A high-fat diet temporarily renders Sod1-deficient mice resistant to an oxidative insult

    A high fat diet temporarily renders Sod1-deficient mice resistant to an oxidative insult Junitsu Ito, Naoki Ishii, Ryusuke Akihara, Jaeyong Lee, Toshihiro Kurahashi, Takujiro Homma, Ryo Kawasaki, Junichi Fujii PII: DOI: Reference:

S0955-2863(16)30652-0 doi: 10.1016/j.jnutbio.2016.10.018 JNB 7677

To appear in:

The Journal of Nutritional Biochemistry

Received date: Revised date: Accepted date:

27 March 2016 18 October 2016 18 October 2016

Please cite this article as: Ito Junitsu, Ishii Naoki, Akihara Ryusuke, Lee Jaeyong, Kurahashi Toshihiro, Homma Takujiro, Kawasaki Ryo, Fujii Junichi, A high fat diet temporarily renders Sod1-deficient mice resistant to an oxidative insult, The Journal of Nutritional Biochemistry (2016), doi: 10.1016/j.jnutbio.2016.10.018

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ACCEPTED MANUSCRIPT A high fat diet temporarily renders Sod1-deficient mice resistant to an

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oxidative insult

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Takujiro Homma1, Ryo Kawasaki2, and Junichi Fujii1,*

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Junitsu Ito1,a, Naoki Ishii1, Ryusuke Akihara1, Jaeyong Lee1, Toshihiro Kurahashi1,b,

Department of Biochemistry and Molecular Biology, Graduate School of Medical

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Science, Yamagata University, 2-2-2 Iidanishi, Yamagata 990-9585, Japan Department of Public Health, Yamagata University Graduate School of Medical

Science, 2-2-2 Iidanishi, Yamagata 990-9585, Japan

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Corresponding author: Junichi Fujii, Ph D

Department of Biochemistry and Molecular Biology, Graduate School of Medical

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Science, Yamagata University, 2-2-2 Iidanishi, Yamagata 990-9585, Japan

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e-mail: [email protected]

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Tel: +81-23-628-5229, Fax: +81-23-628-5230

Running title: Stored lipids protects against oxidative stress

Footnotes a

Present address: Department of Internal Medicine, Yamagata Prefectural Kahoku

Hospital, 111 Aza-Gassando Yachi, Kahoku-chyo, Yamagata 999-3511 Japan b

Present address: Department of Pathology and Cell Regulation, Graduate School of

Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan

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Financial support

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This study was supported by the Takeda Science Foundation

Key words: liver steatosis, high fat diet, lipophagy, oxidative stress, superoxide

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dismutase

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ACCEPTED MANUSCRIPT ABSTRACT Patients with non-alcoholic fatty liver disease (NAFLD) may subsequently develop

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non-alcoholic steatohepatitis (NASH) after suffering from a second insult, such as

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oxidative stress. Aim of this study was to investigate the pathogenesis of the liver injury caused when lipids accumulate under conditions of intrinsic oxidative stress using mice

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that are deficient in superoxide dismutase 1 (SOD1) and the leptin receptor (Lepr). We established Sod1−/−::Leprdb/db mice and carried out analyses of four groups of

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genetically modified mice, namely, wild type, Sod1−/−, Leprdb/db, and Sod1−/−::Leprdb/db mice. Mice with defects in the SOD1 or Lepr gene are vulnerable to developing fatty livers, even when fed a normal diet. Feeding a high-fat diet (HFD) caused an increase in

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the number of lipid droplets in the liver to different extents in each genotypic mouse. A HFD caused the accelerated death of db/db mice, but, contradictory to our expectations,

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the death rates for the Sod1-deficient mice were decreased by feeding HFD. Consistent with the improved probability of survival, liver damage was significantly ameliorated

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by feeding a HFD compared to a normal diet in the mice with an Sod1-deficient

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background. Oxidative stress markers, hyperoxidized peroxiredoxin and lipid peroxidation products, were decreased somewhat in Sod1−/− mice by feeding HFD. We conclude that lipids reacted with reactive oxygen species and eliminated them in the livers of the young mice, which resulted in the alleviation of oxidative stress, but in advanced age oxidized products accumulated, leading to the aggravation of the liver injury and an increase in fatality rate.

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ACCEPTED MANUSCRIPT 1. Introduction Non-alcoholic fatty liver disease (NAFLD) is a liver injury that is associated with

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obesity, diabetes, dyslipidemia, and the metabolic syndrome, and in some instances, can

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degenerate to non-alcoholic steatohepatitis (NASH), a condition associated with insulin resistance and fibrotic liver damage [1]. Leptin is a humoral factor that controls the

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desire for food intake, and hence a dysfunction of the leptin system typically results in obesity under free feeding situations [2]. The db/db mouse that has a defect in the

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receptor for leptin is hyperleptinemic and is prone to developing obesity as the result of excessive food intake. A high fat-containing diet (HFD) is commonly administered to investigate the pathogenesis of NAFLD. Feeding a HFD alone results in fat deposition but usually does not proceed to the development of NASH in rodents, implying that an

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additional factor, a so called “second hit”, is required for the development of NASH [3].

