Endurance training but not high-intensity interval training reduces liver carcinogenesis in mice with hepatocellular carcinogen diethylnitrosamine

Journal Pre-proof Endurance training but not high-intensity interval training reduces liver carcinogenesis in mice with hepatocellular carcinogen diethylnitrosamine

Xue Zhang, Lu Cao, Benlong Ji, Lingxia Li, Zhengtang Qi, Shuzhe Ding PII:

S0531-5565(19)30645-X

DOI:

https://doi.org/10.1016/j.exger.2020.110853

Reference:

EXG 110853

To appear in:

Experimental Gerontology

Received date:

20 September 2019

Revised date:

21 January 2020

Accepted date:

21 January 2020

Please cite this article as: X. Zhang, L. Cao, B. Ji, et al., Endurance training but not high-intensity interval training reduces liver carcinogenesis in mice with hepatocellular carcinogen diethylnitrosamine, Experimental Gerontology(2018), https://doi.org/10.1016/ j.exger.2020.110853

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© 2018 Published by Elsevier.

Journal Pre-proof Endurance training but not high-intensity interval training reduces liver carcinogenesis in mice with hepatocellular carcinogen diethylntrosamine

Xue Zhang 1,2, 3 §, Lu Cao 1,2, Benlong Ji 1,2, Lingxia Li 1,2, Zhengtang Qi 1,2 §*, Shuzhe Ding 1,2*

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The Key Laboratory of Adolescent Health Assessment and Exercise Intervention (Ministry of Education), East China Normal University, Shanghai 200241, China 2

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School of Physical Education and Health, East China Normal University, Shanghai 200241, China Xuhui Campus, Shanghai University of Sports, Shanghai 200237, China

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* Corresponding author. Address: School of Physical Education and Health, East China Normal University, Shanghai 200241, China. Tel: +86-21-54345296 E-mail addresses: [email protected] (Z.Q.), [email protected] (S.D.)

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§ Theses authors contributed equally to this work.

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Running title: ET but not HIT reduces liver carcinogenesis

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Journal Pre-proof Abstract:

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Physical activity may reduce cancer initiation. High-intensity interval training (HIT) has been reported to be superior to moderate continuous endurance training (ET) for maximizing health outcomes in cardiovascular disease, obesity and type 2 diabetes. However, the role of HIT vs. ET in the prevention of liver cancer is poorly understood. This study aimed to determine how HIT vs. ET affects cancer initiation in mice with the hepatocellular carcinogen diethylntrosamine (DEN). C57BL/6 mice were treated with DEN at 3~12 weeks of age and, from 8 to 26 weeks of age, treated with either of exercise modes on treadmill: HIT (85~90% VO2max with intervals) and ET (65~75% VO2max without intervals). We found that mice treated with ET had lower cancer initiation but higher fat mass compared to control DEN-injected mice. In contrast, HIT could not significantly reduce cancer initiation and tumor volumes. Metabolomic analysis in the liver indicated marked differences in cholesterol, palmitic acid, stearic acid, uracil, hydroxypyridine and maltose between HIT- and ET-treated mice, and demonstrated good and obvious separation between ET and DEN control group. Furthermore, mice treated with ET had lower expression of pro-inflammatory cytokines and pro-proliferation genes in liver compared to DEN control group. ET protocol reduced the accumulation of toxic metabolite carbamate, increased the protein level of caspase-1, and reduced JNK phosphorylation in liver. These data indicates that moderate-intensity endurance training may be superior to high-intensity interval training for reducing liver cancer initiation in mice.

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Keywords: liver carcinogenesis; endurance training; high-intensity interval training; inflammation; diethylntrosamine

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Journal Pre-proof 1. Introduction

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Epidemiological studies show that physical activity is inversely associated with cancer initiation and sedentary behaviour is positively associated with tumorigenesis (Kerr et al., 2017). Exercise-mediated changes in body composition (Wall et al., 2017), metabolites (Zhao et al., 2019), systemic inflammation, and immune cell function (Hojman, 2017), were thought to be a possible mechanism for cancer treatment. In addition, chronic exercise prevents muscle wasting in cancer cachexia and thus improving muscle strength, physical function and quality of life (Gould et al., 2013). On the other hand, the metabolic abnormalities and inflammation that accompany cancer support tumor development (Vander and DeBerardinis, 2017). Aerobic exercise mitigated tumor growth and related disorders in rats by improving insulin sensibility, lowering glucose and insulin levels and/or reducing insulin secretion (Moreira et al., 2018). Tumors also disrupt antigen presentation by T-cell or NK-cell activation (Melero et al., 2014). Natural killer (NK) cells are a group of cytotoxic lymphocytes, capable to recognize and eliminate tumor cells in adaptive anti-tumour immunity (Afolabi et al., 2019). Exercise exerts anti-inflammatory effects to control the tumor growth of cancer through intramural NK cell infiltration (Pedersen et al., 2016). In previous studies, endurance and aerobic exercise was used to treat tumor-bearing animals. A smaller number of studies demonstrated that high-intensity training (such as 85% VO2max) increased life span and promoted a reduction of tumor mass in tumor-bearing rats (Bacurau et al., 2007).

