Methods
The effect of THR-ß agonism was investigated in mice with diet-induced obesity and biopsy-confirmed NASH and fibrosis (DIO-NASH model) as described by Kristiansen et al. (2016). All animal experiments were conducted according to the international principles for care and use of laboratory animals and were covered by personal licenses for Jacob Jelsing (2013-15-2934-00784 and 2015-15-0201-00518) issued by the Danish committee for animal research.
Thirty-six male C57BL/6J mice (5 weeks old), obtained from JanVier (JanVier Labs, France), were included in the study. Before treatment with resmetirom, animals had ad libitum access for 34 weeks to a regular rodent diet (Altromin 1324, Brogaarden, Denmark) or a diet high in fat (40%), of these 18% trans-fat, 40% carbohydrates (20% fructose) and 2% cholesterol (D09100301, Research Diet, United States) previously described as the AMLN diet (Clapper, Hendricks, et al, 2013) and tap water.
The study design is illustrated in figure 1a. A baseline liver biopsy was conducted 3 weeks before the intervention for histological assessment of individual fibrosis (stage ≥1) and steatosis (score ≥2), as described (Kristiansen et al., 2016). A week before the intervention the animals were randomized and stratified according to liver Col1a1 quantification into three groups: Group 1, Lean-chow control (n=12); Group 2, DIO-NASH vehicle (n=12); Group 3, DIO-NASH + resmetirom (n=12). Resmetirom (MGL-3196) was purchased from MedChemExpress (catalog no. HY-12216) and administered once daily, in the morning, by oral gavage at a dose of 3 mg/kg. The vehicle used for compound formulation and control injections was 0.6% methyl cellulose with 0.5% Tween 80. The resmetirom concentration in the dosing solution was 0.6 mg/ml, injection volume was 5 ml per kg body weight. The intervention lasted for a period of 8 weeks. At the end of the intervention animals were euthanized and liver tissue and plasma were collected.
Body weight determination, body composition analysis, blood sampling, plasma biochemistry, endotoxin determination and liver tissue biochemistry were performed as previously described (Kristiansen, Veidal, et al, 2016). Statistical significance for the difference to the vehicle control group was assessed using a one-sided ANOVA with Dunnett’s test.
For histology, baseline liver biopsy and terminal samples were collected from the left lateral lobe (about 50-100 mg at baseline and 200 mg at the end) and fixed overnight in 4% paraformaldehyde. Liver tissue was paraffin embedded and sectioned (3μm thickness). Sections were stained with Hematoxylin and Eosin and Sirius Red to assess hepatic steatosis and fibrosis respectively, followed by analysis with Visiomorph software (Visiopharm, Denmark). Collagen 1 alpha 1 (col1a1), α-smooth muscle actin (α-SMA) and galectin-3 were assessed by quantitative immunohistochemistry staining using anti-col1a1 (1:300; Southern Biotech, Birmingham; secondary antibody Bright Vision anti-goat, ImmunoLogic, Netherlands), anti-α-SMA (1:800; Abcam, Cambridge, UK; secondary antibody Envision rabbit, Agilent Technologies, Glostrup, Denmark) or anti-galectin-3 (gal-3; 1:50000; Biolegend, San Diego, United States; secondary antibody anti-rat IgG 1:800, VWR, Soeborg, Denmark; Envision rabbit, Agilent Technologies, Glostrup, Denmark) as described (Kristiansen, Veidal, et al, 2016). A pathologist blinded to the study performed the histological assessment and scoring. NAFLD activity score (NAS) combining steatosis, lobular inflammation and hepatocyte ballooning scores, and fibrosis stage were quantified applying the criteria proposed by Kleiner, Brunt, et al. (2005). Statistical significance for a difference in the number of animals showing improvement or worsening was evaluated using a one-sided Fisher’s exact test with Bonferroni correction with the DIO-NASH vehicle group as comparator.
Liver tissue was harvested from the left lateral lobe, stabilized overnight in RNAlater® solution (Merck KGaA, Darmstadt, Germany) and stored at -80 °C. Total RNA isolation was performed with the RNeasy kit following the instructions of the manufacturer (QIAGEN GmbH, Hilden, Germany). Quantity and integrity of purified RNA was measured with an Agilent RNA 6000 Nano kit using an Agilent 2100 Bioanalyzer (Agilent Technologies Inc, Waldbronn, Germany). Paired-end sequencing was performed using a TruSeq mRNA Library-Kit (Illumina, San Diego, CA, USA) at Atlas Biolabs, Berlin, Germany, with a sequencing depth of 80 to 100 million reads per sample. RNA sequencing raw data were analyzed using a software package and standardized RNA Seq analysis workflow (Array Studio Version 10.1.3.3, Omicsoft Qiagen). This workflow included alignment to a reference mouse gene model, quantification, normalization, and finally detection of differentially expressed genes using the DESeq2 module (Costa-Silva, Domingues & Lopez, 2017). Results were corrected for variable multiplicity and false-discovery rate (FDR) adjusted p-values calculated using the Benjamini-Hochberg correction (Benjamini & Hochberg, 1995). Differentially expressed gene data were further interrogated on pathways and further causal relationship through the use of Ingenuity Pathway Analysis (Ingenuity Qiagen) as described in Krämer, Green, et al. (2014).