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For this instance, a specialized diet with containing special ingredients, such as a methionine-choline-deficient diet and a high-fructose diet, is generally applied to induce

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NASH in a model animal [4, 5].

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Oxidative stress, characterized by elevated levels of reactive oxygen species (ROS) compared to antioxidant levels, is an established causal factor for the exacerbation of liver damage and likely functions as the second hit for NASH development [3,6]. Notably, polyunsaturated lipids are prone to peroxidation that triggers radical chain reactions, resulting in the production of toxic aldehydic derivatives such as malonedialdehyde [7]. Machineries that act against ROS consist of antioxidative enzymes and low molecular antioxidants such as glutathione and vitamins C and E. Among the various antioxidative enzymes, superoxide dismutase (SOD) converts superoxide, a primary ROS, into the less toxic hydrogen peroxide [8]. Three isozymes are translated from genes in mammals and are uniquely localized in the 4

ACCEPTED MANUSCRIPT cytosolic/mitochondrial intermembrane space (SOD1), the mitochondrial matrix (SOD2), and the extracellular space (SOD3). A deficiency in SOD1 leads to elevated

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oxidative stress and the shortening of the life span of mice with increasing incidences of

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abnormalities in lipid metabolism in the liver [9-11] and hepatocarcinogenesis [12-14] and the intestinal epithelium [15].

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It has been reported that an increased ratio of saturated-to-unsaturated fatty acids is related to the progression to NASH in which stress associated with the endoplasmic

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reticulum (ER) appears to play a role [16]. Moreover, a key target of ROS from the view point of liver steatosis also appears to be the ER, the organelle where the oxidative folding of nascent secretory proteins occurs [17]. Very low density lipoprotein (VLDL)

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and chylomicrons are secreted from the liver and intestinal epithelia, respectively. These triglyceride-rich lipoproteins are formed via the oxidative folding of apoprotein B

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followed by accepting lipid droplets carried by the microsomal transfer protein (MTP) in the ER [18, 19]. Hence, a malfunction of the ER would be expected to impair the

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delivery of cellular triglycerides (TG) to the blood, resulting in the deposition of lipid

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droplets in the liver or enterocytes [20]. On another front, upon ER stress, sterol regulatory element-binding proteins (SREBPs), which regulate genes that are involved in steroidogenesis and lipogenesis, are proteolytically activated by the site-1 protease (S1P) and the site-2 protease (S2P) that are localized on Golgi membranes and which stimulate lipid synthesis [21, 22]. When hepatocytes are isolated from the liver and cultured, they are exposed to stronger conditions of oxidative stress than in an in vivo situation, since they are exposed to atmospheric oxygen. As a result, aberrant proteins with non-natural disulfide bridges accumulate, thus triggering ER stress and simultaneously lipogenesis [23]. Based on this scenario, lipid droplets would be formed more extensively in Sod1-deficient 5

ACCEPTED MANUSCRIPT hepatocytes than in ordinary hepatocytes under primary culture conditions [24]. In this study we established a strain of mice with a double deficiency of the leptin

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system and an Sod1 with the expectation that NAFLD would degenerate to NASH by

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the simple administration of a HFD. The results indicated that the double deficient mice showed fibrotic changes to only a limited extent compared to WT mice and most of the

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mice died within one year. Unexpectedly, the administration of a HFD temporarily extended the lifespan of the mice with an Sod1-deficient background while most of the

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db/db mice died early. Based on the ROS-scavenging function of deposited lipids in the liver, we propose a tentative mechanism that explains this complicated response to the

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feeding of a HFD.

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ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. Animals

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C57BL/6 Sod1−/− mice under C57BL/6 background were used as described previously

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[15, 25]. Six female Lepr+/db mice that were purchased from SLC Japan were mated with male Sod1−/− mice resulting in Sod1+/−::Lepr+/db mice. By intercrossing the

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Sod1+/−; Lepr+/db mice and screening by means of a PCR-based genotypic analysis [25, 26], we established four genetic mice groups; wild-type, Sod1−/−, Leprdb/db, and

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Sod1−/−::Leprdb/db mice. After weaning, the male mice were either fed a normal diet (ND; 4.5 kcal%, Picolab Rodent Diet 20, LabDiet) or a high fat diet (HFD; 45 kcal%, D12451, Research Diets) (Supplementary Table 1). The four genetic groups of the mice

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were bred and subjected to observation for up to 52 w. Thus the experiment consisted of 8 groups regarding the genotype and the nutritional conditions. In addition, mice from

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each genetic group fed either a ND or a HFD were sacrificed at the indicated ages, and subjected to autopsy and histological and biochemical analyses. The animal room was

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maintained under specific pathogen-free conditions at a constant temperature of 20–22

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ºC with a 12-hr alternating light-dark cycle. Animal experiments were performed in accordance with the Declaration of Helsinki under the protocol approved by the Animal Research Committee at our institution.

