Running title: Preliminary findings from the EVASYON study 1

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Address for correspondence: 3

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Ascension Marcos, PhD. 5

Immunonutrition Research Group, Department of Metabolism and Nutrition, Institute of Food 6

Science, Technology and Nutrition (ICTAN), Spanish National Research Council (CSIC) 7

C/José Antonio Novais, 10 8

28040 Madrid, Spain 9

Phone: +34 915445607 10

Fax. + 34 91 5493627 11

E-mail: amarcos@if.csic.es 12

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Changes in cardiometabolic risk factors, appetite-controlling 23

hormones and cytokines after a treatment programme in overweight 24

adolescents: preliminary findings from the EVASYON study 25

26

Javier Romeo1, David Martinez-Gomez

1,2, L. Esperanza Diaz

1, Sonia Gómez-Martinez

1, 27

Amelia Marti3, Miguel Martin-Matillas

4, M. Angeles Puertollano

5, Oscar L. Veiga

2, J. 28

Alfredo Martinez3, Julia Wärnberg

6, Belen Zapatera

1, Jesus M. Garagorri

7, Gonzalo 29

Morandé8, Cristina Campoy

9, Luis A Moreno

10, Ascension Marcos

1; EVASYON Study 30

Group* 31

32

1Immunonutrition Research Group, Department of Metabolism and Nutrition, Institute of 33

Food Science, Technology and Nutrition (ICTAN), Spanish National Research Council 34

(CSIC), Madrid, Spain. 2Department of Physical Education, Sport and Human Movement, 35

Facultad de Formación del Profesorado y Educación, Universidad Autonoma de Madrid, 36

Madrid, Spain. 3Department of Physiology and Nutrition. University of Navarra. Pamplona. 37

Spain. 4Department of Physical Education and Sport, School of Physical Activity and Sport 38

Sciences, University of Granada, Granada, Spain. 5Department of Health Sciences. Faculty of 39

Experimental Sciences. University of Jaén. Jaén. Spain. 6Department of Preventive Medicine 40

and Public Health, School of Medicine, University of Navarra, Pamplona, Spain. 7Department 41

of Pediatric, Radiology and Physical Medicine, University of Zaragoza, Spain. 8Servicio de 42

Psiquiatría, Hospital Infantil Universitario Niño Jesús, Madrid, Spain. 9 Faculty of Medicine. 43

University of Granada. Granada. Spain. 10GENUD “Growth; Exercise, Nutrition and 44

Development” research group. University School of Health Sciences, University of Zaragoza, 45

Zaragoza, Spain. 46

47

EVASYON Study Group* 48

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Coordinator: Marcos A. 50

Local clinical treatment teams and researchers (Principal Investigators are italics); Granada: 51

Campoy C., López-Belmonte G., Delgado M., Martín-Matillas M., Aparicio V., Carbonell A., 52

Agil A., Silva D.R., Molina Font J.A., Pérez-Ballesteros C., Piqueras M.J., Chillón P., 53

Tercedor P., Martín-Lagos J.A., Martín-Bautista E., Pérez-Expósito M., Garófano M., Aguilar 54

M.J., Fernández-Mayorga A., Sánchez P.; Madrid: Marcos A., Gómez-Martínez S., Zapatera 55

B., Nova E., Romeo J., Díaz L.E., Pozo T., Wärnberg J., Puertollano M.A., Morandé G., 56

Villaseñor A., Madruga D., Muñoz R., Veiga O.L., Villagra A., Martínez-Gómez D., Garcia 57

R.M., Vaquero M.P., Pérez-Granados A.M., Navas-Carretero S.; Pamplona: Martí A., Azcona 58

C., Moleres A., Rendo T., Marqués M., Martínez J.A.; Santander: Redondo-Figuero C., 59

García-Fuentes M., DeRufino P., González-Lamuño D., Amigo T., Lanza R., Noriega M.J.; 60

Zaragoza: Garagorri J.M., Moreno L.A., Romero P., De Miguel P., Rodríguez G., Bueno G., 61

Mesana Ma.I., Vicente G., Fernández J., Rey-López P., Muro C., Tomás C.; Data 62

management and statistical analysis: Wärnberg J., Calle M.E., Barrios L. 63

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Word count for the manuscript: 2822 67

