Beneficial effects of quercetin–iron complexes on serum and tissue lipids and redox status in obese rats

    Beneficial effects of quercetin iron complexes on serum and tissue lipids and redox status in obese rats Asmahan Imessaoudene, Hafida Merzouk, Farid Berroukeche, Nassima Mokhtari, Bachir Bensenane, Sabri Cherrak, Sid Ahmed Merzouk, Mourad Elhabiri PII: DOI: Reference:

S0955-2863(15)00345-9 doi: 10.1016/j.jnutbio.2015.11.011 JNB 7506

To appear in:

The Journal of Nutritional Biochemistry

Received date: Revised date: Accepted date:

7 June 2015 2 November 2015 20 November 2015

Please cite this article as: Imessaoudene Asmahan, Merzouk Hafida, Berroukeche Farid, Mokhtari Nassima, Bensenane Bachir, Cherrak Sabri, Merzouk Sid Ahmed, Elhabiri Mourad, Beneficial effects of quercetin iron complexes on serum and tissue lipids and redox status in obese rats, The Journal of Nutritional Biochemistry (2015), doi: 10.1016/j.jnutbio.2015.11.011

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ACCEPTED MANUSCRIPT Title: Beneficial effects of quercetin iron complexes on serum and tissue lipids and redox status in obese rats

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Asmahan IMESSAOUDENEa, Hafida MERZOUKa*, Farid BERROUKECHEa, Nassima

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MOKHTARIa, Bachir BENSENANEa, Sabri CHERRAKa, Sid Ahmed MERZOUKb, Mourad ELHABIRIc

Laboratory of Physiology, Physiopathology and Biochemistry of Nutrition, Department of

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a

Belkaïd, Tlemcen 13000, Algeria.

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Biology, Faculty of Natural and Life Sciences, Earth and Universe, University Abou-Bekr b

Department of Technical Sciences, Faculty of

Engineering, University Abou-Bekr Belkaïd, Tlemcen 13000, Algeria.

c

Laboratory of

Bioorganic and Medicinal Chemistry, UMR 7509 CNRS – University of Strasbourg, ECPM,

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25 rue Becquerel Street, 67087 Strasbourg, Cedex 2, France.

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Running title: Quercetin-iron complexes in obesity.

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Key words: Quercetin, iron, obesity, adipose tissue, liver, metabolism, muscle.

Correspondence should be addressed to Professor MERZOUK Hafida, Laboratory of Physiology and Biochemistry of Nutrition, Department of Biology, University ABOU-BEKR BELKAÏD, Tlemcen 13000, Algeria; E-mail: [email protected]; Tel: 00 213 778303645; Fax: 00 213 43212145

ACCEPTED MANUSCRIPT ABSTRACT Obesity is characterized by iron deficiency, carbohydrate and fat alterations as well as

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oxidative stress. Iron status monitoring is recommended because of the conventional oral iron

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preparations which frequently exacerbate the already present oxidative stress. Iron complexation by natural antioxidants can be exploited. We herein investigated the metabolic

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effects of quercetin (25mg/Kg/day), iron (2.5mg Fe/Kg/day) or quercetin-iron complexes (molar ratio 5:1; 25mg/2.5mg/Kg/day) in animal models of obesity. Our results emphasized

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that obese rats displayed metabolic alterations that were worsened by iron supplementation. In contrast, quercetin used alone or as iron complex clearly prevented adipose fat accumulation and alleviated the hyperglycemia, hyperlipidemia, liver steatosis and oxidative stress. In addition, it induced a modulation of lipase activities in obese rats. Interestingly, quercetin–

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iron complexes showed enhanced beneficial effects such as a corrected iron deficiency in

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obese rats when compared to quercetin alone. In conclusion, anti-anemic, hypoglycemic, hypolipidemic and anti-oxidative effects of the

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alterations.

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quercetin-iron complexes shed a light on their beneficial use against obesity related metabolic

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1. INTRODUCTION

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Obesity is a major global health problem. Obesity arises from an imbalance in energy intake

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and expenditure that leads to adiposity with cell hypertrophy and hyperplasia [1], increasing the risk of type 2 diabetes, hypertension, heart disease, dyslipidemia, osteoarthritis,

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gynecological or respiratory problems, infections or cancer, in addition to social stigma [2].

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Obesity is characterized by a state of chronic oxidative stress related to an overproduction of reactive oxygen species (ROS, e.g. hydrogen peroxide, peroxyl, superoxide anion and hydroxyl radicals) and a subsequent decrease in antioxidants levels such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase

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(GPx) activities, vitamins and minerals [3,4]. This oxidative state is associated to metabolic abnormalities, including hyperinsulinemia, carbohydrate and lipid metabolism alterations,

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increased adipose tissue mass and triglyceride storage, elevated blood pressure and increased

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systemic inflammation [5].

Adipose tissue is not only a triglyceride storage organ, it is currently recognized as an

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endocrine organ. It secretes cytokines and adipokines that contribute to inflammation and oxidative stress development [4]. Fat tissue is also a source of many hormones which can act either locally or systemically [6]. The increased oxidative stress in adipocytes might be a cause of obesity-associated metabolic syndrome [7]. Indeed, the adipose tissue role in contributing to obesity-associated cardiovascular and metabolic risks has gained much attention during the last years. A relationship between obesity and iron deficiency has been previously demonstrated [8]. Serum iron levels of obese subjects were significantly lower than those of normal weight subjects [9]. Previous studies have investigated the association between Body Mass Index and iron status in children [10]. Iron deficiency is also common in obese pregnancy associated to

ACCEPTED MANUSCRIPT impaired maternal-fetal iron transfer [11]. Iron deficiency in obese people may be a result of low iron intake, reduced iron absorption, greater iron requirements and chronic low grade

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inflammation state [8,9]. In addition, obesity can lead to chronic over-expression of hepcidin,

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an iron homeostatic regulator, consequent to an excess of fat mass and low-grade chronic inflammation [9]. Increased hepcidin levels may lead to poor iron status by inhibiting iron

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absorption and restricting iron bio-availability. In addition, the liver, which is also the key regulator of iron homeostasis, is characterized by lipid accumulation and oxidative stress for

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obese patients [8,12]. Close monitoring of the iron status with iron supplements are recommended in obese subjects [13]. However, iron supplementation in obese people remains a delicate question since providing free iron, a potent pro-oxidant electroactive metal ion, may further exacerbate the already current oxidative stress [14]. Indeed, conventional oral iron

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preparations, generally iron(II) salts such as iron(II) sulfate, frequently cause gastrointestinal

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side-effects following ROS formation (i.e. in the presence of hydrogen peroxide, Fe(II) catalyzes deleterious hydroxyl radical formation through the Fenton reactions). Thus, strong

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iron complexation by natural compounds derived from dietary constituents capable to (i)

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prevent oxidative stress triggered by Fe(II) and (ii) act as efficient antioxidant to alleviate the current pathological oxidative stress, might be exploited in the development of novel iron supplements in obesity. Today, growing interest is given to the association of specific components of human nutrition and oxidative stress. Flavonoids that are abundant in plant foods (e.g. vegetables and fruits), have recently captured the attention of the general public due to their demonstrated beneficial health effects. They potentially have protective roles against the pathogenesis of multiple diseases associated with oxidative stress such as cancer, coronary heart disease, and atherosclerosis [15,16].