2.2. Blood test After 8 hr of fasting, blood samples were collected directly from the hearts of randomized mice from all 8 experimental groups at 12 or 28 weeks in the absence of an anticoagulant, kept at room temperature for 1 hr, and centrifuged for 15 min at 3,000 rpm in a microcentrifuge. The resulting serum samples were either stored at -80 ºC or used immediately in assays. The levels of TG, alanine aminotransferase (ALT), fasting 7

ACCEPTED MANUSCRIPT blood glucose (FBG), total cholesterol (tCHO), and blood urea nitrogen (BUN) in

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serum were determined using Fuji Dri-chem slides on Fuji Dri-chem 3500V (Fuji film).

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2.3. Histological analyses of the liver

After dissection, the livers of randomized mice from all 8 experimental groups were

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divided and stored either at -80 ºC for protein analyses or fixed in 15% buffered formalin. The samples in formalin fixation were embedded in paraffin. Liver sections (4

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m thick) were subjected to hematoxylin and eosin (H&E) staining. Elastica-Masson (EM) staining was performed for evaluating hepatic fibrosis.

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2.4. Assay for serum insulin

Serum insulin of randomized mice from all 8 experimental groups was assayed using a

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mouse insulin ELISA kit (AKRIN-011T, Shibayagi, Gunma, Japan) according to the manufacture’s protocol. A 10 µl aliquot of serum was plated on an anti-insulin

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monoclonal antibody-coated 96 well plate together with a biotin-bound anti-insulin

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antibody. After incubation with peroxidase-conjugated avidin followed by the chromogenic reaction, the absorbance at 450 nm was measured in a microspectrophotometer (Benchmark Plus, BIRAD). HOMA-IR was obtained by calculation using the following equation that is applicable for humans: HOMA-IR = FBG (mg/dl) x blood insulin concentration (ng/ml) x 26/405.

2.5. Assay for lipid peroxidation products Thiobarbituric acid-reactive substances (TBARS) in the livers of randomized mice from all 8 experimental groups were determined, as described previously [25]. In a typical experiment, about 0.2 g of liver tissue was homogenized in 9 vol. of PBS followed by 8

ACCEPTED MANUSCRIPT centrifugation (9000  g). An aliquot of the supernatant was reacted with thiobarbituric acid. The solution was heated for 1 hr in a heat block, cooled in ice-cold water. After

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the addition of n-butanol (500 µl), the reaction solution was vigorously mixed and

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centrifuged at 300  g for 10 min. The absorbance of 150 µl of the n-butanol fraction was measured at 553 nm in a Valioskan Flash microplate reader (Thermo Fisher

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Scientific). TBARS levels were calculated using an extinction coefficient of 1.56 x 105

2.6. Assay of TG in the liver

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M−1 cm−1.

Liver tissue (< 100 mg) of randomized mice from all 8 experimental groups was

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homogenized using a Physcotron in 10 vol. of PBS. After adequate mixing of 200 l of homogenate with 800 l of chloroform:methanol (2:1, vol/vol), the suspension was

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centrifuged at 700  g for 10 min at room temperature. The lower organic phase was

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transferred to a new tube and the upper layer was extracted with chloroform a second time. The collected organic phases were dried by means of a centrifugal concentrator

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under vacuum and dissolved in 200 l of ethanol containing 1% Triton X-100. The concentrations of TG were measured using a LabAssayTM Triglycride kit (290-637001, Wako).

2.7. Immunoblot analysis of proteins Liver samples of randomized mice from all 8 experimental groups were homogenized in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% (w/v) Nonidet P-40, 0.5% (w/v) Deoxycholate, 0.1% SDS) containing 50 mM NaF, 2.5 mM Na-pyrophosphate, 2 mM sodium orthovanadate, 25 mM -glycerophosphate, 40 M p-aminophenylsulfenyl

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ACCEPTED MANUSCRIPT fluoride, and a protease inhibitor cocktail (169-26063, Wako) and centrifuged at 15,000 rpm for 10 min at 4 ˚C. The protein content in the supernatant was determined using a

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BCA kit (Pierce) followed by immunoblot analyses, as described previously [15]. The

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antibodies used were; homemade (SOD1), hyperoxidized peroxiredoxin (Prx-SO2/3) (ab16830, Abcam), Total OXPHOS Rodent WB Antibody Cocktail (ab110413, Abcam),

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and -actin (sc-69879, Santa Cruz). After incubation with horseradish peroxidase-conjugated 2nd antibodies (Santa Cruz), the bands were detected using

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Immobilon western chemiluminescent HRP substrate (Millipore) on an image analyzer (ImageQuant LAS500, GE Healthcare).

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2.8. Statistical analysis

The results are expressed as the mean ± S.E. For Kaplan-Meier analysis, a log-rank test

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was performed on all 8 experimental groups of mice. To determine the interaction between diet (ND vs. HFD) and genotypes on body weight, we used a linear

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mixed-effects model with restricted maximum likelihood. We performed Kaplan-Meier

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analysis with a log-rank test, and used both logistic regression models and the Cox proportional hazard model for mortality by diet, genotype and the interaction between the diets and genotype. Statistical analyses for other data were performed using two-way ANOVA, followed by the Tukey-Kramer test for multiple groups. A P-value of less than 0.05 was considered to be significant.

