The RNA‐Bee Total‐RNA Isolation Kit (Bio‐Connect, Huissen, the Netherlands) was used for RNA extraction from snap‐frozen liver tissue (left lobe). RNA concentration was determined spectrophotometrically using Nanodrop 1000 (Isogen Life Science, De Meern, Netherlands), and RNA quality was assessed using the 2100 Bioanalyzer (Agilent Technologies, Amstelveen, Netherlands). RNA was then used to generate strand‐specific messenger RNA (mRNA)–seq libraries for next generation sequencing at GenomeScan B.V. (Leiden, the Netherlands). Libraries were multiplexed, clustered, and sequenced on an Illumina NextSeq 500 with a single‐read 75‐cycles sequencing protocol, 13 million reads per sample, and indexing. Differentially expressed genes were determined for HFD versus chow at t = 24 weeks, HFD versus chow at t = 34 weeks, and HFD+OCA versus HFD at t = 34 weeks, using the DESeq‐method34 with statistical cut‐off false discovery rate < 0.05.
To evaluate the representation of human pathophysiological pathways in HFD‐fed Ldlr‐/‐.Leiden mice, murine hepatic gene expression profiles were compared with published data on hepatic gene expression profiles in human NASH: a disease signature for NASH versus control10 that was downloaded from the Gene Expression Omnibus (GEO) with accession number GSE48452 and a gene profile that differentiates severe “progressive” NASH patients (fibrosis stage 3 or 4) from mild NASH patients (fibrosis stage 0 or 1)9 (GEO accession number GSE31803).
The effects of OCA on hepatic gene expression were analyzed by gene enrichment analysis across pathways and biological processes using Ingenuity Pathway Analysis (IPA) suite (www.ingenuity.com, accessed 2016) as described previously.19, 35 The upstream regulator analysis tool of IPA was used to assess the activity of transcription factors as well as other upstream regulators (e.g., receptors, enzymes, metabolites) essentially as reported.36 Gene expression data were used to predict activation or deactivation of upstream regulators (e.g., of TGF‐β or the insulin receptor). A negative z‐score of less than −2 indicates significantly reduced transcriptional activity based on the direction of gene expression changes of target genes. A positive z‐score of greater than 2 indicates significant activation of the upstream regulator. In addition, the effects of OCA on the gene expression profiles for human NASH9, 10 were investigated.