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).