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ACCEPTED MANUSCRIPT 3. Results

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3.1. Properties of Lepr- and/or Sod1-deficient mice fed a high fat-containing diet

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To investigate the coordinate function of hyperlipidemia and oxidative stress in the pathogenesis of the liver, we established 4 genetic groups of male mice; wild-type

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(WT), Sod1−/− (KO), Leprdb/db (db), and Sod1−/−;;Leprdb/db (KO::db) mice with simple designations based on their genotypes in parenthesis. After weaning, they were fed

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either a normal diet (ND) or a high fat diet (HFD) (Table 1), and subjected to long-term surveillance for up to one year (Fig. 1). Because HFD is much higher in calories than ND, the total intake was less in the HFD groups under conditions of free-feeding.

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Across the 4 genotypes (WT, db, KO, KO::db), those on the HFD had a significantly higher body weight than those on ND (P< 0.001). Generally, db and KO:db had

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significantly higher body weight compared to WT (both P<0.001); KO had significantly lower body weight compared to WT (P=0.022). Although there was no significant

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interaction between diets (ND vs. HFD) and db (P=0.818), there were significant

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interactions resulting in the suppression of body weight gain between diet and KO (P=0.024) and diet and KO:db (P=0.043). The mortality of the mice were observed until the mice reached the age of 52 w (Fig. 2). We used the logistic regression model to estimate the odds ratios for cumulative mortality over 48 weeks. KO and KO::db had significantly higher odds for mortality (OR 9.78 and 11.1, respectively); however, there were no significant interactions observed. Then we used the Cox Proportional hazards model to estimate hazard ratios for diets (ND vs. HFD), genotypes, and the interaction between the diets and the genotypes. There were no significant interactions between the diet and genotypes of mice. 11

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3.2. Effects of HFD on the liver

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The liver weight (Supplementary Fig. 1) and ratios of liver/body weights were

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higher in the db and KO::db groups compared to the corresponding WT group (Figs. 3A and 3B). The HFD resulted in a substantial increase in the visceral fat content in both

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the WT and KO mice at 12 w (Fig. 3C), although the differences were less evident at 28 w (Fig. 3D). We performed H&E staining of liver sections at 12 w (data not shown) and

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at 28 w (Fig. 4) to assess the accumulation of lipid droplets and EM staining to diagnose hepatic fibrosis. Lipid droplet-derived vacuoles were evident in the mice with the db background with a characteristic expansion of them to the whole liver. The vacuoles

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were restricted to the perivenular area in the KO mice and their size increased slightly as the result of feeding the HFD. While the sizes of lipid droplets were greatly enlarged

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in the mice with the db background, only a slight expansion of lipid droplets was observed in the KO mice at 28 w. Contrary to our expectation, the fibrous tissue area

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was not expanded in any of the mice livers, even at 28 w of age.

3.3. Evaluation of liver conditions based on blood test Serum TG contents were higher in the mice with db background but their increases were rather suppressed by feeding HFD notably at 28 w (Figs. 5A and 5B). Total cholesterol (tCHO) levels were originally high in mice with the db background and were elevated in the WT, KO, and db mice as the result of feeding the HFD although their difference were small at 28 w (Figs. 5C and 5D). Serum ALT levels were about 10-times higher in KO mice fed with ND but again suppressed by feeding HFD (Figs. 5E and 5F). Mice with db background also showed higher ALT levels and further elevation by feeding HFD. 12

ACCEPTED MANUSCRIPT Fasting blood glucose (FBG) levels were lower in the KO mice than the WT mice and returned to the normal level by feeding HFD (Figs. 6A and 6B). FBG values were

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originally very high in the mice with the db background, although a slight elevation was

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observed in KO::db mice as the result of feeding the HFD (Fig. 6B). To make diagnoses if the mice were insulin resistant or not, we measured serum insulin levels at 12 w (Fig.

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6C). The mice with the db background showed high levels of insulin independent from HFD. HOMA-IR also showed similar profile (Fig. 6D), indicating that severe insulin

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resistance was characteristically observed in the mice with db background.

3.4. TG contents and oxidative damage in the liver

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We measured the TG contents of the livers at 12 w and 28 w (Figs. 7A and 7B). To our surprise, the TG contents were elevated only in the db mouse group and not

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significantly elevated by feeding the HFD at these periods in all of the genotypic mice, although vacuole sizes were markedly expanded in the mice, particularly in the

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HFD-fed mice with the db background (Fig. 4). Total bilirubin contents in the blood

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were also slightly lower in KO and db mice groups (Figs. 7C and 7D), consistent with a partial improvement of liver function. The levels of lipid peroxidation products were somewhat lower in the HFD-fed mice compared to the ND-fed mice, although a significant difference was observed only in the KO::db mice (Fig. 7E). When mitochondrial respiratory chain complexes were examined, there no major difference was detected among the mice groups (Supplementary Fig. 2). We then examined a marker for protein oxidation, hyperoxidized peroxiredoxin (Prx-SO2/3) [27], along with the Prx family proteins Prx 1 to Prx4 (Fig. 8). Several bands were detected using the Prx-SO2/3 antibody, suggesting that hyperoxidation occurred in different Prx isoforms, depending on the nutritional conditions and ages of the mice. The levels of 13

ACCEPTED MANUSCRIPT Prx-SO2/3 were high in the ND-fed KO mice at both 12 w and 28 w, but were low in the HFD-fed mice. In the KO::db mice, the levels of Prx-SO2/3 were slightly higher than the

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corresponding values for the WT or db mice, but lower than that for the KO mice and

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were further suppressed by feeding HFD. These collective data indicate that a HFD suppresses the oxidative stress caused by a Sod1 deficiency, leading to a reduction in

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the level of oxidative hepatic damage.