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Primary liver cancer model is commonly used in mice involving in an intraperitoneal injection of pro-carcinogen diethylnitrosamine (DEN) in neonates. DEN in liver is converted to an active carcinogen that causes DNA alkylation and oxidative damage, leading to development of hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC), resembling poor-prognosis HCC in humans (Santos et al., 2012). In this study, we investigated the effects of exercise mode on liver cancer initiation in DEN-treated mice: high-intensity interval training (HIT, 85~90%VO2max with intervals) and moderate-intensity endurance training (ET, 65~75%VO2max without intervals) (Schefer and Talan, 1996). HIT protocol was reported to reduce cancer initiation and enhance metabolic health in breast cancer patients (Barra et al., 2017); however, it is not well established in HCC. Here, we aimed to determine whether and how HIT, compared to ET, reduces cancer initiation in mice with DEN-induced HCC. In this study, our findings indicate that endurance exercise protocol, but not HIT, contributes to reducing liver cancer initiation in mice treated with DEN. Biological mechanisms, including body composition, metabolomics, inflammatory response, and etc., may explain why HIT cannot reduce liver cancer initiation. High-intensity exercise was shown to be beneficial to health; however, it has to be applied carefully with an individualized prescription of exercise for HCC.

2. Materials and methods 2.1 Mice and DEN treatment 3

Journal Pre-proof Animal breeding and experiments were reviewed and approved by the governmental committee for animal experimentation in East China Normal University, China. Male C57BL/6 mice were purchased from Shanghai SLAC laboratory Animal Co., Ltd (SLAC). Mice were housed and bred in a temperature-controlled room (22℃) on a 12h light/dark cycle in cages and with ad libitum access to food and water. Mice were treated with DEN (25mg/kg) at 21days of age (once a week, 10 weeks) and randomly allocated to DEN, HIT, and ET groups. Negative control (NC) group was treated with saline as negative control. All mice were fed a standard chow diet and sacrificed on 26 weeks of age. 2.2 Exercise intervention

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From 8 weeks of age, mice were assigned to two experimental training protocol and exercise groups were familiarized with a treadmill running for a week. After this period, mice performed HIT or ET exercise protocol 5 days/week for 18 weeks. HIT protocol was performed on a treadmill using 10 running bouts of a 2-minute sprint (25m/min, 85~90%VO2max) interspersed with a 2-minute rest. ET protocol was performed on a treadmill using continuous running at 13m/min (65~75% VO2max) for 40 min once a day. Oxygen consumption at different speeds was calculated by the formula as reported previously (Schefer and Talan, 1996). Mice assigned to NC and DEN group were sedentary and housed in cages.

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2.3 Insulin sensitivity and glucose tolerance testing

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Mice were deprived of food for 16 h and then subjected to glucose or insulin tolerance test. Blood was collected from a small incision in the tip of the tail (time 0) and then 15, 30, 45, 60, 90 and 120 min after an i.p. injection of glucose (1g/kg body weight) or insulin (0.75 U/kg body weight). Blood glucose levels were measured with a blood glucometer (Accu-Check® Active, Roche). 2.4 Tissue collection and tumor analysis Mice were euthanized at 26 weeks of age in the random-fed state. Tumor multiplicity, which represents the number of surface-hemorrhaging tumors per liver, was counted. The diameter of visible tumor (>0.5 mm in diameter) was used to calculate tumor volume. Individual tumor volumes were summed to calculate total liver carcinogenesis per animal. The large lobe of the liver was kept for histology, and the remaining liver was frozen in liquid nitrogen, and stored at -80℃ until further biochemical analyses. The weights of both epididymal and inguinal fat pads were summed and represented as combined adipose weight per animal. 2.5 Liver histology The large lobe of the liver was extracted, further fixed with 4% paraformaldehyde and then embedded with paraffin. Tissue sections were stained with haematoxylin and eosin (H&E) or with Sirius Red. In addition, a part of liver tissues was also embedded in OCT compound and were snap-frozen followed by staining sections with Oil Red O. Slides were digitally scanned using an Aperio ScanScope System to produce 4

Journal Pre-proof high-resolution images. 2.6 Body composition and colorimetric analysis