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ABSTRACT 70

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Objective: We investigated the effects of the EVASYON programme on body fatness, 72

cardiometabolic risk factors, gut appetite-controlling hormones and serum levels of cytokines 73

in adolescents with overweight or obesity (OW/OB). Methods: The study comprised 13 boys 74

(10 obese) and 12 girls (8 obese), aged 13 to 16 years, from a Madrid Hospital. The 75

EVASYON programme was based on a calorie-restricted diet (10-40%), increased physical 76

activity (at least 60 min/day 5 days a week), psychological therapy and nutritional education 77

for 13-month. Anthropometric and blood pressure measurements were measured before and 78

after intervention. Serum glucose, total cholesterol, high-density lipoprotein cholesterol, 79

triglycerides, leptin, total peptide YY and insulin levels were determined before and after 80

intervention. Serum levels of cytokines IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10 and TNF- 81

were also assessed before and after intervention. Results: A decrease in body mass index 82

(BMI), BMI z-score, skinfolds (triceps, biceps, subscapular, thigh, and calf), sum of 6 83

skinfolds and body circumferences (arm relaxed and flexed, waist, hip and proximal thigh) 84

values were observed after the intervention programme (all P<0.05). In addition, diastolic 85

blood pressure also decreased (P<0.05). A decrease in serum leptin levels (–48.4%, P<0.001) 86

was observed after intervention without changes in total peptide PYY and insulin levels. 87

Levels of IL-8, IL-10 and TNF- also decreased (all P<0.05) after the intervention 88

programme. Conclusions: These preliminary results evidence that the EVASYON 89

programme may improve body fat, leptin, and some pro-inflammatory cytokines in 90

adolescents with OW/OB. 91

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Key words: adolescents; obesity; metabolic syndrome; gut hormones; cytokines. 93

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INTRODUCTION 95

Obesity is the most prevalent metabolic disease in adult humans, which has been 96

associated with cardiometabolic risk factors, such as low-grade inflammation, insulin 97

resistance, hypertension and dislipidemia (1,2). Since some of these cardiometabolic risks 98

have been already identified in obese children and adolescents, early obesity underlies the 99

metabolic groundwork for adult cardiovascular diseases (3-6). 100

In this context, adipose tissue is a rich source of many immune-related mediators (e.g. 101

cytokines such as tumour-necrosis factor-alpha [TNF-] and interleukin-6 [IL-6]) that are 102

involved in the inflammatory response (4-6), mediating cardiometabolic disorders (7). 103

Furthermore, adipose tissue is currently regarded as an active endocrine organ that, since in 104

addition to regulating fat mass and energy homeostasis, it releases a large number of bioactive 105

mediators modulating appetite and metabolism (8). Some of these bioactive mediators are 106

hormones called adipokines and include leptin, adiponectin, peptide YY (PYY), among others 107

(9,10). Recent data also suggest that these hormones have immunomodulating effects 108

involving inflammatory processes (8-10). 109

In this sense, the EVASYON (Development, implementation and evaluation of the 110

efficacy of a therapeutic programme for adolescents with overweight and obesity: integral 111

education on nutrition and physical activity) programme comprised a long-term intervention 112

with calorie-restricted diet, physical activity, psychological therapy and nutritional education 113

in adolescents with overweight or obesity (OW/OB) in hospital settings from Spain. Since 114

inflammation associated with obesity declines after weight loss in adults (4), we hypothesized 115

that a programme aimed to lose fat mass might modify specific obesity-associated markers of 116

inflammation in adolescents. Therefore, this preliminary study was performed to characterize 117

the effects of the EVASYON treatment programme on body fat, cardiometabolic risk factors, 118

gut appetite-controlling hormones and cytokines levels in adolescents with OW/OB. 119

METHODS AND PROCEDURES 120

Study design and participants 121

The EVASYON study is a multidisciplinary treatment programme study conducted in 122

5 hospitals from 5 Spanish cities (Granada, Madrid, Pamplona, Santander and Zaragoza) in 123

