Brain, Behavior, and Immunity 26 (2012) 931–941

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Brain, Behavior, and Immunity
journal homepage: www.elsevier.com/locate/ybrbi

Exercise training attenuates hepatic inﬂammation, ﬁbrosis and macrophageinﬁltration during diet induced-obesity in mice
Noriaki Kawanishi a,b,⇑, Hiromi Yano c, Tsubasa Mizokami a, Masaki Takahashi a, Eri Oyanagi d,
Katsuhiko Suzuki eaGraduate School of Sport Sciences, Waseda University, Tokorozawa, Saitama, Japan
Research Fellow of the Japan Society for the Promotion of Sciences, Tokyo, Japan
Department of Health and Sports Science, Kawasaki University of Medical Welfare, Kurashiki, Okayama, Japand
Department of Cytology and Histology, Okayama University Graduate School, Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japane
Faculty of Sport Sciences, Waseda University, Tokorozawa, Saitama, Japanbca r t i c l e

i n f oArticle history:
Received 7 January 2012
Received in revised form 16 April 2012
Accepted 16 April 2012
Available online 23 April 2012
Keywords:
Exercise training
Fatty liver
Hepatic inﬂammation
Hepatic ﬁbrosis
Macrophage
Chemokinea b s t r a c t
Nonalcoholic steatohepatitis, which is considered the hepatic event in metabolic syndrome, was recently
associated with the innate immune system. Although regular exercise reduces hepatic injury markers like
serum alanine aminotransferase (ALT) levels, the mechanisms regulating the effects of exercise on steatohepatitis are unclear. This study aimed to clarify whether exercise training suppresses hepatic injury,
inﬂammation, and ﬁbrosis by suppressing macrophage inﬁltration. Male C57BL/6J (4-week old) mice
were randomly divided into four groups: normal diet (ND) control (n = 7), ND exercise (n = 5), high-fat
diet and high-fructose water (HFF) control (n = 11), and HFF exercise (n = 11) groups. Mice were fed
the ND or HFF from 4 to 20 weeks of age. The exercise groups were trained on a motorized treadmillfor 60 min/day, ﬁve times/week. The nonalcoholic fatty liver disease (NAFLD) activity score and plasma
ALT activity, indicators of liver injury, were increased in HFF control mice but were attenuated in HFF
exercise mice. Hepatic inﬂammation, indicated by hepatic tumor necrosis factor (TNF)-a levels and hepatic resident macrophage inﬁltration, was signiﬁcantly lower in HFF exercise mice than in HFF control
mice. Hepatic ﬁbrosis markers (histological hepatic ﬁbrosis detected by Sirius red and a-smooth muscle
actin staining and tissue inhibitor of matrix metalloproteinase-1 mRNA) were attenuated in HFF exercise
mice compared with HFF control mice. These results suggest that exercise training reduces hepatic
inﬂammation, injury, and ﬁbrosis by suppressing macrophage inﬁltration.
Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction
Nonalcoholic steatohepatitis (NASH) is considered the hepatic
event in metabolic syndrome (Browning and Horton, 2004).
Although the pathogenesis of NASH remains unclear, several factors such as inﬂammation and ﬁbrosis have been proposed (Argoet al., 2009). Inﬂammatory cytokines such as tumor necrosis factor(TNF)-a and reactive oxygen species (ROS) exert a ﬁbrotic effect(Tomita et al., 2006; Minicis et al., 2010). Indeed, TNF-a directly
up-regulates transforming growth factor (TGF)-b and a-smoothmuscle actin (SMA) in hepatocytes (Tomita et al., 2006), andTGF-b subsequently induces tissue inhibitor of metalloproteinase⇑ Corresponding author. Address: Graduate School of Sport Sciences, Waseda
University, 2-579-15 Tokorozawa, Saitama 359-1192, Japan. Tel./fax: +81 4 2947
6753.
E-mail addresses: kawanishinoriaki@akane.waseda.jp (N. Kawanishi), yanohiro@mw.kawasaki-m.ac.jp (H. Yano), o-hisyo.w@akane.waseda.jp (T. Mizokami),masaki-taka@fuji.waseda.jp (M. Takahashi), eri.oyanagi-7@hotmail.co.jp (E. Oyanagi), katsu.suzu@waseda.jp (K. Suzuki).
0889-1591/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bbi.2012.04.006(TIMP) 1, which enhances hepatic ﬁbrosis development via induction of collagen 1a expression (Wang et al., 2009).
The innate immune system is associated with NASH development via activation of hepatic resident macrophages, Kupffer cells(Zhan and An, 2010). Macrophage inﬁltration was induced in thelivers of mice by combined administration of high-fat diet andhigh-fructose water, and Kupffer cells subsequently induced
various pro-inﬂammatory cytokines, which enhance hepatic
inﬂammation and ﬁbrosis (Kohli et al., 2010). Kupffer cells express
toll-like receptors (TLRs) and recognize several molecular patterns
that induce inﬂammatory cytokine production (Szabo et al., 2006).
Seki et al. (2007) recently reported that hepatic inﬂammation andﬁbrosis are attenuated in TLR4-knockout mice. Therefore, the TLRs
in Kupffer cells may play an important role in NASH. Furthermore,
it was suggested that T lymphocytes play an essential role in thedevelopment of adipose tissue inﬂammation (Winer et al., 2009;
Nishimura et al., 2009). Although it is proposed that cytotoxic T
cells are associated with NASH development (Popov and Schuppan,
2010), this has not been conclusively proven.

