2014 early hepatic insult in the offspring of obese maternal mice

8/17/2019 2014 Early Hepatic Insult in the Offspring of Obese Maternal Mice

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Early hepatic insult in the offspring of obese maternal mice

Isabele Bringhenti, Fernanda Ornellas, Marcela Anjos Martins, Carlos

Alberto Mandarim-de-Lacerda, Marcia Barbosa Aguila

PII: S0271-5317(14)00266-8

DOI: doi: 10.1016/j.nutres.2014.11.006

Reference: NTR 7423

To appear in: Nutrition Research

Received date: 30 August 2014

Revised date: 25 November 2014

Accepted date: 27 November 2014

Please cite this article as: Bringhenti Isabele, Ornellas Fernanda, Martins MarcelaAnjos, Mandarim-de-Lacerda Carlos Alberto, Aguila Marcia Barbosa, Early hep-atic insult in the offspring of obese maternal mice, Nutrition Research (2014), doi:10.1016/j.nutres.2014.11.006

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http://dx.doi.org/10.1016/j.nutres.2014.11.006http://dx.doi.org/10.1016/j.nutres.2014.11.006http://dx.doi.org/10.1016/j.nutres.2014.11.006http://dx.doi.org/10.1016/j.nutres.2014.11.006


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ACCEPTED MANUSCRIPT1

Early hepatic insult in the offspring of obese maternal mice

Isabele Bringhenti,

Fernanda Ornellas,

Marcela Anjos Martins,

Carlos Alberto Mandarim-de-Lacerda 1

Marcia Barbosa Aguila

Laboratory of Morphometry, Metabolism and Cardiovascular Disease, Biomedical Center,

State University of Rio de Janeiro, Brazil

1 Corresponding author. Laboratory of Morphometry, Metabolism & Cardiovascular Disease,

Biomedical Center, State University of Rio de Janeiro, Av 28 de setembro 87 fds, Rio de

Janeiro, RJ, 20551-030 Brazil. Phone: +55.21.2868-8316; Fax 2868-8033.

Email: [email protected]; URL: www.lmmc.uerj.br.

mailto:[email protected]://www.lmmc.uerj.br/http://www.lmmc.uerj.br/mailto:[email protected]


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List of abbreviations

AIN-93G - American Institute of Nutrition to support growth during the pregnancy, lactation,

and post-weaning periods

AUC – Area under the curve

BM – Body Mass

CAT - catalase

CPT – Carnitine Palmitoyl-CoA Transferase

DIO – diet-induced obesity

FAS – Fatty Acid Synthase

FFA – Free Fatty Acids

GPx - Glutathione Peroxidase

GRS – Glutathione Reductase

HF – High-fat diet

IR – insulin resistance

MnSOD - Manganese Superoxide Dismutase

NEFA - Non-esterified fatty acids

NAFLD – Nonalcoholic Fatty Liver Disease

OGTT – Oral Glucose Tolerance Test

PPAR – Peroxisome Proliferator Activator Receptor

RT-qPCR – Real Time quantitative Polymerase Chain Reaction

ROS - Reactive oxygen species

SC – Standard chow

SOD – superoxide dismutase

TAG – Triacylglycerol

VLDL – Very low-density lipoprotein

TNF – Tumor Necrosis Factor

Vv – Volume Density


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Abstract

We hypothesized that the maternal obesity initiates metabolic disorders associated with

oxidative stress in the liver of offspring since early life. Mouse's mothers were assigned into

two groups according to the diet offered (n=10 per group): standard chow (SC) or high-fat

diet (HF). The results revealed that HF offspring had an increase in body mass at day 10 (+25

%, P


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1. Introduction

Obesity has become a serious public health problem, affecting individuals of all age ranges,

particularly women in the reproductive stage, setting forth a deleterious maternal nutritional

status [1, 2].

Nonalcoholic fatty liver disease (NAFLD) is associated with numerous physiological

events that are initiated by the increased delivery of nonesterified fatty acids from adipose

tissue to the liver tissue [3]. This established framework has implications in the pathogenesis

of liver steatosis because the input of lipid exceeds the capacity of fatty acids oxidation and/or

export. Although this condition is initially asymptomatic and benign, NAFLD is the

elementary injury for the development of severe pathology such as liver cirrhosis and

hepatocellular carcinoma, which may be associated with increased oxidative stress [4, 5].