OW/OB adolescents. The complete and detailed methodology of the EVASYON Study has 124

been described elsewhere (11). Briefly, the EVASYON treatment programme was conducted 125

in small groups (8-10 participants) during 13-month including twenty visits within two 126

specific stages: 1) Intensive intervention (9 visits): participants visited the hospitals weekly 127

for 2 months and one-week objectives were defined; 2) Extensive intervention (11 visits): 128

participants visited the hospital monthly during 11 months. In this stage, objectives for the 129

adolescents were to be accomplished in one month's time. 130

The EVASYON intervention included calorie-restricted diet (10 to 40%), increased 131

physical activity (at least 60 min/day 5 days a week), psychological therapy and nutritional 132

education for these 13 months. Paediatricians explained the patients several motivational 133

strategies, life and time management strategies including physical activity and sedentary 134

behaviour recommendations, adequate sleep time, nutritional advice, family involvement, 135

among others. 136

Adolescents included in the EVASYON treatment met the inclusion criteria for 137

participating: i) To be aged 13 to 16 years; ii) To be OW/OB in agreement with the 138

International Obesity Task Force age- and sex-specific body mass index (kg/m2, BMI) values 139

(12); iii) To be Spanish or to have been educated in Spain; iv) To be free of other diagnosed 140

disease. Moreover, adolescents in pharmacological treatment or diagnosed with anorexia, 141

bulimia or other eating disorder, except binge eating disorder, were excluded. 142

In order to evaluate the long-term effect of the programme, we analyzed preliminary 143

results corresponding to basal point (before the intervention) and after intensive and extensive 144

phases of interventions (2+11 months) in OW/OB adolescents from the Madrid Hospital 145

(Figure 1). A total of 31 adolescents (13 girls) finished the EVASYON programme in this 146

Hospital. This study encompassed 13 boys and 12 girls (n=25) with valid data on 147

anthropometry and blood samples before intervention and the end of the EVASYON 148

treatment. These adolescents maintained an apparently good health status and did not 149

consume any medications during the study. 150

The study was conducted in accordance with the ethical rules of the Helsinki 151

Declaration (Hong Kong revision, September 1989, in Edinburgh in 2000 and in Korea in 152

2008), following the EEC Good Clinical Practice guidelines (document 111/3976/88 of July 153

1990) and current Spanish law, which regulates clinical research in humans (Royal Decree 154

561/1993 regarding clinical trials). Informed consent was obtained from all adolescents and 155

their parents, and the study was approved by the local ethics committees. 156

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Anthropometry measurements 158

Body weight was measured without shoes and with light clothing to the nearest 0.05 159

kg by using a standard beam balance. Body height was measured using a stadiometer SECA 160

714 (range, 60-200 cm). BMI z-scores were calculated as a function of the subject’s obesity 161

degree when compared with BMI local reference standards (13). Skinfold thicknesses were 162

measured on the left side of the body (14) to the nearest 0.1 mm with a skinfold caliper 163

(Caliper Holtain; Holtain Ltd., Walles, UK) at triceps, biceps, subscapular, suprailiac, thigh, 164

and calf. The five circumferences (arm relaxed, arm flexed, waist, hip and proximal waist) 165

were measured in centimetres with an inelastic tape to the nearest millimetre. All the 166

anthropometric variables were measured in order, three times and averaged. For all the 167

anthropometric measurements, intraobserver reliability was >95% and interobserver 168

reliability was >90%. In the present study, the sum of 6 skinfolds and waist circumference 169

were used as indicators of total and central body fat, respectively. 170

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Resting blood pressure 172

Blood pressure was measured using a validated digital automatic blood pressure 173

monitor (Omron M6, Omron Health Care Co., Ltd., Kyoto, Japan) according to the 174

International Protocol of the European Society of Hypertension (15). 175

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Blood sampling 177

Blood samples were taken twice for each subject before and after the intervention (13-178

month). After an overnight fast (12-hour fast), subjects went to the hospital for blood 179

sampling at 8:00 a.m. Blood samples were collected by puncture of the cubital vein. For 180

biochemical analyses, the blood was collected in SST-Vacutainer (BD) and serum was 181

separated by centrifugation at 3000 rpm for 15 min at 22-24ºC, divided into aliquots, and 182

frozen and stored at –80ºC until withdrawn for analysis. 183

Biochemical parameters. Total cholesterol, HDL-cholesterol, triglycerides, and 184

glucose were analysed by a biochemical autoanalyser (Olympus model AU2700). All serum 185

analysis were performed at the end of the study, in order to have the three samples from each 186

subject analysed in the same run, to avoid systematic errors. Coefficients of variance of lipid 187

variables were 2% for total cholesterol, 2% for HDL-cholesterol and 3% for triacylglycerols. 188