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N. Kawanishi et al. / Brain, Behavior, and Immunity 26 (2012) 931–941

Regular exercise may prevent or attenuate NASH development.
In addition, higher levels of physical activity are negatively associated with the state of ﬁbrosis in patients with NASH (Kistler et al.,2010), and regular exercise reduces the serum alanine aminotransferase (ALT) activity, which is a marker of hepatic injury (Promrat
et al., 2010; Sreenivasa et al., 2006). Furthermore, exercise training
reduces serum ALT activity and hepatic steatosis in obese mice
(Marques et al., 2010; Schultz et al., 2010). However, it is notknown whether exercise training alleviates hepatic injury andﬁbrosis by suppressing inﬂammation in obese mice.
It was recently shown that regular exercise can protect against
the development of several types of chronic inﬂammation (Gleeson
et al., 2011; Pedersen, 2011). In fact, the concentrations of plasma
inﬂammatory cytokines such as TNF-a and IL-6 are lower in physically active subjects than in physically inactive subjects (Pedersen
and Saltin, 2006). Moreover, exercise training is now considered a
crucial event leading to reduced inﬂammation in the adipose tissue. We and another research group have shown that exercise
training enhances down-regulation of TNF-a gene expression in
the adipose tissue of obese mice (Kawanishi et al., 2010; Vieira
et al., 2009). Furthermore, we showed that exercise training attenuates inﬂammatory macrophage inﬁltration into adipose tissue by
suppressing chemokine and adhesion molecules (Kawanishi et al.,2010). Accordingly, it is possible that exercise training attenuatesinﬂammation in liver tissue by suppressing macrophage inﬁltration in diet-induced NASH model mice.
Therefore, the purpose of the present study was to clarify
whether exercise training reduces hepatic injury, inﬂammationand ﬁbrosis in diet-induced NASH model mice. We hypothesizedthat exercise training attenuates hepatic injury, inﬂammationand ﬁbrosis by suppressing the inﬁltration of macrophages.

2. Methods
2.1. Animals, diets and exercise training protocol
Male C57BL/6 mice (4 weeks old) were purchased from Kiwa
Laboratory Animals (Wakayama, Japan). Four animals were housed
together in 1 cage (27  17  13 cm) in a controlled environment
under a light-dark cycle (lights on at 0900 and off at 2100). The
experimental procedures followed the Guiding Principles for theCare and Use of Animals in the Waseda University Institutional
Animal Care and Use Committee (approved number: 2011-A19).
The mice were randomly divided into four groups: normal diet(ND) control (n = 7), ND with exercise training (n = 5), high-fat dietand high-fructose water (HFF) control (n = 11), and HFF with exercise training (n = 11) groups. Ethically, this experiment was started
with the animal number of 8 (Normal diet group) or 12 (High fat
diet group) per respective groups, but the number of exercise training mice were reduced to by three due to injury during the treadmill running. The high-fat diet (D12492; Research Diets, New
Brunswick, NJ) was composed of 60% fat, 20% protein, and 20% carbohydrate (of total calories), and the high-fructose water contained
21% (wt/V) fructose (Sigma, St. Louis, MO).
A mix of high fat and fructose has been shown effective forinducing fatty acid liver disease. Overintake of fructose is also associated with the development of NASH (Lim et al., 2010). Animals
fed a high-fat diet and high-fructose water ad libitum developed
hepatic inﬂammation and ﬁbrosis in mice (Kohli et al., 2010; Sohet
et al., 2009). In contrast, animals fed only a high-fat-diet (about
60% kcal from fat) ad libitum developed steatosis without any hepatic inﬂammation or ﬁbrosis (Kohli et al., 2010). Therefore, feedinghigh-fat diet and high-fructose water is a useful model to develop
fatty acid liver disease and NASH.

The mice in the present study were fed the HFF from 4 to
20 weeks of age. All ND mice were fed a standard normal dietcontaining 10% fat, 20% protein, and 70% carbohydrate (D12450B;
Research Diets, New Brunswick, NJ) and fructose-free water. All
groups had free access to food and water. Body mass was measured
weekly. Food and water intakes per four mice were measured
twice from week 15 to week 16.
Four-week old mice were trained on a motorized treadmill(Natsume, Kyoto, Japan) for 60 min/day during their light phaseat running speeds of 15–20 m/min, ﬁve times/week for 16 weeks.
Exercise speed was set initially from 15 m/min per session during
the ﬁrst four weeks, and increased to 20 m/min per session on andafter 5 weeks. Electric shock was not used during the treadmill run
to avoid noxious stress. Control mice were housed in cages. The
exercise-trained and untrained mice (20-weeks old) were sacriﬁced 3 days after the ﬁnal exercise training session under lightanesthesia with the inhalant isoﬂurane (Abbott, Tokyo, Japan).
Abdominal vein blood samples were collected in heparin tubes,
and plasma was stored at 80 °C. Liver tissue was quickly frozen
in liquid nitrogen and stored at 80 °C.
2.2. Plasma alanine aminotransferase and hepatic triglyceridemeasurements
ALT activity was measured using the L type Wako GPT Kit(Wako, Osaka, Japan). The minimum detectable limit of ALT activity was 2.2 U/L. To examine triglyceride levels in the livers of mice,liver tissue (100 mg) was homogenized in a 1 ml solution containing 5% Triton-X100 in water, and heated to 85 °C for 5 min. Hepatic
triglyceride content was measured using the triglyceride Quantiﬁcation Kit (BioVision, Milpitas, CA). The minimum detectable concentration of Triglyceride was 2 pmol/L.
2.3. Hepatic TNF-a and monocyte chemoattractant protein (MCP)-1
measurement
To examine TNF-a and MCP-1 protein levels in the livers of
mice, liver tissue (100 mg) was homogenized in 500 ll Tissue Protein Extraction Reagent (T-PER) with Protease inhibitor (Thermo,
Rockford, IL). The protein concentration was measured using the
BCA Protein Assay (Thermo). The TNF-a level was measured using
the Mouse TNF-a Quantikine ELISA kit (R&D Systems, Minneapolis,
MN). The MCP-1 level was measured using the Mouse MCP-1
Quantikine ELISA kit (R&D Systems). The minimum detectable concentrations of TNF-a and MCP-1 were 5.1 and 15.6 pg/ml,
respectively.
2.4. Isolation of hepatic mononuclear cells and ﬂow cytometry analysis
We isolated mononuclear cells from the liver using previously
described methods with some modiﬁcations (Kinoshita et al.,2010; Kleiner et al., 2005). After removal of blood from liver tissue,
the liver was minced with scissors. Hanks’ Balanced Salt Solution(HBSS) (10 ml) containing 0.05% collagenase type 4 (Worthington,
Lakewood, NJ) was added to the pieces of liver tissue. The mixture
was shaken for 20 min at 37 °C, and the digested tissue was centrifuged at 500  g for 10 min. The resultant pellet containing themononuclear cells was resuspended in RPMI containing 1% FBS,
ﬁltered through a 70-lm stainless steel mesh, and the digested tissue was centrifuged at 500  g for 10 min. The resultant pellet containing the mononuclear cells was suspended in 12 ml 33% Percoll(Sigma) solution containing 10 U/ml heparin and a 15 min centrifugation step at 500  g was performed at room temperature.
The resultant pellet containing the mononuclear cells was suspended in 5 ml Red Blood Cell Lysing buffer (Sigma) and ﬁlteredthrough a 40-lm nylon mesh. The cells were washed twice with

