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May 5, 2017 - Department of Biochemistry and Molecular Biology II, University of Granada, Granada, Spain. ABSTRACT: To date, no studies are available ...
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Changes in adiposity and body composition during anaemia recovery with goat or cow fermented milks Javier Díaz-Castro, Jorge Moreno-Fernandez, Mario Pulido-Moran, Maria J.M. Alférez, Maria Robles-Rebollo, Julio J. Ochoa, and Inmaculada López-Aliaga J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 05 May 2017 Downloaded from http://pubs.acs.org on May 6, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Graphical abstract 201x106mm (96 x 96 DPI)

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Changes in adiposity and body composition during anaemia recovery with

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goat or cow fermented milks

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Javier Diaz-Castro†,‡,§ , Jorge Moreno-Fernandez†,‡,§, Mario Pulido-Moran‡,#,§,

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María JM Alférez†,‡, María Robles-Rebollo†,‡, Julio J. Ochoa†,‡ and Inmaculada

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López-Aliaga*,†,‡

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Department of Physiology, University of Granada, Granada, Spain.

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Institute of Nutrition and Food Technology “José Mataix Verdú”, University of

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Granada, Granada, Spain.

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#

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Spain.

Department of Biochemistry and Molecular Biology II, University of Granada,

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Corresponding author:

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*Mª Inmaculada López-Aliaga, Department of Physiology, Faculty of Pharmacy,

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Campus Universitario de Cartuja, University of Granada, 18071, Granada, Spain.

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Tel: 34-958-243880, E-mail address: [email protected]

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§

These authors contributed equally to this work.

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Abstract

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To date, no studies are available about the adipose tissue modifications during

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anaemia recovery; therefore the aim of this study is to provide detailed

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information about adipose tissue homeostasis during anaemia recovery with

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fermented milks. Forty male Wistar rats were placed on a pre-experimental period

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of 40 days, divided in two groups (normal-Fe diet and Fe-deficient diet). Then rats

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were fed with fermented goat or cow milk-based diets, with normal-Fe content

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during 30 days.

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Ghrelin and adiponectin decreased in both groups of animals fed on fermented

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goat milk, while leptin and NEFA increased. UCP-1 decreased in anaemic rats

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both fed on fermented milks, and irisin greatly increased in both groups of

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animals fed on fermented goat milk.

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Fermented goat milk reduces adiposity, inducing leptin elevation and ghrelin

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reduction. Conversely plasma adiponectin concentrations decreased in animals fed

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fermented goat milk showing an inverse correlation with NEFA, an important

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marker of lipid mobilization, indicating increased lipolysis. Irisin up-regulation in

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animals fed fermented goat milk contributes to a favourable metabolic profile and

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the browning of adipose tissue during anaemia recovery.

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KEY WORDS: goat or cow fermented milks; anaemia; visceral adipose tissue;

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adipokines; irisin.

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INTRODUCTION

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Currently, in the global scenario, iron deficiency anaemia (IDA) is the most

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frequent derailment of physiology in the world. It is a serious condition in

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industrialized and semi-industrialized countries and it has becomes a very serious

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condition in poor resources countries. IDA is a major public health problem in

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which the losses of Fe or its requirement overcomes the contribution that provides

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the diet, so the Fe storages of the organism get depleted 1.

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While dietary Fe remains the major determinant of Fe status, scientific

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evidence suggests that adiposity could be also an additional determinant of Fe

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status. An inverse relationship between Fe status and body composition suggests a

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need for greater Fe requirements in individuals with a high body mass index, a

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condition that is rapidly escalating throughout the world 2. It is believed that the

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presence of excessive fat can lead to inflammatory responses and affect Fe status3.

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The Fe-containing protein, hepcidin, mediates in this mechanism by which

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inflammation causes Fe deficiency in individuals with high body fat 3.