VLDL-cholesterol and LDL-cholesterol were calculated from existing cholesterol, HDL-189

cholesterol, and triglycerides values (16). 190

Appetite-controlling hormones. Serum levels of leptin, total peptide YY, insulin and 191

adiponectin were measured by Luminex-100 IS (Integrated System: Luminex Corporation, 192

Austin, TX, USA) using the human gut hormone multiplex immunoassay kit (HGT-68K) and 193

Human CVD1 kit (HCVD1-67AK). The intra- and inter-assay precision coefficients of 194

variation were: 7% and 9% respectively for leptin; 5% and 10% respectively for total peptide 195

YY; 4% and 6% respectively for insulin; and 9% and 16% respectively for adiponectin. 196

Serum cytokines. Determination of serum cytokines IL-1β, IL-2, IL-4, IL-5, IL-6, IL-197

8, IL-10 and TNF- was performed on the Luminex-100 IS (Integrated System: Luminex 198

Corporation, Austin, TX, USA) using the multiplex assay kit Linco High Sensitivity Human 199

Cytokine Panel Lincoplex, 96 Well Plate Assay (HSCYTO-60SK), manufactured by Linco 200

Research, Inc., MO, USA. Multianalyte profiling calibration microspheres for classification 201

and reporter readings, as well as sheath fluid were also purchased from Luminex Corporation. 202

Acquired fluorescence data were analyzed by the Luminex-100 IS v.2.3. All analyses were 203

performed according to the manufacturer's protocols. The intra- and inter-assay precision 204

coefficients of variation were: 3% and 2%, respectively for IL-1β; 4% and 8%, respectively 205

for IL-2; 4% and 9%, respectively for IL-4; 5% and 14%, respectively for IL-5; 4% and 5%, 206

respectively for IL-6; respectively for IL-8; 3% and 12%, respectively for IL-10; and 4%, 207

respectively for TNF-. 208

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Statistical analyses 210

Data were assessed for normality and homogeneity of variance, and are expressed as 211

mean ± standard deviation (SD). Student’s t test was used to compare means between before 212

and after intervention (13-month). Due to non-normal distribution, the Wilcoxon test was 213

applied to appetite-controlling hormones and cytokine values to compare means between 214

before and after intervention. Effect sizes (ES) were also calculated using Cohen's d as a 215

measure of ES that reflects the magnitude of the difference between groups in SD units. 216

Cohen's d is computed by subtracting the average score for the control group (before the 217

intervention) from the average score for the treatment group (after the EVASYON treatment 218

programme), and then dividing the difference by the SD on the outcome measure for the 219

sample (17). We used the standard criteria for ES of small (d=0.20), medium (d=0.50), and 220

large effects (d=0.80). We employed these criteria along with the results of the statistical tests 221

in evaluating the impact of the previous results of EVASYON programme presented in the 222

present work. Analysis was performed using SPSS software v.17. Statistical significance was 223

set at P<0.05. 224

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RESULTS 243

Adolescents included in the final analysis were 14.1 ± 1.1 years old, and the 244

prevalence of obesity at baseline was 72% (10 boys and 8 girls). These subjects showed 245

marked differences in body fat characteristics after the intervention programme (Table 1). 246

Thus, significant decreases in BMI mean values (–6.8%) and BMI z-score (–28.0%) were 247

found after the intervention (all P<0.05). Levels of body fat measured by anthropometry at 248

triceps, biceps, subscapular, thigh, and calf also decreased after the intervention (all P<0.05). 249

The main indicators of total and central body fat, that is, the sum of 6 skinfolds (–17.8%) and 250

waist circumference (–3.8%), also significantly decreased after the intervention (both 251

P<0.05). 252

Regarding cardiometabolic risk factors included in the metabolic syndrome definition 253

(Table 2), a more favourable profile was found in all of them, but only diastolic blood 254

pressure (–7.6%) significantly decreased after the intervention programme (P=0.018). No 255

significant changes on adiponectin, total peptide YY and insulin levels were observed after 256

the intervention (Table 3). In contrast, a decrease (P<0.001) on serum leptin levels (–48.4%) 257

was found after the intervention (Table 3). Levels of IL-8 (–34.4%), IL-10 (–39.8%) and 258

TNF- (–21.5%) also significantly decreased (all P<0.05) after the intervention programme 259

(Table 4). 260

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DISCUSSION 268

These preliminary results of the EVASYON treatment programme indicate a clinically 269

relevant body fat reduction after the intervention programme in adolescents with OW/OB. 270