ACCEPTED MANUSCRIPT Among the

vast

family of

polyphenols,

quercetin

is

a

flavonoid

(3′,4′,3,5,7-

pentahydroxyflavone bearing a catechol unit on the B-ring and being one of the most studied

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polyphenolic compound) found in a broad variety of fruits and vegetables including apple,

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citrus fruits, berries, bulbs, cereal grains, onions, cacao, legumes, and tea. Quercetin has been shown to display several pharmacological properties, including antioxidant, anti-

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inflammatory and hepatoprotective effects [17,18]. Quercetin is also able to decrease plasma cholesterol and hepatic lipids, ameliorate diabetes-induced oxidative stress and preserve

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pancreatic beta cell integrity [19]. Indeed, in obese Zucker rats, chronic administration of quercetin markedly improved dyslipidemia, hypertension, and hyperinsulinemia and reduced body weight gain [20]. Metabolic alterations during the development of insulin resistance caused by high-fat feeding are also prevented by dietary quercetin [21].

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The anti-oxidative activities of flavonoids are not only based on their free-radical scavenging

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capacities but also on their chelation properties of transition metal ions. Flavonoid metallic complexes revealed to possess potent biological activities in experiments with cells, tissues,

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and animals thus suggesting perspectives regarding their medical use [22].

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The flavonoid–metal complexes were shown to be more effective free radical scavengers than the free flavonoids alone. In particular, quercetin–iron complexes have been reported to exhibit high lipophilicity and anticancer activities [22]. Considering that obesity is associated to metabolic abnormalities, oxidative stress and iron deficiency, supplementation with quercetin-iron complexes rather than with quercetin alone would constitute an efficient and smart treatment to correct all these alterations. Although independent studies have shown that obesity is associated with increased oxidative stress, inflammation and iron deficiency, and that quercetin-iron complexes exert anti-inflammatory and antioxidant activities, there are, to the best of our knowledge, no reports in the literature of the beneficial effects of quercetin-iron complexes on obesity. Consequently, the importance

ACCEPTED MANUSCRIPT to establish an animal model for investigation of this issue became crucial, due to ethical and methodological limitations in human studies.

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Experimental obesity can be mimicked by dietary manipulations, especially the so-called

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“cafeteria diet” which basically consists of a variety of snack-type foods, normally consumed by humans [23].

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Herein, we have therefore evaluated the metabolic effects and the antioxidant potential of quercetin-iron complexes compared to quercetin or iron alone on obesity induced by

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“cafeteria diet” in rats. Concomitantly, we demonstrated that the deleterious effects of iron supplementation in obesity can be corrected by using quercetin–iron complexes.

2. MATERIALS AND METHODS

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2.1. Dose selection and preparation of quercetin iron complex

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Quercetin (quercetin dihydrate, 97%) and FeSO4 were purchased from Alfa Aesar. All solutions were freshly prepared with ultrapure water before experiments and used

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immediately. Quercetin was dissolved in DMSO/0.9% normal saline so that the final amount

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of DMSO was less than 1%. FeSO4 was dissolved in the same solvent (DMSO/0.9% normal saline). Quercetin-iron complexes were prepared by mixing aqueous stock solutions of quercetin and FeSO4 with quercetin/iron molar ratio of 5:1. Under these conditions, the 5:1 quercetin/iron complex was found to be the most thermodynamically stable species with high lipophilicity [24,25]. UV-Vis measurements were carried out at room temperature using a Lambda 45 model spectrophotometer (Perkin Elmer). The UV-Vis spectra of the free quercetin and the quercetin-Fe(II) complex were measured at pH 7.4. Quercetin is characterized by an intense absorption band whose maximum is centred at 373 nm. Along the spectrophotometric titration of quercetin with Fe(II), the maximum absorption at 373 nm

ACCEPTED MANUSCRIPT underwent a hypochromic shift while a new absorption band emerged at 425 nm and was attributed to the formation of quercetin–iron complexes.

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For the in vivo experiment, the selected quercetin dose was 25 mg/Kg while that of Fe(II)

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sulfate was 2.5 mg/Kg. The dosage and administration of quercetin (25 mg/kg) were assessed based on reported studies showing beneficial metabolic effects at this dose [26,27]. To keep a

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large quercetin/iron molar ratio (equal to 5:1 in the present study), required for complex formation, the iron dose of 2.5 mg/Kg was selected. In the previous studies, 1.5 to 3 mg/Kg

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doses of iron (FeSO4) were commonly used [28]. This iron dose is usually used for iron deficiency in humans [29].

2.2. Animals and experimental protocol

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Male Wistar rats, eight weeks old, weighing between 200 and 230 g and obtained from

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Animal Resource Centre (Algeria), were used in this study. All aspects of the experiments were conducted according to the guidelines provided by the ethical committee of the

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experimental animal care at Tlemcen University. Animals were housed in separate cages (2 -

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3 per cage) at a constant temperature (25 °C) and humidity (60 ± 5%) with light regime identical to natural photo-period (12:12 h light/dark cycle). The rats were randomly divided into two groups of equal average body weight. The first group (Control, C, n=32) was exposed to standard diet (330 kJ/100 g) composed of 25% of energy as protein, 65% of energy as carbohydrate and 10% of energy as lipids (ONAB, Algeria). The second group (Obese, O, n=32) was fed with a cafeteria diet composed of dough, cheese, bacon, potato chips, biscuits and chocolate (in a proportion of 2:2:2:1:1:1, by weight) mixed with standard chow (w/w). The cafeteria diet (420 kJ/100 g) was composed of 23% of energy as protein, 35% of energy as carbohydrates and 42% of energy as lipids. We have previously shown that this cafeteria diet induced hyperphagia and obesity in rats [30,31].

