Exercise training improves liver steatosis in mice

Mice model of fatty liver and exercise training protocol

The non-obese mouse model of fatty liver used in this study involved feeding male
C57BL/6 mice a low-fat, high-sucrose, choline-deficient semi-synthetic diet referred
to as sucrose-enriched choline-deficient diet (SECDD) for five weeks. The composition
of the SECDD is shown in Table 1. Before proceeding to the main study, a pilot experiment using C57BL/6 mice was conducted
to examine the effectiveness of SECDD diet in inducing liver steatosis in comparison
with feeding regular chow.

Table 1. Composition of the sucrose enriched choline deficient diet (SECDD)

For the main study, male C57BL/6 mice were fed the SECDD for five weeks, after which
the mice were divided into an exercise (trained) and a sedentary (untrained) group.
Mice belonging to the trained group did a treadmill-based running exercise (TSE PhenoMaster,
TSE Systems, Germany) for a period of 3 weeks; 5 days/week, 1 h/day. In the first
week, mice ran at 14 m/min with 0° incline and in the following weeks they ran at
a speed of 16 m/min with 2° incline, representing moderate intensity exercise for
the mice. During the exercise training period, the mice continued to be fed on SECDD.
The untrained group of mice were placed on a non-moving treadmill also for a period
of 3 weeks; 5 days/week, 1 h/day. Body weight and food intake were measured weekly
throughout the experiment. After the last bout of exercise, the mice were returned
to their cage and 24 h later, a subset of mice from the trained and untrained groups
were either used to study in-vivo hepatic triglycerides secretion (n?=?7 untrained, n?=?9 trained), sacrificed to collect tissues for biochemical and histological study
(n?=?7 untrained, n?=?9 trained) or sacrificed to collect tissues for various measurements, including
hepatic de novo lipogenesis (DNL) (n?=?12 untrained, n?=?12 trained). A number of measurements were performed in all mice within the last
two cohorts, raising the number of biological replicates to 19 or 21 per group. Prior
to sacrifice, mice were put under anesthesia using isoflurane and blood was drawn
via orbital puncture into EDTA-containing tubes. Plasma was prepared by centrifugation
at 4 °C and stored at ?80 °C. All animal experiments were approved by the animal welfare
committee of Wageningen University.

Plasma and tissue biochemical measurements

Plasma samples of non-fasted mice were analysed for glycerol (Diasys, Germany), triglycerides
(Liquicolor, Germany), non-esterified fatty acids, NEFA (Wako Chemicals, Germany),
cholesterol (Diasys, Germany) and alanine aminotransferase, ALT (Abcam, UK) using
commercially available kits according to manufacturer’s instructions. Tissue glycogen
levels were measured according to a method described previously 24]. Briefly, approximately 50 mg of tissue was digested using 1 N NaOH at 80 °C for
15 min. Glycogen was precipitated from the supernatant by adding 100 % ethanol at
2:1 v/v and the precipitate was pelleted down. Pellet was washed with 80 % ethanol
and solubilised in water by incubating at 37 °C for 15 min. The glycogen sample was
then digested to glucose by incubating at 42 °C for 2 h with amyloglucosidase (Sigma,
USA) in 0.2 M sodium acetate buffer (pH 4.8). The amount of glucose in the digested
samples was measured using a glucose assay kit (DiaSys, Germany).

Liver triglycerides were determined in 5 % liver homogenates prepared in buffer containing
250 mm sucrose, 1 mm EDTA, 10 mm Tris–HCl (pH 7.5) using a commercially available
kit (Liquicolor, Germany). Histological analysis of liver morphology and lipid content
was done using haematoxylin-eosin (HE) and oil-red O (ORO) staining.

De-novo hepatic VLDL triglyceride secretion and lipogenesis

For the VLDL triglyceride secretion test, mice fasted for 4 h were injected via the
orbital plexus with 500 mg/kg bodyweight of the lipoprotein lipase inhibitor Triton
WR1339 (Tyloxapol) as 15 % solution under general anesthesia. Blood was collected
by tail bleeding every 30 min for 2.5 h as the mice remained sedated. Plasma triglyceride
levels were measured in the blood samples collected at different time points using
an enzymatic kit (Liquicolor, Germany). Glucose was measured in the baseline plasma
sample (Diasys, Germany).