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Body composition was measured using NMR(MesoMR23-060H-I, NIUMAG). Serum glucose, T-CHO, TG, glycerol (Nanjing Jianchen Bio, China) and cytokines IL-1β and TNF-α (Shanghai Enzyme-linked Biotechnology, China) were measured following the manufacturers’ instructions. For cytoplasmic and mitochondrial isolation, tissue was homogenized using a glass dounce homogenizer in ice cold isolation buffer (210mM sucrose, 660mM mannitol, 30mM KH2PO4, 15mM MgCl2-6H2O, 3mM EGTA and 75mM MOPS). Homogenization, as well as the following steps, was performed at 4℃ as described previously (Frezza et al., 2007). Finally, protein concentration was measured by Bradford method. For isolated fractions, GPT, GOT, MDA and NADPH were measured using a kit according to the manufacturer’s protocol (Nanjing Jianchen Bio, China). 2.7 Quantitative real-time RT-PCR.

2.8 Western blotting

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RNA was extracted from liver tissues using Trizol (Invitrogen, Singapore) and reverse-transcribed with the ReverTra Ace® qPCR RT Kit (FSQ-101; TOYOBO, Osaka, Japan). cDNA was amplified by real-time PCR in a total reaction volume of 20μl using SYBR Green Realtime PCR Master Mix (QPK-201; TOYOBO, Osaka, Japan). Real-time PCR reactions were cycled in StepOne™ Real-Time PCR System (Applied Biosystems, CA, USA). Primer sets are identified in supplementary Table S2. Target gene expression was normalized to endogenous GAPDH and expressed as 2-ΔΔct relative to the control group.

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Liver tissue was homogenized in RIPA buffer (10 mM Tris pH 8.0, 0.5 mM EGTA, 1% Triton X-100, 0.2% SDS, 100 mM NaCl) with protease inhibitors cocktail and phosphatase inhibitors (Shanghai Beyotime, China). Protein content was quantified using bicinchoninic acid reagents and BSA standards. Proteins were separated by SDS-PAGE and transferred to PVDF membrane and incubated with the primary antibody and horseradish peroxidase-coupled anti-species antibodies. Proteins were visualized by enhanced chemiluminescence. Antibodies used for immunoblotting were: p-AMPKα, AMPK(Cell Signaling 9957S), ERK1/2(abcam ab196883), p-ERK1/2(abcam ab214362), p-JNK1/2/3(abcam ab124956)), TBK1(abcam ab40676), Caspase-1(abcam ab14367) and GAPDH(Santa Cruz 365062). 2.9 Metabolic Profiling GC/MS-based metabolic profiling was performed on the liver tissue extracts with established methods. Liver tissue extracts were derivatized with BSTFA before instrumental analysis. Briefly, 100 μl of 2-chlorophenylalanine (0.3 g/L) served as internal standard. 300 μl of chloroform:methanol (1:3) was added into each sample. The mixture was homogenized for 60 s, followed by incubation at -20°C for 10 min. The samples were then centrifuged for at 12000 × g and 4°C for 15 min. 200 μl of the 5

Journal Pre-proof supernatant was collected individually from each sample into an ampoule bottle and evaporated to dryness under a stream of nitrogen for 2 hrs. 80 μl of a methoxyamine pyridine solution (15 g/L) was subsequently added into the ampoule bottle. The mixture was vortexed for 2 min and incubated at 37°C for 120 min. Then, 80 μl of bis-(trimethylsilyl)-trifluoroacetamide(BSTFA) plus 1% trimethylchlorosilane(TMCS) was added, and the mixture was again vortexed for 30s and incubated at 70°C for 90min. BSTFA with 1% TMCS was purchased from Sigma-Aldrich (St. Louis, MO, USA). Each reaction sample was performed in duplicates. The GC/MS analysis was performed on an Agilent 6980 GC system equipped with a fused-silica capillary column (internal diameter: 30 m × 0.25 mm) and a 0.25-μm HP-5MS stationary phase (Agilent, Shanghai, China). Statistical analyses

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3. Results

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Group results are presented as mean ± SEM and compared by ANOVA followed by Fisher’s PLSD post-hoc test. Significance was accepted at p<0.05. Statistical analyses were performed using GraphPad Prism 6.0. Metabolomic data was analyzed using principle component analysis (PCA) and orthogonal projection to latent structures (OPLS) analysis between groups by SIMCA 15.0. The differential metabolites were selected when they meet the requirements of variable importance in the projection (VIP>1) in OPLS model and p<0.05 from student t-test.

