Free fatty acids repress small heterodimer partner (SHP) activation and adiponectin counteracts bile acid-induced liver injury in superobese patients with nonalcoholic steatohepatitis

Authors

  • Lars P. Bechmann,

    1. Department of Gastroenterology and Hepatology, University Hospital, University Duisburg-Essen, Essen, Germany
    2. Division of Liver Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, NY
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  • Peri Kocabayoglu,

    1. Department of Gastroenterology and Hepatology, University Hospital, University Duisburg-Essen, Essen, Germany
    2. Division of Liver Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, NY
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  • Jan-Peter Sowa,

    1. Department of Gastroenterology and Hepatology, University Hospital, University Duisburg-Essen, Essen, Germany
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  • Svenja Sydor,

    1. Department of Gastroenterology and Hepatology, University Hospital, University Duisburg-Essen, Essen, Germany
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  • Jan Best,

    1. Department of Gastroenterology and Hepatology, University Hospital, University Duisburg-Essen, Essen, Germany
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  • Martin Schlattjan,

    1. Department of Gastroenterology and Hepatology, University Hospital, University Duisburg-Essen, Essen, Germany
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  • Anja Beilfuss,

    1. Department of Gastroenterology and Hepatology, University Hospital, University Duisburg-Essen, Essen, Germany
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  • Johannes Schmitt,

    1. Clinic for Gastroenterology and Hepatology, University Hospital Zurich, Zurich, Switzerland
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  • Rebekka A. Hannivoort,

    1. Division of Liver Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, NY
    2. Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
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  • Alpaslan Kilicarslan,

    1. Department of Gastroenterology and Hepatology, University Hospital, University Duisburg-Essen, Essen, Germany
    2. Department of Internal Medicine, Hacettepe University Hospital Ankara, Ankara, Turkey
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  • Christian Rust,

    1. Clinic for Gastroenterology and Hepatology, University Hospital Munich Grosshadern, Munich, Germany
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  • Frieder Berr,

    1. Department of Medicine I, Paracelsus Medical University/Salzburger Landeskliniken (SALK), Salzburg, Austria
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  • Oliver Tschopp,

    1. Department of Endocrinology & Diabetology, University Hospital Zurich, Zurich, Switzerland
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  • Guido Gerken,

    1. Department of Gastroenterology and Hepatology, University Hospital, University Duisburg-Essen, Essen, Germany
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  • Scott L. Friedman,

    1. Division of Liver Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, NY
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  • Andreas Geier,

    1. Clinic for Gastroenterology and Hepatology, University Hospital Zurich, Zurich, Switzerland
    2. Division of Hepatology, Department of Internal Medicine II, University Hospital Würzburg
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    • These authors equally contributed to this work.

  • Ali Canbay

    Corresponding author
    1. Department of Gastroenterology and Hepatology, University Hospital, University Duisburg-Essen, Essen, Germany
    • Professor of Medicine, University Hospital, University Duisburg-Essen, Hufelandstr. 55, 45122 Essen, Germany===

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    • These authors equally contributed to this work.

    • fax: +49 (201) 723-5719


  • Potential conflict of interest: Dr. Rust is on the speakers' bureau for Falk Foundation.

  • Supported by the Deutsche Forschungsgemeinschaft (DFG, grant 267/4-1 and 267/8-1; to A.C.), Swiss National Foundation (grant 310000-122310/1; to A.G.), the Wilhelm Laupitz Foundation (to A.C.), EASL Sheila Sherlock short-term fellowship (to L.P.B.), IFORES program of the University of Duisburg-Essen (to L.P.B.).

