher in those with NASH.Table 2Baseline Characteristics of the Study GroupsParameterNormal (n = 17)NAFL (n = 21)NASH (n = 21)Age, yr41.5 ± 12.744.1 ± 10.152 ± 11.3aP < .05 vs normal,Sex (female)14 (82.4)16 (76.2)16 (76.2)Ethnicity (Caucasian)13 (76.5)16 (76.2)18 (85.7)BMI (kg/m2)45 ± 6.343.9 ± 10.436.5 ± 9.6aP < .05 vs normal,Waist circumference (cm)128.1 ± 6.5126.4 ± 6.8122.9 ± 7.4Overweight/obese1/162/195/16Hypertension10 (58.8)13 (61.9)13 (61.9)Diabetes5 (28.4)8 (38.1)6 (28.6)Dyslipidemia6 (35.3)6 (28.6)7 (33.3)Features of the metabolic syndrome (≥3)17 (100)21 (100)21 (100)AST (0−65 IU/L)25.6 ± 4.932.8 ± 18.284.2 ± 58.6aP < .05 vs normal,bP < .05 vs NAFL,ALT (0−65 IU/L)32.4 ± 13.553.6 ± 37.9102.8 ± 59.2aP < .05 vs normal,bP < .05 vs NAFL,Alkaline phosphatase (0−110 IU/L)77.3 ± 20.180.7 ± 18.6102.9 ± 25.7aP < .05 vs normal,bP < .05 vs NAFL,Total bilirubin (0.3−0.8 mg/dL)0.5 ± 0.20.6 ± 0.40.6 ± 0.3Albumin (3.5−4.5 g/dL)4.4 ± 0.34.3 ± 0.74.4 ± 0.3Platelets (1000/mm3)295.5 ± 65.8311.8 ± 96.6252.9 ± 70.7Steatosis grade0.0 ± 0.02.1 ± 0.61.9 ± 0.5NAFLD activity score0.0 ± 0.02.19 ± 0.6cP < .05 NAFL vs control.4.33 ± 0.57aP < .05 vs normal,bP < .05 vs NAFL,NOTE. Data are expressed as mean ± SD or absolute number of subjects (n) and percentage in parentheses.BMI, body mass index; NAFL, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis.Across group comparisons by ANOVA:a P < .05 vs normal,b P < .05 vs NAFL,c P < .05 NAFL vs control. Open table in a new tab Activation and Dysregulation of PERK Pathway in NAFLDThe phosphorylated eIF-2α levels were significantly increased in both NAFL and NASH subjects compared with controls (Figure 1A). Despite the increase in phosphorylated eIF-2α, the majority of subjects with NAFL or NASH did not have increased levels of the downstream effectors ATF4 mRNA or protein, CHOP, and GADD34 mRNA (Figures 1B and 2A–C). A few subjects with NAFL or NASH had elevation of ATF4, CHOP, and GADD34 mRNA. CHOP mRNA was proportional to the upstream ATF4 mRNA and downstream GADD34 mRNA. Regardless of the group, GADD34 mRNA levels were also directly proportional to the ATF4 mRNA (r = 0.45, P = .001). Thus, although the upstream element of the PERK pathway, ie, eIF-2α was activated in both NAFL and NASH subjects, the downstream elements (ATF4, CHOP, and GADD34) required for recovery from ER stress22Marciniak S.J. Ron D. Endoplasmic reticulum stress signaling in disease.Physiol Rev. 2006; 86: 1133-1149Crossref PubMed Scopus (774) Google Scholar were not activated in most subjects with either condition.Figure 2Activation status of downstream effectors of the PERK pathway in subjects with the metabolic syndrome and normal liver histology, NAFL, and NASH. ATF4, CHOP, and GADD34 mRNA were measured by quantitative real-time PCR using RNA from the liver of a lean normal individual without the metabolic syndrome as an internal calibrator set at 1 across all studies. The horizontal bars in each study group represent median mRNA fold change. (A) The ATF4 mRNA levels were generally low in the majority of subjects and comparable among the 3 study groups. (B) The data for CHOP were qualitatively similar, although a few subjects with NASH had greater than 2-fold increase. (C) The levels of GADD34 mRNA were also low across all 3 groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Activation and Dysregulation of ATF6 Pathway in NAFLDBiP mRNA levels in subjects with NAFL were comparable with normal histology controls. However, NASH was associated with a significant increase in BiP mRNA compared with NAFL (P = .01) (Figure 3A). Although there was a trend for the median uXBP-1 mRNA to increase in NASH subjects, this was not significant (Figure 3B). However, there was increased uXBP-1 protein in NAFL subjects (Figure 3C). In contrast, despite a trend for increased uXBP-1 mRNA in NASH subjects, the uXBP-1 protein did not rise above levels seen in those with normal histology (Figure 3C).Figure 3Activation status of downstream effectors of the ATF6 pathway in subjects with the metabolic syndrome and normal liver histology, NAFL, and NASH. BiP and unspliced XBP-1 (uXBP-1) mRNA were measured by quantitative real-time PCR using RNA from the liver of a lean normal individual without metabolic syndrome as an internal calibrator set at 1 across all experiments. The horizontal bars in each study group represent median mRNA fold change. (A) NASH was associated with a significant increase in BiP mRNA compared with NAFL (P = .01). (B) There was a trend for the mean uXBP-1 mRNA to increase in NASH, but this did not reach statistical significance. (C) Western blot analysis of uXBP-1 and spliced XBP-1 (sXBP-1) proteins using β-actin as an internal control. Whereas uXBP-1 and sXBP-1 proteins were modestly increased in NAFL, uXBP-1 protein in NASH was comparable with controls with normal histology, and sXBP-1 protein was markedly decreased.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Activation and Dysregulation of IRE-1 Pathway in NAFLDAlthough the median sXBP-1 mRNA levels were not significantly different across the groups, 3 of 21 subjects with NAFL and NASH each had greater than 4.5-fold increase in sXBP-1 mRNA (Figure 4A). There was a significant decrease in sXBP-1 protein in NASH subjects (Figure 3C). Although there was a progressive increase in the median mRNA levels of EDEM from subjects with normal histology to NAFL to NASH, these were not statistically significant (Figure 4B). However, although EDEM mRNA levels were directly and linearly related to sXBP-1 mRNA in those with normal liver histology, the slope of this relationship flattened out in NAFL subjects, and the relationship reversed in subjects with NASH (Figure 4C). The slope of this regression in NASH subjects was significantly different from that for the other groups (P = .04 by ANOVA). Specifically, 3 of 21 subjects with NASH had the lowest quartile EDEM mRNA while having the highest quartile sXBP-1 mRNA. These subjects had the lowest sXBP-1 protein. Interestingly, some subjects with NASH had high EDEM mRNA despite low sXBP-1 protein levels.Figure 4The mRNA expression of sXBP-1 and EDEM and their regulation in subjects with the metabolic syndrome and normal liver histology, NAFL, and NASH was assessed. The horizontal bars in each study group represent median mRNA fold change. (A) All groups showed similar median mRNA levels of sXBP-1 with 2.5-fold increase compared to the internal calibrator set at 1 across all experiments. (B) There was a progressive increase in median EDEM mRNA levels in the NAFLD group compared with normal histology, but this did not reach statistical significance. (C) There was an inverse relationship between sXBP-1 and EDEM mRNA in NASH, and the slope was significantly different when compared with the other groups (P = .04; ANOVA).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Status of Alarm Pathway ActivationThe amount of phosphorylated JNK in those with normal histology was similar to those with NAFL (Figure 5). However, there was a significant increase in phosphorylated JNK levels in those with NASH. In contrast, both subjects with NAFL and NASH had a significant increase in phosphorylated p38 MAPK activity. This was accompanied by a progressive increase in apoptotic activity, measured by TUNEL assay, in NAFL and NASH subjects (Figure 6).Figure 5The total and phosphorylated levels of p38 MAPK and JNK proteins were evaluated by Western blot analysis. The density of individual protein bands was normalized to GAPDH. Wherea