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3.1 Tumor incidence and liver histology

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Fig. 1A illustrates the study design. In brief, C57BL/6 mice were injected with DEN (25 mg/kg i.p.) at 21days of age. Mice were weaned at the same time and randomized to cages with non-littermates to avoid litter bias. From 8 to 26 weeks of age, mice with DEN treatment run on the treadmill according to HIT or ET protocol and were euthanized at 26 weeks of age. We observed that tumor incidence was similar between DEN and HIT; however, it was significantly smaller in ET compared to DEN and HIT (Fig. 1C), and the results of maximal diameters of tumors per mouse showed the same change trends (Fig.1B, P<0.05, F(3,28)=2.275). The mouse with the median cancer initiation for each group is shown as a representative image in Fig.1D. Almost two thirds of DEN-treated mice developed macroscopically identifiable tumors by 23 weeks after DEN administration (Fig.1D, first row), concordant with previous studies (Healy et al., 2015). H&E-stained tumour tissue sections revealed that HCC phenotype was identified from DEN and HIT group; however, it was not significant in ET group (Fig. 1D, second row). In agreement with the H&E-stained sections, the analysis of Sirius red-stained sections revealed that DEN/HIT treatment led to a significant increase in collagen fraction compared to NC group, whereas collagen content was reduced in ET group (Fig. 1D, third row). These findings presented extensive liver cirrhosis with DEN-induced HCC as demonstrated previously (Romualdo et al., 2017). Together, we found marked reductions in tumor incidence 6

Journal Pre-proof and growth, liver fibrosis with moderate-intensity endurance training rather than highintensity interval training. 3.2 Body composition and hepatic lipotoxicity

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Here, MRI scanning analysis showed that DEN treatment significantly reduced fat percentage in all tumorigenic groups compared to NC that did not receive DEN (Fig. 2A, P<0.0001, F(3,28)=4.275). Comparing only DEN-injected mice, ET significantly increased fat percentage (Fig. 2A, P=0.0006, F(3,28)= 3.594), but HIT led to a rise without statistical significance in fat percentage. There were no differences in lean body mass between NC and DEN, but compared to NC, the mice treated with HIT and ET had greater lean body mass (Fig. 2B, HIT: P=0.0075, F(3,28)= 2.749, ET: P=0.0032, F(3,28)= 3.048). Our data suggest a positive effect of exercise on skeletal muscle mass even in the treatment of DEN. Unlike previous concepts in exercise effects on obesity and metabolic diseases, we found that endurance exercise, as well as HIT, increased overall fat percentage in fact. We further observed a quantity of oil-red-O-stained lipid droplets in DEN and HIT group, but it decreased obviously in ET group (Fig.2C). Metabolomic analysis indicated that cholesterol and palmitic acid are common differential metabolites among DEN, HIT and ET, and reduced in ET compared to DEN and HIT (Fig.2E & Table S1). Serum glycerol and inflammatory cytokines have been known as a biomarker of adipose tissue atrophy and fat loss with cancer (Ebadi and Mazurak, 2015). Compared to DEN and HIT, ET elevated serum glycerol (Fig.2D, vs. DEN: P=0.0046, F(3,28)= 1.871, vs. HIT: P=0.0052, F(3,28)= 2.994), and reduced hepatic expression of IL-6 (P=0.022, F(3,28)= 2.323), IL-1b (P=0.013, F(3,28)= 2.537), and IFN-b (Fig.4A, P< 0.0001, F(3,28)= 4.527). Fig. 2F illustrates changes in epididymal, inguinal, and scapular fat pads. Our results indicate that, compared to HIT, ET protocol prevented carcinoma-associated fat loss but reduced DEN-induced hepatic lipotoxicity. These findings suggest that endurance exercise does not always induce fat loss, depending on what fat means to the body and health. 3.3 Glucose homeostasis and AMPK phosphorylation in liver At 12 weeks of age, blood glucose levels were measured in overnight-fasted mice after injection glucose (1g/kg body wt i.p.) (Fig. 3A). There was no difference in glucose tolerance between NC and DEN, but mice in HIT displayed marked glucose intolerance compared to ET (Fig. 3A, P=0.044, F(3,28)= 2.12). At 14 weeks of age, serum glucose levels were measured in 6 hours-fasted mice after injection insulin (0.75U/kg body wt i.p.) (Fig. 3B). There were no differences in insulin tolerance between groups (Fig. 3B). Until sacrifice, there were no differences in serum glucose levels between groups, but compared to NC, mice in HIT and ET had greater serum insulin (Fig. 3C, HIT: P=0.0006, F(3,28)=3.82, ET: P=0.0013, F(3,28)=3.512), whereas mice treated with DEN had a trend for higher insulin levels (Fig. 3C). Consistent with previous study, liver cancer initiation was not independently associated with fasting glucose, insulin, whole-body insulin and glucose tolerance (Healy et al., 2015). 7