Liver mitochondria presents maternal origin and may be an important target in the

investigation of metabolic disorders in the offspring of obese women, corroborated by the fact

that this organelle constitutes a critical site of energy metabolism [6]. Accordingly, liver

mitochondrial dysfunction may be associated with reduced beta oxidation of fatty acids and

decreases in cytochrome C and carnitine palmitoyl-CoA transferase (CPT)-1 enzyme [7].

Additionally, mitochondrial dysfunction leads to excessive lipids in the liver and may

promote the formation of reactive oxygen species, leading to lipid peroxidation,

inflammation, and fibrosis [8].

Previous data support the concept that the offspring of diet-induced obese (DIO) dams

have adverse metabolic programming and develop overweight/obesity and dyslipidemia in

adulthood [9, 10]. However, such offspring have disrupted liver metabolism and develop

NAFLD prior to any notable differences in body mass (BM) or body composition [11], which


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is a phenomenon known as “liver metabolic imprinting” [12, 13]. Nevertheless, it is important

to consider whether the insult in the liver due to maternal obesity occurs sufficiently early to

be detected in the offspring and to allow a preventive intervention.

Although the literature reported a different model of lipotoxity in the fetal liver using

nonhuman primates of obese mothers [14], the field of study is considerably complex and

merits further studies. We were particularly interested here in assessing the hepatic insult in

the very early life of the progeny of DIO mothers, observing the association between the liver

metabolism and oxidative stress.

2. Methods and materials

2.1 Animals and diet

This study was performed in strict accordance with the recommendations of the Guide for the

Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was

approved by the Committee on the Ethics of Animal Experiments of the State University of

Rio de Janeiro (Protocol CEUA 053/10), and all efforts were made to minimize suffering.

Animals were maintained under controlled temperature conditions of 20 ± 2º C; humidity

60 ± 10 % and 12 h/12 h dark/light cycle, and they had free access to food and water. Thus,

four-week-old female C57BL/6 mice were randomly assigned into two groups according to

the diet offered (n=10 per group): standard chow (SC) or high-fat diet (HF). In the SC group,

64 % of energy was derived from carbohydrates, 19 % of energy from protein and 17 % of


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energy from lipids. In the HF group, 32 % of energy was derived from carbohydrates, 19 % of

energy from protein and 49 % of energy from lipids. The vitamin and mineral contents of

both diets were identical and followed the standards for rodent diets recommended by the

American Institute of Nutrition to support growth during the pregnancy, lactation and post-

weaning periods (AIN-93G) (Table 1) [14].

After 8 weeks on the two diets, at twelve weeks of age, females SC and HF were mated

with SC-fed C57BL/6 males of the same age and the females remained on their experimental

diets throughout pregnancy and lactation. Thus, day 0 of gestation was determined by

observing the formation of a vaginal plug. Pups were divided according to the maternal diet:

SC pups were birthed from dams fed SC diet, whereas HF pups were birthed from dams fed

HF diet. One male from each litter was randomly assigned to each of the experimental groups

(the sex was assigned according to the anogenital distance) [15] and was studied at postnatal

day 1 (the birthday) and at postnatal day 10. Figure 1 details the design of the formation of the

groups. We adjusted the litters that were followed until day 10 randomly to seven pups to

provide adequate and standardized nutrition in the period. As a consequence, pups were

divided according to the maternal diets into four groups (n = 5 each group):

a) SC offspring postnatal day 1,

b) HF offspring postnatal day 1,

c) SC offspring postnatal day 10,

d) HF offspring postnatal day 10.

The body mass (BM) of the mothers was measured weekly, during the eight-week before

pregnancy. The BM of the offspring was measured at day 1 and postnatal day 10.


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2.2 Euthanasia and tissue extraction

The newborns were rapidly decapitated, and the blood was collected immediately. At day 10,

puppies were separated from their mothers one hour prior to decapitation, then blood was

collected and the glycemia was measured (Accu-Check Active glucometer; Roche Applied

Science, SP, Brazil), and the liver was carefully and rapidly dissected, weighed and made to

further analyzes. Various fragments were frozen for molecular and biochemical analyzes or

immersed in freshly prepared fixative (1.27 mol/L formaldehyde in 0.1 M phosphate buffer,

pH 7.2) for 48 h at room temperature.