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In mammals, adipose tissue has a key role in whole-body energy

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metabolism. Adipocytes found in adipose depots express divergent physiologies

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throughout lifespan. Considerable research efforts have been focused on adipose

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depot differences in the structure, function and/or regulation of all cells contained

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within the adipose depots and considerable interest has been shown in the

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adipocytes of visceral adipose tissue (VAT) depots due to the production of

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adipokines that appear to function in physiologies such as control of satiety,

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energy homeostasis or body composition. Knowledge pertaining to normal

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regulation, dysfunctional regulation, endocrine/adipokine production, and

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resistance of specific adipose depots to respond to metabolic signals is growing4, 5.

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Diet plays a key role in body weight and body composition, but apart from

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affecting energy balance, we still have limited understanding of the specific foods,

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and nutrients and, in this sense, dairy products comprise a major food group and

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are an important nutrient source in the diet 6. Unfortunately, the consumption of

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dairy products sometimes is discouraged by concerns related to obesity risk and

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metabolic disorders. However, several observational and cross-sectional studies

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have revealed an inverse association between dairy product consumption and

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body composition

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the consumption of these dairy products still remains unclear and not completely

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elucidated. Even in some studies that target the change, management, or

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maintenance of body weight and adiposity, contradictory results have been

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reported 9, 10.

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, but many aspects of the adipose depots physiology during

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Taking into account all these considerations, to date, no studies are

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available about the adipose tissue modifications during anaemia recovery with

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dairy products, therefore the aim of this study is to provide detailed information

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about adipose tissue homeostasis and body composition during anaemia recovery

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with fermented milks. We are particularly interested in the physiological effects of

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these adipose depots by means of several adipokines (leptin and adiponectin) and

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some hormones (ghrelin and insulin), the association of these depots with body

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composition and appetite, protein expression of uncoupling protein 1 (UCP-1) and

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irisin within the VAT. In addition, we will also try to shed light on the effects of

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fermented goat milk consumption on fat tissue, greatly influenced by irisin, as

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well as on the link between the mentioned adipokines and plasma concentrations

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of non-esterified fatty acids (NEFA), as such link works as an important marker of

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lipid mobilization from adipose tissue.

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MATERIAL AND METHODS

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Fermentation and dehydration of the milks

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To prepare the fermented milks, raw cow and goat milk was pasteurized at 77°C

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for 15 min and cooled to room temperature (25ºC). The milk samples were

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transferred to sterile Schott flasks inside a laminar flow chamber, and stored at

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4°C for 24 h before using. Subsequently, both milk types were inoculated with

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traditional yoghurt starters Lactobacillus bulgaricus sub. delbrueckii and

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Streptococcus thermophiles (initial concentration of 1×1011 cfu/mL; 1 %

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inoculum) and incubated at 37°C for approximately 24 h. At the end of

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fermentation, the fermented milks were cooled to 15°C in an ice bath, and the clot

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was broken up with a stainless steel perforated disk with up and down movements

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for approximately 1 min. The fermented milk samples were evaluated for pH by

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using digital pH meter (Crison, Barcelona, Spain) and the fermentation process

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ended when the milks reached pH=4.6. All experiments were carried out in

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triplicates.

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Subsequently, fermented milk samples were subjected to a smooth

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industrial dehydration process in an air tunnel with internal heaters mounted to the

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air in independent chambers that enable a uniform distribution of the temperature

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and a rapid stabilization. In this device, an internal engine turbine with airflow

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improved the process of dehydration. The fermented milks were dehydrated in a

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forced-air tunnel (Conterm Selecta, Barcelona, Spain) at 50 ± 3ºC for 24 h, until

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the final moisture ranged between 2.5% and 4.5%. These conditions prevent the

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loss of nutritional properties of the fermented milk samples.