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To reveal the effects of exercise on metabolic flux, we investigated hepatic metabolic changes induced by DEN in combination with/without exercise in mice using GC/MS-based metabonomics. We found that ET protocol enhanced hepatic levels of glutamic acid (P=0.0046, F(3,28)=3.363) and malonic acid (P=0.0073, F(3,28) = 3.298), and decreased that of galactinol (P=0.036, F(3,28)=2.329) and carbamate (P = 0.0016, F(3,28)=3.875) compared to DEN (Fig. 3E). Compared to HIT, mice with ET had a lower level of stearic acid and maltose in liver (Table S1). To further examine the underlying mechanism for hepatic metabolic changes, we performed Western blot analysis of AMPK in liver. AMP-activated protein kinase (AMPK) is known to be phosphorylated and activated in liver tissue of mice treated with DEN (Shimizu et al., 2011). Here, we observed that AMPK was hyper-phosphorylated and protein level of AMPK beta1/2 was elevated in liver after DEN treatment (Fig. 3D). Both of exercise protocols, which were known to stimulate AMPK phosphorylation, reduced AMPK phosphorylation in liver under DEN treatment (Fig. 3D). However, we did not detect any exercise-specific differences in AMPK phosphorylation and protein expression (Fig. 3D). These data indicate that liver tumorgenesis is not associated with glucose homeostasis, but associated with higher AMPK phosphorylation.

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3.4 Inflammation and innate immune response in liver

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To examine whether exercise-specific inflammation was associated with liver carcinogenesis, we measured mRNA expression of IL-6, IL-1β, IFN-β, and TNFα as markers of inflammation in liver. Although neither exercise protocol significantly reduced liver TNFα expression (Fig. 4A), there was a significant reduction in the expression of IL-6 (P=0.0221, F(3,28)=2.323), IL-1β (P=0.0127, F(3,28)=2.537), IFN-β (P0.0001, F(3,28)=4.527) in ET mice compared to DEN（Fig. 4A）. In contrast, HIT could not reduce the expression of those cytokines in liver (Fig. 4A). To identify deferentially regulated pathways in HCC from running mice, qPCR analysis was performed to detect markers of cellular innate and adaptive immune systems in liver tissue (Fig.4B). Compared to DEN, ET increased NLPR3 expression (P0.0001, F(3,28)=6.785) and decreased AIM2 expression (P=0.0008, F(3,28)=3.470)（Fig.4B）. There were significant differences in NLRP3 (P0.0001, F(3,28)=4.256) and TLR9 expression (P=0.0213, F(3,28)=2.34) between HIT and ET (Fig. 4B). To test the F4/80 expression in macrophage, immunohistochemistry was performed (Fig.4 D&E). F4/80 was decreased significantly in ET compared HIT (Fig. 4 D&E, P=0.0108, F(3,28)=2.661). We next examined the expression of proteins about inflammation, mice treated with DEN had a higher level of TBK1 proteins compared to NC, it decreased when exercise treatment (Fig. 4F). These data indicate that ET protocol has more effects on anti-inflammation and innate immune response in HCC than HIT. 3.5 Cell proliferation and apoptosis in liver Fig. 4C illustrated that ET significantly reduced the mRNA expression of cell proliferation markers including ERK (P=0.0162, F(3,28)=2.440), CSF-1 (P=0.0085, F(3,28)=2.677), STK4 (P=0.0003, F(3,28)=3.698), and cyclinD1 (CCDN1, P=0.0001, F(3,28)=3.950). Of those proliferation markers, CSF-1 (P=0.0043, F(3,28)=2.909), 8

Journal Pre-proof STK4 (P=0.0006, F(3,28)=3.548), and cyclinD1 (P=0.0009, F(3,28)=3.401) were significantly upregulated by DEN treatment only (Fig. 4C). Compared to NC, mice treated with DEN had a higher level of JNK and ERK phosphorylation, but no changes in Caspase-1 protein expression (Fig. 4F). Compared to DEN, mice treated with exercise had a higher level of ERK phosphorylation (Fig.4F). It is noteworthy that HIT further enhanced JNK phosphorylation, and yet ET increased Caspase-1 protein expression (Fig.4F). These results indicate that ET could be more inclined to suppress cell cycle progress and prevent cell proliferation in HCC than HIT. 3.6 Liver metabolomics analysis