Abstract

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries and may proceed to steatohepatitis (NASH). Apoptosis and free fatty acid (FFA)-induced lipotoxicity are important features of NASH pathogenesis. We have shown a hepatoprotective effect of adiponectin in steatotic livers of hepatitis C virus (HCV) patients and recent data links bile acid (BA) metabolism to the pathogenesis of NAFLD. The aim of this study was to identify potential interactions between BA and FFA metabolism in NAFLD. Liver biopsies and serum samples from 113 morbidly obese patients receiving bariatric surgery, healthy individuals, and moderately obese NAFLD patients were studied. Serum FFA, BA, and M30 were increased in NASH versus simple steatosis, while adiponectin was significantly decreased. The NAFLD activity score (NAS) score correlated with BA levels and reversely with adiponectin. Adiponectin reversely correlated with CD95/Fas messenger RNA (mRNA) and hepatocellular apoptosis. The BA transporter high-affinity Na+/taurocholate cotransporter (NTCP) and the BA synthesizing enzyme cholesterol 7 alpha-hydroxylase (CYP7A1) were significantly up-regulated in obese patients and hepatoma cells exposed to FFA. Up-regulation of NTCP and CYP7A1 indicate failure to activate small heterodimer partner (SHP) upon farnesoid X receptor (FXR) stimulation by increasing BA concentrations. In line with the NAS score, adiponectin levels were reversely correlated with BA levels. Adiponectin correlated with NTCP and affects Cyp7A1 expression both in vivo and in vitro. Conclusion: BA synthesis and serum BA levels correlated with disease severity in NAFLD, while adiponectin is reversely correlated. FFA exposure prevented SHP-mediated repression of NTCP and Cyp7A1 expression, which lead to increased BA synthesis and uptake. In NASH, BA accumulation induced hepatocyte cell death and late FXR activation failed to prevent hepatocyte injury due to decreased adiponectin levels. Early treatment with FXR ligands and/or adiponectin-receptor agonists might prevent NASH. (HEPATOLOGY 2013;57:1394–1406)

Nonalcoholic fatty liver disease (NAFLD) as the hepatic manifestation of the metabolic syndrome is recognized as the most prevalent liver disease in Western societies.1 Nonalcoholic steatohepatitis (NASH), the progressive form of NAFLD, is associated with increased morbidity and mortality, as the disease can progress to cirrhosis and liver cancer, requiring liver transplantation in some patients.2 Adipocytokines have recently been identified as important mediators in liver disease and adiponectin has been shown to be hepatoprotective and antiapoptotic.3 As previously shown for diabetes, in NAFLD adiponectin levels are inversely correlated with disease severity.4

Recent publications showed an increase in toxic bile acids (BAs) in liver tissue of NASH patients.5-7 Hepatocellular BA homeostasis is regulated by de novo synthesis of BAs from cholesterol, catalyzed by the key enzyme cholesterol 7 alpha-hydroxylase (CYP7A1), and the hepatocellular transport of BAs from sinusoidal blood into the bile canaliculus.8 BAs from the sinusoidal blood are taken up by the hepatocyte by way of the high-affinity Na+/taurocholate cotransporter (NTCP, SLC10A1) or multispecific organic anion transporters (OATPs). The canalicular secretion is mediated by a variety of transport systems, belonging to the ATP-binding cassette (ABC) family.9 The nuclear receptor for BAs, farnesoid X receptor (FXR), is involved in the feedforward activation of the canalicular BA export pumps BSEP (ABCB11) and MRP2 (ABCC2) and FXR induces the transcriptional repressor small heterodimer partner (SHP), which in turn suppresses transactivation of the human NTCP and CYP7A1 genes.10 This negative feedback regulation of BA uptake and synthesis during cholestasis is regarded as a protective mechanism to prevent excessive BA overload and hepatocellular injury and FXR signaling is emerging as an important mediator in hepatic lipid metabolism.11

In this study we found an increase of serum BAs in NASH as compared to less severe stages of NAFLD, which is inversely correlated with serum adiponectin levels in obese patients who underwent bariatric surgery. In NASH, serum adiponectin levels are decreased and hepatic expression of the adiponectin receptor 2 (ApoR2) is compensatory up-regulated. Repression of Cyp7A1 and NTCP by SHP appears impaired in this cohort and free fatty acid (FFA) treatment of hepatoma cells mimics these effects in vitro.

Abbreviations

ABCB11/BSEP: ATP-binding cassette, subfamily B member 11, bile salt export pump; BMI: body mass index; CD95/Fas: apoptosis-inducing cell surface receptor (advanced nomenclature: TNF superfamily receptor 6); CYP7A1: cholesterol 7 alpha-hydroxylase; FFA: free (nonesterified) fatty acids; FXR: farnesoid X receptor; HSC: hepatic stellate cell; M30: cytokeratin-18 fragment epitope exposed upon cleavage by caspases; NAFL: nonalcoholic fatty liver; NAFLD: nonalcoholic fatty liver disease; NAS, NAFLD activity score; NASH: nonalcoholic steatohepatitis; NTCP: high-affinity Na+/taurocholate cotransporter; qRT-PCR: quantitative real-time polymerase chain reaction; SHP: small heterodimer partner; TGF-β: transforming growth factor β.

Patients and Methods

Patients.