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ACCEPTED MANUSCRIPT 4. Discussion The novel findings of this study are that feeding a HFD reduced liver injuries to

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some extent, in the mice with intrinsic oxidative stress caused by a Sod1 deficiency.

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While feeding an HFD increased the ratio of visceral fat to body weight in the WT mice and KO mice, they were originally very high in the db mice and KO::db mice as

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the result of feeding a ND and these values remained essentially unchanged when a HFD was fed (Fig. 3). While the death rates in the mice with a db background

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increased as the result of feeding a HFD as reported [28], the death rates of the KO mice were somewhat decreased by feeding a HFD (Fig. 2). In case of KO::db mice, the effects of feeding a HFD were complicated, showing characteristics of both the KO

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mice and the db mice; i.e. more mice survived by feeding a HFD in the young grown-up period, but they suddenly died in the advanced period. The double deficient

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mice, most of which died within 50 w, did not show expanded fibrotic lesions (Fig. 4), which is a typical pathological characteristic of NASH [1].

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Analyses of serum and the liver sections at 12 w or 28 w indicated that feeding a

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HFD indeed increased lipid droplet-derived vacuoles in all of the genotypic groups of mice to different extents (Fig. 4). Feeding a HFD aggravated liver damage in the mice with a db background, but in the KO mice, it was suppressed at the young grown-up period (Fig. 5E). Oxidative stress markers, lipid peroxidation products and Prx-SO2/3, were originally high in the KO mice but declined by feeding a HFD (Figs. 7E and 8). Based on these data, we speculate that, excessive lipids that are transiently stored may play protective roles against an oxidative insult but prolonged accumulation eventually results in hepatocyte damage. The production and secretion of triglyceride-rich lipoproteins are impaired by an oxidative insult in the liver and enterocytes [10, 15], which can explain the low 15

ACCEPTED MANUSCRIPT visceral fat levels in the KO mice. ER stress following oxidative damage tends to stimulate lipogenesis by activating fatty acid synthesis and cholesterol synthesis under

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normal feeding conditions [17]. Consistent with these in vivo observations, we

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previously reported that the accumulation of lipid droplets is stimulated to a substantial extent in Sod1-deficient hepatocytes under primary culture conditions due to the

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development of severe oxidative stress triggered by the presence of atmospheric oxygen [24].

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The genes required for fatty acid synthesis and cholesterol synthesis are up-regulated by SREBP1 and SREBP2, respectively [22]. These transcriptional factors undergo proteolytic activation via the specific proteases S1P and S2P, which are

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localized in Golgi membranes. The activation of SREBP1 and SREBP2 is regulated not only by a cholesterol deficiency but also by ER stress [21]. Oxidative stress causes

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ER stress by triggering the accumulation of unfolded proteins, which results in the translocation of SREBPs to the Golgi membrane where S1P and S2P are located and

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the activation of these transcriptional regulatory factors for lipogenesis [20]. Oxidative

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stress also impairs the transfer of lipid droplets to ApoB by MTP and, hence, suppresses the secretion of lipoproteins, which leads to lipid droplets accumulating inside the cells. Thus, oxidative stress would cause an accelerated accumulation of lipid droplets both by activation of the de novo synthesis of fatty acids under normal feeding conditions and by suppressing lipoprotein secretion [15]. Oxidative stress impaired their secretion from enterocytes and hepatocytes, which would result in the accumulation of relatively low levels of lipids in the visceral fat of the KO mice. Consistent with this assumption, serum TG levels were actually lower in the KO mice compared to the other groups (Figs. 5A and 5B). Regarding the mechanism responsible for the improved liver function and 16

ACCEPTED MANUSCRIPT decreased death rates of the KO mice by feeding a HFD, the suppression of ROS toxicity in the liver appears to contribute to the reduced level of liver damage because

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lipid peroxidation products and Prx-SO2/3 were both suppressed in the KO mice as the

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result of feeding a HFD (Figs. 7E and 8). Polyunsaturated fatty acids (PUFA), including essential fatty acids, are preferred targets of ROS [7] and their depletion is associated

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with the development of certain diseases such as NASH [29]. The intake of long-chain omega-3 fatty acids exerts beneficial effects on reducing the effects of NAFLD [30].