ACCEPTED MANUSCRIPT Rats were exposed to the standard or the cafeteria diet for 6 weeks before starting the intragastric administration of quercetin and its iron complex. At the end of the 6th week, cafeteria-

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fed rats were significantly heavier than control-fed ones. Afterwards, the rats in each group

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(control or obese) were divided into four subgroups. The control and the obese groups (C or O, n=8) were gavaged with only DMSO/0.9% normal saline (1 mL per rat, with a final

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concentration of 0.5% DMSO). Quercetin (25 mg/Kg/day) was provided to the CQ and OQ groups (n=8) by intra-gastric administration (1 mL in DMSO/0.9% normal saline/rat). In the

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CFe and OFe groups (n=8), rats were supplemented with FeSO4 (2.5mg Fe/Kg/day) by gavage (1 mL in DMSO/0.9% normal saline/rat). The CQFe and OQFe groups (n=8) were exposed to intra-gastric administration of quercetin/iron complex (molar ratio 5:1; 25mg/2.5mg/Kg/day). Rats were supplemented by quercetin, FeSO4 or complexes via gavage

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for 8 weeks and had free access to their own diet throughout the entire experimental period.

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2.3. Analysis of the total metal contents in the standard/cafeteria diets About 0.5 g of the standard and cafeteria diets were introduced in a mixture of 8 ml of high

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purity concentrated nitric acid (HNO3) and 1 ml of hydrochloric acid (HCl) for 60 minutes to ensure complete dissolution of the samples. The samples were then diluted by a factor of about 40 with deionised water. The diluted digests were analysed for concentrations of iron, copper and zinc on a Varian Inductively Coupled Plasma Optical Emission Spectrometer 735ES.

2.4. Determination of total polyphenols using the Folin-Ciocalteau reagent For the standardization of the spectrophotometric method using the Folin-Ciocalteau reagent, quercetin was used as the reference chemical standard. A quercetin stock solution was prepared at 211 µM in distilled water and was subjected to stepwise dilution. To 200 µL of a

ACCEPTED MANUSCRIPT diluted solution of quercetin, 1 mL of freshly prepared Folin-Ciocalteau reagent was added and the mixture was left for reaction during 4 minutes. The volume (1.2 mL) was completed

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to 2mL with anhydrous sodium carbonate (75g/L) and left for reaction and equilibration for 2

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additional hours. Absorption spectra (400 – 800 nm) were measured with a Varian CARY 50 and the absorbances at 765 nm were plotted as a function of the quercetin concentrations.

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Aliquots of 5 g of the standard and cafeteria diets were placed in an oven at 60°C for 3 days. The samples were weighed and then finely ground. The water contents of the standard and

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cafeteria diets were measured to be at about 12 and 21%, respectively. 50 mL of cold water, 50 mL of hot water and 60 mL of methanol were successively added to 5 g of the dried samples and then vigorously shaken. The samples were then left for equilibration for 3 days at 4°C. The samples were centrifuged for 5 min and the decanted supernatants were taken. The

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total polyphenol contents were determined using the Folin-Ciocalteau reagent similarly to the

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procedure used for the standardization. Polyphenol concentrations were expressed as mg of quercetin equivalents for 100 g of diet dry mass. The cafeteria diet (172 mg equiv.

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quercetin/100 g dry matter) was found to be slightly enriched in total polyphenols with

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respect to the normal diet (150 mg equiv. quercetin/100 g dry matter). It is noteworthy that the Folin-Ciocalteau reagent is a commonly used reducing reagent that will also target nonpolyphenolic compounds that might be present in the two diets.

2.5. Blood and tissue samples At the end of the experimentation period (14 weeks) and after overnight fasting, rats were anaesthetized with intraperitoneal injection of sodium pentobarbital (60 mg/kg of body weight). The blood was drawn from the abdominal aorta into heparinised tubes, and plasma was used for biochemical determinations. After removal of plasma, erythrocytes were washed with isotonic saline. Erythrocytes were lysed with ice-cold distilled water, stored at 4°C for 15

ACCEPTED MANUSCRIPT min and the cell debris was removed by centrifugation (2000 g for 15 min). Erythrocyte lysates were assayed for oxidant/antioxidant markers.

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The liver, gastrocnemius muscle and abdominal adipose tissues were removed, washed with

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ice-cold saline, quickly blotted and weighed. An aliquot of tissues was homogenized in 10 volumes of ice-cold 10 mmol/l phosphate-buffered saline (pH 7.4) containing 1.15% KCl.

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The homogenates were subjected to a 6000 g centrifugation at 4°C for 15 min. The supernatant fractions were collected and used for biochemical and redox markers

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determinations.

A second aliquot of tissues was homogenized in 0.9% (w/v) NaCl containing heparin (Sigma, St. Louis, MO, U.S.A) and used for lipoprotein lipase (LPL) activity. Another aliquot of adipose tissue portion was homogenized in ice cold buffer containing 0.25

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M sucrose, 1 mM dithiothreitol and 1 mM EDTA, pH 7.4, supplemented with 20 mg/ml

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leupeptin, 2 mg/ml antipain and 1 mg/ml pepstatin, and was used for the adipose hormone-

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sensitive lipase (HSL) assay.

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2.6. Determination of hemoglobin and biochemical parameters Blood cell hemoglobin levels were measured using a hemoglobinometer (HemoCue Ltd., Dronfield, United Kingdom). Plasma glucose, plasma and tissue triglyceride and cholesterol were measured using colorimetric enzymatic kits (Sigma, St. Louis, MO). Plasma iron was measured by a direct colorimetric assay based on the generation of an ironferrozine complex without deproteinization (JTC Diagnosemitte UG, Germany). A weak acid buffer dissociates iron from the transferrin-iron complex and is reduced by a reductant. Ferrous ions form a complex with the chromogen ferrozine which the colour intensity is proportional to the iron concentration in the sample.

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2.7. Determination of markers of the oxidant/antioxidant status

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Plasma vitamin C levels were determined using dinitrophenylhydrazine (DPPH) as previously

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described [32].

Nitric oxide (NO) was determined by the method of Guevara et al. [33], after plasma

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deproteinizing procedure (using methanol: diethylether; 3:1 mixture v/v). Nitrite and nitrate levels were measured together; nitrate being previously transformed to nitrite by cadmium

colorimetric method of Griess.

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reduction. Nitrite was assayed directly spectrophotometrically at 492 nm, using the

The determination of the superoxide anion (O2-) was based on Nitro Blue Tetrazolium (NBT) reduction in monofarmazan by O2-. The blue formazan was dissolved using 2 M potassium

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hydroxide and dimethylsulfoxide and its formation was monitored spectrophotometrically at

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560 nm using the molar extinction coefficient (1.5 x 104 M-1 cm-1). The catalase activity (CAT, EC 1.11.1.6) was measured by spectrophotometric analysis of the

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decomposition rate of hydrogen peroxide. Change in absorbance in the presence of

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erythrocyte lysate or tissue homogenate was recorded at 240 nm. Hemolysate and tissue homogenate reduced glutathione (GSH) levels were assayed by a colorimetric method based on the reduction of 5,5-dithiobis-(2- nitrobenzoic) acid (DTNB) by GSH to generate 2-nitro-5-thiobenzoic acid which displays a yellow colour, according to Sigma Aldrich Kit (Saint Louis, MO, USA). Hemolysate and tissue homogenate malondialdehyde (MDA) levels, a marker of lipid peroxidation, were determined by the reaction of MDA with thiobarbituric acid. Hemolysate and tissue homogenate carbonyl proteins (markers of protein oxidation) were assayed by the 2,4-dinitrophenyl hydrazine (DPPH) reaction.