In all, 113 patients suffering from morbid obesity (body mass index [BMI] > 40kg/m2) undergoing bariatric surgery were enrolled in the study (Table 1). Individuals aged <18 or >65 or with liver injuries and pathologies (infectious disease with hepatitis B virus [HBV], hepatitis C virus [HCV], or human immunodeficiency virus [HIV]), history of organ transplantation, history of malignancy within the past 5 years, excessive alcohol consumption indicating alcoholic liver disease (>20 g/day in males or >10 g/day in females) or drug abuse, autoimmunity, genetic disorders, and therapy with immunosuppressive or cytotoxic agents were excluded. Indication for performance of bariatric surgery was made by the surgeon, a dietician, and the primary physician according to National Institutes of Health (NIH) guidelines (BMI >40 kg/m2 or ≥35 kg/m2, plus comorbidities) and patients had to prove unsuccessful attempts to lose weight by lifestyle modification, diet, and exercise. Wedge liver biopsies were taken at the time of bariatric surgery.

Table 1. Demographic and Clinical Data of Controls and NAFLD Patients
 Healthy Control (n=10)NAFL (n = 39)NASH (n = 59)
Female/male3/735/440/19
Age (years)26.0 ± 7.640.22 ± 1.5146.20 ± 1.50*
Weight (kg)69.70 ± 16.05144.41 ± 4.56159.52 ± 3.73*
Height (cm)174.70 ± 11.52167.71 ± 1.21172.49 ± 1.25*
BMI (kg/m2)22.40 ± 2.4651.33 ± 1.5353.52 ± 1.28
AST (U/L)15.20 ± 1.7424.66 ± 1.7539.80 ± 4.25*
ALT (U/L)18.96 ± 3.7728.17 ± 2.5653.17 ± 5.53*
Bilirubin (mg/dL)0.52 ± 0.280.60 ± 0.050.51 ± 0.03
Cholesterol (mg/dL)141.31 ± 17.32194.97 ± 5.39191.97 ± 4.89
LDL 136.94 ± 5.57126.32 ± 4.78
HDL 45.81 ± 2.1343.53 ± 2.03
Fasting blood glucose 95.54 ± 8.46122.9 ± 11.09*
HbA1c 5.54 ± 0.1616.92 ± 4.59*

The control group consisting of 10 healthy volunteers whose blood samples were taken had an average BMI of 22.4 ± 2.46 kg/m2 (Table 1). Control samples of liver specimens were obtained from liver transplantation donors (n = 7). We furthermore assessed serum markers of NAFLD and adiponectin levels from a cohort of 39 moderately obese (BMI of 29.6 ± 1.15 kg/m2) patients with the established diagnosis of NAFLD.

Ethical Considerations.

The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Ethics Committee (Institutional Review Board) of the University Hospital Essen. Patients volunteering were informed about specific risks and benefits of wedge liver biopsy and provided written, informed consent before enrolment in the study.

Sample Preparation and Processing.

Biopsies were processed for histological quantification and RNA was isolated and processed according to standard protocols (see Supporting Material). Analyzed genes and utilized oligonucleotides are listed in Supporting Table 1.

Histology.

Hematoxylin and eosin (H&E) staining was performed according to standard techniques. Samples were investigated and the degree of NAFLD was quantified according to the NASH Scoring System (NAS; Table 2).12 In detail, steatosis (0-3), hepatocellular ballooning (0-2), and lobular inflammation (0-2) were determined. NAS of ≥5 or ≥4 when associated with a score of at least one for ballooning were defined as NASH. The grade of liver fibrosis was assessed using the staging system defined by the NASH clinical research group.

Table 2. Histological Scores for Fibrosis
Fibrosis ScorePatients (Total Number)Percent of Patients
11312%
28373%
433%
n.d.1412%
Total113100%

Enzyme-Linked Immunosorbent Assay (ELISA).

M30 serum concentrations were determined with the M30-Apoptosense (Peviva, Bromma, Sweden) Elisa kit, conducted according to the manufacturers' instructions. M30 is a cytokeratin-18 (CK18) neo-epitope exposed upon apoptotic cleavage by activated caspase-3.13, 14

Assessments of BAs, FFAs, and Cholestenone.

BAs were quantified with a commercially available kit (see Supporting Materials for details). FFA concentrations were measured enzymatically in patients' serum (see Supporting Material). Detection of 7α-hydroxy-4-cholesten-3-one (cholestenone) was conducted according to a published protocol by Axelson et al.15 (see Supporting Material for details).

Cell Culture Experiments.