N. Kawanishi et al. / Brain, Behavior, and Immunity 26 (2012) 931–941
Table 1
Primer sequences for real-time RT-PCR analysis.
Gene

Forward

Reverse

GAPDH
TNF-a
IL-1b
IL-6
IL-10
Collagen
1a
a-SMA
TIMP1
TGF-b
CD11c
TLR4
MCP-1
CXCL14RANTESTGAAGCAGGCATCTGAGGG
CCTCCCTCTCATCAGTTCTACTGGAGAGTGTGGATCCCAAGTAGTCCTTCCTACCCCAATTTCCTGAATTCCCTGGGTGAGAAGGAGCGGAGAGTACTGGATCGCGAAGGTGGAAGAGTGGGAG
ACTTGGTGGTTTGCTACGACGGAAGACACGGATTCCATGGTG
TTGGTCCTTAGCCACTCCTTC
TCACTCTTCACCTGCTCCACT
GCTTCTTTTCCTTGGGGTTCAAACAGGAATACGACGAAGTGGGAAATGCCGCAGATATCGACCCTGCCCCTATATTTGGACTGGATAGCCTTTCTTCTGCTG
ATGGCATGGCTTACACCACC
CTTCTGGGCCTGCTGTTCACCAAGATTCGCTATAGCGACCAGAGGAAAGAGAGAAAGTCCCAGGAATGATTTGGAAAGGATGGGACTTGTGGGCATATCCGCCCGGGTTGTGTTGGTGCACACTGTGTCCGAACTCGAGGCCAATTTTGTCTCCACA
CCAGCCTACTCATTGGGATCACCTGCGCTTCTCGTTCCAGG
CACACGGTGACAGTGCTGG

Stain Buffer (BD Pharmingen, Franklin Lakes, NJ), and the mononuclear cells were resuspended in Stain Buffer. The mononuclear cells(2.5  105 cells) were incubated with Fc-blocker (anti-CD16/CD32)
for 20 min followed by staining with PE-Cy7-CD3e, PE-CD4, and
PE-Cy5-CD8a for 20 min. All of the above mentioned antibodies
were purchased from eBioscience (San Diego, CA). Flow cytometry
was performed using a GuavaÒ EasyCyte™ 6HT (Millipore, Long
Beach, CA) and InCyte software (Millipore). We validated ﬂow
cytometric identiﬁcation of cytotoxic T cells (CD8a+CD3e+) andhelper T cells (CD4+CD3e+). Hepatic resident mononuclear cells
from mice were gated according to side scatter and forward scatterplots. Cytotoxic T cell populations were gated according to CD3eand CD8a on the SSC/FSC plot. Helper T cell populations were gatedaccording to CD3e and CD4 on the SSC/FSC plot.
2.5. Real-time quantitative PCR
A piece of the liver tissue was quickly immersed in RNAlater(Applied Biosystems, Carlsbad, CA) and stored at 80 °C. Total
RNA was extracted from the liver tissue homogenate using Trizol
reagent (Invitrogen, Carlsbad, CA) and the RNeasy Mini Kit (Qiagen,
Valencia, CA) according to the manufacturer’s instructions. The
purity of total RNA was assessed using the NanoDrop system(NanoDrop Technologies, Wilmington, DE). Total RNA was reverse
transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s
instructions. Polymerase chain reactions (PCR) were performed
with the Fast 7500 real-time PCR system (Applied Biosystems)
using the Fast SYBRÒ Green PCR Master Mix (Applied Biosystems).
The thermal proﬁles consisted of 10 min at 95 °C for denaturation
followed by 40 cycles of 95 °C for 3 s and annealing at 60 °C for
15 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAwas used as the housekeeping gene, and all data are represented
relative to its expression (i.e., using standard curve methods) as
fold change based on the values of the ND control group. Speciﬁc
PCR primer pairs for each studied gene are shown in Table 1.
2.6. Histological analysis
A piece of the liver tissue was transferred to a plastic mold, covered with OCT compound, and snap frozen by immersing theclosed in pre-cooled isopentane at 80 °C. Hematoxylin–Eosin(H&E), Oil-Red O, Sirius red, and Masson trichrome staining were
performed. The nonalcoholic fatty liver disease (NAFLD) activity
score is an accepted paradigm for the staging and grading of NASH
development. The NAFLD activity score was determined as933