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The HFD used in this study was not rich in PUFA but the HFD-fed mice were able to ingest more PUFA than the ND-fed mice. A slight consumption of PUFA by reaction with ROS would not be expected to cause serious damage to the liver. Instead,

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accumulated lipids, notably PUFA, probably scavenge ROS, leading to the protection of other biological molecules, such as the ER membrane and DNA, against oxidative

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damage by ROS. Thus, transiently accumulated TG may exert a protective effect against ROS, especially in animals that are under oxidative stress like Sod1-deficient

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mice. Among the various sources, high levels of ROS are produced by the oxygenation

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of xenobiotics via reaction with cytochrome P450 (CYP) [31]. While an excessive intake of acetaminophen causes hepatic injury, feeding mice a HFD has been reported to result in a protective effect against hepatotoxicity caused by an overdose of acetaminophen [32]. Similarly, a hepatic injury triggered by the administration of thioacetamide is also protected by feeding mice a HFD [33]. These reports suggest that accumulated lipid droplets also protect the liver against metabolically produced ROS during the metabolism of xenobiotics. The metabolic degradation of TG in lipid droplets begins with the decomposition of the lipid droplet by autophagy or more specifically lipophagy [34, 35]. Beta oxidation of the released fatty acids follows in mitochondria. Lipid metabolism plays a 17

ACCEPTED MANUSCRIPT major role in supplying energy to certain organs, such as the heart, under fasting conditions but appears to be impaired in the case of KO mice as abnormally high levels

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of lipid droplets accumulate in the livers of Sod1-deficient mice [36]. Therefore, TG

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derived from a HFD also fuel such organs, which may consequently contribute to the decreased death rates of the KO mice.

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In conclusion, the long-term feeding of a HFD causes NAFLD and impaired liver function, but lipid droplets that accumulate, at least temporarily, in the liver, may have a

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beneficial function by way of antioxidation in certain circumstances. This protective action of lipid droplets may rationalize the lipogenesis that is stimulated under oxidative stress [20, 37]. Although a Sod1 deficiency is an extreme case with severe oxidative

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stress and not applicable directly to patients, lipid droplets that transiently accumulate in the liver may exert a protective effect against oxidative stress, which is induced under

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various situations, such as inflammation, drug metabolism, and virus infections.

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Author contribution

JI designed the research study and performed most of the experiments on the mice. NI

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and RA performed triglyceride assays and protein assays. JL and TK partly helped in caring for the mice and biochemical analyses of blood. TH and RK performed statistical analyses of the data and aided in preparing the figures and the manuscript. JF advised the experimental planning, interpreted the data, and wrote the paper.

Conflict of interest The authors declared no conflicts of interest.

Acknowledgement

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ACCEPTED MANUSCRIPT We thank Ms. Junko Takeda for technical assistance in preparing liver sections.

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References

[1] Brown GT, Kleiner DE. Histopathology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Metabolism. 2016 doi: 10.1016/j.metabol.2015.11.008.

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[2] Hamann A, Matthaei S. Regulation of energy balance by leptin. Exp Clin Endocrinol Diabetes. 1996; 104: 293-300.

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[3] Serviddio G, Bellanti F, Vendemiale G. Free radical biology for medicine: Learning from nonalcoholic fatty liver disease. Free Radic Biol Med. 2013; 65: 952–968. [4] Nabeshima A, Yamada S, Guo X, Tanimoto A, Wang KY, Shimajiri S, Kimura S, Tasaki T, Noguchi H, Kitada S, Watanabe T, Fujii J, Kohno K, Sasaguri Y. Peroxiredoxin 4 Protects Against Nonalcoholic Steatohepatitis and Type 2 Diabetes in a Nongenetic Mouse Model. Antioxid. Redox Signal, 2013; 19: 1983-1998.

PT

ED

[5] Sanches SC, Ramalho LN, Augusto MJ, da Silva DM, Ramalho FS. Nonalcoholic Steatohepatitis: A Search for Factual Animal Models. Biomed Res Int. 2015; 574832. doi: 10.1155/2015/574832.

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[6] Cave M, Deaciuc I, Mendez C, Song Z, Joshi-Barve S, Barve S, McClain C. Nonalcoholic fatty liver disease: predisposing factors and the role of nutrition. J Nutr Biochem. 2007;18:184-195.

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[7] Niki E. Role of vitamin E as a lipid-soluble peroxyl radical scavenger: in vitro and in vivo evidence. Free Radic Biol Med. 2014; 66: 3-12. [8] Fridovich I. Superoxide radical and superoxide dismutases. Annu Rev Biochem. 1995; 64: 97–112. [9] Hadjur S, Ung K, Wadsworth L, Dimmick J, Rajcan-Separovic E, Scott RW, Buchwald M, Jirik FR. Defective hematopoiesis and hepatic steatosis in mice with combined deficiencies of the genes encoding Fancc and Cu/Zn superoxide dismutase. Blood. 2001; 98；1003-1011. [10] Uchiyama S, Shimizu T, Shirasawa T. CuZn-SOD deficiency causes ApoB degradation and induces hepatic lipid accumulation by impaired lipoprotein secretion in mice. J Biol Chem. 2006; 281: 31713–31719. [11] Wang L, Jiang Z, Lei XG. Knockout of SOD1 alters murine hepatic glycolysis, gluconeogenesis, and lipogenesis. Free Radic Biol Med. 2012; 53: 1689–1696.