This outcome has been observed mainly in the sum of 6 skinfolds (large ES) as an indicator of 271

total body fat. Adolescence is characterised by an intense growth and development and in this 272

sense, it is important to stress the fact that although body weight did not decrease after 273

intervention, the decrease in BMI values observed is attributable to the increase in the body 274

height observed. These results also point out that in adolescents with OW/OB, the 275

improvement of body composition induced by the EVASYON intervention programme was 276

concomitant with decreased serum concentrations of leptin, IL-8, IL-10 and TNF-. 277

High serum leptin levels have been positively correlated with the components of the 278

metabolic syndrome in obesity during adolescence (18,19). The reduction of leptin levels 279

observed in the present study after body composition improvement is in agreement with other 280

trials (20,21). Although these studies did not evaluate the ES, the leptin decrease observed 281

after the EVASYON treatment programme showed a medium to large ES. Elloumi and co-282

workers (20) evaluated the effect of 2-month weight-loss programme (based on energy 283

restriction and training at the point of maximum lipid oxidation) on plasma levels of 284

adiponectin and leptin, in 21 obese adolescent boys. The authors found that the adolescents 285

who improved their body composition showed an increase in plasma adiponectin 286

concentrations (+74%) and a decrease in plasma leptin levels (–39%). In addition, a decrease 287

in plasma leptin levels (–70%) together with an increase in plasma adiponectin concentrations 288

(+27%) have been found after a 9-month multidisciplinary weight-reduction programme with 289

lifestyle education, moderate energy restriction and regular physical activity (21). Likewise, 290

our results have not reflected the increase in adiponectin levels previously reported by these 291

two studies and others (20-23), and seem to be in disagreement with the favourable body 292

composition changes observed after the intervention programme. 293

On the other hand, high means of BMI and waist circumference values have been 294

highly associated with elevated circulating levels of IL-6 and IL-8 (24). Since elevated levels 295

of pro-inflammatory cytokines correlate with obesity in adolescents confirming the presence 296

of early phases of atherosclerosis risk (25), the decrease in serum IL-8 and TNF- 297

concentrations (medium to large ES) observed in the current study after the intervention, 298

might suggest an attenuation of inflammation related risk factors. 299

To our knowledge, only a small number of studies concerning changes on serum 300

cytokine levels after intervention obesity programmes in adolescents are available. Thus, a 301

significant decrease in circulating IL-6 levels (–48%), associated with weight loss and 302

reduction of fat mass, have been reported (26) after short-term (3 weeks) under energy 303

restriction and exercise programme, in 49 obese adolescents. On the contrary, our results have 304

not reflected a significant decrease in IL-6 levels (–36%) after the intervention programme. 305

This conflicting outcome might be due to, for example, the differences in (i) the duration of 306

the interventions (3-week vs 13-month); (ii) IL-6 levels at baseline (3.9±4.7 vs 3.3±3.0 307

pg/mL); (iii) laboratory methods (ELISA vs Luminex); (iv) and sample sizes (49 vs 25 308

adolescents). 309

Although several authors have found no changes in TNF- in obese adolescents or 310

adults after weight loss (27-29), other studies in adults are in agreement with our results 311

showing decreased TNF- levels after weight loss (30-34). The release of pro-inflammatory 312

biomarkers might be one mechanism through which obesity is linked to increased leptin 313

values (7,10), and the current study concur with this hypothesis since leptin decreases were 314

concurrent with IL-8 and TNF- decreases after intervention. 315

Another conflicting finding in the present study is the decreased IL-10 levels observed 316

after our intervention programme. On the contrary, increased serum IL-10 levels have been 317

found after weight loss in obese adults (35). Since activated adipose tissue increases the 318

synthesis of pro-inflammatory cytokines while regulatory cytokines, such as IL-10 are 319

decreased (36); the decrease in IL-10 values observed after intervention seems to be 320

contradictory. In any case, no studies evaluating IL-10 cytokine after a weight loss 321

programme are available in adolescents. 322

Our results have shown relevant results. Obese subjects go on to display a 323

characteristic profile of hypertension, reduced HDL-cholesterol, and increased levels of LDL-324

cholesterol, triglycerides, and insulin resistance (metabolic syndrome), even during childhood 325