2.3 Liver structure and biochemistry

The fixed liver fragments were then embedded in Paraplast Plus (Sigma-Aldrich Co., St.

Louis, Mo., USA), sectioned at a nominal thickness of 5 µm and stained with hematoxylin

and eosin. At least five microscopic fields per animal were analyzed in a blinded way at

random in digital images (Nikon Eclipse 90i microscope with DS-Ri1 camera, Tokyo, Japan).

We used a test-system made up of 36 test points (PT) produced with the STEPanizer web-

based system (www.stepanizer.com) put over the images for the analysis. Briefly, we

estimated the volume density of liver steatosis using the point-counting technique: Vv[liver,

steatosis] = Pp[steatosis]/PT[liver] (Pp is the number of points that hit the fat drops into the

hepatocyte) [16, 17].

The hepatic triacylglycerol (TAG) levels were measured in 50 mg of frozen liver tissue

placed in an ultrasonic processor with 1 ml of isopropanol. The homogenate was centrifuged


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at 2000 g, and 5 µl of the supernatant was used to measure TAG with a kit and an automated

spectrophotometer (Bioclin System II, Quibasa Ltda., Belo Horizonte, Brazil).

2.4 Antioxidants enzymes activity

Catalase (CAT), glutathione peroxidase (GPx) and manganese superoxide dismutase

(MnSOD) activities were measured in the liver homogenate of the 10 days old offspring. CAT

activity was measured in terms of the rate of decrease in hydrogen peroxide concentration at

240 nm [18]. GPx activity was measured by monitoring the oxidation of NADPH at 340 nm

in the presence of hydrogen peroxide [19]. MnSOD activity was assayed by measuring the

inhibition of adrenaline auto-oxidation at 480 nm [20]. The activity of antioxidant enzymes

was expressed in U/mg protein.

2.5 RT-qPCR.

Total RNA was extracted from approximately 50 mg of liver tissue using Trizol reagent

(Invitrogen, CA, USA). RNA amount was determined using Nanovue (GE Life Sciences)

spectroscopy, and 1 µg of RNA was treated with DNAse I (Invitrogen). Synthesis of the first

strand cDNA was performed using Oligo (dT) primers for mRNA and Superscript III reverse

transcriptase (both Invitrogen). Quantitative real-time PCR (RT-qPCR) was performed using

a Biorad CFX96 cycler and the SYBR Green mix (Invitrogen). Table 2 shows the primers for

qPCR as they were designed using the Primer3 online software.


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3. Results

3.1 Maternal body mass

The BM did not show a significant difference comparing the SC dams with the HF dams

when the study started (SC, 10.46 ± 0.43 g vs. HF, 10.39 ± 0.34 g). However, after eight

weeks in the experimental diets (before pregnancy), we found a greater BM in the HF dams

than in the SC dams (SC, 22.88 ± 0.48 g vs. HF, 29.13 ± 0.22 g; +27 %, P


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3.4 Antioxidant enzymes activity

CAT activity: there was no difference between the SC offspring and the HF offspring in the

postnatal day 10 (SC, 0.037 ± 0.002 U/mg ptn vs. HF, 0.049 ± 0.008 U/mg ptn; P >0.05).

GPx-1 activity: there was no difference between the SC offspring and the HF offspring in

the postnatal day 10 (SC, 9.04 ± 1.20 U/mg ptn vs. HF, 9.24 ± 0.90 U/mg ptn; P >0.05).

MnSOD activity: in postnatal day 10, the MnSOD activity was lower in the HF offspring

than in the SC offspring (HF, 17.34 ± 4.78 U/mg ptn vs.SC, 51.32 ± 13.21 U/mg ptn,

P


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the pups accounted for 11.73 % of the variance of the PPAR-gamma expression ( P


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( P


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reporting that a developing fetus is highly susceptible to excess lipids, and this may increase

the risk of NAFLD [31].