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Animals

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All animal care procedures and experimental protocols were approved by the

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Ethics Committee (Ref. 11022011) of the University of Granada in accordance

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with the European Community guidelines (Declaration of Helsinki; Directive

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2010/63/EU for animal experiments). 40 male Wistar albino breed rats (21 d of

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age and weighing about 42 ± 5 g), purchased from the University of Granada

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Laboratory Animal Service (Granada, Spain) were used during the study. Animal

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assays were carried out in the animal breeding unit of the Centre of Biomedical

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Research of the University of Granada in an area certified as free of pathogens

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and the animals were kept in conditions of high biological safety, with sanitary

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and environmental rigorously controlled parameters.

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During the course of the study, the animals were housed in individual, ventilated,

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thermo-regulated cages with an automatically controlled temperature (23 ± 2ºC),

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humidity (60 ± 5%) and a 12-hour light-dark cycle (9:00 to 21:00 h). Diet intake

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was controlled, pair feeding all the animals (80% of the average intake, calculated

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previously) and deionized water was available ad libitum.

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Design of experiment and diets

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The experimental design is shown in Figure 1. At the beginning of the study 40

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rats were placed on a pre-experimental period (PEP) and divided into two groups:

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the control group receiving a normal-Fe diet (44.6 mg/kg by analysis) 11, and the

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anaemic group receiving a low-Fe diet (6.2 mg/kg by analysis), induced

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experimentally during 40 d by a method previously developed by our research

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group

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caudal vein (with EDTA to measure the haematological parameters) and the rest

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of the blood was centrifuged (1500g, 4ºC, 15 min) without anticoagulant to

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. On day 40 of the study, two blood aliquots per rat were collected from

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separate the serum and subsequent analysis of Fe, total Fe binding capacity

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(TIBC), ferritin and hepcidin.

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After the induction of the anaemia (day 40 of the study), the animals were

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placed on an experimental period (EP) in which both the control and the anaemic

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groups were additionally fed for 30 days either with fermented cow milk or with

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fermented goat milk-based diet, with normal-Fe content (45 mg/kg), prepared

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with fermented cow (Holstein breed) or fermented goat milk (Murciano-granadina

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breed) powder (20% of protein and 10% of fat). The Fe content (mg/kg) in the

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diets by analysis was 42.7 (cow milk-based diet), 43.5 (goat milk-based diet)

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(Table 1). Diet intake was controlled, pair feeding all the animals and deionized

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water was available ad libitum.

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On day 70 of the study , animals were anesthetized intraperitoneally with

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sodium pentobarbital (Sigma-Aldrich Co., St. Louis, MO), totally bled out by

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cannulation of the aorta and blood aliquots with EDTA were analysed to measure

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the haematological parameters. The rest of the blood was centrifuged (1500xg,

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4ºC, 15 min) without anticoagulant to separate the red blood cells from the serum

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and subsequent analysis of Fe, TIBC, ferritin and hepcidin. Aliquots with EDTA as

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anticoagulant were centrifuged to obtain plasma and to measure adipokines (leptin

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and adiponectin), hormones (ghrelin and insulin, thyroid-stimulating hormone,

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triiodothyronine, thyroxine) and NEFA. Perirenal fat adipose depots were

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removed, washed repeatedly with ice-cold deionized water, snap frozen in liquid

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nitrogen and immediately stored at -80°C.

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Assessment of body composition

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Whole body composition (fat and lean tissues) was determined using quantitative

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magnetic resonance (QMR) with an Echo MRI Analyzer system by Echo Medical

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Systems (Houston, TX). All QMR measurements were made during the light

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phase (09:00 A.M. - 6:00 P.M.). The animals were placed into a thin-walled

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plastic cylinder (3mm thick, 6.5 cm inner diameter) with a cylindrical plastic

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insert added to limit movement so that scans could be performed. Meanwhile, in

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the tube, animals were briefly subjected to a low-intensity (0.05 Tesla)

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electromagnetic field to measure fat, lean mass, free water, and total body water.

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Briefly, this system generates a signal that modifies the spin patterns of hydrogen

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atoms within the subject, and uses an algorithm to evaluate the four measured

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components – fat mass, lean muscle mass equivalent, total body water, and free

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water. QMR scans were performed with accumulation times of 2 minutes.