HepG2 cells were kept in cell culture medium (Dulbecco's modified Eagle's medium [DMEM] / high glucose 10% heat-inactivated fetal calf serum [FCS], 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 2 mM L-glutamine) and seeded at a density of ∼1 × 106 cells/cm2. For mimicking a steatosis-like state, cells were incubated with 0.5 mM and 1 mM mixed long-chain FFAs, i.e., 2:1 oleate:palmitate (Sigma-Aldrich, Seelze, Germany). Controls were kept without FFAs.

Statistics.

All data shown are mean ± standard error of the mean (SEM) if not stated otherwise. Differences between FFA concentrations, BA levels, gene expression rates, and M30 neo-epitope concentrations were evaluated by Student t test. For categorical variables, frequencies and percentages were estimated. χ2 or Fisher's exact tests were used for categorical factors. Putative correlations between serum M30 levels with the NASH score or the stage of fibrosis, respectively, were assessed by Spearman's correlation coefficient. P ≤ 0.05 was considered statistically significant. Analyses were performed with SPSS 15.0.1, v. 2006 (Chicago, IL) and GraphPad, v. 5.03 (San Diego, CA).

Results

Elevated Serum Levels of FFAs and BAs Accompany Liver Injury in NASH.

As previously shown by us and other groups, increased lipolysis in visceral fat tissue leads to abundance of FFAs in our cohort of morbidly obese patients.11, 14 FFAs are significantly higher in patients with NASH. Within hepatocytes, FFA-induced lipolysis leads to induction of apoptosis and cell death. Accordingly, serum markers of cell death were increased in patients with simple steatosis without inflammation (nonalcoholic fatty liver [NAFL]; NAS ≤4) and NASH (NAS ≥5) (Fig. 1A). Serum cholesterol levels were higher in obese patients; however, we did not observe changes in serum cholesterol between NAFL and NASH (Table 1; Supporting Fig. 2A). In parallel with alterations in FFAs, BA levels were higher in individuals with high NAS (Fig. 1C). Additionally, we observed a trend towards higher FGF-19 levels in NASH patients, indicating intestinal FXR activation in these individuals (Fig. 1B). Hepatocyte ballooning degeneration is a well-validated histomorphological indicator for hepatocellular injury in NASH and as well a feature of hepatocyte stress in cholestatic liver disease.16 In individuals with advanced ballooning, we found significantly higher serum BA levels (Fig. 2B), a trend towards higher FGF19 levels (Fig. 2F), more apoptosis (Fig. 2C,E), and serum markers of hepatocyte cell death (Fig. 2D).

Figure 1.

Serum FFAs and BAs are increased in NASH progression and adiponectin is inversely correlated with BA levels and liver injury. Serum FFAs are increased in morbidly obese patients, due to increased peripheral lipolysis. FFAs are significantly higher in NASH compared to healthy controls (A). Serum CK18 (M65) and caspase cleaved CK18 (M30) are increased in NAFL and further elevated in NASH as previously published.14 Serum BAs tend to be increased in NASH, compared to NAFL, following histological quantification (NAS)12 (B). Serum concentrations of adiponectin are decreased in NASH compared to simple steatosis, while FGF-19 serum levels are higher in NASH, parallel to increasing BA concentrations. We found a negative correlation between serum BA levels and adiponectin concentration (E), a modest correlation between BAs and the NAS score (C), as well as an inverse correlation of adiponectin and the NAS score (D). *P ≤ 0.05; **P ≤ 0.001.

Figure 2.

Hepatocyte ballooning degeneration is inversely correlated with adiponectin levels and is associated with serum BAs and death receptor expression. Hepatocyte swelling referred to as ballooning is a key histological feature of hepatocellular injury in NAFLD as well as a common finding in BA-induced liver injury in cholestatic liver disease. We found an inverse correlation between adiponectin levels and ballooning (A), and an increase in BA levels in patients with severe ballooning (B). Hepatocellular injury, as assessed by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining (C) and M65 ELISA (D) increased with advanced ballooning. Accordingly, death receptor expression was associated with ballooning (E). In line with an increase in serum BAs, FGF-19 expression increased with ballooning progression (F). *P ≤ 0.05.

Serum Adiponectin Protects from NASH and Is Inversely Correlated With Serum BAs.