A recent study of the 8-week-old offspring of obese dams showed that the offspring were

hyperinsulinemic with significantly increased hepatic oxidative damage markers and with

reduced levels of the antioxidant enzyme GPx-1. Additionally, mitochondrial complex I and

II activities were elevated, whereas the levels of mitochondrial cytochrome c were

significantly reduced, and glutamate dehydrogenase was increased, suggesting mitochondrial

dysfunction. Offspring of obese dams also had significantly greater hepatic lipid content,

associated with increased levels of PPAR-gamma and reduced triglyceride lipase. Liver

glycogen and protein content were concomitantly reduced in the offspring of obese dams [11].

We observed a decrease in mRNA expression of CPT-1 in both ages in this study. CPT-1

is a regulatory enzyme and a target gene of PPAR-alpha in the mitochondria that transfers

fatty acids from the cytosol to the mitochondria prior to beta-oxidation [32, 33].

Consequently, we found the mRNA of FAS to be elevated in both ages of the HF offspring.

FAS is an intermediate enzyme that utilizes acetyl-CoA and malonyl-CoA to form palmitic

acid (C16:0) in the process of fatty acid synthesis. Therefore, FAS plays a role in energy

metabolism and shows augmented levels in association with NAFLD [34, 35].

The disruption in the mRNA expression of antioxidant enzymes may be associated with

hepatic damage [36]. Studies have found that oxidative stress may occur because of increased

reactive oxygen species (ROS) production and/or failure of the antioxidant system. The

antioxidant system is composed of nonenzymatic molecules and antioxidant enzymes such as

MnSOD, CAT and GPx1 that react with and inactivate ROS [37]. Notably, a redox-centered

pathogenesis is associated with NAFLD, whereby the redox state/ROS may modulate lipid

metabolism and insulin sensitivity [38]. Indeed, oxidative stress has been observed to precede

insulin resistance [39].


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It is important to highlight that epigenetic mechanisms comprise several levels of

interconnected and interdependent codes, including methylation of DNA, covalent

modifications of histones, and the gene-regulating and chromatin-organizing activities of non-

coding RNAs [40]. Such processes change the binding of transcription activators and

repressors to specific gene promoters, and/or alter the large-scale conformation and function

of chromatin itself, which modulates gene expression [41]. Additionally, studies have

associated obesity with DNA hypermethylation of enzymes involved in mitochondrial fatty

acid oxidation, gluconeogenesis and lipogenesis in the liver, causing silencing of their

expression and contributing to obesity-induced liver insulin resistance [42, 43].

Although we did not evaluate mitochondrial dysfunction, a recent study reported an

increase in mitochondrial complex I and II activities, resulting in an increase in free radical

generation. The mitochondrial dysfunction associated with reduced antioxidant capacity,

together with reduced GPx1 expression, contributes to the pro-oxidative environment in the

offspring of DIO mothers later in life. Furthermore, increased mitochondrial metabolism is

associated with generated ROS with PPAR-gamma activity and adipocyte differentiation [44].

Our findings demonstrated a decrease in mRNA expression of catalase, MnSOD and GPx1

from the day of birth to day 10 after birth. In addition, the analysis of antioxidant enzyme

activities in the 10 days offspring showed a reduction in the SOD activity, while the activities

of the CAT and GPx were preserved, despite the reduced mRNA level observed to both of the

enzymes. An impairment of the hepatocyte antioxidant defense mechanisms may have

contributed to steatosis development in the offspring since the protective capacity of the

hepatocytes to inhibit degradation induced by free radicals was reduced [36].

In conclusion, the message of the current study is that of DIO in maternal mice may induce

an early oxidative dysfunction coupled with hepatic steatosis in the offspring that is

measurable since the first days of life. Therefore, this time may represent an interventional


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opportunity to prevent liver damage progression and the development of metabolic disorders

later in life, such as insulin resistance.

Acknowledgment

The authors would like to thank Aline Penna and Michele Moura for technical assistance.

Financial support

The research was supported by the National council for scientific research (CNPq, Brazil), the

Rio de Janeiro Foundation for research (Faperj), and the Coordination of Improvement of

Higher Education Personnel (CAPES). The funding sources had no role in study design; in

the collection, analysis and interpretation of data; in the writing of the report; and in the

decision to submit the manuscript for publication.


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References

[1]. Flegal KM, Carroll MD, Kit BK, Ogden CL. Prevalence of obesity and trends in the

distribution of body mass index among US adults, 1999-2010. JAMA. 2012;307:491-7.