(37-39). In another study, after two-months of a physical-endurance and diet-restriction 326

programme in 24 obese adolescent boys, reductions in BMI and waist circumference (–13%) 327

were associated with decreased plasma triglycerides, LDL-cholesterol and total cholesterol 328

levels (40). Although a decrease in diastolic blood pressure was observed, the present study 329

has not reflected changes on cardiometabolic risk factors such as systolic blood pressure and 330

lipid profile after body composition reduction. The different findings found among studies 331

might be partially explained by the specific characteristics of each study (e.g. study duration, 332

sample size) as commented above. For example, other authors have detected an improvement 333

on lipid profile after decreasing BMI in 37 obese children who participated in a 12-month 334

intervention programme called "Obeldicks" (41). Although the duration of this programme 335

was one year, in this case, the age differences of the subjects involved in each study (mean 336

age 11 vs 14 years) could support the differences between their lipid profile results and our 337

current findings. 338

The novelty of the current study is that it is the first long-term study (13-month) 339

concerning the relationships between serum gut appetite-controlling hormones, serum 340

cytokines and changes in body composition in OW/OB adolescents. One limitation of the 341

present study includes the relative small sample size which could reduce the significance of 342

the detected changes and the ES. In addition, body fat was obtained by anthropometric 343

measurements and other methods (e.g. dual energy X-ray absorptiometry, air displacement 344

plethysmography, bioelectric impedance) may be more accurate for assessing body 345

composition changes. In any case, these preliminary results point out to a prominent body fat 346

reduction after the intervention programme, suggesting the potential usefulness of 347

implementing the EVASYON intervention programme for weight management in adolescents 348

with OW/OB. Future studies including the whole sample recruited in the EVASYON study 349

must analyse the role of important determinants (e.g. sexual maturation, socioeconomic 350

status, gene-environmental interactions) on the changes in body fat and cardiometabolic risks 351

during the EVASYON treatment programme. 352

In conclusion, our preliminary results indicate that the EVASYON programme may 353

produce moderate to large treatment effects for body fat reduction in adolescents with 354

OW/OB. Although the body composition improvement did not improve all the classic 355

metabolic syndrome components, these preliminary results suggest that the EVASYON 356

programme may reduce earlier cardiometabolic risk factors such as leptin, IL-8 and TNF- 357

levels, contributing to prevent future cardiovascular events. Nevertheless, further research is 358

required to examine whether this improvement in the inhibition of cardiometabolic risk 359

factors (mainly by an earlier decline in low-grade systemic inflammation) will eventually lead 360

to a clinical benefit associated with an improvement on body composition in children and 361

adolescents. 362

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ACKNOWLEDGEMENTS 366

The authors would like to thank the Spanish Ministry of Health and Consumption 367

(Carlos III Institute of Health. FIS. Grant PI 051579). D. Martínez-Gómez receives a 368

predoctoral fellowship from the Spanish Ministry of Education and Science (AP2006-02464). 369

M.A. Puertollano owned a research contract ("Juan de la Cierva") from the Spanish Ministry 370

of Education and Science. The EVASYON study has received the award from AESAN 371

(Spanish Agency for Food Security and Nutrition) from the Spanish Ministry of Health and 372

Consumption to the best applied research project in 2009. 373

374

DISCLOSURE 375

The authors declare that they have no competing interests. 376

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EXTENSIVE INTERVENTION

(10th-20th visit) 1 visit monthly

(11 months)

INTENSIVE INTERVENTION

(1st-9th visit) 1 visit weekly

(2 months)

RECRUITMENT IN A HOSPITAL FROM MADRID HEALTH

CARE SERVICE

GROUP I n=6

(3 girls)

GROUP II n=7

(2 girls)

GROUP III n=9

(5 girls)

GROUP IV n=9

(3 girls)

EVASYON treatment programme n=35

(15 girls)

EVASYON Inclusion/Exclusion criteria

Adolescents in Madrid with overweight or obesity

Screening N=42

(16 girls)

GROUP I n=6

(3 girls)

GROUP II n=9

(2 girls)

GROUP III n=10

(6 girls)

GROUP IV n=10

(4 girls)

Table 1. Anthropometric measurements before (baseline) and after the intervention (n=25)

Before the

intervention

After the EVASYON

treatment programme

Cohen's d

Weight (kg) 87.0 ± 14.6 84.7 ± 16.5 0.15

Height (cm) 166.3 ± 7.1 170.4 ± 7.7*** 0.56

BMI (kg/m2)