previously described (Kleiner et al., 2005). Scores for steatosis(0–3), lobular inﬂammation (0–3), and hepatocyte ballooning (0–2) were also summed to produce the NAFLD activity score; thus,
the total score ranged from 0 to 8. Oil red O- and Sirius red-staining
positive areas were analyzed on four random low power (100)ﬁelds/slide using ImageJ software (National Institutes of Health,Bethesda, USA).
Immunohistochemical staining was applied to frozen sectionsof liver tissue to examine expression of F4/80 and a-SMA. The 5lm serial sections were incubated in 4% paraformaldehyde for7 min at 4 °C. Endogenous peroxidase was inactivated with 1%hydrogen peroxide in methanol for 30 min at 4 °C. a-Smooth muscle actin (Dako, Carpinteria, CA) and F4/80 (Serotec, Kidlington, UK)primary antibodies were added in 1% BSA solution and sections
were incubated overnight at 4 °C. Secondary anti-rabbit antibody
was added in PBS buffer with normal mouse serum for 30 min atroom temperature. Proteins were visualized using the Vectastain
Elite ABC Kit (anti-rabbit, Vector, Burlingame, CA) for 30 min atroom temperature and further incubation was carried out withdiaminobenzidine (DAB) chromogen. F4/80-positive cells werecounted on 4 random high power (200) ﬁelds/slide using BZ-2
software (KEYNENCE, Osaka, Japan).
Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining was performed on frozen sections of liver tissue.
Sections were digested with 20 lg/mL proteinase K, reacted withterminal deoxynucleotidyl transferase enzyme, and incubated with
anti-digoxigenin conjugate followed by incubation with DAB solution. TUNEL-positive cells were counted on four random highpower (200) ﬁelds/slide using BZ-2 software.
Calculations of the NAFLD activity score, and of the Oil red Oand Sirius red-positive areas, and counts of the F4/80- and TUNEL-positive cell number were performed by three independent
observers, who were blinded to the diagnosis, and the average value for each section was calculated. The coefﬁcients of variation for
the NAFLD activity score was 11.6%.
2.7. Statistical analyses
All data are expressed as means ± SEM. Statistical analyses were
performed using SPSS V17.0 except for the NAFLD activity score.
Body weight changes were analyzed using a general linear model
ANOVA with repeated measures at weekly. The statistical signiﬁcance of differences in body mass, mRNA expression, protein levels
and histological analysis parameters, and T cell populations between groups were determined using 2-way analysis of variance(ANOVA) with diet (ND or HFF) and exercise (control or exercise
training). If signiﬁcant interactions were observed in any of theseanalyses, and comparisons with the Bonferroni correlated
post-hoc test was performed. The NAFLD activity score is a noncontinuous scoring system using 0–8 as possible scores, andnon-parametric tests were employed on NAFLD activity score analysis. The statistical signiﬁcance of differences in the NAFLD activity
score between groups was determined using the Friedman test,
and comparisons with the Steel Dwass post-hoc test were performed subsequently using Ekuseru-Toukei 2010 (Social Survey
Research Information Co. Ltd., Tokyo, Japan). The alpha level was
set at p < 0.05.
3. Results
3.1. Effects of HFF diet and exercise training on characteristic physicalparameters
Fig. 1 illustrates the change in body weight that occurred during
the intervention, using repeated measures ANOVA, and there were934

N. Kawanishi et al. / Brain, Behavior, and Immunity 26 (2012) 931–941

3.3. Effect of HFF diet and exercise training on hepatic inﬂammation

Fig. 1. Body weight change at weekly during intervention. Values representmeans ± SEM. Repeated measures analysis of ANOVA revealed signiﬁcant time,group and time  group effect for body weight gain (boxed text). ⁄⁄p < 0.01.

signiﬁcant time, group and time  group effects on body gain
during the intervention. A statistically signiﬁcant diet  exercise
interaction and exercise effect was not found on body mass at
16 weeks using 2-way ANOVA, but a statistically signiﬁcant effect
was found for diet (F1,30 = 208.67, p < 0.01). A signiﬁcantdiet  exercise interaction was observed for liver mass
(F1,30 = 6.75, p < 0.05). Post-hoc comparisons revealed that HFF
control mice were heavier than ND control and HFF exercise mice(p < 0.01, Table 2). Calorie intake, which was measured in terms ofdiet and water, varied as a main effect of diet (F1,20 = 150.58,
p < 0.01), but the ANOVA did not reveal a statistically signiﬁcantdiet  exercise interaction (Table 2).

3.2. Effect of HFF diet and exercise training on hepatic steatosis
To evaluate hepatic steatosis, we performed Oil-red O staining
and examined hepatic triglyceride content. The Oil-Red O stainingof neutral triglycerides and lipid droplets in the liver (Fig. 2A) was
calculated as the percentage of positive-stained areas per square
millimeter in each section, and ANOVA revealed a signiﬁcantdiet  exercise interaction (F1,28 = 18.9, p < 0.01). The positive area
was signiﬁcantly greater in HFF control mice than in ND control
mice (p < 0.01). However, the positive area in HFF exercise mice
was signiﬁcantly lower than in HFF control mice (p < 0.01,
Fig. 2B). Although the hepatic triglyceride contents varied according to diet (F1,30 = 44.7, p < 0.01) and exercise (F1,30 = 4.08,
p < 0.05), the interaction was not signiﬁcantly different (Fig. 2C).

The TNF-a, which is a marker of hepatic inﬂammation, varied in
the livers as a diet  exercise interaction (F1,30 = 14.64, p < 0.01).
Post-hoc comparisons revealed that the TNF-a levels in both
HFF-control and -exercise groups were higher than in both the
ND control and exercise groups (p < 0.01). In addition, TNF-a wassigniﬁcantly lower in HFF exercise mice than in HFF control mice(p < 0.01, Fig. 3A).
The changes in several cytokine mRNA expression levels areshown in Fig. 3B. The TNF-a mRNA level was not signiﬁcantly affected by exercise and diet  exercise interactions. However, diethad a statistically signiﬁcant effect (F1,30 = 13.29, p < 0.01), andexercise showed a tendency toward having a signiﬁcant effect(F1,30 = 3.56, p = 0.07) on the TNF-a mRNA level. Although dietand/or exercise did not affect IL-1b and IL-6 mRNA levels, themRNA level of IL-10 varied as a diet  exercise interaction(F1,30 = 4.82, p < 0.05). Post-hoc comparisons revealed that the IL10 mRNA level was signiﬁcantly lower in the HFF control mice thanin the ND control mice (p < 0.05). The IL-10 mRNA level was not
signiﬁcantly altered by exercise training (Fig. 3B).
3.4. Effect of HFF diet and exercise training on hepatic injury
We scored NAFLD activity using H&E staining for hepatic injury
assessment. Post-hoc comparisons revealed that the NAFLD activity score, as determined by the degree of steatosis, lobular inﬂammation, and hepatocellular ballooning (Kleiner et al., 2005), wassigniﬁcantly increased in the HFF control mice compared to the
ND control mice (p < 0.01). Compared with HFF control mice, HFF
exercise mice had signiﬁcantly decreased NAFLD activity score(p < 0.01, Fig. 4A and B). In TUNEL-positive cells, a marker of cell
apoptotic death, we found a signiﬁcant diet  exercise interaction(F1,28 = 21.77, p < 0.01). Post-hoc comparisons revealed that the
number of TUNEL-positive cells was signiﬁcantly greater in HFF
control mice compared with ND control mice (p < 0.01). However,the number of positive cells in HFF exercise mice was signiﬁcantly
lower than that in HFF control mice (p < 0.01, Fig. 4A and C). Furthermore, a signiﬁcant diet  exercise interaction in the plasma
ALT level was indicated by ANOVA (F1,30 = 4.91, p < 0.01). The plasma ALT level was signiﬁcantly higher in the HFF control mice compared with the ND control group (p < 0.01). However, the plasma
ALT level in HFF exercise mice was signiﬁcantly lower than thatin HFF control mice (p < 0.05, Fig. 4D).
3.5. Effect of HFF diet and exercise training on hepatic ﬁbrosis
To investigate the effect of exercise training on HFF diet-induced hepatic ﬁbrosis, we examined Sirius red, Masson trichrome,