Since we previously have shown a protective role for adiponectin in hepatic steatosis, and several authors identified adiponectin as an important mediator in NAFLD pathogenesis, we aimed to quantify adiponectin in this cohort.3, 17 As expected, serum adiponectin levels were decreased in NASH compared to NAFL within our cohort of morbidly obese patients who underwent bariatric surgery (Fig. 1B). By comparing NAFL with NASH within the superobese cohort, and focusing solely on the differences between these two groups further on, we acknowledged the fact that obesity itself is reversely correlated with adiponectin levels as demonstrated in the Supporting data (Supporting Fig. 1). Furthermore, as previously described by others, we found an inverse correlation of adiponectin and the NAS (Fig. 1D) and ballooning progression (Fig. 2A), again underscoring the protective effect of adiponectin. Most likely, as a counterregulatory mechanism, we observed an increase in messenger RNA (mRNA) expression of the adiponectin receptor ApoR2 in NASH, which was associated with hepatocellular apoptosis (Figs. 3E,F, 5). Interestingly, in addition to our observation that adiponectin is decreased in NASH and BAs increased with progression of the disease, we found a direct inverse correlation of adiponectin and serum BAs, revealing a potential effect of adiponectin on BA metabolism (Fig. 1E).

Figure 3.

SHP fails to repress NTCP and Cyp7A1 in NAFLD. As previously shown, death receptor expression is increased in NAFLD (A). Surprisingly, Cyp7A1 was dramatically up-regulated in our cohort of morbidly obese patients, regardless of histological stage (B). An even higher up-regulation was found in moderately overweight (“lean”) NAFLD patients. Cyp7A1 and NTCP transactivation is repressed by SHP activation. NTCP expression was up-regulated in patients versus controls and NTCP expression decreased in NASH compared to NAFL. BSEP expression was higher in obese patients versus controls, but remained unchanged between NAFLD groups. Both BA transporters were elevated to a greater extent in the “lean” NAFLD cohort. To confirm the altered NTCP protein expression, western blots for NTCP were conducted for 16 patients in the NAFL and NASH groups, respectively. A representative blot is given in (C). Quantification of all western blots revealed lower protein expression in NASH compared to NAFL (D), with *P < 0.05. FXR and SHP mRNA expression remained unchanged (E) while ApoR2 expression was increased in NASH. Accordingly, ApoR2 mRNA expression was associated with hepatocyte apoptosis (TUNEL (F)) *P ≤ 0.05; **P ≤ 0.001; ***P ≤ 0.0001.

Obesity Induces Expression of Genes Related to BA Synthesis, Import, and Export.

As expected, in NAFLD patients we observed an up-regulation of mRNA expression of death receptors, apoptosis, and fatty acid transport related genes (Fig. 3A). Transcripts of the BA uptake transporter NTCP, which is under physiological conditions repressed by SHP, are up-regulated in obese individuals. However, we observed a decrease in NTCP expression in superobese NAFLD patients compared to “lean” NAFLD. Within the superobese group NASH patients exhibited a further reduction of NTCP in comparison to NAFL, most likely secondary to increased BA levels with FXR and SHP activation (Fig. 3B). These patterns in NTCP expression changes are strikingly clear in mRNA quantification, yet protein quantification with western blot revealed a high interindividual variability in protein expression, as previously described by other groups (Fig. 3C,D).18 Similarly, expression of Cyp7A1, a key gene involved in intrahepatic BA synthesis from cholesterol, which is also repressed by SHP under physiologic conditions, is induced in obese individuals. However, this up-regulation is not attenuated in NASH (Fig. 3B). BA export into the bile canaliculus is mediated by BSEP, a transporter under control of FXR, which is induced in obese individuals (Fig. 3B). The mRNA expression of FXR and SHP remained unchanged compared to healthy controls, but was significantly lower in relation to lean NAFLD patients (Fig. 3E). Other known mediators of BA homeostasis and transcriptional activators of NTCP and Cyp7A1 were slightly increased (HNF4a; MET; LRH1; LXRa; Fig. 4F). Hepatic cholesterol content, which has recently been found to be associated with hepatic steatosis, in our cohort of morbidly obese patients was not related to disease severity of NAFLD (Supporting Fig. 2).19 Similar to our human data, treatment of HepG2 cells with FFAs in vitro lead to transcriptional activation of Cyp7A1 (Supporting Fig. 3A) and NTCP (Supporting Fig. 3B). However, cotreatment with CDCA, a bile salt, which activates FXR significantly attenuated these effects for both genes, NTCP and Cyp7A1. Interestingly, overexpression of adiponectin in HepG2 cells has the same effect as CDCA treatment on Cyp7A1 expression, but does not prevent FFA-induced NTCP up-regulation (Supporting Fig. 3A,B). This indicates a transcriptional repression of Cyp7A1 by adiponectin, independent of FXR activation. In this setting, neither FFA or CDCA treatment nor adiponectin overexpression led to a significant change in cell viability (Supporting Fig. 3F).