31.4 ± 4.4 29.2 ± 5.0*** 0.47

BMI z-score

4.7 ± 1.9 3.4 ± 1.9*** 0.70

Biceps skinfold (mm) 18.6 ± 5.1 13.4 ± 5.5** 0.99

Triceps skinfold (mm) 26.1 ± 3.4 22.6 ± 7.0** 0.64

Subscapular skinfold (mm) 29.0 ± 8.6 25.3 ± 8.3* 0.44

Suprailiac skinfold (mm) 29.3 ± 3.5 27.0 ± 7.2 0.41

Thigh skinfold (mm) 37.2 ± 3.4 30.2 ± 8.1*** 1.14

Calf skinfold (mm) 37.5 ± 4.7 28.2 ± 5.9*** 1.74

Sum of 6 skindfolds (mm) 177.9 ± 18.8 147.0 ± 36.2*** 1.08

Arm circumference (cm) 35.08 ± 3.6 33.4 ± 5.0** 0.40

Flexed arm circumference (cm) 35.8 ± 3.6 34.6 ± 4.9** 0.28

Waist circumference (cm) 101.2 ± 10.4 97.4 ± 13.5* 0.31

Hip circumference (cm) 112.6 ± 11.5 108.6 ± 11.0** 0.36

Proximal thigh circumference (cm) 70.4 ± 6.4 67.8 ± 7.7** 0.36

Values expressed as mean SD values. *P<0.05, **P<0.01, ***P<0.001, denotes statistical

significance between baseline and after the intervention. Student’s t test.

Table 2. Cardiometabolic risk factors before (baseline) and after the intervention (n=25)

Before the

intervention

After the EVASYON

treatment programme

Cohen's d

Fasting glucose (mg/dL) 98.4 ± 8.1 95.9 ± 5.8 0.35

Total cholesterol (mg/dL) 146.2 ± 23.2 143.8 ± 24.6 0.10

Triglycerides (mg/dL) 56.7 ± 22.6 53.3 ± 31.1 0.13

HDL-Cholesterol (mg/dL) 45.1 ± 10.1 45.8 ± 10.4 0.07

LDL- Cholesterol (mg/dL) 89.8 ± 18.4 87.3 ± 17.5 0.14

Systolic blood pressure (mm Hg) 130.4 ± 17.6 127.4 ± 3.8 0.24

Diastolic blood pressure (mm Hg) 70.2 ± 8.8 65.4 ± 5.4* 0.67

Values expressed as mean SD values. *P<0.05, denotes statistical significance between

baseline and after the intervention. Student’s t test.

Table 3. Serum appetite-controlling hormones before (baseline) and after the intervention

(n=25)

Before the

intervention

After the EVASYON

treatment programme

Cohen's d

Leptin (ng/mL) 22.0 ± 12.0 14.1 ± 10.1 * 0.71

Insulin (pg/mL) 488.6 ± 366.7 490.7 ± 444.2 0.01

Total peptide YY (pg/mL) 89.6 ± 47.0 87.6 ± 58.1 0.04

Adiponectin (ng/mL) 5.5 ± 8.7 5.4 ± 8.4 0.01

Values expressed as mean SD values. *P<0.05, denotes statistical significance between

baseline and after the intervention. Wilcoxon test.

Table 4. Serum cytokines before (baseline) and after the intervention (n=25)

Before the

intervention

After the EVASYON

treatment programme

Cohen's d

IL-1β (pg/mL) 1.1 ± 2.8 0.6 ± 1.2 0.21

IL-2 (pg/mL) 3.0 ± 5.3 2.2 ± 4.6 0.17

IL-6 (pg/mL) 3.3 ± 3.0 2.1 ± 1.4 0.50

IL-4 (pg/mL) 5.2 ± 4.7 4.8 ± 3.9 0.10

IL-8 (pg/mL) 10.0 ± 6.2 6.5 ± 3.8 * 0.66

IL-10 (pg/mL) 17.6 ± 9.0 13.4 ± 6.7 * 0.52

TNF-α (pg/mL) 10.2 ± 4.4 8.0 ± 2.7 * 0.60

Values expressed as mean SD values. TNF-α: tumour necrosis factor-α. *P<0.05, denotes

statistical significance between baseline and after the intervention. Wilcoxon test.