Table 2The comparison of body mass, fat tissue mass, liver mass, food intake, water intake and calorie intake between normal (ND) and high fat diets and high fructose water (HFF) in
control and exercise trained mice.
ND

Body mass (g)Liver mass (g)Epididymal fat mass (g)Fat mass/body mass (%)
Food intake (mg/weeks)
Water intake (ml/days)
Calorie intake (kcal/weeks)

HFF

Two-way ANOVAControl

ExerciseControl

Exercise

Diet

ExerciseInteraction

33.2 ± 1.4
1.46 ± 0.05
1.22 ± 0.13
3.61 ± 0.30
20.7 ± 1.0
3.19 ± 0.18
78.8 ± 4.0

32.3 ± 1.2
1.38 ± 0.06
1.21 ± 0.15
3.70 ± 0.39
20.7 ± 0.1
3.33 ± 0.33
79.0 ± 0.2

50.5 ± 0.7
3.24 ± 0.19a
1.76 ± 0.06
3.50 ± 0.13
19.00 ± 0.4
3.39 ± 0.22
118.1 ± 2.1

47.8 ± 1.1
2.26 ± 0.09b,c
1.92 ± 0.11
4.09 ± 0.31
19.6 ± 0.6
3.33 ± 0.15
121.8 ± 4.2

p < 0.01
p < 0.01
NSNS
p < 0.01
NS
p < 0.01

NS
p < 0.01
NS
NS
NS
NS
NS

NS
p < 0.05
NS
NS
NS
NS
NS

Data are means ± SEM. NS, not signiﬁcant.
ap < 0.05, different from ND control.
b
p < 0.05, different from ND exercise.
cp < 0.05, different from HFF control.

N. Kawanishi et al. / Brain, Behavior, and Immunity 26 (2012) 931–941

935

Fig. 2. Effect of exercise training on hepatic steatosis in ND- and HFF-fed mice. (A) Oil Red O staining (red; neutral lipids) of liver sections (n = 32). (B) Oil Red O-positive areas,and (C) hepatic triglyceride contents. Values represent means ± SEM. Analyses were performed using 2-way ANOVA for multiple groups (boxed text). NS; not signiﬁcant,⁄⁄p < 0.01, ⁄p < 0.05. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

Fig. 3. Effect of exercise training on hepatic inﬂammation in ND- and HFF-fed mice.
(A) Hepatic TNF-a protein levels and (B) hepatic mRNA levels of TNF-a, IL-1b, IL-6,
and IL-10. Values represent means ± SEM. Analyses were performed using 2-way
ANOVA for multiple groups (boxed text). NS; not signiﬁcant, ⁄⁄p < 0.01, ⁄p < 0.05.

and a-SMA immunohistochemistry strains (Fig. 5A and B), andmeasured the expression of ﬁbrogenic marker genes in the liver(Fig. 5C). Sirius red and Masson trichrome, which were used tostain tissue collagen ﬁber, revealed that the HFF diet caused deposition of collagen ﬁber. However, HFF exercise trained mice
showed a marked improvement in liver histology, as seen by the
reduction in the amount of detected collagen ﬁbers. a-SMA
immunohistochemistry, which was used to stain activated hepatic
stellate cells, revealed the presence of greater numbers of a-SMApositive cells in HFF control mice throughout the lobule in a pericellular distribution. However, the numbers of a-SMA-positive
cells were attenuated by exercise training in HFF mice. In the Sirius
red-positive area, we found a signiﬁcant diet  exercise interaction(F1,28 = 20.98, p < 0.01). Post-hoc comparisons revealed that the Sirius red-positive area was signiﬁcantly greater in HFF control micethan in ND control mice (p < 0.01). However, the Sirius red area in
HFF exercise mice was signiﬁcantly lower than that in HFF control
mice (p < 0.01, Fig. 5B). With respect to mRNA levels of collagen 1aand TIMP1, which are markers of hepatic ﬁbrosis, we found asigniﬁcant diet  exercise interaction (collagen 1a mRNA;
F1,30 = 15.40, p < 0.01, TIMP1 mRNA; F1,30 = 16.24, p < 0.01). Posthoc comparisons revealed that collagen 1a and TIMP1 mRNA levels
were higher in HFF control mice than in ND control mice (collagen1a and TIMP1 mRNA; p < 0.01, respectively), but these mRNAlevels were signiﬁcantly lower in HFF exercise mice than in HFF
control mice (collagen 1a and TIMP1 mRNA; p < 0.01, respectively,
Fig. 5C). The mRNA levels of a-SMA and TGF-b were also signiﬁcantly affected by both diet (a-SMA mRNA; F1,30 = 30.86, p < 0.01,
TGF-b mRNA; F1,30 = 6.74, p < 0.05) and exercise (a-SMA mRNA ;F1,30 = 7.46, p < 0.05, TGF-b mRNA; F1,30 = 4.85, p < 0.05), but adiet  exercise interaction was not observed.
3.6. Effect of HFF diet and exercise training on macrophage inﬁltration
into the liver
Tissue inﬂammation, which is thought to play a very active rolein hepatic injury and ﬁbrosis, was investigated in this study by the936