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To explore the biomarker that positively correspond with exercise protocols, GC− TOFMS metabolomics analyses was used to identify exercise-induced alterations in liver of mouse with DEN treatment. After data normalization (280 metabolites), PCA was performed on the dataset, which showed a trend of intergroup separation on the scores plot (Supplementary Fig.1A). The data were interrogated using an OPLS-DA model which established intergroup separation between groups (Supplementary Fig.1 B&C). Supplementary Table S1 provided a list of identified discriminant metabolites with the VIP, a measure of their relative influence on the model, along with the fold changes. The discriminant metabolites which account for the intergroup separation were identified by OPLS-DA modols between DEN and ET (R2X(cum) = 0.576, R2Y(cum)= 0.698, Q2(cum)= -0.021), and between HIT and ET (R2X(cum) = 0.468, R2Y(cum)=0.643, Q2(cum)=-0.199). OPLS-DA of liver metabolomic profiles showed that ET is able to reduce the levels of galactinol, timonacic, glycine, cholesterol, carbamate, palmitic acid in liver, and increase the levels of malonic acid, ornithine, glutamic acid. In addition, OPLS-DA analysis suggested that ET levels of cholesterol, palmitic acid, uracil, hydroxypyridine, maltose, and stearic acid were all statistically significantly depressed below HIT levels (Table S1). The data between DEN and HIT was not fitted in the OPLS-DA model, suggesting that there were no discriminant metabolites between DEN and HIT.

4. Discussion

The present study highlights the protective role of endurance exercise with moderate intensity, but not HIT with high-intensity, reduces liver carcinogenesis in mice with DEN. The major finding of the present study regards the specificity of endurance exercise versus HIT to counteract DEN-induced alterations of body composition, inflammation, cell proliferation, metabolomics in tumor-bearing mice, but HIT has no such effects. This study provides strong evidence that the combination of the volume and intensity of exercise is more significant for tumor growth than exercise itself. Recent studies raised up the benefits of HIT and provided practical advices for successful clinical and home-based HIT against cardiovascular diseases, obesity, overweight and diabetes (Karlsen et al., 2017). However, the benefits of HIT have not previously been evaluated in a mouse model of liver cancer. By comparing these 9

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exercise protocols, we observed that fat loss can be linked to primary liver tumor. For example, mice treated with DEN had high cancer initiation and marked lipoatrophy. In addition, mice treated with ET protocol were fatter but had low cancer initiation compared to mice with DEN. It is notable that the DEN-treated mice with ET gained increased fat mass, but did not gain significantly more body mass compared to DEN. Fat loss is a feature of cancer cachexia and also associated with shorter survival and reduced quality of life (Ebadi and Mazurak, 2015). In previous studies, exercisetreated mice have a well-controlled increase in body mass and a reduction in fat percentage (Kelly et al., 2011). In this study, the reason for the fat loss is related to the fact that mice treated with DEN had high cancer initiation and adipose atrophy (Ebadi and Mazurak, 2015). Because ET protocol in our study reduced liver carcinogenesis effectively compared to HIT, the DEN-treated mice with ET did not gain cachexiaassociated fat loss. Our findings demonstrate that ET can prevent tumor development with adipose atrophy.

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Intraperitoneal injections with DEN in rats develops a HCC model starting from liver inflammation, cirrhosis, to HCC stage, thus, hepatic steatosis was considered as ‘bystander phenomenon’ subsequent to inflammatory attacks (Farazi and DePinho, 2006). The accumulation of free fatty acids (FFAs) may lead to the formation of ROS and TNFα, which can induce further liver damage and worsening of inflammation (Tilg and Moschen, 2010). In our study, the OPLS-DA models derived from metabolomic analysis demonstrate good separation between ET and DEN (Fig. S1). Discriminant metabolites about lipid metabolism were palmitic acid, cholesterol and palmitic acid (Table S1). Palmitic acid (PA) is a representative long-chain saturated FFA that is usually observed with an elevation in obesity and in insulin resistant patients (Bigornia et al., 2016) PA increased the expression of the pro-inflammatory cytokines IL-6, TNFα and decreased the mRNA levels of the anti-inflammatory cytokine IL-10 and adiponectin in adipocytes (Palomer et al., 2018). Metabolized FFAs (such as PA) in the hepatocytes lead to the release of triacylglycerol, which is causally related to hepatic inflammation and fibrosis. In this study, endurance training significantly decreased hepatic palmitic acid, cholesterol and glycerol content. In agreement with our results, the previous study showed that exercise training induced the loss of hepatic triglyceride accumulation and reduced hepatic steatosis (Kawanishi et al., 2012). Moreover, monounsaturated PA was identified a biomarker for HCC progression and poorer patient survival (Beyoğlu et al., 2013; Budhu et al., 2013). Thus, the oncogenic effect of HCC is mediated at least by inducing the production of palmitic acid (Xu et al., 2017). Taking together, fat-associated- inflammation may play a crucial role in HCC. Numerous studies have linked the pro-inflammatory cytokines IL-6, IL-1β, and TNFα to liver cancer. Liver inflammation accompanies steatosis in the development of liver cancer in humans (Hashimoto et al., 2009). IL-6 knockout mice were resistant to DEN-induced tumor formation (Naugler et al.). The anti-inflammatory properties of exercise have been demonstrated (Gleeson et al., 2011), but the impact of short-term HIT and ET results in differential effects on anti-inflammatory cytokine function 10