ACCEPTED MANUSCRIPT 2.8. Liver, muscle and adipose tissue lipolytic activities To

estimate

adipose

hormone

sensitive

lipase

(HSL;

EC

3.1.1.3)

activity,

a

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spectrophotometric esterase assay based on the hydrolysis of PNPB (p-nitrophenylbutyrate)

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was used. Hepatic triglyceride lipase (HTGL), muscle and adipose tissue lipoprotein lipase (LPL) activities were assayed in the supernatants containing heparin-releasable lipases. We

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have previously reported details on these enzymatic methods [30,31].

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2.9. Statistical analysis

Results are expressed as means ± SD. The results were tested for normal distribution using the Shapiro–Wilk test. Data not normally distributed were logarithmically transformed. Data were analyzed using a two-way analysis of variance to determine differences between the diets

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(standard or cafeteria), the various treatments (without, iron, quercetin, and iron-quercetin

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complex) with a level of significance of p < 0.05. When significant changes were observed in ANOVA tests, Tukey multiple range test was applied to identify the specific significant

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differences between each pair. These calculations were performed using STATISTICA

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version 4.1 (STATSOFT).

3. RESULTS

3.1. Effects of treatments on body and tissue weights in rats Body weight, weight gain, relative liver and adipose tissue weights were significantly increased in obese rats compared to controls (group O versus group C). Treatment with quercetin as well as with quercetin/iron complexes had no effects on body weight, weight gain and relative tissue weights in control rats (Table 1). In contrast, these treatments induced a significant decrease in body weight, weight gain and relative liver and adipose tissue weights in obese rats. Moreover, iron treatment increased body weight, weight gain and relative tissue

ACCEPTED MANUSCRIPT weights in control rats, with no effects in obese rats. Relative muscle weight didn’t change in obese rats. In addition, treatment with quercetin as well as with quercetin/iron complexes had

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no effects on relative muscle weight.

3.2. Effects of treatments on hemoglobin, plasma iron, glucose and lipid levels in rats

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Hemoglobin levels were significantly low in obese rats compared to controls (Table 2). Treatments had no effects on hemoglobin levels in controls. In obese rats, treatment with

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quercetin had no effects while iron and quercetin/iron complex treatments increased hemoglobin concentrations. Plasma iron concentration was significantly decreased while plasma glucose, cholesterol and triglyceride levels were significantly increased in obese rats compared to controls (Table 2). This is in contrast with the enriched total level of iron

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measured by ICP-OES in the cafeteria diet compared to the standard diet and clearly indicates

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that the iron metabolism is altered in obese rats. The iron/zinc contents in the cafeteria and standard diets were indeed found to be 121±2/60±1 ppm and 90±1/66±1 ppm, respectively.

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The copper levels were found to be lower than the LOD and were not taken into account.

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Although quercetin treatment did not affect plasma iron levels, iron and quercetin/iron complex treatments enhanced plasma iron concentrations in control and obese rats, the highest effect being observed with iron treatment. This indicates that the provided iron either alone or under complexed state with quercetin is easily bioavailable while that originating from the diets is less accessible (bound to proteins or to various chelators). For quercetin or quercetin/iron complex supplementations, plasma glucose and lipid levels were not affected in control rats, while they were significantly reduced in obese rats. Iron treatment induced a significant increase in these parameters in both control and obese rats.

3.3. Effects of treatments on hepatic, muscle and adipose lipid contents in rats

ACCEPTED MANUSCRIPT Liver and adipose tissue triglyceride and cholesterol and muscle triglyceride contents were significantly increased in obese rats when compared to controls (Tables 3 and 4). Both

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quercetin and quercetin/iron complex had no effects on hepatic and muscle triglycerides and

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hepatic and adipose tissue cholesterol contents in control rats. These two treatments caused a significant reduction in muscle and adipose tissue triglycerides in controls, and in liver and

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adipose tissue triglyceride and cholesterol contents in obese rats. However, iron treatment significantly increased liver and adipose tissue lipid contents and muscle triglycerides in both

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control and obese rats. Muscle cholesterol contents did not change with diet or with treatment (Table 3).

3.4. Effects of treatments on hepatic, muscle and adipose lipase activities in rats

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Hepatic lipase and adipose tissue LPL activities were increased while adipose HSL activity

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was decreased in obese rats when compared to controls (Figure 1). Quercetin and quercetin/iron complex treatments have no effects on hepatic lipase and HSL activities, but

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induced a significant reduction in adipose LPL activity in control rats. Iron treatment

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enhanced hepatic and adipose LPL activities in control rats. Obese rats treated with both quercetin and quercetin/iron complex showed reduced hepatic lipase and adipose tissue LPL activities and enhanced adipose HSL activity compared to non treated obese rats. Iron treatment induced a significant rise in hepatic and adipose LPL activities in obese rats. Whatever the diet or the treatment, muscle LPL did not change significantly in the rats (Figure 1).

3.5. Effects of treatments on plasma oxidant/antioxidant markers in rats Plasma vitamin C, erythrocyte GSH levels and catalase activity were reduced in obese rats when compared to controls (Table 5). In control rats, iron, quercetin and quercetin/iron

ACCEPTED MANUSCRIPT complex treatments had no effects on vitamin C concentrations. GSH levels and catalase activity were reduced by iron treatment, and were not affected by quercetin and quercetin/iron

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complex treatments in control rats. In obese rats, iron treatment induced a significant decrease

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in catalase activity while quercetin and quercetin/iron complex treatments caused a significant increase of vitamin C, GSH levels and catalase activity; the effect being more marked with

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quercetin/iron complex.

Plasma O2-, NO, erythrocyte MDA and carbonyl protein levels were enhanced in obese rats

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when compared to controls (Table 4). Iron treatment induced a significant rise in plasma O2-, NO, erythrocyte MDA and carbonyl protein levels in both control and obese rats. Quercetin and quercetin/iron complex treatments significantly decreased plasma O2- concentrations but had no effects on plasma NO, erythrocyte MDA and carbonyl protein levels in control rats. In

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obese rats, all oxidative markers were reduced by quercetin and quercetin/iron complex

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treatments; the effect being more pronounced with quercetin/iron complex.

