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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

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

Changes in Adiposity and Body Composition ... - ACS Publications

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