The rate of liver de novo lipogenesis (DNL) was measured by
²
H NMR using
²
H enrichment of liver triglycerides 25]. At the end of the last exercise training session, a set of mice belonging to both
trained and untrained groups were rested for 5-6 h and given an intraperitoneal injection
of 99.9 %
²
H
₂
O (Sigma-Aldrich, USA) as saline. The mice were then returned to their cage and had
free access to food and water. In order to have constant body water enrichment, the
drinking water was enriched with
²
H
₂
O at 3 %. They were then sacrificed after 16 h, and blood samples were obtained for
the determination of plasma
²
H-enrichment. The livers were excised and hepatic lipids extracted 26]. The extract was dissolved in chloroform and a mixture of
¹
H/
²
H-pyrazine was added as reference. Proton-decoupled
²
H NMR spectra (WALTZ 16-decoupling, 90° hard pulse for excitation) and
¹
H-spectra of lipids were acquired (Bruker Avance III 500 MHz) and the methyl-group
signals of the
¹
H-and
²
H-spectra were quantified relative to the signal of the respective standard. The
²
H-enrichment of body-water was determined as previously described 27]. The ratio between the
²
H-enrichment of the methyl-groups of hepatic triglycerides and body-water represents
the fractional contribution of DNL to hepatic triglycerides.

Hepatic gene and protein expression levels

Total RNA was isolated from the mouse tissues using TRIzol reagent (Invitrogen, Breda,
The Netherlands). RNA was reverse transcribed using RevertAid First strand cDNA synthesis
kit (Thermoscientific). Real-time PCR was carried out using SensiMiX (Bioline) on
a CFX 384 Bio-Rad thermal cycler (Bio-Rad). 36B4 was used as housekeeping gene. Primers
sequences used are shown in Table 2.

Table 2. List of primers

Tissue homogenates were made using RIPA buffer with added protease inhibitor cocktail
and spun to pellet the cell debris. Supernatant was collected and the protein concentration
determined using the BCA method (Pierce BCA protein assay kit). Equal amount of protein
was subjected to a 4-15 % gradient gel electrophoresis and immunoblotted using the
following antibodies for Tubulin (1:1000, SantaCruz), GAPDH (1:1000, Santa Cruz),
LC3 and p62 (1:1000, Novus biologicals); AMPK, pAMPK, Cytochrome C (1:1000, Cell signalling
technology). Blots were further incubated with the appropriate HRP conjugated secondary
antibody (1:5000) and developed using BioRad Clarity (BioRad, USA). The bands were
visualised using ChemiDoc system (BioRad, USA) and quantified with Image Lab software.

Micro-array analysis

After TRIzol, RNA was further purified using RNeasy micro columns (Qiagen, Venlo,
the Netherlands). RNA integrity was checked on an Agilent 2100 bioanalyzer (Agilent
Technologies, Amsterdam, the Netherlands) using 6000 Nano Chips according to the manufacturer’s
instructions. Purified RNA (100 ng) was labeled with the Ambion WT expression kit
(Invitrogen) and hybridized to an Affymetrix Mouse Gene 1.1 ST array plate (Affymetrix,
Santa Clara, CA). Hybridization, washing, and scanning were carried out on an Affymetrix
GeneTitan platform according to the instruction by the manufacturer. Arrays were normalized
using the Robust Multiarray Average method 28], 29]. Probe sets were defined according to Dai et al. 30]. In this method probes are assigned to Entrez IDs as an unique gene identifier. The
P values were calculated using an Intensity-Based Moderated T-statistic (IBMT) 31]. The microarray data were submitted to the Gene Expression Omnibus (accession number
pending). Gene set enrichment analysis (GSEA) was used to find enriched gene sets
in the induced or suppressed genes 32]. Genes were ranked based on the IBMT-statistic and subsequently analyzed for over-
or underrepresentation in predefined gene sets derived from Gene Ontology, KEGG, National
Cancer Institute, PFAM, Biocarta, Reactome and WikiPathways pathway databases. Only
gene sets consisting of more than 15 and fewer than 500 genes were taken into account.
Statistical significance of GSEA results was determined using 1000 permutations.

Statistical analysis

All the results are expressed as mean?±?SEM. Comparisons were made between the trained
and untrained mice. Statistical significance was tested using a two-tailed Student’s
t-test and p??0.05 was considered as significantly different.