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3.6. Effects of treatments on liver and adipose tissue oxidant/antioxidant status in rats

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Liver GSH contents and catalase activity were reduced while liver MDA and carbonyl protein amounts were increased in obese rats compared to control values (Figure 2). In control rats, iron treatment induced a significant reduction in hepatic GSH and catalase activity and a significant increase of hepatic MDA and carbonyl protein contents while quercetin and quercetin/iron complex treatments had virtually no effects. In obese rats, iron supplementation caused a significant fall in hepatic catalase activity and a rise in hepatic MDA and carbonyl proteins while quercetin and quercetin/iron complex treatments increased hepatic GSH and catalase activity and decreased hepatic MDA and carbonyl proteins; the effect being noticeably more pronounced with quercetin/iron complex.

ACCEPTED MANUSCRIPT Adipose tissue GSH contents and catalase activity were reduced while adipose MDA and carbonyl protein amounts were increased in obese rats compared to control values (Figure 2). quercetin

and

quercetin/iron

complex

treatments

affected

adipose

tissue

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Iron,

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oxidant/antioxidant status in a manner similar to that observed for the liver in both control and obese rats. In fact, control rats treated with iron showed a significant reduction in adipose

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tissue GSH and catalase activity and a significant rise in adipose MDA and carbonyl protein contents. Quercetin and quercetin/iron complex treatments induced no effects in controls.

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Obese rats treated with iron displayed decreased adipose catalase activity and increased MDA and carbonyl protein levels. Obese rats treated with quercetin or quercetin/iron complex presented an increase in adipose GSH and catalase activity and a decrease in adipose MDA and carbonyl proteins; the effect being once again more pronounced with quercetin/iron

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complex.

3.7. Effects of treatments on muscle oxidant/antioxidant status in rats

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Muscle catalase activity was reduced while muscle MDA and carbonyl protein amounts were

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increased in obese rats compared to control values (Table 6). Iron treatment induced a significant reduction in catalase activityin control rats and a significant increase in muscle MDA and carbonyl proteins in both control and obese rats. Quercetin and quercetin/iron complex treatments induced a significant increase in catalase activity with a decrease in muscle MDA and carbonyl protein contents.Whatever the diet or the treatment, muscle GSH did not change significantly.

4. DISCUSSION Owing to its unique chemical properties, iron plays a central role in biology, and is an essential trace nutrient for most of the known living organisms. It is involved in many

ACCEPTED MANUSCRIPT fundamental enzymatic functions, such as oxygen metabolism, electron-transfer processes, and synthesis of DNA or RNA. However, even though iron is vital for life, it is also highly

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reactive and can be toxic when present in excess. As a consequence, iron overload,

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encountered for example in the genetic disease hemochromatosis, or in patients submitted to regular blood transfusions can lead to important disorders due to iron-induced oxidative

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stress. This redox metal is also assumed to be involved in Parkinson's disease or Friedrich ataxia due to its ability to catalyze Fenton-type reactions under its ferrous state. On the other

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hand, iron deficiency causes anemia which has been clearly linked to pathological disorders such as obesity [34]. Obesity and iron deficiency are two of the most common nutritional disorders worldwide.

There are strong evidences that polyphenols act as ROS scavengers and as pro-oxidant metals

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chelators (e.g. iron and copper). They have thus the potential to modulate physiological

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reactions involving iron and other transition metals [35]. According to specific structural characteristics, flavonoids (belonging to the polyphenol family) can indeed firmly chelate a

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broad range of metal ions of biological interest (iron, copper and zinc, to cite a few) to form

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complexes that display new pharmacological activities or enhance their intrinsic pharmacological activities. The antioxidant properties of flavonoids are not only based on their free-radical scavenging ability, but also on their abilities to strongly chelate redox-active transition metal ions [36]. Formation of metal complexes plays multiple roles in biological systems. Quercetin complexing capacity, widely used for elucidating the structure of natural flavonoids, can also contribute to the bioactivity of these compounds, by acting as providers/carriers or regulators of metal concentrations [37]. Our results showed that cafeteria induced obese rats displayed increased body weight,adiposity and a metabolic-syndrome-like phenotype characterized by hyperglycemia, hyperlipidemia and oxidative stress, as reported previously [30,31-32]. Regardless of the iron

ACCEPTED MANUSCRIPT supply, they also presented iron deficiency and low haemoglobin concentrations, in agreement with previous studies [13,38,39], indicating that the regulation/homeostatic pathways are

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altered. Indeed, obese rats presented significant increases in hepatic and adipose tissue lipids

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with alterations in lipases activities such as an increase in hepatic and adipose lipase activities and a decrease in adipose hormone sensitive lipase activity. In these obese rats, increased liver

SC

and adipose tissue weight and lipid contents were concomitant with the increase in enzyme activities involved in lipid storage such as LPL, as previously reported [30,31]. Reduced HSL

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was an additional factor for maintenance of increased fat stores. An impaired lipolysis and a reduced HSL expression in adipocytes are observed in obesity [40]. Obese rats also displayed muscle triglyceride accumulation despite normal muscle LPL activity, as previously reported [41].

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In the herein conducted experiments, cafeteria-diet-fed obese rats had an imbalanced

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oxidant/antioxidant system and an enhanced oxidative stress. This is in striking contrast since the total polyphenol content was found to be higher than in the standard diet. Oxidative

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markers such as superoxide anion O2-, nitric oxide NO, MDA and carbonyl proteins were

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elevated in obese rats, while the antioxidant markers such as vitamin C, GSH and catalase activity were significantly reduced. These obese rats presented also an intracellular oxidative stress. In fact, the elevated levels of hepatic, muscle and adipose MDA and protein carbonyls suggested an increased organ lipid peroxidation and protein oxidation, in agreement with previous studies [3,4-12]. In animal models, mitochondrial dysfunction has been reported, which is at least contributory, if not causal, to the development of obesity-related disorders [42]. Oxidative stress in cafeteria fed rats may be generated by exacerbated nutrient oxidation. The levels of tissue glutathione (GSH) and the activity of catalase, one of the most important cellular antioxidant defence mechanisms, were also reduced in obese rats. A fall in GSH contents and in catalase activity in obesity has been previously established [32].