Figure 4.

Serum adiponectin concentration is a negative predictor for NASH and is associated with hepatocellular injury, death receptor expression, and FXR downstream targets. ROC calculations revealed that serum adiponectin concentration predicts simple steatosis versus NASH (A) and a cutoff value of 29.16 ng/mL was identified to differentiate between NAFL and NASH. Patients with adiponectin levels below 29.16 ng/mL had a significantly higher NAS and higher incidence/severity of individual histological NASH features (B). BAs, hyaluronic acid, FFA, and FGF-19 levels were higher in patients with adiponectin below the cutoff (C). FXR downstream targets were also decreased with low adiponectin levels (D). Death receptor expression was increased with low adiponectin levels (E). Other known mediators of BA homeostasis and hepatocellular metabolism remained unchanged between the groups and moderately obese (“lean”) NAFLD patients (F). *P ≤ 0.05; **P ≤ 0.001.

Figure 5.

Expression of NTCP, adiponectin, and ApoR2 in patient liver tissue. Depicted are H&E and immunohistochemical stainings for ApoR2 and NTCP. Left lane shows representative samples of NAFL patients (NAS <4); right lane shows representative samples of NASH patients (NAS ≥5). NASH patients show more steatosis, inflammation, and ballooning in H&E staining, while ApoR2 expression is increased in NASH. NTCP protein expression is down-regulated in NASH compared to NAFL.

Adiponectin Levels Are a Negative Predictor of NASH and Determine BA Metabolism.

Since adiponectin levels were inversely correlated with the NAS, we performed receiver operating characteristic (ROC) calculations to elaborate whether low adiponectin levels might predict NASH. In fact, area under the ROC (AUROC) of adiponectin to predict NAFL versus NASH showed a modest, yet significant prognostic value of adiponectin in this setting (Fig. 4A). We identified an optimal cutoff value for adiponectin to predict NAFL of 29.16 ng/mL, in which patients with lower adiponectin levels were more likely to have NASH than simple steatosis. In fact, patients with adiponectin levels below 29.16 ng/mL had a significantly higher NAS, more steatosis, ballooning, and inflammation (Fig. 4B). Interestingly, BAs and hyaluronic acid, as a noninvasive marker of fibrosis, were significantly higher in patients with adiponectin below this cutoff (Fig. 4C). This observation in combination with the fact that lower adiponectin levels were associated with a lesser degree of steatosis might also account for a potential mechanism of adiponectin in the so-called “burned out” steatosis in patients with advanced NASH.20 FFAs and FGF-19 levels were higher in patients with lower adiponectin levels, yet failed to reach significance. Regarding BA metabolism, Cyp7A1, NTCP, BSEP, and OATP2 were lower in patients with adiponectin levels below the cutoff (Fig. 4D). In contrast, death receptor expression was increased in patients with lower adiponectin levels (Fig. 4E). Various growth factors, regulatory proteins, and (nuclear) receptors were analyzed for mRNA expression (Fig. 4F), although differences were observed for few targets (MET, KLF6/KLF6SV1, and LXRa).

Discussion

The principal findings of this study relate BA transporters to hepatocyte apoptosis in NAFLD and uncover a potential role for adiponectin in BA homeostasis. The observations demonstrate a marked induction of genes involved in hepatocellular BA uptake and synthesis, which are repressed by SHP under physiological conditions, in our cohort of superobese individuals. Treatment of hepatoma cells with FFA induces the same BA uptake and synthesis-related genes in a similar fashion. Adiponectin is inversely correlated with serum BAs and hepatocellular injury, and low adiponectin levels predict simple steatosis as opposed to NASH in obese individuals. Patients with adiponectin levels below 29.16 ng/mL have significantly greater histological features of NASH, higher BA levels, and a lower expression of BA metabolism-related genes, uncovering a novel role for adiponectin and FFA in bile salt metabolism (Fig. 6).

Figure 6.

Model slide of dysregulated intrahepatic BA metabolism. While up-regulation of BSEP indicates FXR activation, possibly due to raised intrahepatic BA levels, it does fail to induce SHP. SHP is a known repressor of NTCP and Cyp7A1, which also were expressed to greater degrees in NAFLD. On the basis of the data presented, we propose a hypothesis suggesting an influence on FXR-mediated induction of SHP by FFA. Thickness of arrows/lines represents the effect of intensity of inhibition/expression.