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(Barry et al., 2018). A critical question we sought to address was whether exercise mode (ET) prevents the development of tumor or reduces pro-inflammatory cytokines. Indeed, we found support for this moderate-intensity continuous exercise, which resulted in lower levels of IL-6, IL-1β and IFNβ expression compared to the non-exercise control group (DEN), but HIT did not reduce the expression of these pro-inflammatory cytokines, consistent with previous findings in the use of exercise as medicine for depression (Paolucci et al., 2018). Moreover, previous studies found that regular moderate-intensity exercise and long-term resistance training both decrease IL-1β level in old adults (BEAVERS et al., 2010; Forti et al., 2016). Although NF-κB was thought a key factor linking inflammation to cancer (Ben-Neriah and Karin, 2011), the significant changes in TNFα between groups were not observed in our study. It reported that high serum glycine levels associated with advanced cancer stage and with poor cancer-specific survival (Sirnio et al., 2019). However, some research indicated that glycine can be used for treatment of inflammation and chemoprevention of carcinoma (Li et al., 2014; Yamashina et al., 2007). In this study, the level of glycine in liver decreased after moderate-intensity exercise. These results suggest that moderate-intensity exercise may be an optimal intensity of exercise to reduce DEN- induced liver inflammation by decreasing IL-6, IL-1β、IFNβ and clycine. This may be due to the higher level of physical stress evoked by the HIT exercise protocol.

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In addition, inflammatory responses are frequently associated with mitogen-activated protein (MAP) kinases, including the cJun NH2-terminal kinase (JNK) signaling pathway that is activated by inflammatory cytokines, endotoxin, and physicalchemical stress (Davis, 2000). It is established that JNK plays a key role in the development of hepatitis and HCC by using JNK-deficient mice. Therefore, JNK inhibition represents a potential mechanism for decreasing hepatic inflammation and preventing hepatitis and HCC (Han et al., 2016). In the present study, we observed that continuous endurance training reduced JNK phosphorylation in liver (Fig. 4E). In contrast, HIT significantly enhanced JNK phosphorylation. It has been reported that reduced JNK signaling decreased expression of the M1-associated pro-inflammatory cytokines IL-6, IL-1β, and TNFα (Han et al., 2013). Furthermore, the roles of JNK in cell proliferation, survival, apoptosis, and necrosis have been reported (Weston and Davis, 2006). Next-generation genome sequencing of human liver cancers has shown massive gene amplification and rearrangements frequently targeting direct regulators of JNK, and JNK mediated cellular proliferation and oncogenic transformation (Iannelli et al., 2014; Schulze et al., 2015). Besides, in present study, we observed that ET protocol increased the protein expression of caspase-1 and reduced the mRNA expression of CCDN (Cyclin D), compared to DEN and HIT. Together, these data support a possible scenario that ET protocol, unlike HIT, leads to a reduction in JNK phosphorylation that consequently inhibits inflammation and/or prevents cell cycle progression at the level of transcription in DEN-induced HCC model. In conclusion, this study demonstrates the powerful influence of moderate-intensity continuous exercise training (ET) on primary liver cancer growth and progression. In 11

Journal Pre-proof contrast, high-intensity interval training (HIT) has no similar anti-tumor effects in the DEN model of murine liver cancer. Compared to HIT, ET exercise protocol prevents carcinogenesis-associated fat loss and HCC-associated metabolic phenotype, and exhibits a greater anti-inflammatory, anti-proliferation effect in liver. If so, these data would provide pre-clinical evidence to support moderate intensity endurance exercise, but not high-intensity interval training, as an optimal adjuvant therapy for patients with primary liver cancer.

Acknowledgments

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We thank Dr. Liu Yumin and Wu Jieli (Shanghai Jiaotong University) for advice and help with body composition analysis and GC-TOFMS metabolomic profiling. Financial support

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This project received financial support from the National Natural Science Foundation of China (Grant No. 31671241, S.Z.), Shanghai Pujiang Program (15PJ032, Z.Q.), and the Key Laboratory Construction Project of Adolescent Health Assessment and Exercise Intervention of Ministry of Education, China (No. 40500-541235- 14203/ 004).