19

ACCEPTED MANUSCRIPT [12] Elchuri S, Oberley TD, Qi W, Eisenstein RS, Jackson Roberts L, Van Remmen H, Epstein CJ, Huang TT. CuZnSOD deficiency leads to persistent and widespread oxidative damage and hepatocarcinogenesis later in life. Oncogene. 2005; 24: 367-380.

RI P

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[13] Busuttil RA, Garcia AM, Cabrera C, Rodriguez A, Suh Y, Kim WH, Huang TT, Vijg J. Organ-specific increase in mutation accumulation and apoptosis rate in CuZn-superoxide dismutase-deficient mice. Cancer Res. 2005; 65: 11271-11275.

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[14] Elchuri S, Naeemuddin M, Sharpe O, Robinson WH, Huang TT. Identification of biomarkers associated with the development of hepatocellular carcinoma in CuZn superoxide dismutase deficient mice. Proteomics. 2007; 7: 2121-2129.

MA NU

[15] Kurahashi T, Konno T, Otsuki N, Kwon M, Tsunoda S, Ito J, Fujii J. A malfunction in triglyceride transfer from the intracellular lipid pool to apoB in enterocytes of SOD1-deficient mice. FEBS Lett. 2012; 586: 4289–4295. [16] Gentile CL, Pagliassotti MJ. The role of fatty acids in the development and progression of nonalcoholic fatty liver disease. J Nutr Biochem. 2008; 19: 567-576.

ED

[17] Gentile CL, Frye M, Pagliassotti MJ. Endoplasmic reticulum stress and the unfolded protein response in nonalcoholic fatty liver disease. Antioxid Redox Signal. 2011; 15: 505-521.

PT

[18] Ginsberg HN, Fisher EA. The ever-expanding role of degradation in the regulation of apolipoprotein B metabolism. J Lipid Res. 2009; 50(Suppl): S162-S166.

CE

[19] Xiao C, Hsieh J, Adeli K, Lewis GF. Gut-liver interaction in triglyceride-rich lipoprotein metabolism. Am J Physiol Endocrinol Metab. 2011; 301: E429-E446.

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[20] Malhotra JD, Kaufman RJ. Endoplasmic reticulum stress and oxidative stress: a vicious cycle or a double-edged sword? Antioxid. Redox Signal. 2007; 9: 2277–2293. [21] Ye J, Rawson RB, Komuro R, Chen X, Davé UP, Prywes R, Brown MS, Goldstein JL. ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Mol. Cell 2000; 6: 1355–1364. [22] Goldstein JL, DeBose-Boyd RA, Brown MS. Protein sensors for membrane sterols. Cell. 2006; 124: 35-46. [23] Fang DL, Wan Y, Shen W, Cao J, Sun ZX, Yu HH, Zhang Q, Cheng WH, Chen J, Ning, B. Endoplasmic reticulum stress leads to lipid accumulation through upregulation of SREBP-1c in normal hepatic and hepatoma cells. Mol Cell Biochem 2013; 381: 127– 137. [24] Lee J, Homma T, Kurahashi T, Kang ES, Fujii J. Oxidative stress triggers lipid droplet accumulation in primary cultured hepatocytes by activating fatty acid synthesis. Biochem. Biophys. Res. Commun., 2015; 464: 229-235.

20

ACCEPTED MANUSCRIPT [25] Iuchi Y, Okada F, Onuma K, Onoda T, Asao H, Kobayashi M, Fujii J. Elevated oxidative stress in erythrocytes due to a SOD1 deficiency causes anaemia and triggers autoantibody production. Biochem J. 2007; 402: 219–227.

RI P

T

[26] Suemizu H, Ohnishi Y, Maruyama C, Tamaoki N. Two-color allele-specific polymerase chain reaction (PCR-SSP) assay of the leptin receptor gene (Leprdb) for genotyping mouse diabetes mutation. Exp Anim. 2001;50: 435-439.

SC

[27] Rhee SG, Woo HA, Kil IS, Bae SH. Peroxiredoxin functions as a peroxidase and a regulator and sensor of local peroxides. J Biol Chem. 2012; 287: 4403–4410.

MA NU

[28] Sataranatarajan K, Ikeno Y, Bokov A, Feliers D, Yalamanchili H, Lee HJ, Mariappan MM, Tabatabai-Mir H, Diaz V, Prasad S, Javors MA, Ghosh Choudhury G, Hubbard GB, Barnes JL, Richardson A, Kasinath BS. Rapamycin Increases Mortality in db/db Mice, a Mouse Model of Type 2 Diabetes. J Gerontol A Biol Sci Med Sci. 2015 pii: glv170. [29] Videla LA, Rodrigo R, Araya J, Poniachik J. Oxidative stress and depletion of hepatic long-chain polyunsaturated fatty acids may contribute to nonalcoholic fatty liver disease. Free Radic Biol Med. 2004; 37: 1499-1507.

ED

[30] Dongiovanni P, Lanti C, Riso P, Valenti L. Nutritional therapy for nonalcoholic fatty liver disease. J Nutr Biochem. 2016; 29: 1-11. doi: 10.1016/j.jnutbio.2015.08.024.