N. Kawanishi et al. / Brain, Behavior, and Immunity 26 (2012) 931–941

Fig. 4. Effect of exercise training on hepatic injury in ND- and HFF-fed mice. (A) H&E (arrowheads; lobular inﬂammation, and arrows; hepatocyte ballooning) and TUNEL staining(arrows; TUNEL-positive cells) of liver sections, (B) NAFLD activity scores, (C) number of TUNEL-positive cells, and (D) plasma ALT activity. Values represent means ± SEM.
Analyses of differences in TUNEL-positive cells and plasma ALT activity between groups was performed using 2-way ANOVA for multiple groups (boxed text). ⁄⁄p < 0.01, ⁄p < 0.05.

assessment of macrophages in the liver by F4/80 immunohistochemistry staining. While administration of the HFF augmented
the number of macrophage clusters preferentially distributed tocell pericentral region, exercise training reduced this inﬁltration(Fig. 6A). In fact, the number of F4/80-positive cell varied as a signiﬁcant diet  exercise interaction (F1,28 = 12.67, p < 0.01). Posthoc comparisons revealed that the number of F4/80-positive cells
was signiﬁcantly higher in HFF control mice compared with ND
control mice (p < 0.01). However, the positive cells in HFF exercise
mice was signiﬁcantly lower than in HFF control mice (p < 0.01,
Fig. 6B). Similarly, we also found a signiﬁcant diet  exercise interaction respect to the mRNA levels of CD11c and TLR4 (CD11cmRNA; F1,30 = 14.78, p < 0.01, TLR4 mRNA; F1,30 = 8.26, p < 0.01).

Post-hoc comparisons revealed that the mRNA levels of CD11c
and TLR4 were signiﬁcantly increased in the HFF control mice compared to the ND control mice (CD11c mRNA; p < 0.01, TLR4 mRNA;p < 0.05). However, these mRNA were signiﬁcantly lower in HFF
exercise mice than in HFF control mice (CD11c and TLR4 mRNA;p < 0.01, respectively, Fig. 6C).
3.7. Effect of HFF diet and exercise training on inﬁltration of T
lymphocytes
To determine T lymphocyte inﬁltration, we examined the populations of cytotoxic and helper T cells. For populations of CD8+
CD3+ and CD4+ CD3+ cells, which are markers of cytotoxic T cells

N. Kawanishi et al. / Brain, Behavior, and Immunity 26 (2012) 931–941

937

Fig. 5. Effect of exercise training on hepatic ﬁbrosis in ND- and HFF-fed mice. (A) Sirius red (arrows; collagen ﬁber), Masson Trichrome (blue; collagen ﬁber), and a-SMA
immunohistochemistry (brown; a-SMA-positive cells) staining of liver sections. (B) The Sirius red-positive areas, and (C) mRNA expression levels of hepatic ﬁbrogenic
markers. Values represent means ± SEM. Analyses were performed using 2-way ANOVA for multiple groups (boxed text). NS; not signiﬁcant, ⁄⁄p < 0.01, ⁄p < 0.05. (Forinterpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)and helper T cells, 2-way ANOVA did not reveal signiﬁcant effects
of diet and exercise and those interactions (Fig. 7).
3.8. Effect of HFF diet and exercise training on macrophage
chemoattractants
We found a signiﬁcant diet  exercise interaction for hepatic
MCP-1, which is a major macrophage chemokine (F1,30 = 6.00,
p < 0.05). Post-hoc comparisons revealed that MCP-1 was signiﬁcantly higher in the HFF control mice than in the ND control mice(p < 0.01). However, MCP-1 was signiﬁcantly lower in HFF exercise
mice than in HFF control mice (p < 0.05, Fig. 8A). Furthermore, we
observed a signiﬁcant effect of diet (F1,30 = 17.82, p < 0.01), but nota diet  exercise interaction with respect to the MCP-1 mRNA level(Fig. 8B). 2-way ANOVA revealed a statistically signiﬁcantdiet  exercise interaction for the CXCL14 mRNA expression level(F1,30 = 19.13, p < 0.01). Post-hoc comparisons revealed a signiﬁcant difference between the HFF control mice and the HFF exercise
mice (p < 0.01). In contrast, diet, exercise and the interaction of dietand exercise did not affect RANTES mRNA expression (Fig. 8B).

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N. Kawanishi et al. / Brain, Behavior, and Immunity 26 (2012) 931–941

Fig. 6. Effect of exercise training on inﬁltration of macrophages into the liver of ND- and HFF-fed mice. (A) F4/80 immunohistochemistry (brown; F480-positive cells) stainingof liver sections, and (B) number of F4/80-positive cells. (C) Hepatic mRNA levels of CD11c and TLR4. Values represent means ± SEM. Analyses were performed using 2-way
ANOVA for multiple groups (boxed text). ⁄⁄p < 0.01, ⁄p < 0.05. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of thisarticle.)4. Discussion
Although recent studies have shown that forced treadmill andswimming exercise training decreases serum ALT activity and hepatic steatosis in obese mice (Marques et al., 2010; Schultz et al.,2010), it remains unclear whether exercise training attenuates
NASH development. In this study, we evaluated the effect of exercise training on HFF induced steatosis, hepatic injury, inﬂammation, and ﬁbrosis (Kohli et al., 2010) with changes in macrophageinﬁltration. Our results indicated that these parameters are attenuated by exercise training in HFF-induced obese mice.
It has been reported that accumulation of hepatic triglycerides
is causally related to hepatic inﬂammation and ﬁbrosis (Day andJames, 1998). Exercise has the potential to prevent the development of hepatic steatosis, depending on the amount of weight loss
(Promrat et al., 2010). In this study, exercise training induced theloss of liver mass and hepatic triglyceride accumulation in HFF
mice, whereas body mass did not change. Therefore, our results
suggest that exercise training may directly attenuate hepatic steatosis regardless of whether exercise results in weight loss. In addition, our results agree with the ﬁndings of recent studies, whichhave reported that exercise training attenuated hepatic steatosis