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Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

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Ethics approval and consent to participate

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The experimental procedures and the animal use and care protocols were approved by the Committee on Ethical Use of Animals of East China Normal University（NO. m20170202）. Authors contributions

Z.Q. and S.D. designed the study and reviewed submission. X.Z., L.C, B.J, and L.L performed experiments. X.Z., and Z.Q. performed data statistics, wrote and edited the manuscript with input from other authors. X.Z., and Z.Q. contributed equally to this work.

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Journal Pre-proof Abbreviations:

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DEN, diethylnitrosamine; HCA, hepatocellular adenoma; HCC, hepatocellular carcinoma; IL-1, interleukin-1β; IL-6, interleukin-6; TNF-, tumor necrosis factor-; IFN-,interferon-; NLRP3, NOD-like receptor family pyrin domain containing 3; TLR9, toll like receptor 9; AIM2, absent in melanoma 2; CSF1, colony stimulating factor 1; MAPK, mitogen-activated protein kinase; ERK, extracellular regulated MAP kinase; JNK, c-Jun NH2-terminal kinase; STK4, serine/threonine kinase 4; CCDN1, cyclin D1; SOD1, superoxide dismutase 1; PTGS2, prostaglandin-endoperoxide synthase 2; GPX4, glutathione peroxidase 4; AMPK, AMP-activated protein kinase; PCA, principle component analysis; OPLS-DA, orthogonal projection to latent structure-discriminant analysis; GC−TOFMS, gas chromatography- time of flight mass spectrometry, ; NMR, nuclear magnetic resonance.

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Journal Pre-proof Figure legend Fig.1 Endurance exercise but not HIT reduces liver tumor incidence in mice treated with DEN. (A) Diagram of study design. (B). Maximal diameters of tumors per mouse. (C) Tumor incidence. (D) Representative macroscopic images (first row), HE-stained (second row) and Sirius Red-stained (third row) liver sections. Data in bar graphs and line graphs presented as mean ± SEM. * Indicates a significant difference, p <0.05 (n = 8).

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Fig.2 Endurance exercise but not HIT reverses DEN-induced fat loss and decreases hepatic steatosis. (A) Fat percentage & (B) lean body mass were measured by NMR. (C) Oil red-stained liver sections. (D) Serum T-CHO, glycerol and TG. (E) Liver cholesterol and palmitic acid. Box plots of discriminant metabolites in groups by PCA and OPLS-DA models of GC−TOFMS spectral data. Ordinates are relative concentrations (peak area/internal standard peak area/mg tissue). (F) Weights of inguinal, epididymal adipose tissue and brown adipose tissue. Data in bar graphs presented as mean ± SEM. *p<0.05, **p <0.01, ***p <0.001, ****p <0.0001, indicates a significant difference (n = 8).

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Fig.3 Endurance exercise suppressed DEN-induced AMPK activation in the liver and had no effects on glucose homeostasis. Integrated area under the curve (AUC) for glucose (A), insulin (B) tolerance and blood glucose levels in mice over time. (C) Serum glucose and insulin. (D) AMPK protein expression in liver. Protein expression was measured in at least 4 independent mice for each group, one representative set is shown. (E) Glutamic acid, ornithine and malonic acid in liver. Box plots of discriminant metabolites in groups by PCA and OPLS-DA models of GC−TOFMS spectral data. Ordinates are relative concentrations (peak area/internal standard peak area/mg tissue). Data in bar graphs and line graphs presented as mean ± SEM. *p<0.05, **p <0.01, ***p <0.001, indicates a significant difference (n = 8). Fig.4 Endurance exercise reduces hepatic gene expression for pro-inflammatory cytokines and cell proliferation. (A) qPCR analysis of inflammatory cytokines and (B) immune cell markers, and(C) proliferation markers (n=8). (D) F4/80 image analysis of immunohistochemistry. (E) Representative macroscopic images of immunohistochemistry. (F) Liver protein expression. Protein expression was measured in at least 4 independent mice for each group, one representative set is shown. Data in bar graphs presented as mean ± SEM. *p<0.05, **p <0.01, ***p <0.001, indicates a significant difference (n = 8).

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Highlights:

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Endurance exercise but not HIT reduces liver tumor incidence in mice treated with DEN. Endurance exercise but not HIT reverses DEN-induced fat loss. Endurance exercise but not HIT enhances hepatic levels of glutamic acid, ornithine and malonic acid. Endurance exercise but not HIT reduces hepatic gene expression for pro-inflammatory cytokines and cell proliferation. Endurance exercise but not HIT increases hepatic gene expression for antioxidative ability.

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