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Haematological test

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All the haematological parameters studied were measured using an automated

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haematology analyser Mythic 22CT (C2 Diagnostics, Grabels, France).

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Serum iron, total iron binding capacity (TIBC) and transferrin saturation

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To calculate the rate of transferrin saturation, serum Fe concentration and TIBC

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were determined by using Sigma Diagnostics Iron and TIBC reagents (Sigma-

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Aldrich Co., St. Louis, MO). The absorbance of samples was read at 550 nm on a

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microplate reader (Bio-Rad Laboratories Inc., Hercules, CA). The percentage of

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transferrin saturation was calculated using the equation:

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Transferrin saturation (%) = serum Fe concentration (µg/L)/TIBC (µg/L) x

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Serum ferritin

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Serum ferritin concentration was determined by using the Rat Ferritin ELISA Kit

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(Biovendor Gmbh, Heidelberg, Germany). The absorbance of the reaction was

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read at 450 nm using a microplate reader (BioTek Instruments, Winooski, VT

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USA). The developed colour intensity was inversely proportional to the

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concentration of serum ferritin.

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Serum hepcidin

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Hepcidin-25 was determined by using a DRG ELISA Kit (DRG Instruments

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GmbH, Marburg, Germany). This kit is a solid phase enzyme-linked

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immunosorbent assay (ELISA) based on the principle of competitive binding. The

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microtiter wells were coated with a monoclonal (mouse) antibody directed

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towards an antigenic site of the Hepcidin-25 molecule. After incubation, the

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unbound conjugate was washed off and a streptavidin-peroxidase enzyme

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complex was added to each well. After incubation, the unbound enzyme complex

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was washed off and substrate solution was added. The reaction was stopped with

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stop solution and the microplate was read at 450nm with a plate reader (Bio-Rad).

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The intensity of colour developed is reverse proportional to the concentration of

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hepcidin in the sample.

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Thyroid hormones, ghrelin, leptin, adiponectin and insulin measurement

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Thyroid-stimulating hormone (TSH), triiodothyronine (T3) and thyroxine (T4)

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were determined by using the RTHYMAG-30K Milliplex MAP Rat Thyroid

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Magnetic Bead Panel; ghrelin (active), leptin and insulin, were determined by

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using the RMHMAG-84K Milliplex MAP Rat Metabolic Hormone Magnetic

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Bead Panel; adiponectin levels were measured using the RADPCMAG-82K

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Milliplex MAP Rat Adipocyte Panel Metabolism Assay (Millipore Corporation,

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St. Charles, MO USA), based on immunoassays on the surface of fluorescent-

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coded beads (microspheres), following the specifications of the manufacturer (50

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events per bead, 50 µl sample, gate settings: 8000-15000, time out 60 seconds,

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melatonin bead set: 34). Plate was read on LABScan 100 analyzer (Luminex

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Corporation, Austin, TX, USA) with xPONENT software for data acquisition.

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Average values for each set of duplicate samples or standards were within 15% of

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the mean. Thyroid hormones, ghrelin, leptin, adiponectin and insulin

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concentrations in plasma samples were determined by comparing the mean of

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duplicate samples with the standard curve for each assay.

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Non-esterified fatty acids (NEFA)

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Non-esterified fatty acids (NEFA) are molecules released from triglycerides by

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the action of the enzyme lipase and are transported in the blood bound to albumin.

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They contribute only to a small proportion of the body’s fat, however they provide

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a large part of the body’s energy. NEFA were measured using a commercial kit

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(Randox Laboratories Ltd., Crumlin, UK). 50µl of standards or serum samples

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were pipetted into eppendorf tubes. Thereafter, 1 ml of the solution R1 was added

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to all tubes. The mixtures were vortexed for 5-10 seconds and incubated at 37ºC

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for 10 minutes. Immediately after, 2 ml of solution R2 were added and the tube

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was mixed and re-incubated at 37ºC. After 10 minutes, the mixture was measured

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for the optical density at 550 nm in a spectrophotometer (Bio-tek,Vermont, USA).