[2]. Borengasser SJ, Kang P, Faske J, Gomez-Acevedo H, Blackburn ML, Badger TM, et al.

High fat diet and in utero exposure to maternal obesity disrupts circadian rhythm and

leads to metabolic programming of liver in rat offspring. PLoS One. 2014;9:e84209.

[3]. Hughes AN, Oxford JT. A lipid-rich gestational diet predisposes offspring to

nonalcoholic fatty liver disease: a potential sequence of events. Hepat Med. 2014;6:15-

23.

[4]. Paredes AH, Torres DM, Harrison SA. Nonalcoholic fatty liver disease. Clin Liver Dis.

2012;16:397-419.

[5]. Shibahara J, Ando S, Sakamoto Y, Kokudo N, Fukayama M. Hepatocellular carcinoma

with steatohepatitic features: a clinicopathological study of Japanese patients.

Histopathology. 2014;64:951-62.

[6]. Bruce KD, Cagampang FR, Argenton M, Zhang J, Ethirajan PL, Burdge GC, et al.

Maternal high-fat feeding primes steatohepatitis in adult mice offspring, involving

mitochondrial dysfunction and altered lipogenesis gene expression. Hepatology.

2009;50:1796-808.

[7]. Sanyal AJ, Campbell-Sargent C, Mirshahi F, Rizzo WB, Contos MJ, Sterling RK, et al.

Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial

abnormalities. Gastroenterology. 2001;120:1183-92.

[8]. Serviddio G, Bellanti F, Giudetti AM, Gnoni GV, Capitanio N, Tamborra R, et al.

Mitochondrial oxidative stress and respiratory chain dysfunction account for liver


21/38

A C C E P

T E D

M A N U S C R I P T


toxicity during amiodarone but not dronedarone administration. Free Radic Biol Med.

2011;51:2234-42.

[9]. Ornellas F, Mello VS, Mandarim-de-Lacerda CA, Aguila MB. Sexual dimorphism in fat

distribution and metabolic profile in mice offspring from diet-induced obese mothers.

Life Sci. 2013;93:454-63.

[10]. Oben JA, Mouralidarane A, Samuelsson AM, Matthews PJ, Morgan ML, McKee C, et

al. Maternal obesity during pregnancy and lactation programs the development of

offspring non-alcoholic fatty liver disease in mice. J Hepatol. 2010;52:913-20.

[11]. Alfaradhi MZ, Fernandez-Twinn DS, Martin-Gronert MS, Musial B, Fowden A,

Ozanne SE. Oxidative stress and altered lipid homeostasis in the programming of

offspring fatty liver by maternal obesity. Am J Physiol Regul Integr Comp Physiol.

2014;307:R26-34.

[12]. Burgueno AL, Cabrerizo R, Gonzales Mansilla N, Sookoian S, Pirola CJ. Maternal

high-fat intake during pregnancy programs metabolic-syndrome-related phenotypes

through liver mitochondrial DNA copy number and transcriptional activity of liver

PPAR-GC1A. J Nutr Biochem. 2013;24:6-13.

[13]. Bayol SA, Simbi BH, Fowkes RC, Stickland NC. A maternal "junk food" diet in

pregnancy and lactation promotes nonalcoholic Fatty liver disease in rat offspring.

Endocrinology. 2010;151:1451-61.

[14]. Reeves PG, Nielsen FH, Fahey GC, Jr. AIN-93 purified diets for laboratory rodents:

final report of the American Institute of Nutrition ad hoc writing committee on the

reformulation of the AIN-76A rodent diet. J Nutr. 1993;123:1939-51.

[15]. Hotchkiss AK, Vandenbergh JG. The anogenital distance index of mice ( Mus musculus

domesticus): an analysis. Contemp Top Lab Anim Sci. 2005;44:46-8.


22/38

A C C E P

T E D

M A N U S C R I P T


[16]. Aguila MB, Pinheiro AR, Parente LB, Mandarim-de-Lacerda CA. Dietary effect of

different high-fat diet on rat liver stereology. Liver Int. 2003;23:363-70.