PT

[31] Yasui H, Hayashi S, Sakurai H. Possible involvement of singlet oxygen species as multiple oxidants in p450 catalytic reactions. Drug Metab Pharmacokinet. 2005: 1-13.

AC

CE

[32] Ito Y, Abril ER, Bethea NW, McCuskey MK, McCuskey RS. Dietary steatotic liver attenuates acetaminophen hepatotoxicity in mice. Microcirculation. 2006; 13: 19-27. [33] Shirai M, Arakawa S, Miida H, Matsuyama T, Kinoshita J, Makino T, Kai K, Teranishi M. Thioacetamide-induced Hepatocellular Necrosis Is Attenuated in Diet-induced Obese Mice. J Toxicol Pathol. 2013; 26: 175-186. [34] Singh R, Kaushik S, Wang Y, Xiang Y, Novak I, Komatsu M, Tanaka K, Cuervo AM, Czaja MJ. Autophagy regulates lipid metabolism. Nature 2009; 458: 1131–1135. [35] Martinez-Lopez N, Singh R. Autophagy and Lipid Droplets in the Liver. Annu. Rev. Nutr. 2015; 35: 215–237. [36] Kurahashi T, Hamashima S, Shirato T, Lee J, Homma T, Kang ES, Fujii J. An SOD1 deficiency enhances lipid droplet accumulation in the fasted mouse liver by aborting lipophagy. Biochem Biophys Res Commun. 2015; 467: 866-871. [37] Sekiya M, Hiraishi A, Touyama M, Sakamoto K. Oxidative stress induced lipid accumulation via SREBP1c activation in HepG2 cells. Biochem Biophys Res Commun. 2008; 375: 602–607. 21

ACCEPTED MANUSCRIPT Figure legends

After weaning,

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Fig. 1. Changes in the body weight of the mice fed a ND or a HFD.

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the WT, db, KO, and KO::db mice were fed either ND (A) or HFD (B). Body weights were measured at the indicated time points. Means ± S.E. n=25–32 in each group. *

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Multivariate analysis of covariance (MANOVA).

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diet-matched WT mice.

Fig. 2. Death rates of mice. Kaplan-Meier analysis followed by a log-rank test was performed for each mouse group fed with ND vs. HFD; the WT, db, KO, and KO::db

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mice fed with either ND (A) or HFD (B). n=25–32 in each group.

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Fig. 3. Fat contents and liver weights of the mice. Mice that were fed a ND or a HFD were sacrificed at 12 w (A, C) and 28 w (B, D). Ratios of liver/body weights (A, B) and

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epididymal fat/body weights (C, D) are shown. Means ± S.E. Numbers of mice were

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shown in each column. Statistical analyses of other data were performed using two-way ANOVA, followed by the Tukey-Kramer test for multiple groups. **P < 0.01, 0.001, ND vs. HFD in each genotypic group.

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Fig. 4. Histology of livers of mice that were fed a ND or a HFD. Liver sections of the mice fed with a ND or a HFD were stained with H&E or EM at 28 w. Bar, 1 mm. Representative data of 3–4 mice in each group were shown.

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performed using two-way ANOVA, followed by the Tukey-Kramer test for multiple groups. Numbers of mice are shown in or above each column.

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ND vs. HFD in each genotypic group. #P < 0.05, ##P < 0.01; ###P < 0.001, each genotypic

Fig. 6. Effects of ND and HFD on fasting blood glucose (FBG), serum insulin levels,

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and HOMA-IR in mice. The levels of fasting blood glucose at 12 w (A) and 28 w (B) and insulin at 12 w (C) were measured. HOMA-IR at 12 w was calculated in the same

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manner as human (D). Means ± S.E. Numbers of mice were shown in or above each column. Statistical analyses for other data were performed using two-way ANOVA,

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followed by the Tukey-Kramer test for multiple groups. #P < 0.05, ##P < 0.01, ###P <

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0.001, each genotypic group vs. WT group mice with ND. †P < 0.05, †††P < 0.001, each genotypic group vs. WT group mice with HFD.

Fig. 7. Effects of a ND and a HFD on TG and lipid peroxidation in the liver. Liver TG and total blood bilirubin were measured at 12 w (A, C) and 28w (B, D). Lipid peroxidation products (TBARS) in the livers were measured at 12 w (E). Means ± S.E. Numbers of mice are shown in each column. Statistical analyses for other data were performed using two-way ANOVA, followed by the Tukey-Kramer test for multiple groups. **P < 0.01, ND vs. HFD in each genotypic group. #P < 0.05, ###P < 0.001, each genotypic group vs. WT group mice with ND. †P < 0.05, ††P < 0.01, †††P < 0.001, each 23

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Fig. 8. Levels of hyperoxidized peroxiredoxin (Prx-SO2/3) and Prx family proteins.

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Protein samples were prepared from mouse livers at 12 w (A, C) or 28 w (B, D) and subjected to immunoblot analyses using the antibodies against Prx-SO2/3, SOD1, and

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-actin (A, B) or Prx1, Prx2, Prx3, Prx4, and -actin (C, D). Representative data for 3

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