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In addition, all the standards, serum samples and assay-control were analysed in

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duplicate. The calculation of sample NEFA concentration fit by using the

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following equation:

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NEFA mmol/L = (Absorbance of sample/Absorbance of standard) x concentration

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of standard.

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Western-Blot analysis and immunohistochemistry

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50 mg of fat samples were added to a Potter–Elvehjem glass apparatus on ice, and

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whole cell extracts were obtained from the tissue by homogenisation in 1:20 (w/v)

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of tissue to T-PER Reagent (Thermo Scientific Inc., Hanover Park, IL, USA). It

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was centrifuged to pellet tissue debris. Protease inhibitor (1:200 dilution; Sigma-

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Aldrich, St. Louis, MO, USA) was added, avoiding protein degradation. The Total

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protein concentration was determined in extract by using a Thermo Scientific

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Pierce BCA Protein Assay Kit (Thermo Scientific Inc., Hanover Park, IL, USA).

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12 µg of total protein from the extract were loaded in 4–20% Criterion TGX (Tris-

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Glycine extended) gels (Mini-PROTEAN TGX Precast Gels, 15 µL; 15 wells;

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Bio-Rad Laboratories, Inc., Hercules, CA, USA). The electrophoresis was carried

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out at 250 V in a vertical electrophoresis tank (Mini-PROTEAN® System; Bio-

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Rad Laboratories, Inc.) for 20 min.

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A Fermentas PageRuler Plus Prestained Protein Ladder was used as

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molecular weight marker (Thermo Scientific Inc., Hanover Park, IL, USA).

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Subsequently, proteins were transferred from gel onto PVDF membrane (Bio-Rad

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Laboratories, Inc.) by wet transfer for 60 min at 120V with transfer buffer

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comprising 250 mM Trizma HCl, 200 mM glycine, and 6% methanol, pH 8.3

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(Sigma-Aldrich). Membranes were then washed 3 times in TBS, and they were

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finally incubated with rabbit polyclonal to anti-UCP1 antibody [(Abcam, UK

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(dilution 1:1000)], rabbit monoclonal anti-FNDC5 RabMAb (Irisin) antibody

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[Abcam, UK (dilution 1:800)] and mouse monoclonal to beta Actin antibody

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[Abcam, UK (dilution 1:1000)] as primary antibodies, in 5% dry milk in TTBS

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overnight at 4 °C with shaking. β-actin was used as a control for total protein

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loaded.

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Blots were then washed 3 times for 5 min each in TTBS and they were

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incubated with the appropriate secondary conjugated antibody [ImmunStar Goat

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Anti-Mouse (GAM)-HRP; 1:80,000 and Immun-Star Goat Anti-Rabbit (GAR)-

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HRP; Bio-Rad Laboratories Inc.; 1:50,000] in TTBS for 1 h at room temperature.

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The membranes were visualised with Luminata forte western HRP Substrate

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(Merck KGaA, Darmstadt, Germany). Signal quantification and recording

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densitometry of each band were performed with chemiluminescence in

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ImageQuant LAS 4000 (Fujifilm Life Science Corporation, Stamford, CT, USA).

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All results were analysed with Image J software.

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Statistical analysis

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Data are reported as means ± standard error of the mean (SEM). Statistical

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analyses were performed with the SPSS computer program (version 22.0, 2013,

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SPSS Inc., Chicago, IL). Differences between groups fed on normal-Fe- or low-

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Fe-content diets during the PEP were tested for statistical significance with

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Student’s t test. Variance analysis by Two-way ANOVA was used to assess the

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effect of the anaemia and to compare the different diets supplied to the animals.

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Following a significant F-test (P < 0.05), individual means were tested by

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pairwise comparison with the Tukey’s multiple comparison test, when main

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effects and interactions were significant. The level of significance was set at P