[17]. Catta-Preta M, Mendonca LS, Fraulob-Aquino J, Aguila MB, Mandarim-de-Lacerda

CA. A critical analysis of three quantitative methods of assessment of hepatic steatosis

in liver biopsies. Virchows Arch. 2011;459:477-85.

[18]. Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121-6.

[19]. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of

erythrocyte glutathione peroxidase. J Lab Clin Med. 1967;70:158-69.

[20]. Bannister JV, Calabrese L. Assays for superoxide dismutase. Methods Biochem Anal.

1987;32:279-312.

[21]. Magliano DC, Bargut TC, de Carvalho SN, Aguila MB, Mandarim-de-Lacerda CA,

Souza-Mello V. Peroxisome proliferator-activated receptors-alpha and gamma are

targets to treat offspring from maternal diet-induced obesity in mice. PLoS One.

2013;8:e64258.

[22]. Sullivan EL, Smith MS, Grove KL. Perinatal exposure to high-fat diet programs energy

balance, metabolism and behavior in adulthood. Neuroendocrinology. 2011;93:1-8.

[23]. Srinivasan M, Katewa SD, Palaniyappan A, Pandya JD, Patel MS. Maternal high-fat

diet consumption results in fetal malprogramming predisposing to the onset of

metabolic syndrome-like phenotype in adulthood. Am J Physiol Endocrinol Metab.

2006;291:E792-9.

[24]. Brion MJ, Ness AR, Rogers I, Emmett P, Cribb V, Davey Smith G, et al. Maternal

macronutrient and energy intakes in pregnancy and offspring intake at 10 y: exploring

parental comparisons and prenatal effects. Am J Clin Nutr. 2010;91:748-56.


23/38

A C C E P

T E D

M A N U S C R I P T


[25]. Arentson-Lantz EJ, Buhman KK, Ajuwon K, Donkin SS. Excess pregnancy weight gain

leads to early indications of metabolic syndrome in a swine model of fetal

programming. Nutr Res. 2014;34:241-9.

[26]. Gregorio BM, Souza-Mello V, Carvalho JJ, Mandarim-de-Lacerda CA, Aguila MB.

Maternal high-fat intake predisposes nonalcoholic fatty liver disease in C57BL/6

offspring. Am J Obstet Gynecol. 2010;203:495 e1-8.

[27]. Cederroth CR, Nef S. Fetal programming of adult glucose homeostasis in mice. PLoS

ONE. 2009;4:e7281.

[28]. Cerf ME, Louw J. High fat programming induces glucose intolerance in weanling

Wistar rats. Horm Metab Res. 2010;42:307-10.

[29]. Okamura M, Inagaki T, Tanaka T, Sakai J. Role of histone methylation and

demethylation in adipogenesis and obesity. Organogenesis. 2010;6:24-32.

[30]. Shankar K, Kang P, Harrell A, Zhong Y, Marecki JC, Ronis MJ, et al. Maternal

overweight programs insulin and adiponectin signaling in the offspring. Endocrinology.

2010;151:2577-89.

[31]. McCurdy CE, Bishop JM, Williams SM, Grayson BE, Smith MS, Friedman JE, et al.

Maternal high-fat diet triggers lipotoxicity in the fetal livers of nonhuman primates. J

Clin Invest. 2009;119:323-35.

[32]. Huang YY, Gusdon AM, Qu S. Nonalcoholic fatty liver disease: molecular pathways

and therapeutic strategies. Lipids Health Dis. 2013;12:171.

[33]. Lee HJ, Choi SS, Park MK, An YJ, Seo SY, Kim MC, et al. Fenofibrate lowers

abdominal and skeletal adiposity and improves insulin sensitivity in OLETF rats.

Biochem Biophys Res Commun. 2002;296:293-9.


24/38

A C C E P

T E D

M A N U S C R I P T


[34]. Kohjima M, Enjoji M, Higuchi N, Kato M, Kotoh K, Yoshimoto T, et al. Re-evaluation

of fatty acid metabolism-related gene expression in nonalcoholic fatty liver disease. Int

J Mol Med. 2007;20:351-8.

[35]. Morgan K, Uyuni A, Nandgiri G, Mao L, Castaneda L, Kathirvel E, et al. Altered

expression of transcription factors and genes regulating lipogenesis in liver and adipose

tissue of mice with high fat diet-induced obesity and nonalcoholic fatty liver disease.