The pathogenesis of NAFLD is widely known to be associated with hepatocyte steatosis and FFA-induced lipotoxicity followed by the secretion of proinflammatory cytokines and stellate cell (HSC) activation, which in the end results in disease progression and fibrosis.21, 22 Since our group and others observed increasing BA concentrations in NASH, in addition to lipotoxicity, BAs, as products of endogenous hepatic synthesis, may themselves contribute to liver injury in NAFLD.5 In this context, accumulation of BAs in hepatocytes causes hepatocyte death, giant cell hepatitis, and progressive liver damage in hereditary disorders requiring liver transplantation at a young age.23 The mutagenic potential of BAs may even explain the early development of hepatocellular carcinoma in children with hereditary BSEP deficiency.24

Hepatobiliary transport systems are regulated at a transcriptional and posttranscriptional level.9, 25 Nuclear receptors have been identified to function as regulators for positive and negative feedback pathways orchestrating bile formation under different clinical conditions.26 The nuclear BA receptor FXR plays a central role in BA homeostasis and regulates Na+-dependent (NTCP) BA uptake, apart from canalicular excretion (BSEP), as well as the rate-limiting step of BA formation (CYP7A1).27–30 Upon activation by BAs, FXR represses BA uptake and synthesis (NTCP, CYP7A1) by way of SHP and simultaneously activates BA efflux (BSEP).31 In our cohort, we found significant elevations of CYP7A1, consistent with lowered SHP activity, despite only a modest decrease in SHP mRNA expression. CYP7A1 represents the key enzyme catalyzing BA biosynthesis. In one human study, investigating patients with obstructive cholestasis, hepatic CYP7A1 mRNA tended to be increased, similar to the results in our fatty liver patients.32 High serum cholestenone levels confirm the increased CYP7A1 mRNA expression in our study at the functional level.

SHP also represses hepatocyte BA uptake by transcriptional repression of NTCP.33 In experimental obstructive cholestasis in rodents, NTCP is down-regulated by activation of FXR and subsequently induction of the repressor SHP. This mechanism prevents excessive BA transport from portal blood and uptake into the hepatocytes.10 Similar mechanisms are in action for human NTCP where SHP has been shown to suppress the glucocorticoid receptor-mediated transactivation of the human NTCP promoter.34 In human fatty livers, we observed an increase in NTCP expression, which in contrast to Cyp7A1 decreased with progression to NASH. The observed attenuation of baseline SHP activity under steatotic conditions was confirmed in our in vitro experiments. FFA treatment led to a significant up-regulation of NTCP and Cyp7A1. Similar to our findings in NASH regarding NTCP expression, BA (CDCA) treatment attenuated these effects, most likely by way of FXR-SHP activation. This is consistent with recent data from ob/ob mice where SHP-induction and down-regulation of NTCP is absent despite retention of BAs.35 In a recent study, Wanninger et al.36 observed an up-regulation of hepatic CYP7A1 mRNA expression, in line with elevated serum triglyceride levels in adiponectin knockout mice. We also observed lower adiponectin levels with advanced stages of NAFLD and our in vitro data also confirm a negative regulation of Cyp7A1 by adiponectin. As Aranha et al.6 have shown, BAs within the liver are elevated in patients with steatohepatitis. In agreement with our data, this would suggest an increased BA uptake and production. Elevated intrahepatic BA amounts would lead to activation of FXR and subsequently enhanced BSEP expression, which we also observed.29 On the other hand, impaired adaptation of BSEP expression may lead to increased BA accumulation in hepatocytes and enhanced cell death. Interestingly, steatotic hepatocytes were shown to be more susceptible to BA-induced apoptosis.37 Even low elevations of BAs normally not considered harmful could enhance hepatocyte apoptosis in patients with NAFLD. However, in our in vitro studies we did not observe alterations in viability upon FFA and CDCA cotreatment.