in obese mice (Marques et al., 2010; Schultz et al., 2010). Recent
studies have shown that hepatic ceramide plays an important rolein NASH development (Chocian et al., 2010; Alkhouri et al., 2009;
Malhi and Gores, 2008). It will be necessary to examine whether
exercise training reduces ceramide content in the liver duringdiet-induced obesity in mice.
The anti-inﬂammatory properties of exercise have been demonstrated (Gleeson et al., 2011; Pedersen, 2011). Recently, we reported that exercise training attenuates TNF-a mRNA expressionin adipose tissue of high fat-induced obese mice (Kawanishi
et al., 2010). In this study, exercise training decreased hepatic
TNF-a in diet-induced obese mice. Additionally, TNF-a mRNA
expression in the liver was also attenuated by exercise in HFFfed mice. It is well known that the HFF diet leads to the productionof TNF-a and induces hepatic inﬂammation with high expressionof TNF-a mRNA in the liver (Roth et al., 2011; Sohet et al., 2009;
Tetri et al., 2008). Therefore, exercise training may improve hepatic
inﬂammation by inhibiting the HFF diet-induced TNF-a mRNA
expression in obese mice.
TNF-a plays a critical role in the development of NASH by
inducing hepatic injury and ﬁbrosis. Li et al. (2003) reported thatanti-TNF antibody treatment improves fatty liver and hepatic

N. Kawanishi et al. / Brain, Behavior, and Immunity 26 (2012) 931–941

Fig. 7. Effect of exercise training on inﬁltration of T lymphocytes in ND- and HFFfed mice. Populations of CD8+ CD3+ and CD4+ CD3+ cells in hepatic mononuclear
cells were determined using ﬂow cytometry. Values represent means ± SEM.
Analyses were performed using 2-way ANOVA for multiple groups (boxed text).
NS; not signiﬁcant.

Fig. 8. Effect of exercise training on macrophage chemoattractants in ND- and HFFfed mice. (A) Hepatic MCP-1, and (B) mRNA expression levels of hepatic macrophage-speciﬁc chemokines. Values represent means ± SEM. Analyses were performed using 2-way ANOVA for multiple groups (boxed text). NS; not signiﬁcant,⁄⁄p < 0.01, ⁄p < 0.05.

injury in NASH model mice. Furthermore, Tomita et al. (2006) reported that TNF-a receptor deﬁcient mice show lower hepatocyte
apoptosis than wild-type mice. We also observed that exercise
training in obese mice attenuated TUNEL-positive cells in the liver,and reduced ALT activity in plasma, and reduced the TNF-a level inthe liver. Several studies reported that inﬂammatory cytokines like
TNF-a and IL-1b in the liver induce cell apoptosis via activation of
pro-apoptotic proteins in NASH model mice (Zhang et al., 2010;
Miura et al., 2010; Kudo et al., 2009). Furthermore, exercise

939

training decreased collagen deposition along with reduction of
the TGF-b and TIMP1 mRNA expression in the liver, and attenuatedactivated hepatic stellate cells in the present study. Tomita et al.
(2006) reported that TNF-a induces up-regulation of TGF-b and
TIMP1 via activation of hepatic stellate cells, which enhances collagen accumulation. Therefore, exercise training may suppress
TNF-a, which is an important factor for NASH due to HFF-induced
obesity.
Interestingly, it has been shown that Kupffer cells are associatedwith NASH development (Zhang et al., 2010). Recent studies
showed enhanced inﬁltration of CD11b+ and F4/80+ macrophages
in HFF diet-induced obese mice (Kohli et al., 2010; Karlmark
et al., 2009). Kupffer cells produce inﬂammatory cytokines suchas TNF-a, which induce TGF-b and TIMP-1 production in activatedhepatic stellate cells (Tomita et al., 2006). Imamura et al. (2005)
suggested that reducing macrophage inﬁltration into the liver suppresses activation of hepatic stellate cells and hepatic ﬁbrosis.
Therefore, the induction of TNF-a production by hepatic resident
macrophages may play a key role in the pathogenesis of hepatic
inﬂammation and ﬁbrosis. Indeed, this study showed that thealteration in F4/80 positive cells in the liver was similar to the altered pattern of hepatic TNF-a and collagen 1a mRNA levels in theliver. Therefore, suppression of macrophage inﬁltration may be
associated with an improvement of hepatic inﬂammation andﬁbrosis in obese mice. Furthermore, exercise training bothdecreased TNF-a levels and F4/80 positive cells in the liver and decreased the CD11b+ and F4/80+ cell population in the hepaticmononuclear cells of obese mice (data not shown). Therefore, theability of exercise to reduce TNF-a and hepatic ﬁbrosis might becaused by depression of macrophage inﬁltration.
Macrophage activation has been operationally classiﬁed into two
separate polarization states: M1 and M2 (Gordon and Taylor, 2005).
M1 macrophages strongly express TLR4, which produces inﬂammatory cytokines such as TNF-a. In contrast, M2 macrophages produce
IL-10, which suppresses inﬂammation and oxidative stress. Interestingly, mice with high-fat diet-induced obesity show a phenotypic
switching from M2 macrophages to M1 macrophages in adipose tissue, resulting in an induced inﬂammatory state (Lumeng et al.,2007). Interestingly, we previously demonstrated that exercise
training reduced the expression of inﬂammatory cytokines andM1 macrophage markers in the adipose tissue of obese mice (Kawanishi et al., 2010). Accordingly, it is possible that exercise training
attenuates inﬂammation in the liver by both suppressing macrophage inﬁltration and accelerating phenotypic switching from M1
to M2 macrophages in obese mice. M1 macrophages strongly express CCR2, which modulates macrophage inﬁltration to the several
tissues (Murray and Wynn, 2011). In various liver disease models,
CCR2 deﬁcient mice exhibited reduced macrophage inﬁltrationand ﬁbrosis compared to wild-type mice. (Seki et al., 2009; Dambach et al., 2002). Furthermore, Tamura et al. (2010) reported thathigh expression of TNF-a mRNA after the administration of a highfat and sucrose diet was attenuated by CCR2 inhibitor treatment
in obese mice. Obesity-induced M1 macrophage inﬁltration into
the liver is an important factor for TNF-a production and hepaticﬁbrosis. Exercise training attenuated both mRNA expression andcell surface expression of CD11c, which is a marker of M1 macrophages, and did not change CD206 mRNA expression, which is amarker of M2 macrophages in the liver (data not shown).
A recent study proposed that TLR4 plays a pathogenic role in
NASH, because TLR4-knockout mice showed both lower hepatic
inﬂammation and ﬁbrosis than wild-type mice (Seki et al., 2007).
In this study, we observed that exercise training reduced the mRNA
expression of both TLR4 and hepatic TNF-a. Therefore, the exercise
training-induced reduction of TNF-a in the liver might be regulatedby the down-regulation of TLR4. Collectively, our ﬁndings indicatethat exercise training suppresses the inﬁltration of inﬂammatory

