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. Hematoxylin and eosin (H&E) staining was performed according to standard techniques. Samples were investigated and the degree of NAFLD was quantified according to Crenolanib in vivo 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. 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 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 DMXAA according to a published protocol by Axelson et al.15 (see Supporting Material for details). 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. All data shown are mean ± standard error of the mean (SEM) if not stated otherwise. Differences between FFA concentrations, BA levels, gene expression
check details 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). 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.