ACCEPTED MANUSCRIPT All these alterations were worsened by the combined effects of cafeteria diet–induced obesity and iron supplementation because OFe rats presented the highest glucose and lipid values

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compared with the other groups of rats. It has been shown that the high-fat diet and iron

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supplementation acted synergistically on glucose and lipid metabolisms [43]. In fact, iron supplementation induced its own accumulation in target tissues such as liver, muscle,

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pancreas and adipose tissue with generation of ROS, decreased insulin-stimulated glucose transport and glucose uptake because of tissue damage, reduced glucose oxidation in skeletal

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muscle, increased hepatic glucose output, and affected pancreas insulin synthesis and secretion and led to insulin resistance. All these abnormalities contribute to increase blood glucose [43,44]. Oxidative stress was also worsened by iron supplementation in obese rats. The pro-oxidant properties of iron have been well established by previous studies [45]. Our

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findings further support the pro-oxidant effects of iron in the liver, the muscle and the adipose

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tissue. Iron supplementation increased lipid and protein oxidation and reduction of the GSH levels and catalase activity in these organs. Iron treatment has been reported to cause lipid

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peroxidation in rat tissue [46]. The liver is the organ most likely to be distressed by iron

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overload. It is clear that iron overload induced oxidative stress by promoting both ROS production and reduced expression of antioxidant enzymes as previously reported [43]. It is noteworthy that iron treatment also induced lipid and redox alterations in control rats, but at a lesser extent than those observed in obese rats. These finding confirmed previous studies showing that both high fat diet and iron supplementation affected unfavourably lipid and glucose metabolism [43]. Iron excess is believed to generate oxidative stress, consequently to an increase in the steady state concentration of ROS and RNS [46]. In our study, daily treatment with iron increased superoxide anion O2- and NO concentrations in both control and obese rats. It has been hypothesized that hydroxyl radicals may be formed via the Fenton reaction in the presence of

ACCEPTED MANUSCRIPT free redox active iron and react immediately with any surrounding biomolecules, such as lipid and protein forming protein carbonyl and MDA thereby increasing total oxidant stress [47].

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Increased NO generation has been also evidenced in the liver under conditions of acute iron

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overload [48]. Taken together, our finding confirmed previous studies demonstrating that excess iron intake could be a causal factor for the development of Type 2 diabetes and

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metabolic syndrome [43]. Indeed, several clinical and experimental studies have reported an increased level of oxidative stress induced by iron therapy with ferrous sulphate [49]. On the

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other hand, we observed that both quercetin alone and quercetin-iron complex prevented the body weight gain and adipose fat accumulation and alleviated the hyperglycemia, the hyperlipidemia, liver steatosis and muscle triglyceride accumulation in cafeteria fed obese rats. These results were in agreement with previous studies [50]. It has been reported that

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quercetin reduced lipid accumulation in 3T3-L1 adipocytes [18,51]. Quercetin also attenuated

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lipid accumulation through down-regulation of PPARγ and C/EBPα and induced apoptosis through suppression of ERK1/2 phosphorylation and activation of the mitochondria pathway

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[51]. In mice fed with a Western diet, quercetin reduced fat accumulation in the liver and the

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expression of steatosis-related genes [50]. Although quercetin has never been shown to promote the excretion of sterol, it may also affect the intestinal lipid absorption [50]. There is growing evidence that quercetin acts by altering hepatic cholesterol absorption and triglyceride assembly and secretion as well as through inhibition of phosphodiesterase in both adipose tissue and liver [20]. It has been reported that at high dose, quercetin has antiinflammatory effects in the visceral adipose tissue and reduced body weight gain in obese Zucker rats [20]. In addition, quercetin improved glycemic control by the reduction of intestinal glucose absorption at the level of glucose transporters (GLUT), by the increase of glucokinase activity converting glucose into glucose-6-phosphate, a metabolite destined to glycogen synthesis, by the increase in the number of pancreatic islets improving insulin

ACCEPTED MANUSCRIPT secretion, and by metal chelating activity thereby removing a causal factor for the development of free radicals [18,20,52].

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The high contents of liver TG and TC could be referred to the ability of ROS to block TG

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secretion into the plasma and disturb cholesterol catabolism into bile acids. Quercetin or quercetin-iron complexes supplementation reduced liver TG, liver TC and almost normalized

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the lipid profile in obese rats. Indeed, quercetin alone and quercetin-iron complex induced a decrease in liver and adipose LPL and an increase in adipose HSL, suggesting a reduction in

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fat storage and an enhancement in lipolysis. Quercetin–iron complexes showed enhanced beneficial effects compared to quercetin taken alone [53]. In addition, iron deficiency in obese rats was clearly corrected by quercetin –iron complexes.

Our finding demonstrated that treatments with either quercetin or quercetin-iron complexes

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alleviated oxidative stress in obese rats, by reducing oxidant markers and increasing

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antioxidant defence. This elevation in antioxidants may be due to neutralization of ROS as quercetin has been found to scavenge superoxide anions and other free radicals in vitro [54].

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In addition it has been reported, that quercetin decreased the levels of NO through inhibition

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of inducible nitric oxide synthase (iNOS) over-expression [55]. Quercetin and quercetin-iron complexes administration prevented the increase of liver, muscle and adipose MDA and carbonyl protein levels and the depletion of tissue GSH content in obese rats. Furthermore, quercetin and quercetin-iron complexes restored the reduced activities of hepatic, muscle and adipose catalase in these obese rats. Quercetin is recognized to have a strong scavenging activity of oxygen radicals and protection against lipid and protein oxidation which has been primarily attributed to its flavonoid fraction [56]. Quercetin influences important regulators of fat accumulation and metabolic disorders, especially, by offsetting mitochondrial dysfunction and oxidative stress [50].

ACCEPTED MANUSCRIPT Metal ion chelation has generated increased interest, as experimental data have shown that phenols within metal complexes exhibit greater antioxidant activity than the free flavonoids

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[53]. Iron- quercetin complexes have marked impact on the reduction of toxic metals

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bioavailability, and decreased oxidative stress throughout the whole body. These complexes are nontoxic and readily cross the cell membranes via GLUTs, so they easily permeate into

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the cell cytosol. However, quercetin at high concentration inhibited both its own membrane transport and then its scavenging activity. Similarly albumin, which binds quercetin, is known

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to increase the rate of quercetin loss from cells by preventing reuptake. These mechanisms did not exist with complexes. This may improve the efficacy of complexes [57,58]. Our finding showed that in addition to increasing plasma iron contents, quercetin-iron complexes can reduce obesity-related metabolic alterations and, in doing so, reduce iron

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redox activity. To the extent that supplementation with iron in the form of ferrous sulfate in

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iron deficiency induced several alterations implied that iron at therapeutic levels also can exaggerate long-term obesity related complications. The use of quercetin-iron complexes

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rather than ferrous sulfate can constitute a smart alternative and might confer an advantage by

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correcting iron deficiency and by reducing obesity related complications. Indeed, the use of quercetin-iron complexes can also improve several iron metabolism parameters in obesity such as transferrin, ferritin and hepcidin levels. Further studies are needed to investigate this issue. In conclusion, the data presented herein showed a protective role of quercetin alone and quercetin-iron

complex

against

obesity related

metabolic

alterations

through

its

hypoglycemic, hypolipidemic and antioxidative effects. However, the effects of quercetiniron complex were stronger in addition to the correction of iron deficiency without iron toxicity. The prophylactic effect of quercetin-iron complex highlights a promising strategy for obesity treatment.

ACCEPTED MANUSCRIPT

ACKNOWLEDGEMENTS

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This work was supported by the French-Algerian Cooperation Program PHC Tassili

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International Research Extension Grant TASSILI 13MDU 892 and the French Foreign Office (Campus France). This work was partly supported by the CNRS (UMR 7509), the

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University of Strasbourg and the University Abou-Bekr Belkaïd. The authors thank Dr. Anne-Boos (UMR 7178 CNRS-Unistra, Strasbourg, France) for the ICP-MS measurements.

