Changes in Adiposity and Body Composition during Anemia Recovery

Subscriber access provided by Georgetown University | Lauinger and Blommer Libraries

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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.

Page 1 of 36

Journal of Agricultural and Food Chemistry

Graphical abstract 201x106mm (96 x 96 DPI)

ACS Paragon Plus Environment


1

Changes in adiposity and body composition during anaemia recovery with

2

goat or cow fermented milks

3 4

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

5

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

6

López-Aliaga*,†,‡

7 8 9

†

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

10

‡

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

11

Granada, Granada, Spain.

12

#

13

Spain.

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

14 15 16

Corresponding author:

17

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

18

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

19

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

20 21

§

These authors contributed equally to this work.

22 23 24 25


Page 2 of 36

Page 3 of 36


26

Abstract

27 28

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

29

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

30

information about adipose tissue homeostasis during anaemia recovery with

31

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

32

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

33

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

34

during 30 days.

35

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

36

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

37

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

38

animals fed on fermented goat milk.

39

Fermented goat milk reduces adiposity, inducing leptin elevation and ghrelin

40

reduction. Conversely plasma adiponectin concentrations decreased in animals fed

41

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

42

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

43

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

44

the browning of adipose tissue during anaemia recovery.

45 46 47

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

48

adipokines; irisin.

49 50



51

INTRODUCTION

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59

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

60

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

61

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

62

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

63

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

64

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

65

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

66

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

67

metabolism. Adipocytes found in adipose depots express divergent physiologies

68

throughout lifespan. Considerable research efforts have been focused on adipose

69

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

70

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

71

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

72

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

73

energy homeostasis or body composition. Knowledge pertaining to normal

74

regulation, dysfunctional regulation, endocrine/adipokine production, and

75

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


Page 4 of 36

Page 5 of 36


76

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

77

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

78

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

79

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

80

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

81

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

82

have revealed an inverse association between dairy product consumption and

83

body composition

84

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

85

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

86

maintenance of body weight and adiposity, contradictory results have been

87

reported 9, 10.

7, 8

, but many aspects of the adipose depots physiology during

88

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

89

available about the adipose tissue modifications during anaemia recovery with

90

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

91

about adipose tissue homeostasis and body composition during anaemia recovery

92

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

93

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

94

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

95

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

96

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

97

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

98

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

99

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

100

lipid mobilization from adipose tissue.



101

MATERIAL AND METHODS

102

Fermentation and dehydration of the milks

103

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

104

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

105

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

106

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

107

traditional yoghurt starters Lactobacillus bulgaricus sub. delbrueckii and

108

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

109

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

110

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

111

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

112

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

113

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

114

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

115

triplicates.

116

Subsequently, fermented milk samples were subjected to a smooth

117

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

118

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

119

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

120

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

121

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

122

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

123

loss of nutritional properties of the fermented milk samples.

124

Animals


Page 6 of 36

Page 7 of 36


125

All animal care procedures and experimental protocols were approved by the

126

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

127

with the European Community guidelines (Declaration of Helsinki; Directive

128

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

129

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

130

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

131

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

132

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

133

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

134

and environmental rigorously controlled parameters.

135

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

136

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

137

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

138

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

139

previously) and deionized water was available ad libitum.

140 141

Design of experiment and diets

142

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

143

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

144

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

145

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

146

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

147

group

148

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

149

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

12

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



150

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

151

(TIBC), ferritin and hepcidin.

152

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

153

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

154

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

155

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

156

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

157

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

158

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

159

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

160

water was available ad libitum.

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

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

170

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

171

nitrogen and immediately stored at -80°C.

172 173 174


Page 8 of 36

Page 9 of 36


175

Assessment of body composition

176

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

177

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

178

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

179

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

180

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

181

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

182

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

183

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

184

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

185

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

186

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

187

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

188 189

Haematological test

190

All the haematological parameters studied were measured using an automated

191

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

192 193

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

194

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

195

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

196

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

197

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

198

transferrin saturation was calculated using the equation:



199

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

200

100

201 202

Serum ferritin

203

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

204

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

205

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

206

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

207

concentration of serum ferritin.

208 209

Serum hepcidin

210

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

211

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

212

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

213

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

214

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

215

unbound conjugate was washed off and a streptavidin-peroxidase enzyme

216

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

217

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

218

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

219

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

220

hepcidin in the sample.

221 222 223


Page 10 of 36

Page 11 of 36


224

Thyroid hormones, ghrelin, leptin, adiponectin and insulin measurement

225

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

226

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

227

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

228

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

229

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

230

Milliplex MAP Rat Adipocyte Panel Metabolism Assay (Millipore Corporation,

231

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

232

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

233

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

234

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

235

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

236

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

237

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

238

concentrations in plasma samples were determined by comparing the mean of

239

duplicate samples with the standard curve for each assay.

240 241

Non-esterified fatty acids (NEFA)

242

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

243

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

244

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

245

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

246

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

247

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

248

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



249

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

250

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

251

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

252

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

253

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

254

following equation:

255

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

256

of standard.

257 258

Western-Blot analysis and immunohistochemistry

259

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

260

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

261

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

262

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

263

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

264

protein concentration was determined in extract by using a Thermo Scientific

265

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

266

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

267

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

268

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

269

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

270

Rad Laboratories, Inc.) for 20 min.

271

A Fermentas PageRuler Plus Prestained Protein Ladder was used as

272

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

273

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


Page 12 of 36

Page 13 of 36


274

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

275

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

276

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

277

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

278

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

279

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

280

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

281

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

282

loaded.

283

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

284

incubated with the appropriate secondary conjugated antibody [ImmunStar Goat

285

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

286

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

287

The membranes were visualised with Luminata forte western HRP Substrate

288

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

289

densitometry of each band were performed with chemiluminescence in

290

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

291

All results were analysed with Image J software.

292 293

Statistical analysis

294

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

295

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

296

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

297

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

298

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



Page 14 of 36

299

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

300

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

301

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

302

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

Changes in Adiposity and Body Composition during Anemia Recovery

Recommend Documents