An altered intestinal FGF19 production and/or altered hepatic responsiveness to FGF19 may accordingly contribute to the dysregulation of BA homeostasis observed in our patients with NAFLD, as has previously been shown that a rather moderate increase in BA flux may superimpose an influence of FGF15.38 In human hepatocytes, the FGF19-mediated phosphorylation of the hepatic FGF receptor (FGFR) 4 with subsequent activation of the Erk1/2 pathway inhibits CYP7A1 independently of SHP.39 The central role of CYP7A1 inhibition in hepatic dyslipidemia becomes evident from studies in mice with transgenic expression of Cyp7A1 in the liver, which prevents high-fat diet-induced obesity and insulin resistance.40 The same group observed a glucose-mediated epigenetic modification of the CYP7A1 promoter region, leading to CYP7A1 induction, independent of FXR activation, linking glucose metabolism to BA synthesis.41 In a recent study the hepatic response to FGF19 was impaired in 20 NAFLD patients with insulin resistance compared to 15 healthy controls, while plasma FGF19 levels appeared not significantly different between these groups.42 In our cohort, we observed a modest increase in plasma FGF19 levels in NAFL compared to the NASH subgroup. This finding might be in accordance with blunted repression of CYP7A1 and an increased cholestenone production and may also play a functional role in the natural course of the disease.

Toxic effects of BAs are not solely the result of detergent effects, but derive from activation of cell death pathways.43, 44 It is well known that BAs exhibit proapoptotic effects in a Fas- and tumor necrosis factor (TNF)-related apoptosis-inducing ligand dependent way in vitro45, 46 as well as by way of mitochondrial pathways, while the mechanism seems to be caspase-dependent.47 High intracellular BA levels promote hepatocyte apoptosis, markers of which we found both at the transcriptional level within the liver (NOXA, CD95/Fas, FasL) and in the systemic circulation (M30). Interestingly, in a recent study van der Poorten et al.20 identified increased BA levels in NASH as responsible for the activation of adipocytes to produce adiponectin, which supports the importance of a crosstalk between adipose tissue and the liver in NAFLD. In our cohort, adiponectin was inversely correlated with NAFLD disease progression and serum BA levels. We have previously shown that adiponectin prevents CD95/Fas up-regulation and that ApoR2 is associated with steatosis in HCV.3 Adiponectin and other adipokines in the pathogenesis of NASH have recently been widely studied and adiponectin has been evaluated as a prognostic marker for NASH.48 Kaser et al.49 also found decreased expression levels of hepatic adiponectin in patients with NASH as opposed to simple steatosis in obese individuals. In that study, patients with a similar BMI as in our study were investigated and hepatic adiponectin as well as ApoR2 expression were decreased in individuals with NASH. While we observed a similar pattern for adiponectin, hepatic ApoR2 expression in our cohort was lower in obese patients compared to lean patients, but in NASH ApoR2 expression was again increased compared to those patients with an NAS <5. While Kaser et al. defined patients with steatosis and lobular or portal inflammation as NASH, we utilized the NAS system,12 which is a cumulative score, consisting of steatosis, ballooning, and inflammation, to differentiate our NAFL versus NASH groups. This might account for the observed differences in ApoR2 expression, revealing a delicate role for adiponectin in hepatic inflammation, ballooning, and apoptosis. In this context, TNF-α is known to repress adiponectin expression and, among other mechanisms, ApoR2 activation induces phosphorylation of AMP-activated protein kinase (AMPK), increases phosphorylation of c-Jun-N-terminal-kinase (JNK), and activates peroxisome proliferator-activated receptor α (PPARα) signaling.50, 51

Our study failed to show a prognostic value for adiponectin to predict NASH, but adiponectin levels were significantly decreased in NASH and AUROC calculations revealed a modest, yet significant diagnostic value for adiponectin. Analysis of data related to an optimal cutoff value to determine further proved an effect of adiponectin on CD95/Fas, histological features of NASH, as well as BA transport related genes. Several studies observed alterations in BA and adiponectin levels, yet to our knowledge, we are the first to demonstrate a potential direct effect of adiponectin and its receptor on BA homeostasis in NASH patients.52

In conclusion, our results show that serum levels of BAs are increasing in NASH and BA transport, as well as synthesis is markedly dysregulated in NAFLD. The up-regulation of the BA importer NTCP and the key enzyme in synthesis CYP7A1 in NAFLD and hepatoma cells treated with FFAs indicates a dysfunctional repression of target genes by SHP. We could also show that adiponectin is inversely correlated with serum BAs and hepatocellular death and a potential effect of adiponectin on BA homeostasis-related genes, especially CYP7A1. While we provide a hint connecting BA metabolism, hepatocellular cell death, and adipocytokines, the exact mechanisms remain unknown. Further studies will aim to identify the involved pathways and distinct points of application to disrupt the vicious cycle of hepatic steatosis and its sequelae.

Acknowledgements

We thank Mrs. Mechthild Beste and Claudia Gottier for technical expertise and determination of bile acid concentrations.

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