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The authors also warmly thank Dr Elisabeth Davioud-Charvet (UMR 7509 CNRS-Unistra, Strasbourg, France) for the fruitful discussions and interesting comments on this manuscript. The authors thank Dr Meriem Saker (University of Tlemcen) for linguistic help with the

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manuscript.

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COMPETING INTERESTS STATEMENT

[1]

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The authors have no conflicting financial interests.

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ACCEPTED MANUSCRIPT Table 1. Body weight, Weight gain, liver, muscle and adipose tissue relative weight in the rats Body weight

Relative

Relative

Relative

Adipose tissue

muscle

weight

weight

liver weight

C

286.25±29.72 c

56.54±5.28 d

3.44±0.14 c

0.81±0.03 c

0.68±0.05

CQ

312.75±27.92 c

54.26±3.41 d

3.41±0.16 c

0.82±0.02 c

0.67±0.05

CFe

364.75±19.14 b

108.11± 8.75 c

3.85±0.11 b

1.17±0.05 b

0.63±0.06

CQFe

300.25±20.27 c

58.55 ± 5.21 d

3.57±0.12 c

0.80±0.03 c

0.66±0.04

O

496±15 a

254.32±14.34 a

4.85±0.10 a

1.98±0.11a

0.66±0.04

OQ

341.50±17.78 b

157.29±13.44 b

3.39±0.23 c

0.99±0.04 b

0.64±0.04

OFe

470 ±20.08 a

240.45±15 a

4.51±0.21 a

1.94±0.12 a

0.67±0.03

OQFe

352.50±28.43 b

160.47±12.50 b

3.46±0.11 c

1.08±0.05 b

0.66±0.05

Diet

0.005

0.004

0.010

0.008

0.122

Treat

0.004

0.005

0.010

0.006

0.136

(ANOVA)

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P

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Obese rats

ment

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rats

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Control

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T

(g)

Weight gain (g)

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Values are presented as means ± SD. C: control rats; CQ: control – quercetin treated rats; CFe: control – iron treated rats; CQFe: control – quercetin/iron complex treated rats; O: obese

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rats; OQ: obese – quercetin treated rats; CFe: obese – iron treated rats; CQFe: obese – quercetin/iron complex treated rats. Data were tested by two-way ANOVA and Tukey post hoc tests. Values with different superscript letters (a, b, c, d, ……) are significantly different according Tukey test at P < 0.05.

ACCEPTED MANUSCRIPT Table 2. Hemoglobin, plasma iron, glucose and lipid levels in the rats

0.74±0.15 d

142.74±19.58 d

98.56±6.98 e

CQ

11.89±0.86 a

172.24±14.33 c

0.69±0.14 d

159.54±17.07 d

89.72±6.72 e

CFe

12.66±1.28 a

243±14.08 a

1.40±0.15 b

208.22±18.48 b

133.02±8.44 c

CQFe

12.74±1.33 a

198.07±15.75b

1.08±0.23 d

160.41± 10.48 d 88.72±8.84 e

8.33±0.64 c

102±15.45 d

1.55±0.11 b

219.17±11.64 b

172.17±11.72 b

8.65±0.83 c

114.09±13.22d

1.26±0.13 c

192.21±10.29 c

114.07±13.13 d

10.84±1.05 b

208.14±15.06b

1.78±0.15 a

242.15±16.51 a

209.66±17.80 a

187±18.25 c

1.21±0.14 c

190.15±16.85 c

112.44±18.18 d

0.010

0.001

0.006

0.008

0.006

0.007

0.002

0.004

0.005

0.006

OFe

ED

rats

OQ

12.44±1.25 a

PT

OQFe

(ANOV

Treatment

AC

A)

Diet

CE

P

RI P

175.87±17 c

O

Obese

(mg/dL)

12.5±1.43 a

SC

rats

Triglycerides

C

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Control

Iron(µg/dL)

Total cholesterol (mg/dL)

T

Hb (g/dL)

Glucose (g/L)

Values are presented as means ± SD. C: control rats; CQ: control – quercetin treated rats; CFe: control – iron treated rats; CQFe: control – quercetin/iron complex treated rats; O: obese rats; OQ: obese – quercetin treated rats; CFe: obese – iron treated rats; CQFe: obese – quercetin/iron complex treated rats. Data were tested by two-way ANOVA and Tukey post hoc tests. Values with different superscript letters (a, b, c, d, ……) are significantly different according Tukey test at P < 0.05.

ACCEPTED MANUSCRIPT

21.80±1.62 e

21.56±2.21 c

CQ

20.09±1.33 e

20.43±1.49 c

15.50±2.30

CFe

42.21±2.56 b

53.64±2.91 b

18.17±2.24

CQFe

21.73±1.89 e

20.78±1.12 c

16.70±1.22

O

35.99±1.59 c

55.80±1.89 b

15.27±1.75

OQ

26.48±1.49 d

20.88±1.87 c

16.68±1.46

OFe

48.50±1.95 a

62.76±1.31 a

15.11±2.48

OQFe

28.82±2.51 d

21.54±1.12 c

15.75±2.21

0.001

0.003

0.145

0.003

0.005

0.187

Obese rats

P

Treatment

PT

Diet

(ANOVA)

Muscle Cholesterol (mg/g)

RI P

T

C

SC

rats

AT Cholesterol (mg/g)

MA NU

Control

Liver Cholesterol (mg/g)

ED

Table 3. Cholesterol contents of liver, muscle and adipose tissue in the rats

16.45±1.46

CE

Values are presented as means ± SD. C: control rats; CQ: control – quercetin treated rats; CFe: control – iron treated rats; CQFe: control – quercetin/iron complex treated rats; O: obese

AC

rats; OQ: obese – quercetin treated rats; CFe: obese – iron treated rats; CQFe: obese – quercetin/iron complex treated rats. Data were tested by two-way ANOVA and Tukey post hoc tests. Values with different superscript letters (a, b, c, d, ……) are significantly different according Tukey test at P < 0.05.