Eur J Gastroenterol Hepatol. 2008;20:843-54.

[36]. Rolo AP, Teodoro JS, Palmeira CM. Role of oxidative stress in the pathogenesis of

nonalcoholic steatohepatitis. Free Radic Biol Med. 2012;52:59-69.

[37]. Roberts CK, Sindhu KK. Oxidative stress and metabolic syndrome. Life Sci.

2009;84:705-12.

[38]. Serviddio G, Bellanti F, Vendemiale G. Free radical biology for medicine: learning

from nonalcoholic fatty liver disease. Free Radic Biol Med. 2013;65:952-68.

[39]. Matsuzawa-Nagata N, Takamura T, Ando H, Nakamura S, Kurita S, Misu H, et al.

Increased oxidative stress precedes the onset of high-fat diet-induced insulin resistance

and obesity. Metabolism. 2008;57:1071-7.

[40]. Goldberg AD, Allis CD, Bernstein E. Epigenetics: a landscape takes shape. Cell.

2007;128:635-8.

[41]. Gluckman PD, Hanson MA, Buklijas T, Low FM, Beedle AS. Epigenetic mechanisms

that underpin metabolic and cardiovascular diseases. Nat Rev Endocrinol. 2009;5:401-

8.

[42]. Jiang M, Zhang Y, Liu M, Lan MS, Fei J, Fan W, et al. Hypermethylation of hepatic

glucokinase and L-type pyruvate kinase promoters in high-fat diet-induced obese rats.

Endocrinology. 2011;152:1284-9.


25/38

A C C E P

T E D

M A N U S C R I P T


[43]. Sookoian S, Rosselli MS, Gemma C, Burgueno AL, Fernandez Gianotti T, Castano GO,

et al. Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease:

impact of liver methylation of the peroxisome proliferator-activated receptor gamma

coactivator 1alpha promoter. Hepatology. 2010;52:1992-2000.

[44]. Tormos KV, Anso E, Hamanaka RB, Eisenbart J, Joseph J, Kalyanaraman B, et al.

Mitochondrial complex III ROS regulate adipocyte differentiation. Cell Metab.

2011;14:537-44.


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Figure legends

Figure 1. Sampling design for the study. For the composition of experimental groups, one

male pup of each litter was considered in this study, totaling a sample of five animals per

group.

Figure 2. Body mass.

Data are presented as means and standard error of the means (n = 5 per group). Significant

differences are indicated (one-way ANOVA and post-hoc Holm-Sydak tests).

Abbreviations: SC, standard chow group; HF, high-fat diet group.

Symbols: †, significantly different from SC counterpart; ‡, different from day 1.

Figure 3. Blood glucose levels.




Symbols: †, significantly different from SC counterpart; ‡, significantly different from day 1.


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Figure 4. Photomicrographs of liver tissue (hematoxylin-eosin stain, same magnification for

all photomicrographs): (A) SC day 1; (B) HF day 1; (C) SC day 10; (D) HF day 10; (E)

triacylglycerol content; and (F) volume density of steatosis.




Symbols: †, significantly different from SC counterpart.

Figure 5. Correlation of liver steatosis (variable y) and liver triacylglycerol levels (variable x).

The coefficient of correlation of Pearson, r, and P values are indicated.


Figure 6. Relative mRNA expression of FAS, PPAR-gamma, CPT1 and PPAR-alpha.


differences are indicated (one-way ANOVA and post-hoc Holm-Sydak tests). (A) FAS

relative expression; (B) PPAR-gamma relative expression; (C) CPT-1 relative expression; (D)

PPAR-alpha relative expression. An endogenous control (beta-actin) was used to normalize

the expression of the selected genes.

Abbreviations: SC, standard chow group; HF, high-fat diet group. FAS, fatty acid synthase;

PPAR, peroxisome-proliferator activator receptor; and CPT, carnitine palmitoyltransferase.



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Figure 7. Relative mRNA expression of catalase, MnSOD, GPx-1 and GRS An endogenous

control (beta-actin) was used to normalize the expression of the selected genes.


differences are indicated (one-way ANOVA and post-hoc Holm-Sydak tests). (A) Catalase

relative expression; (B) MnSOD relative expression; (C) GPx-1 relative expression; (D) GRS

relative expression.