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N. Kawanishi et al. / Brain, Behavior, and Immunity 26 (2012) 931–941

macrophages in obese mice and that the reduction in hepatic
inﬂammation, ﬁbrosis, and injury by exercise training is associatedwith suppression of inﬂammatory macrophage inﬁltration.
The factors affecting the inhibition of macrophage inﬁltration
into the liver by exercise training are presently unknown. A recent
study reported that cytotoxic T cells produce RANTES, which modulates the activation and inﬁltration of macrophages in adipose tissue, and CD8a deﬁcient mice also show lower number ofinﬁltrating inﬂammatory macrophages than control mice following administration of a high-fat diet (Nishimura et al., 2009). On
the other hand, helper T cells produce cytokines such as IL-4 andIL-10, which suppress macrophage inﬁltration (Odegaard et al.,2007). In this study, exercise training and HFF did not change thepopulation of cytotoxic T (CD3+ CD8+) cells and helper T (CD3+
CD4+) cells in the liver. Furthermore, exercise training and the
HFF diet did not alter the gene expression of RANTES. Therefore,
cytotoxic T cells and helper T cells might not be associated with
suppression of macrophage inﬁltration. However, helper T cell activation has been operationally deﬁned as several separate polarization states such as regulatory T and Th17 cells, which areassociated with the induction of inﬂammation (Winer et al.,2009). Regulatory T cells have also been shown to inhibit macrophage activation (Tiemessen et al., 2007). In contrast, Th17 cells
produce IL-17, which induces chemokines such as MCP-1 (Parket al., 2005). Therefore, it will be necessary to examine whether
exercise training modulates Th17 and regulatory T cell populationsin the near future.
Macrophage-speciﬁc chemokines such as MCP-1 and CXCL14
are associated with adipose tissue inﬂammation, and mice deﬁcient in these chemokine did not exhibit macrophage inﬁltration
despite their obesity (Kanda et al., 2006; Nara et al., 2007). Interestingly, Baeck et al. (2012) also reported that MCP-1 deﬁcient
mice exhibited reduced macrophage inﬁltration, and hepatic
inﬂammation, injury compared to control mice following administration of a methionine and choline deﬁcient diet. In the present
study, exercise training reduced MCP-1 and CXCL14 mRNA levels.
Therefore, exercise training-induced depression of macrophageinﬁltration results from the suppression of chemokine production
such as MCP-1 and CXCL14.
A limitation of this study was that citrate synthase activity was
not evaluated in relation to lipid metabolism. In addition, exercise
intensity was not based on the aerobic capacity of the animal.
However, this type of exercise training has been demonstrated toincrease maximal oxygen uptake max and citrate synthase activity
in skeletal muscle of mice (Röckl et al., 2007). Because of increased
citrate synthase activity to promote lipolysis, it is considered that
this increase might have effects on the development of fatty liver.
However, Machado et al. (2012) have reported that citrate synthase activity in skeletal muscle does not correlate to fatty liver,
hepatic inﬂammation and ﬁbrosis. On the other hand, it is unclear
whether citrate synthase activity in the liver is associated with thedevelopment of NASH. Therefore, future study is necessary toinvestigate the effect of exercise training on citrate synthase activity in the liver and skeletal muscle. Moreover, future study is necessary to investigate the relationship between aerobic capacity andimprovement of inﬂammation and ﬁbrosis.
Another limitation was that we did not examine the reproducibility of plasma ALT activity, hepatic TNF-a and MCP-1 levels. In
this study, the liver was used in the analysis for a variety of histological, ﬂow cytometric and PCR analyses, etc., and the amount ofliver that could be used for protein extraction was small. In termsof hepatic MCP-1 and TNF levels observed in this study, it was necessary to measure in multiple.
In conclusion, we have demonstrated that exercise training
markedly reduces the hepatic TNF-a level and hepatic ﬁbrosis
markers in the liver. Furthermore, exercise training attenuates

F4/80-positive cells and chemokine expression in the liver. Taken
together, our results suggest that the development of hepatic
inﬂammation, ﬁbrosis and macrophage inﬁltration caused byingestion of high-fat diet and high-fructose water is attenuatedby exercise training.
Information about the contributions of each author
Noriaki Kawanishi designed research, analyzed data (all analysis) or performed statistical analysis, wrote manuscript. Hiromi
Yano analyzed data (histological analysis). Tsubasa Mizokami analyzed data (ﬂow cytometry analysis and histological analysis).
Masaki Takahashi analyzed data (real-time PCR analysis and histological analysis). Eri Oyanagi analyzed data (histological analysis).
Katsuhiko Suzuki had primary responsibility for ﬁnal content.
Acknowledgments
This work was supported by a Grant-in-Aid for the Global COE(Centers of Excellence) Program ‘‘Sport Sciences for the Promotionof Active Life’’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a Grant-in-Aid for the Japan
Society for the Promotion of Science Fellows. We thank the support
staff in the Tissue and Electromagnetic Microscopy Center of the
Kawasaki Medical School.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bbi.2012.04.006.
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