ACCEPTED MANUSCRIPT

53.60±3.38 e

112.83±5.40 e

CQ

54.78±4.04 e

84.31±2.07 f

36.50±2.35

CFe

78.49±4.71 c

153.83±7.38 c

63.72±3.98

CQFe

55.13±3.48 e

86.29±1.56 f

34.83±2.55

O

96.83±5.76 b

236.83±23.32 b

62.17±4.13

OQ

76.41±2.39 c

121.83±6.69 d

47.08±1.12

OFe

119.05±6.58 a

278.17±28.44 a

83.18±2.18

OQFe

67.04±2.91 d

108±8.71 e

40.74±1.32

0.004

0.001

0.005

0.001

0.001

0.004

Obese rats

P

RI P

34±2.23

e

e

b

e

b c a

d

CE

Treatment

PT

Diet

(ANOVA)

Muscle Triglycerides (mg/g)

T

C

SC

rats

AT Triglycerides (mg/g)

MA NU

Control

Liver Triglycerides (mg/g)

ED

Table 4. Triglyceride contents of liver, muscle and adipose tissue in the rats

AC

Values are presented as means ± SD. C: control rats; CQ: control – quercetin treated rats; CFe: control – iron treated rats; CQFe: control – quercetin/iron complex treated rats; O: obese rats; OQ: obese – quercetin treated rats; CFe: obese – iron treated rats; CQFe: obese – quercetin/iron complex treated rats. Data were tested by two-way ANOVA and Tukey post hoc tests. Values with different superscript letters (a, b, c, d, ……) are significantly different according Tukey test at P < 0.05.

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Table 5. Plasma and erythrocyte antioxidant/oxidant markers in the rats NO

MDA

(µmol/L)

T

(µmol/L)

(µmol/g protein)

4.24±0.35 d

1.44±0.24 d

3.60±0.41 d

4.04±0.93 d

1.51±0.25 d

3.85±0.38 d

19.77±1.02 c

7.43±0.56 c

2.87±0.13 c

4.45±0.30 c

5.29±0.53 f

4.37±0.53 d

1.34±0.23 d

3.70±0.25 d

25.67±1.47 b

9.84±0.66 b

4.98±0.33 b

5.30±0.22 b

4.99±0.28 d

2.59±0.32 c

4.06±0.24 c

12.43±0.33a

6.20±0.43 a

6.87±0.23 a

3.25±0.37e

1.41±0.28 d

3.88±0.20 d

(U/gHb)

C

20.98±1.74 c

7.72±0.51 a

159.53±15.64 a

8.11±0.62 e

CQ

19.31±1.23 c

7.87±0.89 a

155.14±13.71 a

5.88±0.75 f

CFe

20.09±1.81 c

3.13±0.36 c

102±11.96 b

CQFe

22.61±2.55 c

7.93±0.79 a

153.02±10.34 a

O

16.71±1.40 d

2.81±0.48 c

OQ

30.96±1.47 b

5.20±0.40 b

OFe

15.71±1.79 d

2.61±0.45 c

OQFe

38.54±1.27 a

0.001

Treatmen

0.002

75.11±7.09 c

0.005 0.001

158.51±12.23 a

14.85±1.19 d 30.83±1.17 a

12.02±1.03 e

0.004

0.002

0.004

0.003

0.005

0.004

0.001

0.005

0.001

0.004

CE

t

6.89±0.85 a

PT

P (ANOVA) Diet

148.66±12.23a

ED

Obese rats

(µmol/L)

RI P

(µmol/gHb)

96.67±8.64 b

CARP

O2-

SC

rats

Catalase

MA NU

Control

GSH

Vitamin C (µmol/L)

Values are presented as means ± SD. C: control rats; CQ: control – quercetin treated rats; CFe: control – iron treated rats; CQFe: control – quercetin/iron complex treated rats; O: obese

AC

rats; OQ: obese – quercetin treated rats; CFe: obese – iron treated rats; CQFe: obese – quercetin/iron complex treated rats; CARP: carbonyl proteins; GSH: reduced glutathione; MDA: malondialdehyde; NO: nitric oxide; O2-: superoxide anion. Data were tested by two-way ANOVA and Tukey post hoc tests. Values with different superscript letters (a, b, c, d, ……) are significantly different according Tukey test at P < 0.05.

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Muscle CAPR (nmol/g)

C

33.77±2.12b

1.33±0.12

1.32±0.07d

1.25±0.04e

CQ

38.18±1.01a

1.45±0.13

1.27±0.06d

0.87±0.06f

CFe

18.55±1.04c

1.08±0.21

1.68±0.05c

1.71±0.05d

CQFe

38.66±1.21a

1.43±0.13

1.02±0.06e

1.09±0.03f

O

15.94±1.51d

1.12±0.22

2.50±0.10b

3.30±0.28b

OQ

40.64±2.19a

1.39±0.15

1.75±0.05c

1.97±0.06c

OFe

16.58±1.08d

1.17±0.15

3.61±0.11a

3.88±0.27a

OQFe

40.75±2.01a

1.50±0.17

1.14±0.07e

1.32±0.08e

Diet

0.004

0.123

0.003

0.003

0.008

0.145

0.004

0.004

P (ANOVA)

SC

PT

Treatment

T

Muscle MDA (nmol/g)

MA NU

Obese rats

Muscle GSH (nmol/g)

ED

Control rats

Muscle Catalase (U/g)

RI P

Table 6. Muscle antioxidant/oxidant markers in the rats

CE

Values are presented as means ± SD. C: control rats; CQ: control – quercetin treated rats; CFe: control – iron treated rats; CQFe: control – quercetin/iron complex treated rats; O: obese

AC

rats; OQ: obese – quercetin treated rats; CFe: obese – iron treated rats; CQFe: obese – quercetin/iron complex treated rats; CARP: carbonyl proteins; GSH: reduced glutathione; MDA: malondialdehyde; NO: nitric oxide; O2-: superoxide anion. Data were tested by two-way ANOVA and Tukey post hoc tests. Values with different superscript letters (a, b, c, d, ……) are significantly different according Tukey test at P < 0.05.

PT

ED

MA NU

SC

RI P

T

ACCEPTED MANUSCRIPT

CE

Figure 1. Liver, muscle and adipose tissue lipase activities in the rats Values are presented as means ± SD. C: control rats; CQ: control – quercetin treated rats; CFe: control – iron treated rats; CQFe: control – quercetin/iron complex treated rats; O: obese

AC

rats; OQ: obese – quercetin treated rats; CFe: obese – iron treated rats; CQFe: obese – quercetin/iron complex treated rats. Data were tested by two-way ANOVA and Tukey post hoc tests. Values with different superscript letters (a, b, c, d, ……) are significantly different according Tukey test at P < 0.05.

AC

CE

PT

ED

MA NU

SC

RI P

T

ACCEPTED MANUSCRIPT

Figure 2. Liver and adipose tissue oxidant/antioxidant markers in the rats Values are presented as means ± SD. C: control rats; CQ: control – quercetin treated rats; CFe: control – iron treated rats; CQFe: control – quercetin/iron complex treated rats; O: obese rats; OQ: obese – quercetin treated rats; CFe: obese – iron treated rats; CQFe: obese – quercetin/iron complex treated rats. Data were tested by two-way ANOVA and Tukey post hoc tests. Values with different superscript letters (a, b, c, d, ……) are significantly different according Tukey test at P < 0.05.