Abbreviations: SC, standard chow group; HF, high-fat diet group; MnSOD, manganese

superoxide dismutase; GPx, glutathione peroxidase; GRS, glutathione reductase.



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Figure 1


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Figure 2


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Figure 3


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Figure 4


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Figure 5


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Figure 6


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Figure 7


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Table 1. Ingredient and proximate analysis of diets fed to mice

Abbreviations: SC: standard chow; HF: high-fat diet.

Nutrients (g/kg) SC HF

Casein 190.00 230.00

Corn starch 539.50 299.50

Sucrose 100.00 100.00

Soybean oil 70.00 70.00

Lard - 200.00

Fiber 50.00 50.00

Vitamin Mixture (mg) 10.00 10.00

Mineral Mixture (mg) 35.00 35.00

Cysteine 3.00 3.00

Choline 2.50 2.50

Antioxidants 0.01 0.01

Total mass (g) 1.00 1.00

Energy (kcal/kg) 3.95 4.95

Carbohydrates (%) 64.00 32.00

Protein (%) 19.00 19.00

Lipids (%) 17.00 49.00


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Table 2. RT-qPCR primers and respective sequences

Abbreviations: Carnitine palmitoyltransferase I (CPT-1); Catalase (CAT); Fatty acid synthase

(FAS); Glutathione peroxidase (GPx1); Glutathione reductase (GSR); Manganese Superoxide

Dismutase (MnSOD); Peroxisome proliferator activator receptor (PPAR ).

Name 5-3’ Primers

FAS FW CACCCACTGGAAGCTGGTAT

FAS RV TCGAGGAAGGCACTACACCT

CPT-1 FW AAGGAATGCAGGTCCACATC

CPT-1 RV CCAGGCTACAGTGGGACATT

PPAR alpha FW TCGAGGAAGGCACTACACCT

PPAR alpha RV TCTTCCCAAAGCTCCTTCAA

PPAR gamma2 FW ACGATCTGCCTGAGGTCTGT

PPAR gamma2 RV CATCGAGGACATCCAAGACA

MnSOD FW GGTCTCCAACATGCCTCTCT

MnSOD RV AACCATCCACTTCGAGCAGA

GPx1

GPx1

FW

RV

TCTGCAGATCGTTCATCTCG

GTCCACCGTGTATGCCTTCT

CAT

CAT

FW

RV

CAAGTTTTTGATGCCCTGGGT

ACATGGTCTGGGACTTCTGG

GSR

GSR

FW

RV

CAGCATAGACGCCTTTGACA

CACGACCATGATTCCAGATG

Beta-actin FW CTCCGGCATGTGCAA

Beta-actin RV CCCACCATCACACCCT


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Table 3. Two-way ANOVA analyzing high-fat diet and age.

Abbreviations: CAT (catalase), CPT (carnitine palmitoyl transferase), FAS (fatty acid

synthase), GPx (glutathione peroxidase), GRS (glutathione reductase), MnSOD (manganese

superoxide dismutase), PPAR (peroxisome-proliferator activator receptor), TAG

(triacylglycerol).

Two-Way ANOVA

% of the variation and significance test

Interaction Diet Age

% P≤ % P≤ % P≤

Body Mass 1.63 0.0001 2.25 0.0001 95.65 0.0001

CAT 1.24 NS 67.76 0.0001 1.38 NS

CPT-1 0.38 NS 62.94 0.0001 2.30 NS

FAS 4.29 NS 52.22 0.0001 10.91 0.05

Glycemia 2.59 0.01 4.81 0.001 88.77 0.0001

GPx-1 22.87 0.0001 22.87 0.0001 39.68 0.0001

GRS 12.36 NS 34.81 0.01 0.97 NS

Hepatic TAG 0.29 NS 69.66 0.0001 0.44 NS

Liver steatosis 0.87 NS 79.23 0.0001 0.13 NS

MnSOD 4.86 NS 62.47 0.0001 9.54 0.05

PPAR-alpha 4.69 NS 55.04 0.001 2.53 NS

PPAR-gamma 0.057 NS 61.51 0.0001 11.73 0.05

2014 early hepatic insult in the offspring of obese maternal mice

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